ABSTRACT
Novel molecules that directly target the neonatal Fc receptor (FcRn) and/or Fc gamma receptors (FcγRs) are emerging as promising treatments for immunoglobulin G (IgG)-dependent autoimmune pathologies. Mutated Fc regions and monoclonal antibodies that target FcRn are currently in clinical development and hold promise for reducing the levels of circulating IgG. Additionally, engineered structures containing multimeric Fc regions allow the dual targeting of FcRn and FcγRs; however, their tolerance needs to first be validated in phase I clinical studies. Here, for the first time, we have developed a modified monomeric recombinant Fc optimized for binding to all FcRns and FcγRs without the drawback of possible tolerance associated with FcγR cross-linking. A rational approach using Fc engineering allowed the selection of LFBD192, an Fc with a combination of six mutations that exhibits improved binding to human FcRn and FcγR as well as mouse FcRn and FcγRIV. The potency of LFBD192 was compared with that of intravenous immunoglobulin (IVIg), an FcRn blocker (Fc-MST-HN), and a trimeric Fc that blocks FcRn and/or immune complex-mediated cell activation through FcγR without triggering an immune reaction in several in vitro tests and validated in three mouse models of autoimmune disease.
Subject(s)
Antirheumatic Agents/pharmacology , Arthritis, Experimental/prevention & control , Autoimmunity/drug effects , Immunoglobulin Fc Fragments/pharmacology , Receptors, Fc/antagonists & inhibitors , Receptors, IgG/antagonists & inhibitors , Animals , Antirheumatic Agents/metabolism , Arthritis, Experimental/genetics , Arthritis, Experimental/immunology , Arthritis, Experimental/metabolism , Binding, Competitive , Complement C5a/metabolism , Female , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/metabolism , Humans , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/immunology , Immunoglobulin Fc Fragments/metabolism , Interleukin-2/metabolism , Jurkat Cells , Kinetics , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Phagocytosis/drug effects , Platelet Aggregation/drug effects , Protein Binding , Protein Engineering , Receptors, Fc/genetics , Receptors, Fc/immunology , Receptors, Fc/metabolism , Receptors, IgG/genetics , Receptors, IgG/immunology , Receptors, IgG/metabolism , Secretory Pathway , Signal Transduction , THP-1 CellsABSTRACT
The interaction of the Fc region of therapeutic antibodies and antibody-drug conjugates with Fcγ receptors (FcγRs) can lead to unpredictable and severe side effects. Over the last decades several strategies have been developed to overcome this drawback, including extensive Fc- and glycoengineering and antibody isotype switching. However, these approaches result in permanently Fc-silenced antibody derivates which partially or completely lack antibody-mediated effector functions. Nevertheless, for a majority of antibody-based drugs, Fc-mediated effector functions, like antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) as well as complement-dependent cytotoxicity (CDC), represent the most substantial modes of action. We argued that a new strategy combining the beneficial properties of Fc-silencing and controlled activation of effector functions can pave the way to potent antibody therapeutics, reducing the FcγRs-mediated off-target toxicity. We present a novel Fc-tamed antibody format, where the FcγR-binding sites of antibodies are blocked by anti-isotypic masking units, hindering the association of FcγR and complement component 1 (c1q) to the Fc domain. The masking units were genetically fused to trastuzumab, including a protease-addressable peptide-liker. Our Fc-tamed antibodies demonstrated completely abolished interaction to soluble high-affinity Fcγ-Receptor I and c1q. In reporter cell-based ADCC assays, our Fc-tamed antibodies exhibited a 2,700 to 7,100-fold reduction in activation, compared to trastuzumab. Upon demasking by a tumor-associated protease, the Fc-activated antibodies demonstrated restored FcγR-binding, c1q-binding and the ability to induce potent ADCC activation. Furthermore, cell killing assays using donor-derived NK cells were performed to validate the functionality of the Fc-tamed antibody variants. To our knowledge, this approach represents the first non-permanently Fc-silenced antibody, which can be re-activated by a tumor-associated protease, eventually extending the field of novel antibody formats.
Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Drug-Related Side Effects and Adverse Reactions , Immunoglobulin Fc Fragments/immunology , Peptide Hydrolases/metabolism , Animals , Antibody-Dependent Cell Cytotoxicity , Antineoplastic Agents, Immunological/administration & dosage , Antineoplastic Agents, Immunological/adverse effects , Biomarkers , Cell Line, Tumor , Chickens , Humans , Hydrolysis , Matrix Metalloproteinase 9/metabolism , Receptors, IgG/antagonists & inhibitors , Receptors, IgG/metabolism , Single-Chain Antibodies/administration & dosage , Single-Chain Antibodies/adverse effects , Single-Chain Antibodies/pharmacology , Trastuzumab/administration & dosage , Trastuzumab/adverse effects , Trastuzumab/pharmacologyABSTRACT
Bone erosion is one of the primary features of inflammatory arthritis and is caused by excessive differentiation and activation of osteoclasts. Fc gamma receptors (FcγRs) have been implicated in osteoclastogenesis. Our recent studies demonstrate that joint-deposited lupus IgG inhibited RANKL-induced osteoclastogenesis. FcγRI is required for RANKL-induced osteoclastogenesis and lupus IgG-induced signaling transduction. We reviewed the results of studies that analyzed the association between FcγRs and bone erosion in inflammatory arthritis. The analysis revealed the dual roles of FcγRs in bone destruction in inflammatory arthritis. Thus, IgG/FcγR signaling molecules may serve as potential therapeutic targets against bone erosion.
