Antibody-opsonized Cells Research Articles

There Is Strength in Numbers: Quantitation of Fc Gamma Receptors on Murine Tissue-Resident Macrophages.

Many of the effector functions of antibodies rely on the binding of antibodies/immune complexes to cellular Fcγ receptors (FcγRs). Since the majority of innate immune effector cells express both activating and inhibitory Fc receptors, the outcome of the binding of immune complexes to cells of a given population is influenced by the relative affinities of the respective IgG subclasses to these receptors, as well as by the numbers of activating and inhibitory FcγRs on the cell surface. A group of immune cells that has come into focus more recently is the various subsets of tissue-resident macrophages. The central functions of FcγRs on tissue macrophages include the clearance of opsonized pathogens, the removal of small immune complexes from the circulation and the depletion of antibody-opsonized cells in the therapy of autoimmunity and cancer. Despite these essential functions of FcγRs on tissue-resident macrophages, an in-depth quantification of FcγRs is lacking. Thus, the aim of our current study was to quantify the various Fcγ receptors on macrophages in murine liver, lung, kidney, brain, skin and spleen. Our study identified a pronounced heterogeneity between FcγR expression patterns of the different tissue macrophages, which may reflect their specialized functions within their unique niches in different organ environments.

CD32a receptor in health and disease

Low-affinity Fcγ-receptors that recognize the Fc portion of immunoglobulin (Ig) molecules, usually being in antigen-bound state, thus representing a link between innate and adaptive immunity. They play a significant role in inflammatory and infectious diseases. Among them, a separate FcγRII family (CD32) is discerned, which is characterized by transmission of intracellular signal independently of the common γ-chain, they have one α-chain containing two extracellular immunoglobulin-like domains. FcγRII receptors are present in almost all cells of the innate immune system: monocytes and macrophages, neutrophils, eosinophils, dendritic cells, as well as on B-lymphocytes and platelets. They perform two main functions: target recognition, facilitation of phagocytosis and destruction of antibody-opsonized cells by monocytes/ macrophages (including pathogenic cells). In parallel, the phagocytes are activated via the cytokine synthesis stimulation. The FcγRIIA (CD32a) and FcγRIIC (CD32c) activating receptors, like as FcγRIIB (CD32b) inhibiting receptors are present among the members of the FcγRII family. The low-affinity FcγRII receptors bind to IgG, with immune complexes being their natural ligands. High levels of immune complexes are usually found in both chronic viral infections and autoimmune diseases. There are shown polymorphic variants of the CD32a gene, which can affect the receptor function, and, thereby, causing susceptibility for different infections, influence the development of autoimmune diseases and primary immunodeficiencies. Activation of the CD32a receptor induces the production of pro-inflammatory cytokines, including TNFα and interferons, that are involved into inflammation in systemic lupus erythematosus, Kawasaki disease, Graves’ disease and rheumatoid arthritis. It has been shown that antibacterial activity of platelets is carried out via the CD32a receptor. The study of CD32a expression in people The CD32a receptor is considered a biomarker of cells that are a reservoir of HIV infection. At the present time, however, many questions remain regarding the mechanisms of CD32a expression of on HIV-infected cells and the role of CD32a in the formation of an HIV reservoir and/or development of appropriate resistance. In addition to HIV infection, the significance of FcγR receptors is shown in other infectious diseases, for example, with influenza and dengue virus infections. Better understanding of the CD32a structure and function will help to assess its role in immunopathogenesis of different conditions. This review focuses on the role of CD32a in development of the normal immune response in normal state and various diseases.

Size-Dependent Segregation Controls Macrophage Phagocytosis of Antibody-Opsonized Targets

Macrophages protect the body from damage anddisease by targeting antibody-opsonized cells for phagocytosis. Though antibodies can be raised against antigens with diverse structures, shapes, and sizes, it is unclear why some are more effective at triggering immune responses than others. Here,we define an antigen height threshold that regulates phagocytosis of both engineered and cancer-specificantigens by macrophages. Using a reconstituted model of antibody-opsonized target cells, we find that phagocytosis is dramatically impaired for antigens that position antibodies >10nm from the target surface. Decreasing antigen height drives segregation of antibody-bound Fc receptors from the inhibitory phosphatase CD45 inan integrin-independent manner, triggering Fc receptor phosphorylation and promoting phagocytosis. Our work shows that close contact between macrophage and target is a requirement for efficient phagocytosis, suggesting that therapeutic antibodies should target short antigens in order to trigger Fc receptor activation through size-dependent physical segregation.

