FcγRI plays a critical role in patients with ulcerative colitis relapse.

Ulcerative colitis (UC) is a disease that frequently relapses and affects more than 0.1% general population; the underlying mechanism is poorly understood. Published data show that polymorphonuclear neutrophils (PMN) contribute to the pathogenesis of UC. This study aims to identify antigen (Ag)-specific PMNs and investigate their role in UC relapse. In this study, the correlation between PMN activities and UC relapse was assessed in a group of UC patients. A UC mouse model was developed to expand the findings of UC patient study. The results showed that a positive correlation was detected between the high PMN activities and the food Ag-specific IgG amounts in colon biopsies of UC patients. UC patient-derived Ag-specific PMNs could be activated upon exposure to food specific Ag. The Ag/FcγRI complexes were detected on the surface of PMNs in UC patients. Re-exposure of sensitized PMNs to specific Ag triggered PMN activation and induced UC-like inflammation in the mouse colon. We conclude that FcγRI plays a critical role in UC relapse. Inhibition of FcγRI can efficiently inhibits experimental UC.

The many faces of FcγRI: implications for therapeutic antibody function.

Fcγ receptor I (FcγRI or CD64) is the sole human Fc receptor with high affinity for monovalent IgG. While it contains an immunoreceptor tyrosine-based activation motif in its cytoplasmic domain, binding of FcγRI can result in a complex array of activating and inhibitory outcomes. For instance, binding of monomeric IgG provides a low-intensity tonic signal through FcγRI that is necessary for full interferon γ receptor signaling in the same cell. Interaction of FcγRI with larger high-avidity complexes can result in phagocytosis, the generation of reactive oxygen species, as well as the synthesis and release of inflammatory cytokines. However, numerous reports also document potent anti-inflammatory effects brought about by FcγRI engagement with immune complexes such as the inhibition of IFNγ and TLR4 signaling, and secretion of interleukin-10. This has led to conflicting hypotheses regarding the function of FcγRI, especially with regard to its role in the efficacy of several therapeutic monoclonal antibodies. While many of these issues are still unclear, continued characterization of the regulation and context dependence of FcγRI function, as well as the molecular mechanisms responsible for these various outcomes, will improve our understanding of FcγRI biology as well as the therapeutic strategies designed to harness or constrain its actions.

The binding of an anti-PD-1 antibody to FcγRΙ has a profound impact on its biological functions.

Antibodies targeting PD-1 have been demonstrated durable anti-cancer activity in certain cancer types. However, the anti-PD-1 antibodies are less or not efficacious in many situations, which might be attributed to co-expression of multiple inhibitory receptors or presence of immunosuppressive cells in the tumor microenvironment. Most of the anti-PD-1 antibodies used in clinical studies are of IgG4 isotype with the S228P mutation (IgG4S228P). The functional impact by the interaction of anti-PD-1 IgG4S228P antibody with Fc gamma receptors (FcγRs) is poorly understood. To assess the effects, we generated a pair of anti-PD-1 antibodies: BGB-A317/IgG4S228P and BGB-A317/IgG4-variant (abbreviated as BGB-A317), with the same variable regions but two different IgG4 Fc-hinge sequences. There was no significant difference between these two antibodies in binding to PD-1. However, BGB-A317/IgG4S228P binds to human FcγRI with high affinity and mediates crosslinking between PD-1 and FcγRI. In contrast, BGB-A317 does neither. Further cell-based assays showed that such crosslinking could reverse the function of an anti-PD-1 antibody from blocking to activating. More importantly, the crosslinking induces FcγRI+ macrophages to phagocytose PD-1+ T cells. In a mouse model transplanted with allogeneic human cancer cells and PBMCs, BGB-A317 showed significant tumor growth inhibition, whereas BGB-A317/IgG4S228P had no such inhibition. Immunohistochemistry study revealed an inverse correlation between FcγRI+ murine macrophage infiltration and the density of CD8+PD-1+ human T cells within tumors in the BGB-A317/IgG4S228P-treated group. These evidences suggested that FcγRI+ binding and crosslinking had negative impact on the anti-PD-1 antibody-mediated anti-cancer activity.

