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.

Fc engineered ACE2-Fc is a potent multifunctional agent targeting SARS-CoV2.

Joining a function-enhanced Fc-portion of human IgG to the SARS-CoV-2 entry receptor ACE2 produces an antiviral decoy with strain transcending virus neutralizing activity. SARS-CoV-2 neutralization and Fc-effector functions of ACE2-Fc decoy proteins, formatted with or without the ACE2 collectrin domain, were optimized by Fc-modification. The different Fc-modifications resulted in distinct effects on neutralization and effector functions. H429Y, a point mutation outside the binding sites for FcγRs or complement caused non-covalent oligomerization of the ACE2-Fc decoy proteins, abrogated FcγR interaction and enhanced SARS-CoV-2 neutralization. Another Fc mutation, H429F did not improve virus neutralization but resulted in increased C5b-C9 fixation and transformed ACE2-Fc to a potent mediator of complement-dependent cytotoxicity (CDC) against SARS-CoV-2 spike (S) expressing cells. Furthermore, modification of the Fc-glycan enhanced cell activation via FcγRIIIa. These different immune profiles demonstrate the capacity of Fc-based agents to be engineered to optimize different mechanisms of protection for SARS-CoV-2 and potentially other viral pathogens.

Corrigendum: Fc engineered ACE2-Fc is a potent multifunctional agent targeting SARS-CoV2.

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Fc Binding by FcγRIIa Is Essential for Cellular Activation by the Anti-FcγRIIa mAbs 8.26 and 8.2.

FcγR activity underpins the role of antibodies in both protective immunity and auto-immunity and importantly, the therapeutic activity of many monoclonal antibody therapies. Some monoclonal anti-FcγR antibodies activate their receptors, but the properties required for cell activation are not well defined. Here we examined activation of the most widely expressed human FcγR; FcγRIIa, by two non-blocking, mAbs, 8.26 and 8.2. Crosslinking of FcγRIIa by the mAb F(ab')2 regions alone was insufficient for activation, indicating activation also required receptor engagement by the Fc region. Similarly, when mutant receptors were inactivated in the Fc binding site, so that intact mAb was only able to engage receptors via its two Fab regions, again activation did not occur. Mutation of FcγRIIa in the epitope recognized by the agonist mAbs, completely abrogated the activity of mAb 8.26, but mAb 8.2 activity was only partially inhibited indicating differences in receptor recognition by these mAbs. FcγRIIa inactivated in the Fc binding site was next co-expressed with the FcγRIIa mutated in the epitope recognized by the Fab so that each mAb 8.26 molecule can contribute only three interactions, each with separate receptors, one via the Fc and two via the Fab regions. When the Fab and Fc binding were thus segregated onto different receptor molecules receptor activation by intact mAb did not occur. Thus, receptor activation requires mAb 8.26 Fab and Fc interaction simultaneously with the same receptor molecules. Establishing the molecular nature of FcγR engagement required for cell activation may inform the optimal design of therapeutic mAbs.

Boosting of Markers of Fcγ Receptor Function in Anti-HIV Antibodies During Structured Treatment Interruption.

Anti-HIV envelope (Env) antibodies elicit important Fc receptor functions, including FcγRIIIa-mediated natural killer cell killing of opsonized infected targets. How these antibodies evolve during HIV infection and treatment remains poorly understood. We describe changes in anti-HIV Env IgG using longitudinal samples from seroconverter subjects treated soon after infection and later during periods of structured treatment interruption (STI). Our well-validated dimeric rsFcγR binding assays combine effects of opsonizing antibody subclasses, epitopes, and geometries to provide a measure of FcγR (Fcγ receptor)-mediated functionality. IgG1 anti-Env titers diminished rapidly during antiretroviral therapy (ART; t1/2 3.0 ± 0.8 months), while the dimeric rsFcγRIIIa activity persisted longer (t1/2 33 ± 11 months), suggesting that there is maintenance of functional antibody specificities within the diminished pool of anti-HIV Env Abs. The initial antibody response to infection in two subjects was characterized by approximately fivefold higher FcγRIIIa compared with FcγRIIa binding activity. Uncoupling of FcγRIIa and FcγRIIIa activities may be a distinct feature of the early antibody response that preferentially engages FcγRIIIa-mediated effector functions. Two to three STI cycles, even with low viremia, were sufficient to boost dimeric FcγR activity in these seroconverter subjects. We hypothesize that increased humoral immunity induced by STI is a desirable functional outcome potentially achievable by therapeutic immunization during ART. We conclude that controlled viral antigen exposure under the protection of suppressive ART may be effective in eliciting FcγR-dependent function in support of viral reactivation and kill strategies.