New roles and controls of mast cells.

Mast cells are innate immune cells implicated in immune surveillance and defense. They are filled with secretory granules where a vast array of molecules endowed with multiple biological activities are stored. The process of granule secretion, named degranulation, is a tightly controlled biological phenomenon that allows mast cells to rapidly and efficiently release bioactive mediators in response to extracellular stimuli. MC degranulation allows fighting pathogens, limiting envenomation and contributes to tissue homeostasis. However, it is also a potentially harmful response that plays a key role in the development of allergy and inflammatory diseases. Recent findings revealed that MC degranulation is a complex modular process that can be controlled at multiple levels. First, mast cells can decode different activation stimuli into two main patterns of degranulation that differently impact inflammatory responses. Second, mast cells in contact with antibody-opsonized cells or parasites form antibody-dependent degranulatory synapse for dedicated secretion and defense. Third, IL-33 fine-tunes FcR-mediated degranulation at the single cell level. Together these recent findings show how mast cells adapt their degranulation responses to environmental cues and highlight the remarkable functional plasticity of these cells.

Abstract 4601: Mechanisms of action for therapeutic antibody variants of human IgG3 isotype: Enhancing the CDC activity of cetuximab and rituximab

Abstract Objectives. Human IgG3 differs from other antibody isotypes by its extended hinge region and by its higher affinity for C1q. The ability of complement to promote lysis of antibody-opsonized cells is well-established and may represent a main mechanism in antibody mediated tumor therapy. The aim of this project was to investigate effector mechanisms of tumor- directed IgG3 antibodies in more detail. Experimental procedures. Therapeutic antibodies against EGFR (cetuximab, 225) and CD20 (rituximab, C2B8) were produced as IgG1 and IgG3 isotype variants by co-transfecting CHO cells with the respective de novo synthesized VH and VL domains fused to the respective heavy chain containing vectors. A Fab fragment of C2B8-IgG3 was crystallized and analyzed by X-ray structure analysis at PETRA III (DESY, Hamburg, Germany). Based on the resolved structure selected mutations were introduced into the CH1 domain of IgG3 aiming to improve or abolish CDC. Purified antibodies were assessed for complement fixation in ELISA and on tumor cells. Complement- and antibody-dependent cellular cytotoxicity (CDC and ADCC) activities were measured using 51chromium release assays. The significance of complement regulatory factors on target cells was investigated by siRNA mediated knock-down. A peritoneal tumor model was used to investigate the in vivo efficacy of IgG3 in comparison to IgG1 antibodies. Results. Switching isotypes from IgG1 to IgG3 did neither affect target antigen binding nor the proliferation inhibition by EGFR antibodies. Unexpectedly, the IgG3 antibodies were not very active in ADCC, in spite of their strong FcγRIII (CD16) binding affinity. However, for target cells expressing low antigen levels, the IgG3 isotype improved CDC against CD20 and enabled CDC against EGFR expressing tumor cells. The complement-regulatory factor CD55 in particular mitigated the IgG3 induced CDC activity. Structural comparison of Fab fragments of IgG1 and IgG3 demonstrated significant differences in the CH1 domains of the respective heavy chains, which contain the disulfide bond to the light chain and also harbor the docking interface for the complement factors C3b and C4b. Preliminary results from an peritoneal tumor model demonstrated that 225-IgG3 was effective in killing EGFR- expressing cells. This in vivo activity was abolished by cobra venom factor treatment. Conclusion. Switching the isotype of a therapeutic IgG1 antibody to IgG3 represents a promising approach to enable complement-mediated killing of tumor cells. Protein engineering of the C3b/C4b interaction interface in the CH1 domain may constitute a novel strategy to modulate the CDC activity against tumor target cells. Together, these approaches may enhance the CDC efficacy of CD20- and EGFR- directed antibodies. Citation Format: Ralf Schwanbeck, Anna Skof, Thies Rösner, Marco Jansen, Anina Räuchle, Anna Kretschmer, Axel Scheidig, Jeanette Leusen, Stefanie Derer, Thomas Valerius. Mechanisms of action for therapeutic antibody variants of human IgG3 isotype: Enhancing the CDC activity of cetuximab and rituximab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4601. doi:10.1158/1538-7445.AM2017-4601