Lentivirus-mediated knockdown of FcγRI (CD64) attenuated lupus nephritis via inhibition of NF-κB regulating NLRP3 inflammasome activation in MRL/lpr mice.

Lupus nephritis, one of the most serious complications of systemic lupus erythematosus (SLE), has been confirmed in a large number of clinical surveys. Current studies have suggested that inflammatory situation is generally considered to facilitate the occurrence and development of lupus nephritis. Previous research found that Fcγ receptor I (FcγRI) was compulsory for several autoimmune and inflammatory diseases, and it might be involved in the treatment of lupus nephritis. Furthermore, the possible molecular mechanism of the role of FcγRI in lupus nephritis still needs a further study. In the present study, in order to evaluate the effect of FcγRI on kidney function in lupus-prone MLR/lpr mice, FcγRI knockdown was implemented utilizing FcγRI-RNAi lentivirus. We reported that the administration of FcγRI-RNAi lentivirus (1) mainly inhibited FcγRI expression on macrophage of the kidneys, lowered the levels of urinary protein and serum anti-dsDNA antibody and prevented the impairment of renal function; (2) reduced the renal inflammatory cytokines (IL-1ß and IL-18); (3) decreased NF-κB p65 nuclear migration, suppressed NOD-like receptor protein 3 (NLRP3) inflammasome activation, and finally inhibited renal inflammation. Together, these results showed the role of FcγRI on macrophages to involve in renal inflammatory response, potentially via regulating the NLRP3 inflammasome-associated signaling.

Human serum substitution by artificial sera of scalable allergen reactivity based on polyclonal antibodies and chimeras of human FcγRI and IgE domains.

Human sera are the first choice as controls for diagnostic applications such as immunoassays, but are limited regarding availability, varying quality, and high costs. In this study, we aimed to circumvent these limitations by the use of a chimeric adaptor molecule comprising the extracellular domains of the human FcγRI (CD64) fused with human IgE Fc domains (CD64-IgE Fc). Allergen-specific antibodies were produced in rabbits using eight different allergens, extracts, and allergen mixtures including mites, pollen, drugs, and food. Preincubation of polyclonal IgG with CD64-IgE Fc established allergen-specific artificial sera that showed comparable results for more than 20 allergens and allergen extracts in three diagnostic systems for the determination of specific IgE. The agreement for these artificial sera is within ±1 radioallergosorbent test (RAST) class. Hence, rabbit IgG complexed with the IgG-specific CD64-IgE Fc adaptor molecule could provide a substitute for human reference sera with specificity for virtually any protein of interest.

Structural insights into the interaction of human IgG1 with FcγRI: no direct role of glycans in binding.

The three-dimensional structure of a human IgG1 Fc fragment bound to wild-type human FcγRI is reported. The structure of the corresponding complex was solved at a resolution of 2.4 Šusing molecular replacement; this is the highest resolution achieved for an unmutated FcγRI molecule. This study highlights the critical structural and functional role played by the second extracellular subdomain of FcγRI. It also explains the long-known major energetic contribution of the Fc `LLGG' motif at positions 234-237, and particularly of Leu235, via a `lock-and-key' mechanism. Finally, a previously held belief is corrected and a differing view is offered on the recently proposed direct role of Fc carbohydrates in the corresponding interaction. Structural evidence is provided that such glycan-related effects are strictly indirect.

Broadening anticancer spectrum by preprocessing and treatment of T- lymphocytes expressed FcγRI and monoclonal antibodies for refractory cancers.