Subject(s)
Arthritis/metabolism , Bone Remodeling , Bone and Bones/metabolism , Osteoclasts/metabolism , Osteogenesis , Receptors, IgG/metabolism , Animals , Anti-Inflammatory Agents/therapeutic use , Antibodies, Monoclonal/therapeutic use , Arthritis/drug therapy , Arthritis/immunology , Arthritis/pathology , Bone Remodeling/drug effects , Bone and Bones/drug effects , Bone and Bones/immunology , Bone and Bones/pathology , Humans , Immunoglobulin G/metabolism , Immunotherapy , Osteoclasts/drug effects , Osteoclasts/immunology , Osteoclasts/pathology , Osteogenesis/drug effects , RANK Ligand/metabolism , Receptors, IgG/antagonists & inhibitors , Receptors, IgG/immunology , Signal TransductionABSTRACT
Epidermal growth factor receptor (EGFR)-targeted cancer therapy such as anti-EGFR monoclonal antibodies and tyrosine kinase inhibitors have demonstrated clinical efficacy. However, there remains a medical need addressing limitations of these therapies, which include a narrow therapeutic window mainly due to skin and organ toxicity, and primary and secondary resistance mechanisms of the EGFR-signaling cascade (e.g., RAS-mutated colorectal cancer). Using the redirected optimized cell killing (ROCK®) antibody platform, we have developed AFM24, a novel bispecific, IgG1-scFv fusion antibody targeting CD16A on innate immune cells, and EGFR on tumor cells. We herein demonstrate binding of AFM24 to CD16A on natural killer (NK) cells and macrophages with KD values in the low nanomolar range and to various EGFR-expressing tumor cells. AFM24 was highly potent and effective for antibody-dependent cell-mediated cytotoxicity via NK cells, and also mediated antibody-dependent cellular phagocytosis via macrophages in vitro. Importantly, AFM24 was effective toward a variety of EGFR-expressing tumor cells, regardless of EGFR expression level and KRAS/BRAF mutational status. In vivo, AFM24 was well tolerated up to the highest dose (75 mg/kg) when administered to cynomolgus monkeys once weekly for 28 days. Notably, skin and other toxicities were not observed. A transient elevation of interleukin-6 levels was detected at all dose levels, 2-4 hours post-dose, which returned to baseline levels after 24 hours. These results emphasize the promise of bispecific innate cell engagers as an alternative cancer therapy and demonstrate the potential for AFM24 to effectively target tumors expressing varying levels of EGFR, regardless of their mutational status.Abbreviations: ADA: antidrug antibody; ADCC: antibody-dependent cell-mediated cytotoxicity; ADCP: antibody-dependent cellular phagocytosis; AUC: area under the curve; CAR: chimeric-antigen receptor; CD: Cluster of differentiation; CRC :colorectal cancer; ECD: extracellular domain; EGF: epidermal growth factorEGFR epidermal growth factor receptor; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; Fc: fragment, crystallizableFv variable fragment; HNSCC: head and neck squamous carcinomaIL interleukinm; Ab monoclonal antibody; MOA: mechanism of action; NK :natural killer; NSCLC: non-small cell lung cancer; PBMC: peripheral blood mononuclear cell; PBS: phosphate-buffered saline; PD: pharmacodynamic; ROCK: redirected optimized cell killing; RSV: respiratory syncytial virus; SABC: specific antibody binding capacity; SD: standard deviation; TAM: tumor-associated macrophage; TKI: tyrosine kinase inhibitor; WT: wildtype.
Subject(s)
Antibodies, Bispecific , Antineoplastic Agents, Immunological , Killer Cells, Natural/immunology , Macrophages/immunology , Neoplasm Proteins , Neoplasms/drug therapy , Phagocytosis/drug effects , Receptors, IgG , A549 Cells , Animals , Antibodies, Bispecific/immunology , Antibodies, Bispecific/pharmacology , Antineoplastic Agents, Immunological/immunology , Antineoplastic Agents, Immunological/pharmacology , Drug Screening Assays, Antitumor , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/immunology , HCT116 Cells , HT29 Cells , Humans , Killer Cells, Natural/pathology , MCF-7 Cells , Macaca fascicularis , Macrophages/pathology , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/immunology , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/pathology , Receptors, IgG/antagonists & inhibitors , Receptors, IgG/immunologyABSTRACT
BACKGROUND: Palmitic acid (PA) promotes brain pathologies including Alzheimer's disease (AD)-related proteins, neuroinflammation, and microglial activation. The activation of neurons and microglia via their Fc gamma receptors (FcγRs) results in producing inflammatory cytokines. OBJECTIVE: To investigate the expression of FcγRs, FcγR signaling proteins, AD-related proteins, proinflammatory cytokines, and cell viability of neurons and microglia in association with PA exposure as well as the effects of FcγR blockade on these parameters in response to PA. METHODS: 200 and 400µM PA-conjugated BSA were applied to SH-SY5Y and HMC3 cells for 24âh. For FcγR blockage experiment, both cells were exposed to FcγR blocker before receiving of 200 and 400µM of PA-conjugated BSA for 24âh. RESULTS: PA significantly increased AD-related proteins, including Aß and BACE1, as well as increasing TNFα, IL-1ß, and IL-6 in SH-SY5Y and HMC3 cells. However, the p-Tau/Tau ratio was only increased in SH-SY5Y cells. These results were associated with an increase in FcγRs activation and a decrease in cell viability in both cell types. FcγRs blockage diminished the activation of FcγR in SH-SY5Y and HMC3 cells. Interestingly, blocking FcγRs before PA exposure reduced the increment of AD-related proteins, proinflammatory cytokines caused by PA. FcγRs blocking also inhibits cell death for 23%of SH-SY5Y cells and 64%of HMC3 cells, respectively. CONCLUSION: These findings suggest that PA is a risk factor for AD via the increased AD-related pathologies, inflammation, FcγRs activation, and brain cell death, while FcγR blockage can alleviate these effects.
