Differential effects of FcRn antagonists on the subcellular trafficking of FcRn and albumin.

The homeostasis of immunoglobulin G (IgG) is maintained by the neonatal Fc receptor, FcRn. Consequently, antagonism of FcRn to reduce endogenous IgG levels is an emerging strategy for treating antibody-mediated autoimmune disorders using either FcRn-specific antibodies or an engineered Fc fragment. For certain FcRn-specific antibodies, this approach has resulted in reductions in the levels of serum albumin, the other major ligand transported by FcRn. Cellular and molecular analyses of a panel of FcRn antagonists have been carried out to elucidate the mechanisms leading to their differential effects on albumin homeostasis. These analyses have identified two processes underlying decreases in albumin levels during FcRn blockade: increased degradation of FcRn and competition between antagonist and albumin for FcRn binding. These findings have potential implications for the design of drugs to modulate FcRn function.

Characterization of SARS-CoV-2 Convalescent Patients' Serological Repertoire Reveals High Prevalence of Iso-RBD Antibodies.

While our understanding of SARS-CoV-2 pathogenesis and antibody responses following infection and vaccination has improved tremendously since the outbreak in 2019, the sequence identities and relative abundances of the individual constituent antibody molecules in circulation remain understudied. Using Ig-Seq, we proteomically profiled the serological repertoire specific to the whole ectodomain of SARS-CoV-2 prefusion-stabilized spike (S) as well as to the receptor binding domain (RBD) over a 6-month period in four subjects following SARS-CoV-2 infection before SARS-CoV-2 vaccines were available. In each individual, we identified between 59 and 167 unique IgG clonotypes in serum. To our surprise, we discovered that ∼50% of serum IgG specific for RBD did not recognize prefusion-stabilized S (referred to as iso-RBD antibodies), suggesting that a significant fraction of serum IgG targets epitopes on RBD inaccessible on the prefusion-stabilized conformation of S. On the other hand, the abundance of iso-RBD antibodies in nine individuals who received mRNA-based COVID-19 vaccines encoding prefusion-stabilized S was significantly lower (∼8%). We expressed a panel of 12 monoclonal antibodies (mAbs) that were abundantly present in serum from two SARS-CoV-2 infected individuals, and their binding specificities to prefusion-stabilized S and RBD were all in agreement with the binding specificities assigned based on the proteomics data, including 1 iso-RBD mAb which bound to RBD but not to prefusion-stabilized S. 2 of 12 mAbs demonstrated neutralizing activity, while other mAbs were non-neutralizing. 11 of 12 mAbs also bound to S (B.1.351), but only 1 maintained binding to S (B.1.1.529). This particular mAb binding to S (B.1.1.529) 1) represented an antibody lineage that comprised 43% of the individual's total S-reactive serum IgG binding titer 6 months post-infection, 2) bound to the S from a related human coronavirus, HKU1, and 3) had a high somatic hypermutation level (10.9%), suggesting that this antibody lineage likely had been elicited previously by pre-pandemic coronavirus and was re-activated following the SARS-CoV-2 infection. All 12 mAbs demonstrated their ability to engage in Fc-mediated effector function activities. Collectively, our study provides a quantitative overview of the serological repertoire following SARS-CoV-2 infection and the significant contribution of iso-RBD antibodies, demonstrating how vaccination strategies involving prefusion-stabilized S may have reduced the elicitation of iso-RBD serum antibodies which are unlikely to contribute to protection.

Evidence of variable human Fcγ receptor-Fc affinities across differentially-complexed IgG.

Antibody-mediated effector functions are widely considered to unfold according to an associative model of IgG-Fcγ receptor (FcγR) interactions. The associative model presupposes that Fc receptors cannot discriminate antigen-bound IgG from free IgG in solution and have equivalent affinities for each. Therefore, the clustering of Fcγ receptors (FcγR) in the cell membrane, cross-activation of intracellular signaling domains, and the formation of the immune synapse are all the result of avid interactions between the Fc region of IgG and FcγRs that collectively overcome the individually weak, transient interactions between binding partners. Antibody allostery, specifically conformational allostery, is a competing model in which antigen-bound antibody molecules undergo a physical rearrangement that causes them to stand out from the background of free IgG by virtue of greater FcγR affinity. Various evidence exists in support of this model of antibody allostery, but it remains controversial. We report observations from multiplexed, label-free kinetic experiments in which the affinity values of FcγR were characterized for covalently immobilized, captured, and antigen-bound IgG. Across the strategies tested, receptors had greater affinity for the antigen-bound mode of IgG presentation. This phenomenon was observed across multiple FcγRs and generalized to multiple antigens, antibody specificities, and subclasses. Furthermore, the thermodynamic signatures of FcγR binding to free or immune-complexed IgG in solution differed when measured by an orthogonal label-free method, but the failure to recapitulate the trend in overall affinity leaves open questions as to what additional factors may be at play.