Background: Chimeric antigen receptor T (CAR-T) cell therapies have achieved remarkable success in the treatment of hematological tumors. However, given the distinct features of solid tumors, particularly heterogeneity, metabolic aggressiveness, and fewer immune cells in tumor microenvironment (TME), the practical utility of CAR-T cells for solid tumors remains as a challenging issue. Meanwhile, although anti-PD-1 monoclonal antibody (mAb) has shown clinical efficacy, most mAbs also show limited clinical benefits for solid tumors due mainly to the issues associated with the lack of immune cells in TME. Thus, the infiltration of targeted immunological active cells into TME could generate synergistic efficacy for mAbs. Methods: We present a combinational strategy for solid tumor treatment, which combines armored-T cells to express Fc-gamma receptor I (FcγRI) fragment on the surfaces for targeting various tumors with therapeutically useful mAbs. Choosing CD20 and HER-2 as the targets, we characterized the in vitro and in vivo efficacy and latent mechanism of the combination drug by using flow cytometry, ELISA and other methods. Results: The combination and preprocessing of armored T-cells with corresponding antibody of Rituximab and Pertuzumab exerted profound anti-tumor effects, which is demonstrated to be mediated by synergistically produced antibody-dependent cellular cytotoxicity (ADCC) effects. Meanwhile, mAb was able to carry armored-T cell by preprocessing for the infiltration to TME in cell derived xenograft (CDX) model. Conclusions: This combination strategy showed a significant increase of safety profiles from the reduction of antibody doses. More importantly, the present strategy could be a versatile tool for a broad spectrum of cancer treatment, with a simple pairing of engineered T cells and a conventional antibody.

Combining Cellular Immunization and Phage Display Screening Results in Novel, FcγRI-Specific Antibodies.

Antibodies that specifically bind to individual human fragment crystallizable γ receptors (FcγRs) are of interest as research tools in studying immune cell functions, as well as components in bispecific antibodies for immune cell engagement in cancer therapy. Monoclonal antibodies for human low-affinity FcγRs have been successfully generated by hybridoma technology and are widely used in pre-clinical research. However, the generation of monoclonal antibodies by hybridoma technology that specifically bind to the high-affinity receptor FcγRI is challenging. Monomeric mouse IgG2a, IgG2b, and IgG3 bind human FcγRI with high affinity via the Fc part, leading to an Fc-mediated rather than a fragment for antigen binding (Fab)-mediated selection of monoclonal antibodies. Blocking the Fc-binding site of FcγRI with an excess of human IgG or Fc during screening decreases the risk of Fc-mediated interactions but can also block the potential epitopes of new antibody candidates. Therefore, we replaced hybridoma technology with phage display of a single-chain fragment variable (scFv) antibody library that was generated from mice immunized with FcγRI-positive cells and screened it with a cellular panning approach assisted by next-generation sequencing (NGS). Seven new FcγRI-specific antibody sequences were selected with this methodology, which were produced as Fc-silent antibodies showing FcγRI-restricted specificity.

Cellular Membrane-Derived Nanovesicles Expressing hCD64 for Targeting Prostate Cancer.

Extracellular vesicles are ideal therapeutic potentiators for various diseases. However, they commonly lack targeting capability and are rapidly cleared by phagocytes. This requires appropriate administration at high doses, which can lead to toxic and adverse reactions. To overcome these limitations, we developed bleb nanovesicles containing human Fcγ receptor I (hCD64), known for their strong affinity to monomeric IgG. In this study, we focused on prostate cancer, which has a specific membrane antigen. We have utilized the hCD64-expressing bleb nanovesicles attaching anti-prostate-specific membrane antigen (PSMA) antibodies and confirmed their targeting ability in PSMA-related cell lines and prostate cancer xenograft models. Our findings underscore the promising potential of nanovesicle Fcγ receptor-IgG as a platform for cancer diagnosis and therapy systems, inspiring further research.

Systematic analysis of Fc mutations designed to enhance binding to Fc-gamma receptors.

A critical attribute of therapeutic antibodies is their ability to engage with humoral or cellular effector mechanisms, and this depends on the ability of the Fc region to bind to complement (C1q) or Fc receptors. Investigators have sought to optimize these effects by engineering the Fc region to bind to a greater or lesser extent to individual receptors. Different approaches have been used in the clinic, but they have not been systematically compared. We have now produced a matched set of anti-CD20 antibodies representing a range of variants and compared their activity in cell-based assays for complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent phagocytosis using a range of individual Fc receptors. We have also compared the thermal stability of the variants by differential scanning fluorimetry (DSF). The results reveal a spectrum of activities which may be appropriate for different applications.

Systematic analysis of Fc mutations designed to reduce binding to Fc-gamma receptors.