Subject(s)
Microglia/metabolism , Neurons/metabolism , Palmitic Acid/toxicity , Receptors, IgG/antagonists & inhibitors , Receptors, IgG/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/physiology , Enzyme Inhibitors/toxicity , Humans , Microglia/drug effects , Neurons/drug effectsABSTRACT
Natural killer cell engagers gained enormous interest in recent years due to their potent anti-tumor activity and favorable safety profile. Simultaneously, chicken-derived antibodies entered clinical studies paving the way for avian-derived therapeutics. In this study, we describe the affinity maturation of a common light chain (cLC)-based, chicken-derived antibody targeting EGFR, followed by utilization of the same light chain for the isolation of CD16a- and PD-L1-specific monoclonal antibodies. The resulting binders target their respective antigen with single-digit nanomolar affinity while blocking the ligand binding of all three respective receptors. Following library-based humanization, bispecific and trispecific variants in a standard 1 + 1 or a 2 + 1 common light chain format were generated, simultaneously targeting EGFR, CD16a, and PD-L1. The trispecific antibody mediated an elevated antibody-dependent cellular cytotoxicity (ADCC) in comparison to the EGFR×CD16a bispecific variant by effectively bridging EGFR/PD-L1 double-positive cancer cells with CD16a-positive effector cells. These findings represent, to our knowledge, the first detailed report on the generation of a trispecific 2 + 1 antibodies exhibiting a common light chain and illustrate synergistic effects of trispecific antigen binding. Overall, this generic procedure paves the way for the engineering of tri- and oligospecific therapeutic antibodies derived from avian immunizations.
Subject(s)
Antibodies, Bispecific/pharmacology , Antibodies, Monoclonal, Humanized/pharmacology , Drug Design , Epitopes , Immune Checkpoint Inhibitors/pharmacology , Immunoglobulin Light Chains/pharmacology , Killer Cells, Natural/drug effects , Skin Neoplasms/drug therapy , Animals , Antibodies, Bispecific/immunology , Antibodies, Monoclonal, Humanized/immunology , Antibody Specificity , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/immunology , B7-H1 Antigen/metabolism , Cell Line, Tumor , Chickens , Cytotoxicity, Immunologic/drug effects , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/immunology , ErbB Receptors/metabolism , Immune Checkpoint Inhibitors/immunology , Immunization , Immunoglobulin Light Chains/immunology , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Receptors, IgG/antagonists & inhibitors , Receptors, IgG/immunology , Receptors, IgG/metabolism , Skin Neoplasms/immunology , Skin Neoplasms/metabolism , Skin Neoplasms/pathologyABSTRACT
Soft tissue sarcoma (STS) constitutes a rare group of heterogeneous malignancies. Effective treatment options for most subtypes of STS are still limited. As a result, especially in metastatic disease, prognosis is still dismal. The ligands for the activating immunoreceptor NKG2D (NKG2DL) are commonly expressed in STS, but generally absent in healthy tissues. This provides the rationale for utilization of NKG2DL as targets for immunotherapeutic approaches. We here report on the preclinical characterization of bispecific fusion proteins (BFP) consisting of the extracellular domain of the NKG2D receptor fused to Fab-fragments directed against CD3 (NKG2D-CD3) or CD16 (NKG2D-CD16) for treatment of STS. After characterization of NKG2DL expression patterns on various STS cell lines, we demonstrated that both NKG2D-CD16 and NKG2D-CD3 induce profound T and NK cell reactivity as revealed by analysis of activation, degranulation and secretion of IFNγ as well as granule associated proteins, resulting in potent target cell lysis. In addition, the stimulatory capacity of the constructs to induce T and NK cell activation was analyzed in heavily pretreated STS patients and found to be comparable to healthy donors. Our results emphasize the potential of NKG2D-CD3 and NKG2D-CD16 BFP to target STS even in an advanced disease.