A fully recombinant human IgG1 Fc multimer (GL-2045) inhibits complement-mediated cytotoxicity and induces iC3b

AbstractGL-2045 is a recombinant human immunoglobulin G1 (IgG1)–based Fc multimer designed to recapitulate the anti-inflammatory activities of intravenous immunoglobulin (IVIG) on the innate and adaptive immune responses. We used functional in vitro studies to determine if GL-2045 could mimic the modulatory activity of IVIG on complement activation. GL-2045, at log-order lower concentrations than heat-aggregated IgG (HAGG) and IVIG, protected antibody-opsonized cells from complement-dependent cytotoxicity. These protective effects were completely mediated by the higher order multimer fractions of GL-2045 and were partially dependent upon sequestration of C1q. Exposure of serum to GL-2045 and, to a lesser extent, IVIG, resulted in high levels of C4a, limited levels of C3a, and no C5a. In contrast, HAGG induced high levels of C4a, C3a, and C5a. The means by which GL-2045 governed complement activation was dependent on its ability to augment the function of factor H, alone and in combination with factor I, to indirectly limit the alternative form of C3 convertase, with resultant increases in the anti-inflammatory molecule, the “inactive” form of C3b, called iC3b. Although IVIG, like GL-2045, potentiated factor H function, it also directly inhibited the alternative form of C3 convertase. Our findings help elucidate how IVIG, GL-2045, and HAGG regulate complement function. Furthermore, the capacity of GL-2045 to sequester C1q and augment factor H activity, in combination with its ability to generate activation-induced immunomodulatory complement split products, such as iC3b, make it a viable drug candidate for the treatment of diverse complement-mediated diseases.

Highlights of This Issue

Current treatment of medulloblastoma often relies on the use of radiation, causing severe side effects that can greatly compromise the quality of life for these children. Markowitz, Powell, and colleagues identified the protein kinase MRK/ZAK as a radiosensitization target for medulloblastoma. The authors also designed an irreversible inhibitor of MRK/ZAK, M443 that effectively radiosensitizes patient-derived medulloblastoma cells both in vitro and in vivo. Further development of M443 may allow for lowering radiation doses delivered to patients, while preserving therapeutic efficacy.In this study, Miller and colleagues developed a new class of DNA alkylating cytotoxic agents for use as payloads in antibody–drug conjugates (ADC), designed to extend the types of cancer potentially treatable with ADC therapeutics. Through innovative chemical design, a novel DNA-interacting compound was transformed from acting as a DNA cross-linker to an alkylator. Through this fundamental modification, and via linker optimization, DNA alkylating ADCs characterized by a high therapeutic index were created. These findings represent a major advancement in ADC technology, designed to ultimately offer cancer patients a more tolerable and highly active therapeutic option.Trogocytosis involves the ingestion of fragments of antibody-opsonized cells by effector cells such as macrophages through a “gnawing”-like process. The effects of this process on the survival of tumor cells are not well defined. Velmurugan and colleagues use a combination of advanced microscopy and cellular assays to demonstrate that persistent trogocytosis by macrophages can result in death of HER2-positive breast cancer cells in the presence of the HER2-specific antibody trastuzumab. This study extends the antitumor activities of macrophages to trogocytosis and has relevance to the use of therapeutic antibodies in cancer.A novel HER4 mutation was identified in a head and neck squamous cell carcinoma cell line. This mutation is an activating oncogenic mutation with a transformation ability and tumorigenicity, and the growth of this cell line is dependent on this mutation. Afatinib exhibited dramatic anticancer effect against this cell line by inhibiting the HER4 signal, both in vitro and in vivo. These results provide a novel insight regarding afatinib as a HER4 inhibitor for HER4-mutated cancers, and a HER4 mutation as a potential predictive biomarker for afatinib.