Evidence for the Role of a Second Fc-Binding Receptor in Placental IgG Transfer in Nonhuman Primates.

Transplacental transfer of maternal antibodies provides the fetus and newborn with passive protection against infectious diseases. While the role of the highly conserved neonatal Fc receptor (FcRn) in transfer of IgG in mammals is undisputed, recent reports have suggested that a second receptor may contribute to transport in humans. We report poor transfer efficiency of plant-expressed recombinant HIV-specific antibodies, including engineered variants with high FcRn affinity, following subcutaneous infusion into rhesus macaques close to parturition. Unexpectedly, unlike those derived from mammalian tissue culture, plant-derived antibodies were essentially unable to cross macaque placentas. This defect was associated with poor Fcγ receptor binding and altered Fc glycans and was not recapitulated in mice. These results suggest that maternal-fetal transfer of IgG across the three-layer primate placenta may require a second receptor and suggest a means of providing maternal antibody treatments during pregnancy while avoiding fetal harm. IMPORTANCE This study compared the ability of several human HIV envelope-directed monoclonal antibodies produced in plants with the same antibodies produced in mammalian cells for their ability to cross monkey and mouse placentas. We found that the two types of antibodies have comparable transfer efficiencies in mice, but they are differentially transferred across macaque placentas, consistent with a two-receptor IgG transport model in primates. Importantly, plant-produced monoclonal antibodies have excellent binding characteristics for human FcRn receptors, permitting desirable pharmacokinetics in humans. The lack of efficient transfer across the primate placenta suggests that therapeutic plant-based antibody treatments against autoimmune diseases and cancer could be provided to the mother while avoiding transfer and preventing harm to the fetus.

Functional consequences of allotypic polymorphisms in human immunoglobulin G subclasses.

Heritable polymorphisms within the human IgG locus, collectively termed allotypes, have often been linked by statistical associations, but rarely mechanistically, to a wide range of disease states. One potential explanation for these associations is that IgG allotype alters host cell receptors' affinity for IgG, dampening or enhancing an immune response depending on the nature of the change and the receptors. In this work, a panel of allotypic antibody variants were evaluated using multiplexed, label-free biophysical methods and cell-based functional assays to determine what effect, if any, human IgG polymorphisms have on antibody function. While we observed several differences in FcγR affinity among allotypes, there was little evidence of dramatically altered FcγR-based effector function or antigen recognition activity associated with this aspect of genetic variability.

Boosting of cross-reactive antibodies to endemic coronaviruses by SARS-CoV-2 infection but not vaccination with stabilized spike.

Preexisting antibodies to endemic coronaviruses (CoV) that cross-react with SARS-CoV-2 have the potential to influence the antibody response to COVID-19 vaccination and infection for better or worse. In this observational study of mucosal and systemic humoral immunity in acutely infected, convalescent, and vaccinated subjects, we tested for cross-reactivity against endemic CoV spike (S) protein at subdomain resolution. Elevated responses, particularly to the ß-CoV OC43, were observed in all natural infection cohorts tested and were correlated with the response to SARS-CoV-2. The kinetics of this response and isotypes involved suggest that infection boosts preexisting antibody lineages raised against prior endemic CoV exposure that cross-react. While further research is needed to discern whether this recalled response is desirable or detrimental, the boosted antibodies principally targeted the better-conserved S2 subdomain of the viral spike and were not associated with neutralization activity. In contrast, vaccination with a stabilized spike mRNA vaccine did not robustly boost cross-reactive antibodies, suggesting differing antigenicity and immunogenicity. In sum, this study provides evidence that antibodies targeting endemic CoV are robustly boosted in response to SARS-CoV-2 infection but not to vaccination with stabilized S, and that depending on conformation or other factors, the S2 subdomain of the spike protein triggers a rapidly recalled, IgG-dominated response that lacks neutralization activity.

Antibody attributes that predict the neutralization and effector function of polyclonal responses to SARS-CoV-2.