Subject(s)
CD3 Complex/antagonists & inhibitors , NK Cell Lectin-Like Receptor Subfamily K/genetics , Receptors, IgG/antagonists & inhibitors , Recombinant Fusion Proteins/therapeutic use , Sarcoma/drug therapy , Adult , Aged , Antibodies, Bispecific/genetics , Antibodies, Bispecific/pharmacology , CD3 Complex/metabolism , Cell Degranulation/drug effects , Cell Degranulation/immunology , Cell Line, Tumor , Female , GPI-Linked Proteins/antagonists & inhibitors , GPI-Linked Proteins/metabolism , Humans , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Lymphocyte Activation/drug effects , Male , Middle Aged , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Primary Cell Culture , Protein Domains/genetics , Receptors, IgG/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/pharmacology , Sarcoma/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Young AdultABSTRACT
Multispecific antibodies that bridge immune effector and tumor cells have shown promising preclinical and clinical efficacies. Here, we isolated and characterized novel llama single-domain antibodies (sdAbs) against CD16. One sdAb, NRC-sdAb048, bound recombinant human and cynomolgus monkey CD16 ectodomains with equivalent affinity (KD: 1 nM) but did not recognize murine CD16. Binding was similar for human CD16a expressed on NK cells and CD16b (NA2) expressed on neutrophils but dramatically weaker (KD: â¼6 µM) for the CD16b (NA1) allotype. The sdAb stained primary human peripheral blood NK cells. Irrespective of fusion orientation and linker length, bispecific sdAb-sdAb and sdAb-scFv dimers (anti-CD16/EGFR, anti-CD16/HER2, and anti-CD16/CD19) retained full binding affinity for each target, coengaged both antigens simultaneously, elicited ADCC against target antigen-expressing tumor cells in a reporter bioassay, and triggered target-specific activation and degranulation of primary NK cells as measured via interferon-γ and CD107a expression. These molecules may have applications in cancer immunotherapy.
Subject(s)
Antibodies, Bispecific/metabolism , Killer Cells, Natural/transplantation , Neoplasms/therapy , Recombinant Fusion Proteins/metabolism , Single-Domain Antibodies/metabolism , Animals , Antibodies, Bispecific/genetics , Antibody-Dependent Cell Cytotoxicity , Antigens, Neoplasm/metabolism , Biological Assay , Camelids, New World , GPI-Linked Proteins/antagonists & inhibitors , GPI-Linked Proteins/genetics , Humans , Immunotherapy/methods , Jurkat Cells , Killer Cells, Natural/metabolism , Macaca fascicularis , Mice , Neoplasms/immunology , Primary Cell Culture , Protein Domains/genetics , Receptors, IgG/antagonists & inhibitors , Receptors, IgG/genetics , Recombinant Fusion Proteins/genetics , Single-Domain Antibodies/geneticsABSTRACT
[Figure: see text].
Subject(s)
Antibodies/pharmacology , Antihypertensive Agents/pharmacology , Blood Pressure/drug effects , Hypertension/prevention & control , Immunoglobulin G/blood , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/drug effects , Receptors, IgG/antagonists & inhibitors , Animals , Case-Control Studies , Disease Models, Animal , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , HEK293 Cells , Humans , Hypertension/genetics , Hypertension/metabolism , Hypertension/physiopathology , Male , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Muscle, Smooth, Vascular/physiopathology , Myocytes, Smooth Muscle/metabolism , Rats , Receptor, Angiotensin, Type 1/metabolism , Receptors, IgG/genetics , Receptors, IgG/metabolism , Signal Transduction , Vascular Remodeling/drug effectsABSTRACT
Human cytomegalovirus (HCMV) is endowed with multiple highly sophisticated immune evasion strategies. This includes the evasion from antibody mediated immune control by counteracting host Fc-gamma receptor (FcγR) mediated immune control mechanisms such as antibody-dependent cellular cytotoxicity (ADCC). We have previously shown that HCMV avoids FcγR activation by concomitant expression of the viral Fc-gamma-binding glycoproteins (vFcγRs) gp34 and gp68. We now show that gp34 and gp68 bind IgG simultaneously at topologically different Fcγ sites and achieve efficient antagonization of host FcγR activation by distinct but synergizing mechanisms. While gp34 enhances immune complex internalization, gp68 acts as inhibitor of host FcγR binding to immune complexes. In doing so, gp68 induces Fcγ accessibility to gp34 and simultaneously limits host FcγR recognition. The synergy of gp34 and gp68 is compelled by the interfering influence of excessive non-immune IgG ligands and highlights conformational changes within the IgG globular chains critical for antibody effector function.
Human cytomegalovirus is a type of herpes virus that rarely causes symptoms in healthy people but can cause serious complications in unborn babies and in people with compromised immune systems, such as transplant recipients. The virus has found ways to successfully evade the immune system, and once infected, the body retains the virus for life. It deploys an arsenal of proteins that bind to antibodies, specialized proteins the immune system uses to flag virus-infected cells for destruction. This prevents certain cells of the immune system, the natural killer cells, from recognizing and destroying virus-infected cells. These immune-evading proteins are called viral Fc-gamma receptors, or vFcγRs. While it has been previously shown that these receptors are able to evade the immune system, it remained unknown how exactly they prevent natural killer cells from recognizing infected cells. Now, Kolb et al. show that the cytomegalovirus deploys two vFcγRs called gp34 and gp68, which work together to block natural killer cells. The latter reduces the ability of natural killer cells to bind to antibodies on cytomegalovirus-infected cells. This paves the way for gp34 to pull virus proteins from the surface of the infected cell, making them inaccessible to the immune system. Neither protein fully protects virus-infected cells on its own, but together they are highly effective. The experiments reveal further details about how cytomegalovirus uses two defense mechanisms simultaneously to outmaneuver the immune system. Understanding this two-part viral evasion system may help scientists to develop vaccines or new treatments that can protect vulnerable people from diseases caused by the cytomegalovirus.