Elimination of the binding of immunoglobulin Fc to Fc gamma receptors is highly desirable for the avoidance of unwanted inflammatory responses to therapeutic antibodies and fusion proteins. Many different approaches have been used in the clinic, but they have not been systematically compared. We have now produced a matched set of anti-CD20 antibodies with different Fc subclasses and variants and compared their activity for binding to C1q, Fc-gamma receptors and in cell-based assays. Most of the variants still have significant levels of activity in one or more of these assays and many of them have impaired temperature stability compared with the corresponding wild-type antibody.

FcγRI FG-loop functions as a pH sensitive switch for IgG binding and release.

Understanding the molecular mechanism underlying the hierarchic binding between FcγRs and IgG antibodies is critical for therapeutic antibody engineering and FcγR functions. The recent determination of crystal structures of FcγRI-Fc complexes, however, resulted in two controversial mechanisms for the high affinity receptor binding to IgG. Here, we describe high resolution structures of a bovine FG-loop variant of FcγRI in complex with the Fc fragment of IgG1 crystallized in three different conditions at neutral pH, confirming the characteristic FG loop-Fc interaction is critical to the high affinity immunoglobulin binding. We showed that the FcγRI D2-domain FG-loop functioned as a pH-sensing switch for IgG binding. Further live cell imaging of FcγRI-mediated internalization of immune complexes showed a pH sensitive temporal-spatial antibody-antigen uptake and release. Taken together, we demonstrate that the structures of FcγRI-Fc crystallized at neutral and acidic pH, respectively, represent the high and low affinity binding states of the receptor for IgG uptake and release. These results support a role for FcγRI in antigen delivery, highlight the importance of Fc glycan in antibody binding to the high affinity receptor and provide new insights to future antibody engineering.

SARS-CoV-2: Immunity, Challenges with Current Vaccines, and a Novel Perspective on Mucosal Vaccines.

The global rollout of COVID-19 vaccines has played a critical role in reducing pandemic spread, disease severity, hospitalizations, and deaths. However, the first-generation vaccines failed to block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and transmission, partially due to the limited induction of mucosal immunity, leading to the continuous emergence of variants of concern (VOC) and breakthrough infections. To meet the challenges from VOC, limited durability, and lack of mucosal immune response of first-generation vaccines, novel approaches are being investigated. Herein, we have discussed the current knowledge pertaining to natural and vaccine-induced immunity, and the role of the mucosal immune response in controlling SARS-CoV2 infection. We have also presented the current status of the novel approaches aimed at eliciting both mucosal and systemic immunity. Finally, we have presented a novel adjuvant-free approach to elicit effective mucosal immunity against SARS-CoV-2, which lacks the safety concerns associated with live-attenuated vaccine platforms.

A CD64/FcγRI-mediated mechanism hijacks PD-1 from PD-L1/2 interaction and enhances anti-PD-1 functional recovery of exhausted T cells.

Therapeutic monoclonal antibodies (mAb) targeting the immune checkpoint inhibitor programmed cell death protein 1 (PD-1) have achieved considerable clinical success in anti-cancer therapy through relieving T cell exhaustion. Blockade of PD-1 interaction with its ligands PD-L1 and PD-L2 is an important determinant in promoting the functional recovery of exhausted T cells. Here, we show that anti-PD-1 mAbs act through an alternative mechanism leading to the downregulation of PD-1 surface expression on memory CD4+ and CD8+ T cells. PD-1 receptor downregulation is a distinct process from receptor endocytosis and occurs in a CD14+ monocyte dependent manner with the CD64/Fcγ receptor I acting as the primary factor for this T cell extrinsic process. Importantly, downregulation of surface PD-1 strongly enhances antigen-specific functional recovery of exhausted PD-1+CD8+ T cells. Our study demonstrates a novel mechanism for reducing cell surface levels of PD-1 and limiting the inhibitory targeting by PD-L1/2 and thereby enhancing the efficacy of anti-PD-1 Ab in restoring T cell functionality.

A Pathogenic Role for FcγRI in the Immune Response against Chlamydial Respiratory Infection.