Abstract PR08: Macrophage-mediated trogocytosis leads to death of antibody-opsonized tumor cells

Abstract The interplay between innate immune cells and antibodies to control cancerous growth is of central importance for mediating direct anti-tumor effects and the induction of long-lived anti-tumor immunity. Phagocytosis of antibody-opsonized cells by macrophages through Fcgamma receptor interactions is well characterized and leads to cell death through target cell engulfment into phagosomes. By contrast, the contribution of the related process called trogocytosis, involving the ingestion of limited amounts of the target cell by phagocytic cells, to cell attrition is less certain. In the current study we have developed a high throughput, quantitative assay to distinguish whole cell phagocytosis (WCP) and trogocytosis for different macrophage:breast cancer cell combinations. These analyses, combined with long term microscopic imaging, demonstrate that trogocytosis leads to target cell death. The implementation of multifocal plane microscopy to investigate trogocytic events at high spatiotemporal resolution reveals that these processes involve tubular extensions of opsonized tumor cells that are subsequently pinched off by the recipient macrophage. Of relevance to the design of second generation antibodies with improved efficacy, antibody engineering to enhance Fcgamma receptor interactions results in increased trogocytic activity. Collectively, these studies have significance to the rapidly expanding use of antibodies to treat cancer. Citation Format: Ramraj Velmurugan, Dilip K. Challa, Sripad Ram, Raimund J. Ober, E. Sally Ward. Macrophage-mediated trogocytosis leads to death of antibody-opsonized tumor cells. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR08.

Abstract B089: Macrophage-mediated trogocytosis leads to death of antibody-opsonized tumor cells

Abstract The interplay between innate immune cells and antibodies to control cancerous growth is of central importance for mediating direct anti-tumor effects and the induction of long-lived anti-tumor immunity. Phagocytosis of antibody-opsonized cells by macrophages through Fc gamma receptor interactions is well characterized and leads to cell death through target cell engulfment into phagosomes. By contrast, the contribution of the related process called trogocytosis, involving the ingestion of limited amounts of the target cell by phagocytic cells, to cell attrition is less certain. In the current study we have developed a high throughput, quantitative assay to distinguish whole cell phagocytosis and trogocytosis for different macrophage:breast cancer cell combinations. These analyses, combined with long term microscopic imaging, demonstrate that trogocytosis leads to death of HER2-overexpressing breast tumor cells. The implementation of multifocal plane microscopy to investigate trogocytic events at high spatiotemporal resolution reveals that these processes involve tubular extensions of opsonized tumor cells that are subsequently pinched off by the recipient macrophage. Of relevance to the design of second generation antibodies with improved efficacy, antibody engineering to enhance Fc gamma receptor interactions results in increased trogocytic activity. Collectively, these studies have significance to the rapidly expanding use of antibodies to treat cancer. This research was supported in part by grants from the Cancer Prevention and Research Institute of Texas (CPRIT, RP 110069) and the National Institutes of Health (R01 GM85575). Citation Format: Ramraj Velmurugan, Dilip Challa, Sripad Ram, Raimund Ober, E. Sally Ward. Macrophage-mediated trogocytosis leads to death of antibody-opsonized tumor cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B089.

Reticuloendothelial Saturation Dictates the Development of RBC Resistance to Antibody-Mediated Clearance Following Incompatible Transfusion