Subject(s)
Cytomegalovirus/immunology , Immunoglobulin G/metabolism , Receptors, IgG/antagonists & inhibitors , Antibody-Dependent Cell Cytotoxicity , Carrier Proteins/metabolism , Cell Line , Cytomegalovirus/metabolism , Glycoproteins/metabolism , Humans , Immune Evasion , Killer Cells, Natural/metabolism , Membrane Glycoproteins/metabolism , Protein Binding , Receptors, IgG/immunology , Receptors, IgG/metabolism , Viral Proteins/metabolismABSTRACT
Bispecific antibodies (bsAbs) have emerged as promising therapeutics. A bispecific diabody (bsDb) is a small bsAb consisting of two distinct chimeric single-chain components, with two possible arrangements of the domains. We previously reported the effect of domain order on the function of a humanized bsDb targeting the epidermal growth factor receptor (EGFR) on cancer cells, and CD3 on T cells. Notably, the co-localization of a T-cell receptor (TCR) with CD3 is bulky, potentially affecting the cross-linking ability of bsDbs, due to steric hindrance. Here, we constructed and evaluated humanized bsDbs, with different domain orders, targeting EGFR and CD16 on natural killer (NK) cells (hEx16-Dbs). We predicted minimal effects due to steric hindrance, as CD16 lacks accessory molecules. Interestingly, one domain arrangement displayed superior cytotoxicity in growth inhibition assays, despite similar cross-linking abilities for both domain orders tested. In hEx16-Dbs specifically, domain order might affect the agonistic activity of the anti-CD16 portion, which was supported by a cytokine production test, and likely contributed to the superiority of one of the hEx16-Dbs. Our results indicate that both the target antigen and mode of action of an antibody must be considered in the construction of highly functional bsAbs.
Subject(s)
Antibodies, Bispecific/immunology , Killer Cells, Natural/immunology , Neoplasms/therapy , Receptors, IgG/immunology , Antibodies, Bispecific/pharmacology , Antineoplastic Agents/immunology , Antineoplastic Agents/pharmacology , CD3 Complex/immunology , Cell Line, Tumor , Cell Proliferation/drug effects , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/immunology , Humans , Immunotherapy , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Neoplasms/immunology , Neoplasms/pathology , Receptors, IgG/antagonists & inhibitors , T-Lymphocytes/immunologyABSTRACT
Antibody-dependent enhancement (ADE) is a mechanism by which the pathogenesis of certain viral infections is enhanced in the presence of sub-neutralizing or cross-reactive non-neutralizing antiviral antibodies. In vitro modelling of ADE has attributed enhanced pathogenesis to Fcγ receptor (FcγR)-mediated viral entry, rather than canonical viral receptor-mediated entry. However, the putative FcγR-dependent mechanisms of ADE overlap with the role of these receptors in mediating antiviral protection in various viral infections, necessitating a detailed understanding of how this diverse family of receptors functions in protection and pathogenesis. Here, we discuss the diversity of immune responses mediated upon FcγR engagement and review the available experimental evidence supporting the role of FcγRs in antiviral protection and pathogenesis through ADE. We explore FcγR engagement in the context of a range of different viral infections, including dengue virus and SARS-CoV, and consider ADE in the context of the ongoing SARS-CoV-2 pandemic.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Antibodies, Viral/administration & dosage , Antibody-Dependent Enhancement/drug effects , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Leukocytes/drug effects , Pneumonia, Viral/drug therapy , Receptors, IgG/immunology , Antibodies, Monoclonal/adverse effects , Antibodies, Monoclonal/biosynthesis , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/adverse effects , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/adverse effects , Antibodies, Viral/biosynthesis , Betacoronavirus/immunology , Betacoronavirus/pathogenicity , COVID-19 , Coronavirus Infections/immunology , Coronavirus Infections/virology , Dengue/drug therapy , Dengue/immunology , Dengue/virology , Dengue Virus/drug effects , Dengue Virus/immunology , Dengue Virus/pathogenicity , Gene Expression Regulation , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/immunology , Humans , Leukocytes/immunology , Leukocytes/virology , Pandemics , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Receptors, IgG/antagonists & inhibitors , Receptors, IgG/genetics , Severe acute respiratory syndrome-related coronavirus/drug effects , Severe acute respiratory syndrome-related coronavirus/immunology , Severe acute respiratory syndrome-related coronavirus/pathogenicity , SARS-CoV-2 , Severe Acute Respiratory Syndrome/drug therapy , Severe Acute Respiratory Syndrome/immunology , Severe Acute Respiratory Syndrome/virology , Signal Transduction , Virus Internalization/drug effectsABSTRACT
Despite the successes achieved with molecular targeted inhibition of the oncogenic driver Bcr-Abl in chronic myeloid leukemia (CML), the majority of patients still require lifelong tyrosine kinase inhibitor (TKI) therapy. This is primarily caused by resisting leukemic stem cells (LSCs), which prevent achievement of treatment-free remission in all patients. Here we describe the ITIM (immunoreceptor tyrosine-based inhibition motif)-containing Fc gamma receptor IIb (FcγRIIb, CD32b) for being critical in LSC resistance and show that targeting FcγRIIb downstream signaling, by using a Food and Drug Administration-approved BTK inhibitor, provides a successful therapeutic approach. First, we identified FcγRIIb upregulation in primary CML stem cells. FcγRIIb depletion caused reduced serial re-plaiting efficiency and cell proliferation in malignant cells. FcγRIIb targeting in both a transgenic and retroviral CML mouse model provided in vivo evidence for successful LSC reduction. Subsequently, we identified BTK as a main downstream mediator and targeting the Bcr-Abl-FcγRIIb-BTK axis in primary CML CD34+ cells using ibrutinib, in combination with standard TKI therapy, significantly increased apoptosis in quiescent CML stem cells thereby contributing to the eradication of LSCs.. As a potential curative therapeutic approach, we therefore suggest combining Bcr-Abl TKI therapy along with BTK inhibition.