BACKGROUND: While antibodies can provide significant protection from SARS-CoV-2 infection and disease sequelae, the specific attributes of the humoral response that contribute to immunity are incompletely defined. METHODS: We employ machine learning to relate characteristics of the polyclonal antibody response raised by natural infection to diverse antibody effector functions and neutralization potency with the goal of generating both accurate predictions of each activity based on antibody response profiles as well as insights into antibody mechanisms of action. RESULTS: To this end, antibody-mediated phagocytosis, cytotoxicity, complement deposition, and neutralization were accurately predicted from biophysical antibody profiles in both discovery and validation cohorts. These models identified SARS-CoV-2-specific IgM as a key predictor of neutralization activity whose mechanistic relevance was supported experimentally by depletion. CONCLUSIONS: Validated models of how different aspects of the humoral response relate to antiviral antibody activities suggest desirable attributes to recapitulate by vaccination or other antibody-based interventions.

Boosting of Cross-Reactive Antibodies to Endemic Coronaviruses by SARS-CoV-2 Infection but not Vaccination with Stabilized Spike.

Pre-existing antibodies to endemic coronaviruses (CoV) that cross-react with SARS-CoV-2 have the potential to influence the antibody response to COVID-19 vaccination and infection for better or worse. In this observational study of mucosal and systemic humoral immunity in acutely infected, convalescent, and vaccinated subjects, we tested for cross reactivity against endemic CoV spike (S) protein at subdomain resolution. Elevated responses, particularly to the ß-CoV OC43, were observed in all natural infection cohorts tested and were correlated with the response to SARS-CoV-2. The kinetics of this response and isotypes involved suggest that infection boosts preexisting antibody lineages raised against prior endemic CoV exposure that cross react. While further research is needed to discern whether this recalled response is desirable or detrimental, the boosted antibodies principally targeted the better conserved S2 subdomain of the viral spike and were not associated with neutralization activity. In contrast, vaccination with a stabilized spike mRNA vaccine did not robustly boost cross-reactive antibodies, suggesting differing antigenicity and immunogenicity. In sum, this study provides evidence that antibodies targeting endemic CoV are robustly boosted in response to SARS-CoV-2 infection but not to vaccination with stabilized S, and that depending on conformation or other factors, the S2 subdomain of the spike protein triggers a rapidly recalled, IgG-dominated response that lacks neutralization activity.

Biophysical Evaluation of Rhesus Macaque Fc Gamma Receptors Reveals Similar IgG Fc Glycoform Preferences to Human Receptors.

Rhesus macaques are a common non-human primate model used in the evaluation of human monoclonal antibodies, molecules whose effector functions depend on a conserved N-linked glycan in the Fc region. This carbohydrate is a target of glycoengineering efforts aimed at altering antibody effector function by modulating the affinity of Fcγ receptors. For example, a reduction in the overall core fucose content is one such strategy that can increase antibody-mediated cellular cytotoxicity by increasing Fc-FcγRIIIa affinity. While the position of the Fc glycan is conserved in macaques, differences in the frequency of glycoforms and the use of an alternate monosaccharide in sialylated glycan species add a degree of uncertainty to the testing of glycoengineered human antibodies in rhesus macaques. Using a panel of 16 human IgG1 glycovariants, we measured the affinities of macaque FcγRs for differing glycoforms via surface plasmon resonance. Our results suggest that macaques are a tractable species in which to test the effects of antibody glycoengineering.

Antibody Attributes that Predict the Neutralization and Effector Function of Polyclonal Responses to SARS-CoV-2.

While antibodies provide significant protection from SARS-CoV-2 infection and disease sequelae, the specific attributes of the humoral response that contribute to immunity are incompletely defined. In this study, we employ machine learning to relate characteristics of the polyclonal antibody response raised by natural infection to diverse antibody effector functions and neutralization potency with the goal of generating both accurate predictions of each activity based on antibody response profiles as well as insights into antibody mechanisms of action. To this end, antibody-mediated phagocytosis, cytotoxicity, complement deposition, and neutralization were accurately predicted from biophysical antibody profiles in both discovery and validation cohorts. These predictive models identified SARS-CoV-2-specific IgM as a key predictor of neutralization activity whose mechanistic relevance was supported experimentally by depletion. Validated models of how different aspects of the humoral response relate to antiviral antibody activities suggest desirable attributes to recapitulate by vaccination or other antibody-based interventions.