The Rare Anaphylaxis-Associated FcγRIIa3 Exhibits Distinct Characteristics From the Canonical FcγRIIa1.

FcγRIIa is an activating FcγR, unique to humans and non-human primates. It induces antibody-dependent proinflammatory responses and exists predominantly as FcγRIIa1. A unique splice variant, we designated FcγRIIa3, has been reported to be associated with anaphylactic reactions to intravenous immunoglobulins (IVIg) therapy. We aim to define the functional consequences of this FcγRIIa variant associated with adverse responses to IVIg therapy and evaluate the frequency of associated SNPs. FcγRIIa forms from macaque and human PBMCs were investigated for IgG-subclass specificity, biochemistry, membrane localization, and functional activity. Disease-associated SNPs were analyzed by sequencing genomic DNA from 224 individuals with immunodeficiency or autoimmune disease. FcγRIIa3 was identified in macaque and human PBMC. The FcγRIIa3 is distinguished from the canonical FcγRIIa1 by a unique 19-amino acid cytoplasmic insertion and these two FcγRIIa forms responded distinctly to antibody ligation. Whereas FcγRIIa1 was rapidly internalized, FcγRIIa3 was retained longer at the membrane, inducing greater calcium mobilization and cell degranulation. Four FCGR2A SNPs were identified including the previously reported intronic SNP associated with anaphylaxis, but in only 1 of 224 individuals. The unique cytoplasmic element of FcγRIIa3 delays internalization and is associated with enhanced cellular activation. The frequency of the immunodeficiency-associated SNP varies between disease populations but interestingly occurred at a lower frequency than previously reported. None-the-less enhanced FcγRIIa3 function may promote a proinflammatory environment and predispose to pathological inflammatory responses.

Immunomodulation of inflammatory leukocyte markers during intravenous immunoglobulin treatment associated with clinical efficacy in chronic inflammatory demyelinating polyradiculoneuropathy.

OBJECTIVE: The objective of the study was to profile leukocyte markers modulated during intravenous immunoglobulin (IVIg) treatment, and to identify markers and immune pathways associated with clinical efficacy of IVIg for chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) with potential for monitoring treatment efficacy. METHODS: Response to IVIg treatment in newly diagnosed IVIg-naïve and established IVIg-experienced patients was assessed by changes in expression of inflammatory leukocyte markers by flow cytometry. The adjusted INCAT disability and Medical Research Council sum scores defined clinical response. RESULTS: Intravenous immunoglobulin modulated immunopathogenic pathways associated with inflammatory disease in CIDP. Leukocyte markers of clinical efficacy included reduced CD185+ follicular helper T cells, increased regulatory markers (CD23 and CD72) on B cells, and reduction in the circulating inflammatory CD16+ myeloid dendritic cell (mDC) population and concomitant increase in CD62L and CD195 defining a less inflammatory lymphoid homing mDC phenotype. A decline in inflammatory CD16+ dendritic cells was associated with clinical improvement or stability, and correlated with magnitude of improvement in neurological assessment scores, but did not predict relapse. IVIg also induced a nonspecific improvement in regulatory and reduced inflammatory markers not associated with clinical response. CONCLUSIONS: Clinically effective IVIg modulated inflammatory and regulatory pathways associated with ongoing control or resolution of CIDP disease. Some of these markers have potential for monitoring outcome.