Subject(s)
Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Fusion Proteins, bcr-abl/antagonists & inhibitors , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Neoplastic Stem Cells/metabolism , Protein Kinase Inhibitors/pharmacology , Receptors, IgG/antagonists & inhibitors , Signal Transduction/drug effects , Animals , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Mice , Mice, Inbred C57BL , Receptors, IgG/geneticsABSTRACT
In relapsed/refractory Hodgkin lymphoma (R/R HL), immunotherapies such as the anti-programmed death-1 inhibitor pembrolizumab have demonstrated efficacy as monotherapy and are playing an increasingly prominent role in treatment. The CD30/CD16A-bispecific antibody AFM13 is an innate immune cell engager, a first-in-class, tetravalent antibody, designed to create a bridge between CD30 on HL cells and the CD16A receptor on natural killer cells and macrophages, to induce tumor cell killing. Early studies of AFM13 have demonstrated signs of efficacy as monotherapy for patients with R/R HL and the combination of AFM13 with pembrolizumab represents a rational new treatment modality. Here, we describe a phase 1b, dose-escalation study to assess the safety and preliminary efficacy of AFM13 in combination with pembrolizumab in patients with R/R HL. The primary objective was estimating the maximum tolerated dose; the secondary objectives were to assess safety, tolerability, antitumor efficacy, pharmacokinetics, and pharmacodynamics. In this heavily pretreated patient population, treatment with the combination of AFM13 and pembrolizumab was generally well tolerated, with similar safety profiles compared to the known profiles of each agent alone. The combination of AFM13 with pembrolizumab demonstrated an objective response rate of 88% at the highest treatment dose, with an 83% overall response rate for the overall population. Pharmacokinetic assessment of AFM13 in the combination setting revealed a half-life of up to 20.6 hours. This proof-of-concept study holds promise as a novel immunotherapy combination worthy of further investigation. This phase 1b study was registered at www.clinicaltrials.gov as NCT02665650.
Subject(s)
Antigens, Neoplasm/immunology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Hodgkin Disease/drug therapy , Immunotherapy , Ki-1 Antigen/antagonists & inhibitors , Receptors, IgG/antagonists & inhibitors , Adolescent , Adult , Aged , Antibodies, Bispecific/administration & dosage , Antibodies, Bispecific/adverse effects , Antibodies, Bispecific/pharmacokinetics , Antibodies, Monoclonal, Humanized/administration & dosage , Antibodies, Monoclonal, Humanized/adverse effects , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Combined Modality Therapy , Dose-Response Relationship, Immunologic , Female , Half-Life , Hematopoietic Stem Cell Transplantation , Hodgkin Disease/therapy , Humans , Immunity, Innate/drug effects , Ki-1 Antigen/immunology , Male , Maximum Tolerated Dose , Middle Aged , Proof of Concept Study , Receptors, IgG/immunology , Recurrence , Transplantation, Autologous , Young AdultABSTRACT
In recent years, the success of immunotherapy targeting immunoregulatory receptors (immune checkpoints) in cancer have generated enthusiastic support to target these receptors in a wide range of other immune related diseases. While the overwhelming focus has been on blockade of these inhibitory pathways to augment immunity, agonistic triggering via these receptors offers the promise of dampening pathogenic inflammatory responses. V-domain Ig suppressor of T cell activation (VISTA) has emerged as an immunoregulatory receptor with constitutive expression on both the T cell and myeloid compartments, and whose agonistic targeting has proven a unique avenue relative to other checkpoint pathways to suppress pathologies mediated by the innate arm of the immune system. VISTA agonistic targeting profoundly changes the phenotype of human monocytes towards an anti-inflammatory cell state, as highlighted by striking suppression of the canonical markers CD14 and Fcγr3a (CD16), and the almost complete suppression of both the interferon I (IFN-I) and antigen presentation pathways. The insights from these very recent studies highlight the impact of VISTA agonistic targeting of myeloid cells, and its potential therapeutic implications in the settings of hyperinflammatory responses such as cytokine storms, driven by dysregulated immune responses to viral infections (with a focus on COVID-19) and autoimmune diseases. Collectively, these findings suggest that the VISTA pathway plays a conserved, non-redundant role in myeloid cell function.