FcγRI is an important cell surface receptor reported to be involved in multiple immune responses, although it has not yet been extensively studied in intracellular bacterial infections. Here, using a mouse model of C. muridarum respiratory infection, we were able to determine how FcγRI regulates the host resistance against chlamydial invasion. According to our findings, the chlamydial loads and pulmonary pathology were both reduced in FcγRI deficient (Fcgr1-/-) animals. Being infected, monocytes, macrophages, neutrophils, DCs, CD4+/CD8+ T cells, and effector Th1 subsets displayed increased FcγRI expression patterns. Altered infiltration of these cells in the lungs of Fcgr1-/- mice further demonstrated the regulation of FcγRI in the immune system and identified Th1 cells and macrophages as its target cell populations. As expected, we observed that the Th1 response was augmented in Fcgr1-/- mice, while the pro-inflammatory M1 macrophage polarization was constrained. These findings might indicate FcγRI as a potential regulator for host immunity and inflammatory response during chlamydial infection.

Sensory Neuron Expressed FcγRI Mediates Postinflammatory Arthritis Pain in Female Mice.

Persistent arthritis pain after resolution of joint inflammation represents a huge health burden in patients with rheumatoid arthritis (RA). However, the underling mechanisms are poorly understood. We and other groups recently revealed that FcγRI, a key immune receptor, is functionally expressed in joint nociceptors. Thus, we investigated a potential role of sensory neuron expressed FcγRI in postinflammatory arthritis pain in a mouse model of collagen antibody-induced arthritis (CAIA). Here, we show that global deletion of Fcgr1 significantly attenuated mechanical hyperalgesia in the ankle and hind paw of female mice in both inflammatory and postinflammatory phases of CAIA. No obvious differences in cartilage destruction were observed after resolution of joint inflammation between genotypes. In situ hybridization (ISH) revealed that a larger proportion of dorsal root ganglion (DRG) neurons expressed Fcgr1 mRNA signal in the late phase of CAIA. Conditional deletion of Fcgr1 in primary sensory neurons produced similar analgesic effects without affecting joint swelling. Knockdown of Fcgr1 expression within DRG in the postinflammatory phase of CAIA alleviated persistent pain. Inflammation within DRG after resolution of joint inflammation in the CAIA model was evidenced by T cell and neutrophil infiltration and upregulated mRNA expression of numerous inflammatory mediators. Yet, such changes were not altered by genetic deletion of Fcgr1. We suggest that neuroinflammation within the DRG after resolution of joint inflammation might upregulate FcγRI signaling in DRG neurons. Sensory neuron expressed FcγRI thus merits exploration as a potential target for the treatment of arthritis pain that persists in RA patients in remission.

Activation of activating Fc gamma receptors down-regulates the levels of interferon ß, interferon γ and interferon λ1 in porcine alveolar macrophages during PRRSV infection.

Porcine activating Fc gamma receptors (FcγRI and FcγRIII) have been cloned and characterized for many years. However, their roles in interferon (IFN) antiviral immune response to porcine reproductive and respiratory syndrome virus (PRRSV) infection have not yet been investigated extensively. In this study, PRRSV infection assay showed that PRRSV increased significantly the transcription of IFN-ß, IFN-γ and IFN-λ1 in porcine alveolar macrophages (PAMs) in early infection and decreased significantly the transcription of IFN-ß, IFN-γ and IFN-λ1 in PAMs in late infection. Activation assay showed that specific activation of FcγRI or FcγRIII in PAMs decreased significantly the transcription of IFN-ß, IFN-γ and IFN-λ1 and increased significantly the transcription of transforming growth factor ß1 (TGF-ß1). PRRSV infection assay mediated by FcγRI and FcγRIII showed that specific activation of FcγRI or FcγRIII in PAMs during PRRSV infection decreased significantly the transcription of IFN-ß, IFN-γ and IFN-λ1, but increased significantly the transcription of TGF-ß1 and enhanced significantly viral replication. In conclusion, our studies suggested that activating FcγR signaling inhibited the transcriptional levels of IFN-ß, IFN-γ and IFN-λ1 in PAMs in response to PRRSV infection.