Functional Homology for Antibody-Dependent Phagocytosis Across Humans and Rhesus Macaques.

Analyses of human clinical HIV-1 vaccine trials and preclinical vaccine studies performed in rhesus macaque (RM) models have identified associations between non-neutralizing Fc Receptor (FcR)-dependent antibody effector functions and reduced risk of infection. Specifically, antibody-dependent phagocytosis (ADP) has emerged as a common correlate of reduced infection risk in multiple RM studies and the human HVTN505 trial. This recurrent finding suggests that antibody responses with the capability to mediate ADP are most likely a desirable component of vaccine responses aimed at protecting against HIV-1 acquisition. As use of RM models is essential for development of the next generation of candidate HIV-1 vaccines, there is a need to determine how effectively ADP activity observed in RMs translates to activity in humans. In this study we compared ADP activity of human and RM monocytes and polymorphonuclear leukocytes (PMN) to bridge this gap in knowledge. We observed considerable variability in the magnitude of monocyte and PMN ADP activity across individual humans and RM that was not dependent on FcR alleles, and only modestly impacted by cell-surface levels of FcRs. Importantly, we found that for both human and RM phagocytes, ADP activity of antibodies targeting the CD4 binding site was greatest when mediated by human IgG3, followed by RM and human IgG1. These results demonstrate that there is functional homology between antibody and FcRs from these two species for ADP. We also used novel RM IgG1 monoclonal antibodies engineered with elongated hinge regions to show that hinge elongation augments RM ADP activity. The RM IgGs with engineered hinge regions can achieve ADP activity comparable to that observed with human IgG3. These novel modified antibodies will have utility in passive immunization studies aimed at defining the role of IgG3 and ADP in protection from virus challenge or control of disease in RM models. Our results contribute to a better translation of human and macaque antibody and FcR biology, and may help to improve testing accuracy and evaluations of future active and passive prevention strategies.

Markers of Polyfunctional SARS-CoV-2 Antibodies in Convalescent Plasma.

Convalescent plasma is a promising therapy for coronavirus disease 2019 (COVID-19), but the antibody characteristics that contribute to efficacy remain poorly understood. This study analyzed plasma samples from 126 eligible convalescent blood donors in addition to 15 naive individuals, as well as an additional 20 convalescent individuals as a validation cohort. Multiplexed Fc Array binding assays and functional antibody response assays were utilized to evaluate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody composition and activity. Donor convalescent plasma samples contained a range of antibody cell- and complement-mediated effector functions, indicating the diverse antiviral activity of humoral responses observed among recovered individuals. In addition to viral neutralization, convalescent plasma samples contained antibodies capable of mediating such Fc-dependent functions as complement activation, phagocytosis, and antibody-dependent cellular cytotoxicity against SARS-CoV-2. Plasma samples from a fraction of eligible donors exhibited high activity across all activities evaluated. These polyfunctional plasma samples could be identified with high accuracy with even single Fc Array features, whose correlation with polyfunctional activity was confirmed in the validation cohort. Collectively, these results expand understanding of the diversity of antibody-mediated antiviral functions associated with convalescent plasma, and the polyfunctional antiviral functions suggest that it could retain activity even when its neutralizing capacity is reduced by mutations in variant SARS-CoV-2.IMPORTANCE Convalescent plasma has been deployed globally as a treatment for COVID-19, but efficacy has been mixed. Better understanding of the antibody characteristics that may contribute to its antiviral effects is important for this intervention as well as offer insights into correlates of vaccine-mediated protection. Here, a survey of convalescent plasma activities, including antibody neutralization and diverse effector functions, was used to define plasma samples with broad activity profiles. These polyfunctional plasma samples could be reliably identified in multiple cohorts by multiplex assay, presenting a widely deployable screening test for plasma selection and investigation of vaccine-elicited responses.

Antibody responses to endemic coronaviruses modulate COVID-19 convalescent plasma functionality.