The high-affinity receptor for IgG, FcγRI, of humans and non-human primates.

Non-human primate (NHP) models, especially involving macaques, are considered important models of human immunity and have been essential in preclinical testing for vaccines and therapeutics. Despite this, much less characterization of macaque Fc receptors has occurred compared to humans or mice. Much of the characterization of macaque Fc receptors so far has focused on the low-affinity Fc receptors, particularly FcγRIIIa. From these studies, it is clear that there are distinct differences between the human and macaque low-affinity receptors and their interaction with human IgG. Relatively little work has been performed on the high-affinity IgG receptor, FcγRI, especially in NHPs. This review will focus on what is currently known of how FcγRI interacts with IgG, from mutation studies and recent crystallographic studies of human FcγRI, and how amino acid sequence differences in the macaque FcγRI may affect this interaction. Additionally, this review will look at the functional consequences of differences in the amino acid sequences between humans and macaques.

Polymorphisms and interspecies differences of the activating and inhibitory FcγRII of Macaca nemestrina influence the binding of human IgG subclasses.

Little is known of the impact of Fc receptor (FcR) polymorphism in macaques on the binding of human (hu)IgG, and nothing is known of this interaction in the pig-tailed macaque (Macaca nemestrina), which is used in preclinical evaluation of vaccines and therapeutic Abs. We defined the sequence and huIgG binding characteristics of the M. nemestrina activating FcγRIIa (mnFcγRIIa) and inhibitory FcγRIIb (mnFcγRIIb) and predicted their structures using the huIgGFc/huFcγRIIa crystal structure. Large differences were observed in the binding of huIgG by mnFcγRIIa and mnFcγRIIb compared with their human FcR counterparts. MnFcγRIIa has markedly impaired binding of huIgG1 and huIgG2 immune complexes compared with huFcγRIIa (His(131)). In contrast, mnFcγRIIb has enhanced binding of huIgG1 and broader specificity, as, unlike huFcγRIIb, it avidly binds IgG2. Mutagenesis and molecular modeling of mnFcγRIIa showed that Pro(159) and Tyr(160) impair the critical FG loop interaction with huIgG. The enhanced binding of huIgG1 and huIgG2 by mnFcγRIIb was shown to be dependent on His(131) and Met(132). Significantly, both His(131) and Met(132) are conserved across FcγRIIb of rhesus and cynomolgus macaques. We identified functionally significant polymorphism of mnFcγRIIa wherein proline at position 131, also an important polymorphic site in huFcγRIIa, almost abolished binding of huIgG2 and huIgG1 and reduced binding of huIgG3 compared with mnFcγRIIa His(131). These marked interspecies differences in IgG binding between human and macaque FcRs and polymorphisms within species have implications for preclinical evaluation of Abs and vaccines in macaques.

A conserved host and pathogen recognition site on immunoglobulins: structural and functional aspects.

A common site in the constant region (Fc) of immunoglobulins is recognized by host receptors and is a frequent target of proteins expressed by pathogens. This site is located at the junction of two constant domains in the antibody heavy chains and produces a large shallow cavity formed by loops of the CH2 and CH3 domains in IgG and IgA (CH3 and CH4 domains in IgM). Crystal structures have been determined for complexes of IgG-Fc and IgA-Fc with a structurally diverse set of host, pathogen and in vitro selected ligands. While pathogen proteins may directly block interactions with the immunoglobulins thereby evading host immunity, it is likely that the same pathogen molecules also interact with other host factors to carry out their primary biological function. Herein we review the structural and functional aspects of host and pathogen molecular recognition of the common site on the Fc of immunoglobulins. We also propose that some pathogen proteins may promote virulence by affecting the bridging between innate and adaptive immunity.

Structural basis for Fc gammaRIIa recognition of human IgG and formation of inflammatory signaling complexes.

The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. FcγRIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of FcγRIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of FcγRIIa (FcγRIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence FcγRIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for FcγRIIa (IV.3), FcγRIIb (X63-21), and a pan FcγRII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of FcγRIIa and FcγRIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of FcγRIIa-HR binds Ag-Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly.