Antibody-Mediated Porcine Reproductive and Respiratory Syndrome Virus Infection Downregulates the Production of Interferon-α and Tumor Necrosis Factor-α in Porcine Alveolar Macrophages via Fc Gamma Receptor I and III.

Antibody-dependent enhancement (ADE) contributes to the pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV)-persistent infection. However, the mechanisms of PRRSV-ADE infection are still confusing. A clear understanding of the event upon virus infection by the ADE pathway has become crucial for developing efficient intervention of the PRRSV infection. In this study, an ADE assay showed that PRRSV-ADE infection in porcine alveolar macrophages (AMs) significantly decreased the production of interferon-α (IFN-α) and tumor necrosis factor-α (TNF-α), and significantly increased the production of interleukine-10 (IL-10). A gene knockdown assay based on small interfering RNA (siRNA) showed that both Fc gamma receptor I (FcγRI) and FcγRIII in porcine AMs were involved in PRRSV-ADE infection. An activation assay showed that specific activation of FcγRI or FcγRIII in porcine AMs during PRRSV infection not only significantly decreased the production of IFN-α and TNF-α, but also significantly increased the production of IL-10 and significantly facilitated PRRSV replication. In conclusion, our studies suggested that ADE downregulated the production of IFN-α and TNF-α in porcine AMs maybe via FcγRI and FcγRIII, thereby leading to enhanced PRRSV infection.

Taking the Hinge off: An Approach to Effector-Less Monoclonal Antibodies.

A variety of Fc domain engineering approaches for abrogating the effector functions of mAbs exists. To address some of the limitations of the current Fc domain silencing approaches, we are exploring a less commonly considered option which relies on the deletion of the hinge. Removal of the hinge domain in humanized IgG1 and IgG4 mAbs obliterates their ability to bind to activating human Fc gamma receptors I and IIIA, while leaving their ability to engage their target antigen intact. Deletion of the hinge also reduces binding to the Fc neonatal receptor, although Fc engineering allows partial recovery of affinity. Engineering of the CH3 domain, stabilizes hinge deleted IgG4s and prevents Fab arm exchange. The faster clearing properties together with the pacified Fc make modality of the hinge deleted mAb an appealing solution for therapeutic and diagnostic applications.

The Impact of FcγRI Binding on Immuno-PET.

Antibodies are promising vectors for PET imaging. However, the high uptake of radioimmunoconjugates in nontarget tissues such as the liver and spleen hampers their performance as radiotracers. This off-target uptake can lead to suboptimal tumor-to-background activity concentration ratios, decreasing the contrast of images and reducing their diagnostic utility. A possible cause of this uptake is the sequestration of radioimmunoconjugates by immune cells bearing Fc-γ-receptors (FcγR) that bind to the Fc regions of antibodies. Methods: Since the heavy chain glycans influence the affinity of FcγR for the Fc domain, we set out to investigate whether radioimmunoconjugates with truncated glycans would exhibit altered binding to FcγRI and, in turn, improved in vivo performance. Using the HER2-targeting antibody trastuzumab, we synthesized a series of desferrioxamine-bearing immunoconjugates with differing glycosylation states and interrogated their FcγRI binding via surface plasmon resonance, enzyme-linked immunosorbent assay, and flow cytometry. Furthermore, we labeled these immunoconjugates with 89Zr and explored their biodistribution in athymic nude, NSG, and humanized NSG mice bearing human epidermal growth factor receptor 2-expressing human breast cancer xenografts. Results: We observed a strong correlation between the impaired in vitro FcγRI binding of deglycosylated immunoconjugates and significant decreases in the in vivo off-target uptake of the corresponding 89Zr-labeled radioimmunoconjugates (i.e., liver activity concentrations are reduced by ∼3.5-fold in humanized NSG mice). These reductions in off-target uptake were accompanied by concomitant increases in the tumoral activity concentrations of the glycoengineered radioimmunoconjugates, ultimately yielding improved tumor-to-healthy organ contrast and higher quality PET images. Conclusion: Our findings suggest that the deglycosylation of antibodies represents a facile strategy for improving the quality of immuno-PET in animal models as well as in certain patient populations.