Coming together at the hinges: Therapeutic prospects of IgG3.

The human IgG3 subclass is conspicuously absent among the formats for approved monoclonal antibody therapies and Fc fusion protein biologics. Concern about the potential for rapid degradation, reduced plasma half-life, and increased immunogenicity due to marked variation in allotypes has apparently outweighed the potential advantages of IgG3, which include high affinity for activating Fcγ receptors, effective complement fixation, and a long hinge that appears better suited for low abundance targets. This review aims to highlight distinguishing features of IgG3 and to explore its functional role in the immune response. We present studies of natural immunity and recombinant antibody therapies that elucidate key contributions of IgG3 and discuss historical roadblocks that no longer remain clearly relevant. Collectively, this body of evidence motivates thoughtful reconsideration of the clinical advancement of this distinctive antibody subclass for treatment of human diseases. Abbreviations: ADCC - Antibody-Dependent Cell-mediated CytotoxicityADE - Antibody-dependent enhancementAID - Activation-Induced Cytidine DeaminaseCH - Constant HeavyCHF - Complement factor HCSR - Class Switch RecombinationEM - Electron MicroscopyFab - Fragment, antigen bindingFc - Fragment, crystallizableFcRn - Neonatal Fc ReceptorFcγR - Fc gamma ReceptorHIV - Human Immunodeficiency VirusIg - ImmunoglobulinIgH - Immunoglobulin Heavy chain geneNHP - Non-Human Primate.

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.

Enrichment of high affinity subclasses and glycoforms from serum-derived IgG using FcγRs as affinity ligands.

As antibodies continue to gain predominance in drug discovery and development pipelines, efforts to control and optimize their activity in vivo have matured to incorporate sophisticated abilities to manipulate engagement of specific Fc binding partners. Such efforts to promote diverse functional outcomes include modulating IgG-Fc affinity for FcγRs to alternatively potentiate or reduce effector functions, such as antibody-dependent cellular cytotoxicity and phagocytosis. While a number of natural and engineered Fc features capable of eliciting variable effector functions have been demonstrated in vitro and in vivo, elucidation of these important functional relationships has taken significant effort through use of diverse genetic, cellular and enzymatic techniques. As an orthogonal approach, we demonstrate use of FcγR as chromatographic affinity ligands to enrich and therefore simultaneously identify favored binding species from a complex mixture of serum-derived pooled polycloncal human IgG, a load material that contains the natural repertoire of Fc variants and post-translational modifications. The FcγR-enriched IgG was characterized for subclass and glycoform composition and the impact of this bioseparation step on antibody activity was measured in cell-based effector function assays including Natural Killer cell activation and monocyte phagocytosis. This work demonstrates a tractable means to rapidly distinguish complex functional relationships between two or more interacting biological agents by leveraging affinity chromatography followed by secondary analysis with high-resolution biophysical and functional assays and emphasizes a platform capable of surveying diverse natural post-translational modifications that may not be easily produced with high purity or easily accessible with recombinant expression techniques.

Going native: Direct high throughput screening of secreted full-length IgG antibodies against cell membrane proteins.

Gel microdroplet - fluorescence activated cell sorting (GMD-FACS) is an innovative high throughput screening platform for recombinant protein libraries, and we show here that GMD-FACS can overcome many of the limitations associated with conventional screening methods for antibody libraries. For example, phage and cell surface display benefit from exceptionally high throughput, but generally require high quality, soluble antigen target and necessitate the use of anchored antibody fragments. In contrast, the GMD-FACS assay can screen for soluble, secreted, full-length IgGs at rates of several thousand clones per second, and the technique enables direct screening against membrane protein targets in their native cellular context. In proof-of-concept experiments, rare anti-EGFR antibody clones were efficiently enriched from a 10,000-fold excess of anti-CCR5 clones in just three days. Looking forward, GMD-FACS has the potential to contribute to antibody discovery and engineering for difficult targets, such as ion channels and G protein-coupled receptors.

Multiplexed Fc array for evaluation of antigen-specific antibody effector profiles.