Background: Immune-mediated destruction of red blood cells (RBCs) normally occurs following incompatible transfusion, however some RBCs may remain in circulation after initial cellular clearance despite the presence of RBC specific antibodies. Recent studies suggest that antibody-induced “antigen-modulation” may in part be responsible for this cellular resistance to antibody-mediated removal following the initial phase of RBC clearance. However, the factors that dictate whether cells undergo clearance or antigen modulation immediately following incompatible RBC transfusion remain unknown. As previous studies suggest that reticuloendothelial phagocytic capacity is sometimes surpassed upon engagement and removal of antibody-opsonized cells, we hypothesized that rapid change in the phagocytic burden of reticuloendothelial cells may contribute to the development of RBC resistance following incompatible RBC transfusion.Methods: Anti-Fy3 immunized or non-immunizedC57BL/6 recipient mice were first transfused with HOD.FVB RBCs expressing the HEL, OVA and Duffy chimeric antigen (HOD), followed by a second HOD.FVB RBC transfusion at various time intervals following the initial transfusion. Following transfusion, mice were evaluated at 10 min, 1h, 2h and 4h post-transfusion for HOD.FVB RBC survival, detectable HOD antigen, and RBC bound antibody by flow cytometric analysis. To examine the potential impact of RBC age on the sensitivity of HOD.FVB to antibody-induced clearance, HOD.FVB mice were injected with N-hydroxysulfosuccinimide (NHS) biotin 35 days prior to RBC isolation, to aid in differentiation of older from younger transfused RBCs, and then likewise transfused into anti-Fy3 immunized or non-immunizedC57BL/6 recipients and similarly evaluated for clearance, bound antibody and detectable antigen on biotin positive or negative transfused cells using flow cytometric analysis.Results: While HOD.FVB RBCs transfused into anti-Fy3-immunized C57BL/6 recipients displayed a rapid clearance following the initial transfusion, the degree of HOD.FVB RBC clearance significantly decreased following secondary transfusions that occurred within 2 hours of the initial transfusion. However, 2 days following the initial transfusion, recipients cleared HOD.FVB RBCs at the same rate as immunized recipients not previously exposed to HOD.FVB RBCs. Examination of the HOD antigen and bound antibody demonstrated that antigen levels likewise displayed the most significant decreases during the same time period in which RBC clearance decreased. Removal of HOD RBCs during the initial clearance phase did not appear to reflect preferential clearance of older RBCs, as transfused biotinylated and non-biotinylated RBCs harvested 1 day or 35 days following biotinylation displayed equal levels of antibody-induced clearance.Conclusion: Alterations in recipient clearance capacity following incompatible RBC exposure suggests that rapid phagocytic removal of RBCs may temporarily staturate the reticuloendothelial system immediately following RBC transfusion. However, antigen modulation appears to continue even while congestion of the reticuloendothelial system has halted RBC clearance. RBCs that escape the initial wave of clearance appear to become protected from antibody-mediated removal once the phagocytic capability of the recipient is restored, as the level of detectable antigen has dropped below that required for triggering phagocytic removal. The initial phase of clearance does not appear to reflect preferential clearance of older RBCs within the transfused units, but instead likely reflects stochastic removal of antibody bound RBCs, as RBC clearance occurred independent of RBC age. DisclosuresNo relevant conflicts of interest to declare.

The role of complement in mAb-based therapies of cancer

The ability of complement to promote lysis of antibody-opsonized cells is well-established. Virtually all of the molecular details of this reaction have been elucidated and numerous points of regulation have also been delineated. Use of this information, along with the techniques that were first applied in the fundamental studies of complement, has allowed for investigations of the role of complement in mAb-based immunotherapies of cancer. These studies, which have often combined in vitro investigations with parallel correlative clinical measurements, have revealed that several FDA-approved mAbs make use of complement as an effector function in promoting opsonization and killing of targeted malignant cells. We describe the key methods used in this work, and discuss how the results of these studies provide rational approaches for making more effective use of complement in mAb-based cancer immunotherapy.

Within Peripheral Blood Mononuclear Cells, Antibody-Dependent Cellular Cytotoxicity of Rituximab-Opsonized Daudi cells Is Promoted by NK Cells and Inhibited by Monocytes due to Shaving

Treatment of chronic lymphocytic leukemia patients with anti-CD20 mAb rituximab (RTX) leads to substantial CD20 loss on circulating malignant B cells soon after completion of the RTX infusion. This CD20 loss, which we term shaving, can compromise the therapeutic efficacy of RTX, and in vitro models reveal that shaving is mediated by effector cells which express Fc gammaRI. THP-1 monocytes and PBMC promote shaving, but PBMC also kill antibody-opsonized cells by antibody-dependent cellular cytotoxicity (ADCC), a reaction generally considered to be due to NK cells. We hypothesized that within PBMC, monocytes and NK cells would have substantially different and competing activities with respect ADCC or shaving, thereby either enhancing or inhibiting the therapeutic action of RTX. We measured ADCC and RTX removal from RTX-opsonized Daudi cells promoted by PBMC, or mediated by NK cells and monocytes. NK cells take up RTX and CD20 from RTX-opsonized B cells, and mediate ADCC. PBMC depleted of NK cells show little ADCC activity, whereas PBMC depleted of monocytes have greater ADCC than the PBMC. Pre-treatment of RTX-opsonized B cells with THP-1 cells or monocytes suppresses NK cell-mediated ADCC, and blockade of Fc gammaRI on monocytes or THP-1 cells abrogates their ability to suppress ADCC. Our results indicate NK cells are the principal cells in PBMC that kill RTX-opsonized B cells, and that monocytes can suppress ADCC by promoting shaving. These results suggest that RTX-based immunotherapy of cancer may be enhanced based on paradigms which include infusion of compatible NK cells and inhibition of monocyte shaving activity.