SARS-CoV-2 (CoV2) antibody therapies, including COVID-19 convalescent plasma (CCP), monoclonal antibodies, and hyperimmune globulin, are among the leading treatments for individuals with early COVID-19 infection. The functionality of convalescent plasma varies greatly, but the association of antibody epitope specificities with plasma functionality remains uncharacterized. We assessed antibody functionality and reactivities to peptides across the CoV2 and the 4 endemic human coronavirus (HCoV) genomes in 126 CCP donations. We found strong correlation between plasma functionality and polyclonal antibody targeting of CoV2 spike protein peptides. Antibody reactivity to many HCoV spike peptides also displayed strong correlation with plasma functionality, including pan-coronavirus cross-reactive epitopes located in a conserved region of the fusion peptide. After accounting for antibody cross-reactivity, we identified an association between greater alphacoronavirus NL63 antibody responses and development of highly neutralizing antibodies against CoV2. We also found that plasma preferentially reactive to the CoV2 spike receptor binding domain (RBD), versus the betacoronavirus HKU1 RBD, had higher neutralizing titer. Finally, we developed a 2-peptide serosignature that identifies plasma donations with high anti-spike titer, but that suffer from low neutralizing activity. These results suggest that analysis of coronavirus antibody fine specificities may be useful for selecting desired therapeutics and understanding the complex immune responses elicited by CoV2 infection.

Structure and Fc-Effector Function of Rhesusized Variants of Human Anti-HIV-1 IgG1s.

Passive transfer of monoclonal antibodies (mAbs) of human origin into Non-Human Primates (NHPs), especially those which function predominantly by a Fc-effector mechanism, requires an a priori preparation step, in which the human mAb is reengineered to an equivalent NHP IgG subclass. This can be achieved by changing both the Fc and Fab sequence while simultaneously maintaining the epitope specificity of the parent antibody. This Ab reengineering process, referred to as rhesusization, can be challenging because the simple grafting of the complementarity determining regions (CDRs) into an NHP IgG subclass may impact the functionality of the mAb. Here we describe the successful rhesusization of a set of human mAbs targeting HIV-1 envelope (Env) epitopes involved in potent Fc-effector function against the virus. This set includes a mAb targeting a linear gp120 V1V2 epitope isolated from a RV144 vaccinee, a gp120 conformational epitope within the Cluster A region isolated from a RV305 vaccinated individual, and a linear gp41 epitope within the immunodominant Cys-loop region commonly targeted by most HIV-1 infected individuals. Structural analyses confirm that the rhesusized variants bind their respective Env antigens with almost identical specificity preserving epitope footprints and most antigen-Fab atomic contacts with constant regions folded as in control RM IgG1s. In addition, functional analyses confirm preservation of the Fc effector function of the rhesusized mAbs including the ability to mediate Antibody Dependent Cell-mediated Cytotoxicity (ADCC) and antibody dependent cellular phagocytosis by monocytes (ADCP) and neutrophils (ADNP) with potencies comparable to native macaque antibodies of similar specificity. While the antibodies chosen here are relevant for the examination of the correlates of protection in HIV-1 vaccine trials, the methods used are generally applicable to antibodies for other purposes.

Antibody responses to endemic coronaviruses modulate COVID-19 convalescent plasma functionality.

COVID-19 convalescent plasma, particularly plasma with high-titer SARS-CoV-2 (CoV2) antibodies, has been successfully used for treatment of COVID-19. The functionality of convalescent plasma varies greatly, but the association of antibody epitope specificities with plasma functionality remains uncharacterized. We assessed antibody functionality and reactivities to peptides across the CoV2 and the four endemic human coronavirus (HCoV) genomes in 126 COVID-19 convalescent plasma donations. We found strong correlation between plasma functionality and polyclonal antibody targeting of CoV2 spike protein peptides. Antibody reactivity to many HCoV spike peptides also displayed strong correlation with plasma functionality, including pan-coronavirus cross-reactive epitopes located in a conserved region of the fusion peptide. After accounting for antibody cross-reactivity, we identified an association between greater alphacoronavirus NL63 antibody responses and development of highly neutralizing antibodies to SARS-CoV-2. We also found that plasma preferentially reactive to the CoV2 receptor binding domain (RBD), versus the betacoronavirus HKU1 RBD, had higher neutralizing titer. Finally, we developed a two-peptide serosignature that identifies plasma donations with high anti-S titer but that suffer from low neutralizing activity. These results suggest that analysis of coronavirus antibody fine specificities may be useful for selecting therapeutic plasma with desired functionalities.

SARS-CoV-2 antibody signatures robustly predict diverse antiviral functions relevant for convalescent plasma therapy.