Interaction of human, rat, and mouse immunoglobulin A (IgA) with Staphylococcal superantigen-like 7 (SSL7) decoy protein and leukocyte IgA receptor.

Host survival depends on an effective immune system and pathogen survival on the effectiveness of immune evasion mechanisms. Staphylococcus aureus utilizes a number of molecules to modulate host immunity, including the SSL family of which SSL7 binds IgA and inhibits Fcα receptor I (FcαRI)-mediated function. Other Gram-positive bacterial pathogens produce IgA binding proteins, which, similar to SSL7, also bind the Fc at the CH2/CH3 interface (the junction between constant domains 2 and 3 of the heavy chain). The opposing activities of the host FcαRI-IgA receptor ligand pair and the pathogen decoy proteins select for host and pathogen variants, which exert stronger protection or evasion, respectively. Curiously, mouse but not rat IgA contains a putative N-linked glycosylation site in the center of this host receptor and pathogen-binding site. Here, we demonstrate that this site is glycosylated and that the effect of amino acid changes and glycosylation of the CH2/CH3 interface inhibits interaction with the pathogen IgA binding protein SSL7, while maintaining binding of pIgR, essential to the biosynthesis and transport of SIgA.

Structural basis for evasion of IgA immunity by Staphylococcus aureus revealed in the complex of SSL7 with Fc of human IgA1.

Infection by Staphylococcus aureus can result in severe conditions such as septicemia, toxic shock, pneumonia, and endocarditis with antibiotic resistance and persistent nasal carriage in normal individuals being key drivers of the medical impact of this virulent pathogen. In both virulent infection and nasal colonization, S. aureus encounters the host immune system and produces a wide array of factors that frustrate host immunity. One in particular, the prototypical staphylococcal superantigen-like protein SSL7, potently binds IgA and C5, thereby inhibiting immune responses dependent on these major immune mediators. We report here the three-dimensional structure of the complex of SSL7 with Fc of human IgA1 at 3.2 A resolution. Two SSL7 molecules interact with the Fc (one per heavy chain) primarily at the junction between the Calpha2 and Calpha3 domains. The binding site on each IgA chain is extensive, with SSL7 shielding most of the lateral surface of the Calpha3 domain. However, the SSL7 molecules are positioned such that they should allow binding to secretory IgA. The key IgA residues interacting with SSL7 are also bound by the leukocyte IgA receptor, FcalphaRI (CD89), thereby explaining how SSL7 potently inhibits IgA-dependent cellular effector functions mediated by FcalphaRI, such as phagocytosis, degranulation, and respiratory burst. Thus, the ability of S. aureus to subvert IgA-mediated immunity is likely to facilitate survival in mucosal environments such as the nasal passage and may contribute to systemic infections.

A common site of the Fc receptor gamma subunit interacts with the unrelated immunoreceptors FcalphaRI and FcepsilonRI.

The transmembrane (TM) region of the Fc receptor-gamma (FcRgamma) chain is responsible for the association of this ubiquitous signal transduction subunit with many immunoreceptor ligand binding chains, making FcRgamma key to a number of leukocyte activities in immunity and disease. Some receptors contain a TM arginine residue that interacts with Asp-11 of the FcRgamma subunit, but otherwise the molecular basis for the FcRgamma subunit interactions is largely unknown. This study reports residues in the TM region of the FcRgamma subunit are important for association with the high affinity IgE receptor FcepsilonRI and a leukocyte receptor cluster member, the IgA receptor FcalphaRI. FcRgamma residue Leu-21 was essential for surface expression of FcepsilonRIalpha/gamma2 and Tyr-8, Leu-14, and Phe-15 contributed to expression. Likewise, detergent-stable FcRgamma association with FcalphaRI was also dependent on Leu-14 and Leu-21 and in addition required residues Tyr-17, Tyr-25, and Cys-26. Modeling the TM regions of the FcRgamma dimer indicated these residues interacting with both FcalphaRI and FcepsilonRI are near the interface between the two FcRgamma TM helices. Furthermore, the FcRgamma residues interacting with FcalphaRI form a leucine zipper-like interface with mutagenesis confirming a complementary interface comprising FcalphaRI residues Leu-217, Leu-220, and Leu-224. The dependence of these two nonhomologous receptor interactions on FcRgamma Leu-14 and Leu-21 suggests that all the associated Fc receptors and the activating leukocyte receptor cluster members interact with this one site. Taken together these data provide a molecular basis for understanding how disparate receptor families assemble with the FcRgamma subunit.