Subject(s)
B7 Antigens/agonists , COVID-19/pathology , Cytokine Release Syndrome/prevention & control , Myeloid Cells/immunology , Animals , Antigen Presentation/immunology , B7 Antigens/antagonists & inhibitors , B7 Antigens/immunology , CD4-Positive T-Lymphocytes/immunology , Cytokine Release Syndrome/immunology , Cytokine Release Syndrome/pathology , GPI-Linked Proteins/antagonists & inhibitors , Humans , Immunotherapy , Interferon Type I/antagonists & inhibitors , Lipopolysaccharide Receptors/antagonists & inhibitors , Lymphocyte Activation/immunology , Mice , Receptors, IgG/antagonists & inhibitors , SARS-CoV-2/immunologyABSTRACT
Medial vascular calcification occurs during the aging process and is strongly accelerated by chronic kidney disease (CKD). Elevated C-reactive protein (CRP) levels are associated with vascular calcification, cardiovascular events and mortality in CKD patients. CRP is an important promoter of vascular inflammation. Inflammatory processes are critically involved in initiation and progression of vascular calcification. Thus, the present study explored a possible impact of CRP on vascular calcification. We found that CRP promoted osteo-/chondrogenic transdifferentiation and aggravated phosphate-induced osteo-/chondrogenic transdifferentiation and calcification of primary human aortic smooth muscle cells (HAoSMCs). These effects were paralleled by increased cellular oxidative stress and corresponding pro-calcific downstream-signaling. Antioxidants or p38 MAPK inhibition suppressed CRP-induced osteo-/chondrogenic signaling and mineralization. Furthermore, silencing of Fc fragment of IgG receptor IIa (FCGR2A) blunted the pro-calcific effects of CRP. Vascular CRP expression was increased in the klotho-hypomorphic mouse model of aging as well as in HAoSMCs during calcifying conditions. In conclusion, CRP augments osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells through mechanisms involving FCGR2A-dependent induction of oxidative stress. Thus, systemic inflammation may actively contribute to the progression of vascular calcification.
Subject(s)
Aging/metabolism , Aging/pathology , C-Reactive Protein/metabolism , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Vascular Calcification/metabolism , Vascular Calcification/pathology , Animals , Cell Transdifferentiation/physiology , Cells, Cultured , Chondrogenesis/physiology , Disease Models, Animal , Glucuronidase/genetics , Glucuronidase/metabolism , Humans , Klotho Proteins , Mice , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Osteogenesis/physiology , Oxidative Stress , RNA, Small Interfering/genetics , Receptors, IgG/antagonists & inhibitors , Receptors, IgG/genetics , Receptors, IgG/metabolism , Renal Insufficiency, Chronic/complications , Renal Insufficiency, Chronic/metabolism , Renal Insufficiency, Chronic/pathology , Signal Transduction , Vascular Calcification/etiologyABSTRACT
Ezetimibe (EZE) and glucuronidated EZE (EZE-Glu) differentially target Niemann-Pick C1-like 1 (NPC1L1) and CD13 (aminopeptidase-N) to inhibit intestinal cholesterol absorption and cholesterol processing in other cells, although the precise molecular mechanisms are not fully elucidated. Cellular effects of EZE, EZE-Glu, and the low-absorbable EZE-analogue S6130 were investigated on human monocyte-derived macrophages upon loading with atherogenic lipoproteins. EZE and S6130, but not EZE-Glu disturbed the colocalization of CD13 and its coreceptor CD64 (Fcγ receptor I) in membrane microdomains, and decreased the presence of both receptors in detergent-resistant membrane fractions. Biotinylated cholesterol absorption inhibitor C-5 (i.e., derivative of EZE) was rapidly internalized to perinuclear tubular structures of cells, resembling endoplasmic reticulum (ER), but CD13 was detected on extracellular sites of the plasma membrane and endolysosomal vesicles. Administration of EZE, but not of EZE-Glu or S6130, was associated with decreased cellular cholesteryl ester content, indicating the sterol-O acyltransferase 1 (SOAT1)-inhibition by EZE. Furthermore, EZE decreased the expression of molecules involved in cholesterol uptake and synthesis, in parallel with increased apolipoprotein A-I-mediated cholesterol efflux and upregulation of efflux-effectors. However, NPC1L1 the other claimed molecular target of EZE, was not detected in macrophages, thereby excluding this protein as target for EZE in macrophages. Thus, EZE is very likely a CD13-linked microdomain-disruptor and SOAT1-inhibitor in macrophages leading to in vitro anti-atherosclerotic effects through a decrease of net cellular cholesterol content. © 2019 International Society for Advancement of Cytometry.
