Monomeric IgG Research Articles

Stellabody: A novel hexamer-promoting mutation for improved IgG potency.

Advances in antibody engineering are being directed at the development of next generation immunotherapeutics with improved potency. Hexamerisation of IgG is a normal physiological aspect of IgG biology and recently described mutations that facilitate this process have a substantial impact upon monoclonal antibody behavior resulting in the elicitation of dramatically enhanced complement-dependent cytotoxicity, Fc receptor function, and enhanced antigen binding effects, such as targeted receptor agonism or microbe neutralization. Whereas the discovery of IgG hexamerisation enhancing mutations has largely focused on residues with exposure at the surface of the Fc-Fc and CH2-CH3 interfaces, our unique approach is the engineering of the mostly buried residue H429 in the CH3 domain. Selective substitution at position 429 forms the basis of Stellabody technology, where the choice of amino acid results in distinct hexamerisation outcomes. H429F results in monomeric IgG that hexamerises after target binding, so called "on-target" hexamerisation, while the H429Y mutant forms pH-sensitive hexamers in-solution prior to antigen binding. Moreover, Stellabody technologies are broadly applicable across the family of antibody-based biologic therapeutics, including conventional mAbs, bispecific mAbs, and Ig-like biologics such as Fc-fusions, with applications in diverse diseases.

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.

DNA Nanostructure-Templated Antibody Complexes Provide Insights into the Geometric Requirements of Human Complement Cascade Activation.

The classical complement pathway is activated by antigen-bound IgG antibodies. Monomeric IgG must oligomerize to activate complement via the hexameric C1q complex, and hexamerizing mutants of IgG appear as promising therapeutic candidates. However, structural data have shown that it is not necessary to bind all six C1q arms to initiate complement, revealing a symmetry mismatch between C1 and the hexameric IgG complex that has not been adequately explained. Here, we use DNA nanotechnology to produce specific nanostructures to template antigens and thereby spatially control IgG valency. These DNA-nanotemplated IgG complexes can activate complement on cell-mimetic lipid membranes, which enabled us to determine the effect of IgG valency on complement activation without the requirement to mutate antibodies. We investigated this using biophysical assays together with 3D cryo-electron tomography. Our data revealed the importance of interantigen distance on antibody-mediated complement activation, and that the cleavage of complement component C4 by the C1 complex is proportional to the number of ideally spaced antigens. Increased IgG valency also translated to better terminal pathway activation and membrane attack complex formation. Together, these data provide insights into how nanopatterning antigen-antibody complexes influence the activation of the C1 complex and suggest routes to modulate complement activation by antibody engineering. Furthermore, to our knowledge, this is the first time DNA nanotechnology has been used to study the activation of the complement system.

Abstract 6346: IgG engineered to target PIGR-mediated transcytosis specifically targets intracellular oncoproteins and abrogated tumor growth

Abstract Despite their long half-life, therapeutic antibodies are considered ineffective against intracellular antigens due to their perceived inability to penetrate epithelial cells. Using recombinant antibodies targeting common mutations in oncogenes, we have demonstrated that dimeric IgA, but not the same antibody on an IgG backbone, penetrates human epithelial cancer cells through PIGR-dependent directional transcytosis, specifically neutralizing mutated oncodrivers and expelling antigens outside the cell, bound to secretory IgA. Accordingly, targeting of KRASG12D abrogated tumor cell proliferation in cell culture assays. In vivo, KRASG12D-specific dIgA1 limited the growth of RASG12D-mutated ovarian and lung carcinomas, including in syngeneic tumor-bearing immunocompetent mice. Moreover, dIgA targeting of KRASG12D restricted tumor growth more effectively than small-molecule KRASG12D inhibitors, supporting the potential of this approach for the treatment of human cancers. Since producing dimeric IgA is more challenging than producing monomeric IgG, and because the half-life of IgA is shorter (4-7 days) than IgG (21 days), we have developed a new approach combining KRASG12D specific IgG with a peptide that could join PIGR and penetrates tumor cells through transcytosis, that showed the same effect than IgA controlling tumor growth. Our results provide a rationale for developing modified IgG cell-penetrating antibodies to effectively target common mutations in intracellular oncogenes that drive many aggressive and frequent human cancers. In addition, further experiments suggest that these antibodies could be effectively used to target tumors that have become resistant to existing KRAS inhibitors. Citation Format: Carmen Maria Anadon Galindo, Luis U. Lopez-Bailon, Ricardo A. Chaurio, Jessica A. Mine, Noah E. Plappert, Jose R. Conejo-Garcia. IgG engineered to target PIGR-mediated transcytosis specifically targets intracellular oncoproteins and abrogated tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6346.