Fc receptor gamma chain residues at the interface of the cytoplasmic and transmembrane domains affect association with FcalphaRI, surface expression, and function.

The assembly of multiple subunit immunoreceptors is dependent on transmembrane interactions. The Fc receptor gamma (FcR-gamma) chain is a ubiquitous immune receptor tyrosine-based activation motif-containing dimeric subunit, gamma(2), which in humans associates with both the activating members of the leukocyte receptor cluster, including the IgA receptor FcalphaRI, and the classical Fc receptors, including the IgE receptor FcepsilonRI. This study identifies a new site in the transmembrane region of FcR-gamma that affects receptor assembly and surface expression with FcalphaRI but not with FcepsilonRI. The wild type complex, FcalphaRI-gamma(2)WT, remains robustly associated in both Brij-96 and Thesit detergent conditions. However, mutation of either Tyr(25) or Cys(26) of FcR-gamma, near the interface of the transmembrane and cytoplasmic regions, resulted in impaired FcR-gamma association with FcalphaRI. This association was disrupted in the presence of the detergent Brij-96 but was preserved in milder conditions using the detergent Thesit. Ligand-mediated cross-linking of the FcalphaRI-gamma(2)Y25F mutant receptor resulted in diminished signal transduction, including an abnormal calcium response, compared with the FcalphaRI-gamma(2)WT receptor. Furthermore, the FcalphaRI-gamma(2)Y25F mutant receptor was expressed at the cell surface at approximately 10% of that of the wild type, whereas the surface expression of FcepsilonRI-gamma(2)Y25F was not significantly different from the wild type. In contrast, although the FcalphaRI-gamma(2)C26S mutant was also less stably associated, it was not reduced in surface expression or function. Thus, these TM residues of FcR-gamma are important for association with FcalphaRI and probably other activating LRC members but not with the classical FcR, FcepsilonRI.

Mutagenesis within human FcepsilonRIalpha differentially affects human and murine IgE binding.

Soluble fragments of the alpha-chain of FcepsilonRI, the high-affinity receptor for IgE, compete with membrane-bound receptors for IgE and may thus provide a means to combat allergic responses. Mutagenesis within FcepsilonRIalpha is used in this study, in conjunction with the crystal structure of the FcepsilonRIalpha/IgE complex, to define the relative importance of specific residues within human FcepsilonRIalpha for IgE binding. We have also compared the effects of these mutants on binding to both human and mouse IgE, with a view to evaluating the mouse as an appropriate model for the analysis of future agents designed to mimic the human FcepsilonRIalpha and attenuate allergic disease. Three residues within the C-C' region of the FcepsilonRIalpha2 domain and two residues within the alpha2 proximal loops of the alpha1 domain were selected for mutagenesis and tested in binding assays with human and mouse IgE. All three alpha2 mutations (K117D, W130A, and Y131A) reduced the affinity of human IgE binding to different extents, but K117D had a far more pronounced effect on mouse IgE binding, and although Y131A had little effect, W130A modestly enhanced binding to mouse IgE. The mutations in alpha1 (R15A and F17A) diminished binding to both human and mouse IgE, with these effects most likely caused by disruption of the alpha1/alpha2 interface. Our results demonstrate that the effects of mutations in human FcepsilonRIalpha on mouse IgE binding, and hence the inhibitory properties of human receptor-based peptides assayed in rodent models of allergy, may not necessarily reflect their activity in a human IgE-based system.