Convalescent plasma has emerged as a promising COVID-19 treatment. However, the humoral factors that contribute to efficacy are poorly understood. This study functionally and phenotypically profiled plasma from eligible convalescent donors. In addition to viral neutralization, convalescent plasma contained antibodies capable of mediating such Fc-dependent functions as complement activation, phagocytosis and antibody-dependent cellular cytotoxicity against SARS-CoV-2. These activities expand the antiviral functions associated with convalescent plasma and together with neutralization efficacy, could be accurately and robustly from antibody phenotypes. These results suggest that high-throughput profiling could be used to screen donors and plasma may provide benefits beyond neutralization.

Features and Functions of Systemic and Mucosal Humoral Immunity Among SARS-CoV-2 Convalescent Individuals.

Understanding humoral immune responses to SARS-CoV-2 infection will play a critical role in the development of vaccines and antibody-based interventions. We report systemic and mucosal antibody responses in convalescent individuals who experienced varying disease severity. Robust antibody responses to diverse SARS-CoV-2 antigens and evidence of elevated responses to endemic CoV were observed among convalescent donors. SARS-CoV-2-specific IgA and IgG responses were often negatively correlated, particularly in mucosal samples, suggesting subject-intrinsic biases in isotype switching. Assessment of antibody-mediated effector functions revealed an inverse correlation between systemic and mucosal neutralization activity and site-dependent differences in the isotype of neutralizing antibodies. Serum neutralization correlated with systemic anti-SARS-CoV-2 IgG and IgM response magnitude, while mucosal neutralization was associated with nasal SARS-CoV-2-specific IgA. These findings begin to map how diverse Ab characteristics relate to Ab functions and outcomes of infection, informing public health assessment strategies and vaccine development efforts.

Fc-mediated effector function contributes to the in vivo antiviral effect of an HIV neutralizing antibody.

Treatment of HIV infection with either antiretroviral (ARV) therapy or neutralizing monoclonal antibodies (NAbs) leads to a reduction in HIV plasma virus. Both ARVs and NAbs prevent new rounds of viral infection, but NAbs may have the additional capacity to accelerate the loss of virus-infected cells through Fc gamma receptor (FcγR)-mediated effector functions, which should affect the kinetics of plasma-virus decline. Here, we formally test the role of effector function in vivo by comparing the rate and timing of plasma-virus clearance in response to a single-dose treatment with either unmodified NAb or those with either reduced or augmented Fc function. When infused into viremic simian HIV (SHIV)-infected rhesus macaques, there was a 21% difference in slope of plasma-virus decline between NAb and NAb with reduced Fc function. NAb engineered to increase FcγRIII binding and improve antibody-dependent cellular cytotoxicity (ADCC) in vitro resulted in arming of effector cells in vivo, yet led to viral-decay kinetics similar to NAbs with reduced Fc function. These studies show that the predominant mechanism of antiviral activity of HIV NAbs is through inhibition of viral entry, but that Fc function can contribute to the overall antiviral activity, making them distinct from standard ARVs.

Hinge length contributes to the phagocytic activity of HIV-specific IgG1 and IgG3 antibodies.

Antibody functions such as neutralization require recognition of antigen by the Fab region, while effector functions are additionally mediated by interactions of the Fc region with soluble factors and cellular receptors. The efficacy of individual antibodies varies based on Fab domain characteristics, such as affinity for antigen and epitope-specificity, and on Fc domain characteristics that include isotype, subclass, and glycosylation profile. Here, a series of HIV-specific antibody subclass and hinge variants were constructed and tested to define those properties associated with differential effector function. In the context of the broadly neutralizing CD4 binding site-specific antibody VRC01 and the variable loop (V3) binding antibody 447-52D, hinge truncation and extension had a considerable impact on the magnitude of phagocytic activity of both IgG1 and IgG3 subclasses. The improvement in phagocytic potency of antibodies with extended hinges could not be attributed to changes in either intrinsic antigen or antibody receptor affinity. This effect was specific to phagocytosis and was generalizable to different phagocytes, at different effector cell to target ratios, for target particles of different size and composition, and occurred across a range of antibody concentrations. Antibody dependent cellular cytotoxicity and neutralization were generally independent of hinge length, and complement deposition displayed variable local optima. In vivo stability testing showed that IgG molecules with altered hinges can exhibit similar biodistribution and pharmacokinetic profiles as IgG1. Overall, these results suggest that when high phagocytic activity is desirable, therapeutic antibodies may benefit from being formatted as human IgG3 or engineered IgG1 forms with elongated hinges.