Subject(s)
CD13 Antigens/ultrastructure , Cholesterol/isolation & purification , Flow Cytometry , Membrane Transport Proteins/genetics , Receptors, IgG/ultrastructure , Atherosclerosis/genetics , Biological Transport/drug effects , CD13 Antigens/antagonists & inhibitors , Cholesterol/metabolism , Ezetimibe/pharmacology , Glucuronates/genetics , Humans , Macrophages/metabolism , Macrophages/ultrastructure , Membrane Microdomains/drug effects , Membrane Microdomains/ultrastructure , Membrane Transport Proteins/metabolism , Monocytes/metabolism , Monocytes/ultrastructure , Receptors, IgG/antagonists & inhibitorsABSTRACT
Human NK cell antitumor activities involve Ab-dependent cell-mediated cytotoxicity (ADCC), which is a key mechanism of action for several clinically successful tumor-targeting therapeutic mAbs. Human NK cells exclusively recognize these Abs by the Fcγ receptor CD16A (FcγRIIIA), one of their most potent activating receptors. Unlike other activating receptors on NK cells, CD16A undergoes a rapid down-regulation in expression by a proteolytic process following NK cell activation with various stimuli. In this review, the role of a disintegrin and metalloproteinase-17 (ADAM17) in CD16A cleavage and as a regulatory checkpoint is discussed. Several studies have examined the effects of inhibiting ADAM17 or CD16A cleavage directly during NK cell engagement of Ab-coated tumor cells, which resulted in strengthened Ab tethering, decreased tumor cell detachment, and enhanced CD16A signaling and cytokine production. However, the effects of either manipulation on ADCC have varied between studies, which may be due to dissimilar assays and the contribution of different killing processes by NK cells. Of importance is that NK cells under various circumstances, including in the tumor microenvironment of patients, down-regulate CD16A and this appears to impair their function. Considerable progress has been made in the development of ADAM17 inhibitors, including human mAbs that have advantages of high specificity and increased half-life in vivo. These inhibitors may provide a therapeutic means of increasing ADCC potency and/or antitumor cytokine production by NK cells in an immunosuppressive tumor microenvironment, and if used in combination with tumor-targeting Abs or NK cell-based adoptive immunotherapies may improve their efficacy.
Subject(s)
ADAM17 Protein , Adoptive Transfer , Antineoplastic Agents, Immunological/therapeutic use , Killer Cells, Natural , Neoplasm Proteins , Neoplasms , Receptors, IgG , ADAM17 Protein/antagonists & inhibitors , ADAM17 Protein/immunology , Animals , Antibody-Dependent Cell Cytotoxicity/drug effects , Humans , Killer Cells, Natural/immunology , Killer Cells, Natural/transplantation , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/immunology , Neoplasms/immunology , Neoplasms/therapy , Receptors, IgG/antagonists & inhibitors , Receptors, IgG/immunology , Signal Transduction/drug effects , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunologyABSTRACT
Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune response largely mediated by natural killer (NK) cells that can lyse target cells and combat tumors and viral infections. However, the role of ADCC in response to primary HIV infection is poorly understood. In the present study, we explored the ADCC response and evaluated its characteristics in 85 HIV-infected individuals, including 42 with primary infections. Our results showed that ADCC occurs during acute infection, and the earliest ADCC response to a single peptide was detected at 52 days. Primary HIV-infected individuals exhibiting ADCC responses had lower viral set points than those with no ADCC response, and functional analyses demonstrated that the ADCC response could significantly inhibit viral infection during primary HIV infection. HIV epitopes that provoked the ADCC response were determined and three relatively conserved epitopes (HNVWATYACVPTDPNPQE, TSVIKQACPKISFDPIPI, and VVSTQLLLNGSLAEEEII) from the surface of the three-dimensional structure of the HIV Env protein were identified. Overall, our data indicate that ADCC responses may be significant for the control of HIV from an early stage during infection. These findings merit further investigation and will facilitate improvements in vaccines or therapeutic interventions against HIV infection.
Subject(s)
Antibody-Dependent Cell Cytotoxicity/immunology , HIV Antibodies/immunology , HIV Infections/immunology , HIV Infections/virology , HIV-1/immunology , Viral Load , Adult , Amino Acid Sequence , Antibodies, Monoclonal/pharmacology , Antibody-Dependent Cell Cytotoxicity/drug effects , Antigens, Viral/chemistry , Antigens, Viral/immunology , Antiretroviral Therapy, Highly Active , CD4 Lymphocyte Count , Conserved Sequence , Epitope Mapping , Epitopes/chemistry , Epitopes/immunology , Female , HIV Infections/diagnosis , HIV Infections/drug therapy , Host-Pathogen Interactions/immunology , Humans , Immunophenotyping , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Male , Models, Molecular , Peptides/immunology , Protein Conformation , Receptors, IgG/antagonists & inhibitors , Structure-Activity Relationship , Time Factors , Treatment OutcomeABSTRACT
Patients with acute myeloid leukemia (AML) often relapse after initial therapy because of persistence of leukemic stem cells that frequently express the IL-3 receptor alpha chain CD123. Natural killer (NK) cell-based therapeutic strategies for AML show promise and we explore the NK cell lines, NK-92 and CD16+ NK-92, as a treatment for AML. NK-92 has been tested in phase I clinical trials with minimal toxicity; irradiation prior to infusion prevents risk of engraftment. The CD16 negative NK-92 parental line was genetically modified to express the high affinity Fc gamma receptor, enabling antibody-dependent cell-mediated cytotoxicity, which we utilized in combination with an anti-CD123 antibody to target leukemic stem cells. NK-92 was preferentially cytotoxic against leukemic stem and progenitor cells compared with bulk leukemia in in vitro assays, while CD16+ NK-92 in combination with an anti-CD123 mAb mediated antibody-dependent cell-mediated cytotoxicity against CD123+ leukemic targets. Furthermore, NK-92 infusions (with or without prior irradiation) improved survival in a primary AML xenograft model. Mice xenografted with primary human AML cells had a superior survival when treated with irradiated CD16+NK-92 cells and an anti-CD123 monoclonal antibody (7G3) versus treatment with irradiated CD16+NK-92 cells combined with an isotype control antibody. In this proof-of-principle study, we show for the first time that a CD16+NK-92 cell line combined with an antibody that targets a leukemic stem cell antigen can lead to improved survival in a relevant pre-clinical model of AML.