Antibodies are widely considered to be a frequent primary and often mechanistic correlate of protection of approved vaccines; thus evaluating the antibody response is of critical importance in attempting to understand and predict the efficacy of novel vaccine candidates. Historically, antibody responses have been analyzed by determining the titer of the humoral response using measurements such as an ELISA, neutralization, or agglutination assays. In the simplest case, sufficiently high titers of antibody against vaccine antigen(s) are sufficient to predict protection. However, antibody titer provides only a partial measure of antibody function, which is dependent on both the variable region (Fv) to bind the antigen target, and the constant region (Fc) to elicit an effector response from the innate arm of the immune system. In the case of some diseases, such as HIV, for which an effective vaccine has proven elusive, antibody effector function has been shown to be an important driver of monoclonal antibody therapy outcomes, of viral control in infected patients, and of vaccine-mediated protection in preclinical and clinical studies. We sought to establish a platform for the evaluation of the Fc domain characteristics of antigen-specific antibodies present in polyclonal samples in order to better develop insights into Fc receptor-mediated antibody effector activity, more fully understand how antibody responses may differ in association with disease progression and between subject groups, and differentiate protective from non-protective responses. To this end we have developed a high throughput biophysical platform capable of simultaneously evaluating many dimensions of the antibody effector response.

Biophysical and Functional Characterization of Rhesus Macaque IgG Subclasses.

Antibodies raised in Indian rhesus macaques [Macaca mulatta (MM)] in many preclinical vaccine studies are often evaluated in vitro for titer, antigen-recognition breadth, neutralization potency, and/or effector function, and in vivo for potential associations with protection. However, despite reliance on this key animal model in translation of promising candidate vaccines for evaluation in first in man studies, little is known about the properties of MM immunoglobulin G (IgG) subclasses and how they may compare to human IgG subclasses. Here, we evaluate the binding of MM IgG1, IgG2, IgG3, and IgG4 to human Fc gamma receptors (FcγR) and their ability to elicit the effector functions of human FcγR-bearing cells, and unlike in humans, find a notable absence of subclasses with dramatically silent Fc regions. Biophysical, in vitro, and in vivo characterization revealed MM IgG1 exhibited the greatest effector function activity followed by IgG2 and then IgG3/4. These findings in rhesus are in contrast with the canonical understanding that IgG1 and IgG3 dominate effector function in humans, indicating that subclass-switching profiles observed in rhesus studies may not strictly recapitulate those observed in human vaccine studies.

Functional polarization of tumour-associated macrophages by tumour-derived lactic acid.

Macrophages have an important role in the maintenance of tissue homeostasis. To perform this function, macrophages must have the capacity to monitor the functional states of their 'client cells': namely, the parenchymal cells in the various tissues in which macrophages reside. Tumours exhibit many features of abnormally developed organs, including tissue architecture and cellular composition. Similarly to macrophages in normal tissues and organs, macrophages in tumours (tumour-associated macrophages) perform some key homeostatic functions that allow tumour maintenance and growth. However, the signals involved in communication between tumours and macrophages are poorly defined. Here we show that lactic acid produced by tumour cells, as a by-product of aerobic or anaerobic glycolysis, has a critical function in signalling, through inducing the expression of vascular endothelial growth factor and the M2-like polarization of tumour-associated macrophages. Furthermore, we demonstrate that this effect of lactic acid is mediated by hypoxia-inducible factor 1α (HIF1α). Finally, we show that the lactate-induced expression of arginase 1 by macrophages has an important role in tumour growth. Collectively, these findings identify a mechanism of communication between macrophages and their client cells, including tumour cells. This communication most probably evolved to promote homeostasis in normal tissues but can also be engaged in tumours to promote their growth.

Beyond pattern recognition: NOD-like receptors in dendritic cells.

Innate instruction of adaptive immunity was proposed more than 20 years ago as a mechanism by which long-lived lymphocyte responses are targeted to appropriate antigens. At the time Charles Janeway proposed this theory, most of the innate immune receptors were unknown, and the pivotal role of the dendritic cell in instructing T cell priming was debated. There is now overwhelming evidence that the innate and adaptive branches of the immune system must interact to generate immunity. Much of this work has focused on families of innate immune receptors called pattern recognition receptors (PRRs) on dendritic cells, which translate these inflammatory triggers into productive T cell responses. Nevertheless, we are only beginning to understand how these defence molecules shape the generation of immunity. We review the varied roles of one class of PRRs, the NOD-like receptors (NLRs), in immune responses and propose a new model in which adaptive immunity requires coordinated PRR activation within the dendritic cell.