IPSC-derived natural killer cells expressing the FcγR fusion CD64/16A can be armed with antibodies for multitumor antigen targeting

BackgroundAntibody therapies can direct natural killer (NK) cells to tumor cells, tumor-associated cells, and suppressive immune cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). This antigen-specific effector function of human NK...

An FcRn-targeted mucosal vaccine against SARS-CoV-2 infection and transmission

SARS-CoV-2 is primarily transmitted through droplets and airborne aerosols, and in order to prevent infection and reduce viral spread vaccines should elicit protective immunity in the airways. The neonatal Fc receptor (FcRn) transfers IgG across epithelial barriers and can enhance mucosal delivery of antigens. Here we explore FcRn-mediated respiratory delivery of SARS-CoV-2 spike (S). A monomeric IgG Fc was fused to a stabilized spike; the resulting S-Fc bound to S-specific antibodies and FcRn. Intranasal immunization of mice with S-Fc and CpG significantly induced antibody responses compared to the vaccination with S alone or PBS. Furthermore, we intranasally immunized mice or hamsters with S-Fc. A significant reduction of virus replication in nasal turbinate, lung, and brain was observed following nasal challenges with SARS-CoV-2 and its variants. Intranasal immunization also significantly reduced viral airborne transmission in hamsters. Nasal IgA, neutralizing antibodies, lung-resident memory T cells, and bone-marrow S-specific plasma cells mediated protection. Hence, FcRn delivers an S-Fc antigen effectively into the airway and induces protection against SARS-CoV-2 infection and transmission.

Comparisons of beta2-microglobulin, apolipoprotein A1, and immunoglobulins (IgG and IgM) detected in the serum and urine from individual cats

Detection of serum and urinary proteins is important for normal conditions, but comparison of individual serum and urine proteins is rarely performed. The aim of this study was to examine beta2-microglobulin (beta2-MG), apolipoprotein A-I (ApoA-I), and immunoglobulins (IgG and IgM) in the serum and urine of cats with chronic kidney disease and lower urinary tract disease (LUTD), in addition to healthy cats. Serum and urine samples were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by immunoblotting for beta2-MG, ApoA-I, IgG, and IgM. The molecular weight of serum beta2-MG was greater than the predicted molecular weight (11,472 Da), and different types of modified beta2-MGs were detected in the urine of healthy and diseased cats including original type in addition to grycocylated and partially digested types. Serum and urinary ApoA-I molecular weights were lower than the predicted molecular weight (28,943 Da), and high levels of urinary ApoA-I were detected in LUTD cats, although urinary ApoA-I was not detected in healthy cats. Under non-reducing conditions, H-chains of urinary IgM pentamers and IgG monomers were detected in healthy cats. These results suggest that urinary beta2-MG is modified in a different manner from serum beta2-MG, urinary ApoA-I is a potential marker of LUTD, and urinary IgM pentamer, IgG monomer, and their H-chains are found after glomerular filtration even in healthy conditions.

FcγRIIIA activation-mediated up-regulation of glycolysis alters MDSCs modulation in CD4+ T cell subsets of Sjögren syndrome

Our and other researchers’ previous studies found that myeloid-derived suppressor cells (MDSCs) were increased, and these MDSCs, supposed to play immunosuppressive roles, showed significant pro-inflammatory effects in Sjögren’s syndrome (SS). However, the key factors and potential mechanisms leading MDSCs to be inflammatory remain unclear. In this study, we found that MDSCs from SS patients were positively correlated with the percentages of Th17 cells, disease activity and serum autoantibodies, and showed higher levels of Fc gamma receptor (FcγR) IIIA and glycolysis. Most importantly, SS MDSCs or heat-aggregated IgG (HAIG)-treated MDSCs down-regulated Th1/Th2 ratio and up-regulated Th17/Treg ratio, which could be obviously rescued by IgG monomer or glycolysis inhibitor 2-DG. As well, the levels of FcγRIV and glycolysis in MDSCs and the ratio of Th17/Treg were increased, and the ratio of Th1/Th2 was decreased in SS-like NOD mice. Our study indicated that MDSCs showed pro-inflammatory phenotypes by disturbing CD4+ T-cell balances in SS. The pro-inflammatory effects of MDSCs might be directly linked to the enhanced glycolysis mediated by FcγRIIIA activation.

Protection of cell therapeutics from antibody-mediated killing by CD64 overexpression

Allogeneic cell therapeutics for cancer therapy or regenerative medicine are susceptible to antibody-mediated killing, which diminishes their efficacy. Here we report a strategy to protect cells from antibody-mediated killing that relies on engineered overexpression of the IgG receptor CD64. We show that human and mouse iPSC-derived endothelial cells (iECs) overexpressing CD64 escape antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity from IgG antibodies in vitro and in ADCC-enabled mice. When CD64 expression was combined with hypoimmune genetic modifications known to protect against cellular immunity, B2M−/−CIITA−/− CD47/CD64-transgenic iECs were resistant to both IgG antibody-mediated and cellular immune killing in vitro and in humanized mice. Mechanistic studies demonstrated that CD64 or its intracellularly truncated analog CD64t effectively capture monomeric IgG and occupy their Fc, and the IgG bind and occupy their target antigens. In three applications of the approach, human CD64t-engineered thyroid epithelial cells, pancreatic beta cells and CAR T cells withstood clinically relevant levels of graft-directed antibodies and fully evaded antibody-mediated killing.

IgM antibodies derived from memory B cells are potent cross-variant neutralizers of SARS-CoV-2.

Humoral immunity to SARS-CoV-2 can be supplemented with polyclonal sera from convalescent donors or an engineered monoclonal antibody (mAb) product. While pentameric IgM antibodies are responsible for much of convalescent sera's neutralizing capacity, all available mAbs are based on the monomeric IgG antibody subtype. We now show that IgM mAbs derived from immune memory B cell receptors are potent neutralizers of SARS-CoV-2. IgM mAbs outperformed clonally identical IgG antibodies across a range of affinities and SARS-CoV-2 receptor-binding domain epitopes. Strikingly, efficacy against SARS-CoV-2 viral variants was retained for IgM but not for clonally identical IgG. To investigate the biological role for IgM memory in SARS-CoV-2, we also generated IgM mAbs from antigen-experienced IgM+ memory B cells in convalescent donors, identifying a potent neutralizing antibody. Our results highlight the therapeutic potential of IgM mAbs and inform our understanding of the role for IgM memory against a rapidly mutating pathogen.

Targeting the high affinity receptor, FcγRI, in autoimmune disease, neuropathy, and cancer.

The Fc gamma receptor I (FcγRI or CD64) is the only human Fc receptor with a high affinity for monomeric IgG. It plays a crucial role in immunity, as it mediates cellular effector functions of antibodies including phagocytosis, antigen presentation, and cytokine production. FcγRI is constitutively saturated with monomeric IgG and this feeds the dogma that it has no role in immune responses. However, recent findings have implicated a role for FcγRI in various autoimmune disorders, neuropathies, and antibody therapy in tumor models. By a process known as 'inside-out' signaling, stimulation of myeloid cells with cytokines such as tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ) enhances FcγRI binding to immune complexes (ICs), including antibody-opsonized pathogens or tumor cells. This review focuses on the current knowledge on interaction of FcγRI with IgG and ICs and the effect of inside-out signaling on FcγRI functioning. Additionally, this review will address potential clinical applications of targeting FcγRI, and the tools that can be used to overcome IC-mediated autoimmune diseases on the one hand, and to enhance antibody-based anti-cancer therapy on the other.

Cross-reactivity of IgM anti-modified protein antibodies in rheumatoid arthritis despite limited mutational load

BackgroundAnti-modified protein antibodies (AMPA) targeting citrullinated, acetylated and/or carbamylated self-antigens are hallmarks of rheumatoid arthritis (RA). Although AMPA-IgG cross-reactivity to multiple post-translational modifications (PTMs) is evident, it is unknown whether the first responding B cells, expressing IgM, display similar characteristics or if cross-reactivity is crucially dependent on somatic hypermutation (SHM). We now studied the reactivity of (germline) AMPA-IgM to further understand the breach of B cell tolerance and to identify if cross-reactivity depends on extensive SHM. Moreover, we investigated whether AMPA-IgM can efficiently recruit immune effector mechanisms.MethodsPolyclonal AMPA-IgM were isolated from RA patients and assessed for cross-reactivity towards PTM antigens. AMPA-IgM B cell receptor sequences were obtained by single cell isolation using antigen-specific tetramers. Subsequently, pentameric monoclonal AMPA-IgM, their germline counterparts and monomeric IgG variants were generated. The antibodies were analysed on a panel of PTM antigens and tested for complement activation.ResultsPentameric monoclonal and polyclonal AMPA-IgM displayed cross-reactivity to multiple antigens and different PTMs. PTM antigen recognition was still present, although reduced, after reverting the IgM into germline. Valency of AMPA-IgM was crucial for antigen recognition as PTM-reactivity significantly decreased when AMPA-IgM were expressed as IgG. Furthermore, AMPA-IgM was 15- to 30-fold more potent in complement-activation compared to AMPA-IgG.ConclusionsWe provide first evidence that AMPA-IgM are cross-reactive towards different PTMs, indicating that PTM (cross-)reactivity is not confined to IgG and does not necessarily depend on extensive somatic hypermutation. Moreover, our data indicate that a diverse set of PTM antigens could be involved in the initial tolerance breach in RA and suggest that AMPA-IgM can induce complement-activation and thereby inflammation.

The FcγRIII Engagement Augments PMA-Stimulated Neutrophil Extracellular Traps (NETs) Formation by Granulocytes Partially via Cross-Talk between Syk-ERK-NF-κB and PKC-ROS Signaling Pathways.

Polymorphonuclear neutrophils (PMNs) are the most abundant white blood cell in the circulation capable of neutrophil extracellular traps (NETs) formation after stimulation. Both NADPH oxidase-dependent and -independent pathways are involved in NET formation. The IgG is the most abundant immunoglobulin in human serum. However, the impact of the circulating IgG on NET formation is totally unexplored. In this study, the all-trans retinoic acid (ATRA)-induced mature granulocytes (dHL-60) were pre-treated with monomeric human IgG, papain-digested Fab fragment, crystallizable IgG Fc portion, rituximab (a human IgG1), or IgG2. The NET formation of the dHL-60 in the presence/absence of phorbol 12-myristate 13-acetate (PMA) stimulation was then measured by the fluorescent area after SYTOX green nucleic acid stain. The intracellular reactive oxygen species (ROS) generation was measured by flow cytometry. Total and phosphorylated Syk, SHP-1, and ERK were detected by immunoblot. We found that human monomeric IgG and its subclasses IgG1 and IgG2 per se induced negligible NET formation of dHL-60, but the FcγRIII engagement by these IgG subclasses and Fc portion augment PMA-stimulated dHL-60 NET formation in a dose-dependent manner. Furthermore, we found that increased Syk and ERK phosphorylation, intracellular ROS generation, and pro-inflammatory cytokines, IL-8 and TNF-α, production could be induced after FcγRIII engagement. Blocking FcγRIII engagement by a specific antibody diminished the augmented NET formation. In conclusion, we discovered that cross-talk between FcγRIII engagement-induced Syk-ERK and PMA-induced PKC signaling pathways augment NET formation of dHL-60 via increased ROS generation and pro-inflammatory cytokines, IL-8 and TNF-α, production.

FcRn-Targeted Mucosal Vaccination against Influenza Virus Infection.

The respiratory tract is constantly exposed to various airborne pathogens. Most vaccines against respiratory infections are designed for the parenteral routes of administration; consequently, they provide relatively minimal protection in the respiratory tract. A vaccination strategy that aims to induce the protective mucosal immune responses in the airway is urgently needed. The FcRn mediates IgG Ab transport across the epithelial cells lining the respiratory tract. By mimicking this natural IgG transfer, we tested whether FcRn delivers vaccine Ags to induce a protective immunity to respiratory infections. In this study, we designed a monomeric IgG Fc fused to influenza virus hemagglutinin (HA) Ag with a trimerization domain. The soluble trimeric HA-Fc were characterized by their binding with conformation-dependent HA Abs or FcRn. In wild-type, but not FcRn knockout, mice, intranasal immunization with HA-Fc plus CpG adjuvant conferred significant protection against lethal intranasal challenge with influenza A/PR/8/34 virus. Further, mice immunized with a mutant HA-Fc lacking FcRn binding sites or HA alone succumbed to lethal infection. Protection was attributed to high levels of neutralizing Abs, robust and long-lasting B and T cell responses, the presence of lung-resident memory T cells and bone marrow plasma cells, and a remarkable reduction of virus-induced lung inflammation. Our results demonstrate for the first time, to our knowledge, that FcRn can effectively deliver a trimeric viral vaccine Ag in the respiratory tract and elicit potent protection against respiratory infection. This study further supports a view that FcRn-mediated mucosal immunization is a platform for vaccine delivery against common respiratory pathogens.

Process development exploiting competitive adsorption-based displacement effects in monoclonal antibody aggregate removal-A new high-throughput screening procedure for membrane chromatography.

High-throughput screening (HTS) approaches are commonly used to accelerate downstream process development. Although most HTS approaches use batch isothermal data (KP screen) or bind and elute mode as screening procedure, different or new process designs are rarely investigated. In this paper, a mechanistic model case study for the separation of two different two-component solutions was conducted and confirmed prior evidence. With these outcomes, a novel HTS screening procedure was developed including the determination of competitive adsorption-based displacement effects and key parameter identification. The screening procedure employing an overload bind and elute (OBE) mode is presented in a case study dealing with IgG aggregate removal in a typical monoclonal antibody purification step, applying a Sartobind®S membrane adsorber (MA). Based on a MA scale down device, the OBE mode allows the determination of classical process parameters and dynamic effects, such as displacement effects. Competitive adsorption-based displacement effects are visualized by introducing a displacement identifier leading to a displacement process map. Based on this map, the approach is transferred to and confirmed by the OBE recycle experiments with 4.6 and 8.2ml benchtop scsale devices resulting in 45% reduced IgG monomer and 88% increased higher molecular weight species binding capacities.

Production and characterization of a human antisperm monoclonal antibody against CD52g for topical contraception in women.

BackgroundApproximately 40% of human pregnancies are unintended, indicating a need for more acceptable effective contraception methods. New antibody production systems make it possible to manufacture reagent-grade human monoclonal antibodies (mAbs) for clinical use. We used the Nicotiana platform to produce a human antisperm mAb and tested its efficacy for on-demand topical contraception.MethodsHeavy and light chain variable region DNA sequences of a human IgM antisperm antibody derived from an infertile woman were inserted with human IgG1 constant region sequences into an agrobacterium and transfected into Nicotiana benthamiana. The product, an IgG1 mAb [“Human Contraception Antibody” (HCA)], was purified on Protein A columns, and QC was performed using the LabChip GXII Touch protein characterization system and SEC-HPLC. HCA was tested for antigen specificity by immunofluorescence and western blot assays, antisperm activity by sperm agglutination and complement dependent sperm immobilization assays, and safety in a human vaginal tissue (EpiVaginal™) model.FindingsHCA was obtained at concentrations ranging from 0.4 to 4 mg/ml and consisted of > 90% IgG monomers. The mAb specifically reacted with a glycan epitope on CD52g, a glycoprotein produced in the male reproductive tract and found in abundance on sperm. HCA potently agglutinated sperm under a variety of relevant physiological conditions at concentrations ≥ 6.25 µg/ml, and mediated complement-dependent sperm immobilization at concentrations ≥ 1 µg/ml. HCA and its immune complexes did not induce inflammation in EpiVaginal™ tissue.InterpretationHCA, an IgG1 mAb with potent sperm agglutination and immobilization activity and a good safety profile, is a promising candidate for female contraception.FundingThis research was supported by grants R01 HD095630 and P50HD096957 from the National Institutes of Health.

Association of IgG1 Antibody Clearance with FcγRIIA Polymorphism and Platelet Count in Infliximab-Treated Patients.

The FcγRIIA/CD32A is mainly expressed on platelets, myeloid and several endothelial cells. Its affinity is considered insufficient for allowing significant binding of monomeric IgG, while its H131R polymorphism (histidine > arginine at position 131) influences affinity for multimeric IgG2. Platelet FcγRIIA has been reported to contribute to IgG-containing immune-complexe clearance. Given our finding that platelet FcγRIIA actually binds monomeric IgG, we investigated the role of platelets and FcγRIIA in IgG antibody elimination. We used pharmacokinetics analysis of infliximab (IgG1) in individuals with controlled Crohn’s disease. The influence of platelet count and FcγRIIA polymorphism was quantified by multivariate linear modelling. The infliximab half-life increased with R allele number (13.2, 14.4 and 15.6 days for HH, HR and RR patients, respectively). It decreased with increasing platelet count in R carriers: from ≈20 days (RR) and ≈17 days (HR) at 150 × 109/L, respectively, to ≈13 days (both HR and RR) at 350 × 109/L. Moreover, a flow cytometry assay showed that infliximab and monomeric IgG1 bound efficiently to platelet FcγRIIA H and R allotypes, whereas panitumumab and IgG2 bound poorly to the latter. We propose that infliximab (and presumably any IgG1 antibody) elimination is partly due to an unappreciated mechanism dependent on binding to platelet FcγRIIA, which is probably tuned by its affinity for IgG2.

PAT for Continuous Chromatography Integrated into Continuous Manufacturing of Biologics towards Autonomous Operation

This study proposes a reliable inline PAT concept for the simultaneous monitoring of different product components after chromatography. The feed for purification consisted of four main components, IgG monomer, dimer, and two lower molecular weight components of 4.4 kDa and 1 kDa molecular weight. The proposed measurement setup consists of a UV–VIS diode-array detector and a fluorescence detector. Applying this system, a R2 of 0.93 for the target component, a R2 of 0.67 for the dimer, a R2 of 0.91 for the first side component and a R2 of 0.93 for the second side component is achieved. Root mean square error for IgG monomer was 0.027 g/L, for dimer 0.0047 g/L, for side component 1 0.016 g/L and for the side component 2 0.014 g/L. The proposed measurement concept tracked component concentration reliably down to 0.05 g/L. Zero-point fluctuations were kept within a standard deviation of 0.018 g/L for samples with no IgG concentration but with side components present, allowing a reliable detection of the target component. The main reason inline concentration measurements have not been established yet, is the false-positive measurement of target components when side components are present. This problem was eliminated using the combination of fluorescence and UV–VIS data for the test system. The use of this measurement system is simulated for the test system, allowing an automatic fraction cut at 0.05 g/L. In this simulation a consistent yield of >99% was achieved. Process disturbances for processed feed volume, feed purity and feed IgG concentration can be compensated with this setup. Compared to a timed process control, yield can be increased by up to 12.5%, if unexpected process disturbances occur.

Multimeric antibodies from antigen-specific human IgM+ memory B cells restrict Plasmodium parasites.

Multimeric immunoglobulin-like molecules arose early in vertebrate evolution, yet the unique contributions of multimeric IgM antibodies to infection control are not well understood. This is partially due to the difficulty of distinguishing low-affinity IgM, secreted rapidly by plasmablasts, from high-affinity antibodies derived from later-arising memory cells. We developed a pipeline to express B cell receptors (BCRs) from Plasmodium falciparum-specific IgM+ and IgG+ human memory B cells (MBCs) as both IgM and IgG molecules. BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity. Crystallization of one IgM+ MBC-derived antibody complexed with antigen defined a linear epitope within a conserved Plasmodium protein. In its physiological multimeric state, this antibody displayed exponentially higher antigen binding than a clonally identical IgG monomer, and more effectively inhibited P. falciparum invasion. Forced multimerization of this IgG significantly improved both antigen binding and parasite restriction, underscoring how avidity can alter antibody function. This work demonstrates the potential of high-avidity IgM in both therapeutics and vaccines.

Engineered IgG1-Fc Molecules Define Valency Control of Cell Surface Fcγ Receptor Inhibition and Activation in Endosomes.

The inhibition of Fcγ receptors (FcγR) is an attractive strategy for treating diseases driven by IgG immune complexes (IC). Previously, we demonstrated that an engineered tri-valent arrangement of IgG1 Fc domains (SIF1) potently inhibited FcγR activation by IC, whereas a penta-valent Fc molecule (PentX) activated FcγR, potentially mimicking ICs and leading to Syk phosphorylation. Thus, a precise balance exists between the number of engaged FcγRs for inhibition versus activation. Here, we demonstrate that Fc valency differentially controls FcγR activation and inhibition within distinct subcellular compartments. Large Fc multimer clusters consisting of 5-50 Fc domains predominately recruited Syk-mScarlet to patches on the plasma membrane, whereas PentX exclusively recruited Syk-mScarlet to endosomes in human monocytic cell line (THP-1 cells). In contrast, SIF1, similar to monomeric Fc, spent longer periods docked to FcγRs on the plasma membrane and did not accumulate and recruit Syk-mScarlet within large endosomes. Single particle tracking (SPT) of fluorescent engineered Fc molecules and Syk-mScarlet at the plasma membrane imaged by total internal reflection fluorescence microscopy (SPT-TIRF), revealed that Syk-mScarlet sampled the plasma membrane was not recruited to FcγR docked with any of the engineered Fc molecules at the plasma membrane. Furthermore, the motions of FcγRs docked with recombinant Fc (rFc), SIF1 or PentX, displayed similar motions with D ~ 0.15 μm2/s, indicating that SIF1 and PentX did not induce reorganization or microclustering of FcγRs beyond the ligating valency. Multicolor SPT-TIRF and brightness analysis of docked rFc, SIF1 and PentX also indicated that FcγRs were not pre-assembled into clusters. Taken together, activation on the plasma membrane requires assembly of more than 5 FcγRs. Unlike rFc or SIF1, PentX accumulated Syk-mScarlet on endosomes indicating that the threshold for FcγR activation on endosomes is lower than on the plasma membrane. We conclude that the inhibitory effects of SIF1 are mediated by stabilizing a ligated and inactive FcγR on the plasma membrane. Thus, FcγR inhibition can be achieved by low valency ligation with SIF1 that behaves similarly to FcγR docked with monomeric IgG.