Biophysical differences in IgG1 Fc-based therapeutics relate to their cellular handling, interaction with FcRn and plasma half-life.

Antibody-based therapeutics (ABTs) are used to treat a range of diseases. Most ABTs are either full-length IgG1 antibodies or fusions between for instance antigen (Ag)-binding receptor domains and the IgG1 Fc fragment. Interestingly, their plasma half-life varies considerably, which may relate to how they engage the neonatal Fc receptor (FcRn). As such, there is a need for an in-depth understanding of how different features of ABTs affect FcRn-binding and transport behavior. Here, we report on how FcRn-engagement of the IgG1 Fc fragment compare to clinically relevant IgGs and receptor domain Fc fusions, binding to VEGF or TNF-α. The results reveal FcRn-dependent intracellular accumulation of the Fc, which is in line with shorter plasma half-life than that of full-length IgG1 in human FcRn-expressing mice. Receptor domain fusion to the Fc increases its half-life, but not to the extent of IgG1. This is mirrored by a reduced cellular recycling capacity of the Fc-fusions. In addition, binding of cognate Ag to ABTs show that complexes of similar size undergo cellular transport at different rates, which could be explained by the biophysical properties of each ABT. Thus, the study provides knowledge that should guide tailoring of ABTs regarding optimal cellular sorting and plasma half-life.

Antibody variable sequences have a pronounced effect on cellular transport and plasma half-life.

Monoclonal IgG antibodies are the fastest growing class of biologics, but large differences exist in their plasma half-life in humans. Thus, to design IgG antibodies with favorable pharmacokinetics, it is crucial to identify the determinants of such differences. Here, we demonstrate that the variable region sequences of IgG antibodies greatly affect cellular uptake and subsequent recycling and rescue from intracellular degradation by endothelial cells. When the variable sequences are masked by the cognate antigen, it influences both their transport behavior and binding to the neonatal Fc receptor (FcRn), a key regulator of IgG plasma half-life. Furthermore, we show how charge patch differences in the variable domains modulate both binding and transport properties and that a short plasma half-life, due to unfavorable charge patches, may partly be overcome by Fc-engineering for improved FcRn binding.

An engineered human albumin enhances half-life and transmucosal delivery when fused to protein-based biologics.

Needle-free uptake across mucosal barriers is a preferred route for delivery of biologics, but the efficiency of unassisted transmucosal transport is poor. To make administration and therapy efficient and convenient, strategies for the delivery of biologics must enhance both transcellular delivery and plasma half-life. We found that human albumin was transcytosed efficiently across polarized human epithelial cells by a mechanism that depends on the neonatal Fc receptor (FcRn). FcRn also transported immunoglobulin G, but twofold less than albumin. We therefore designed a human albumin variant, E505Q/T527M/K573P (QMP), with improved FcRn binding, resulting in enhanced transcellular transport upon intranasal delivery and extended plasma half-life of albumin in transgenic mice expressing human FcRn. When QMP was fused to recombinant activated coagulation factor VII, the half-life of the fusion molecule increased 3.6-fold compared with the wild-type human albumin fusion, without compromising the therapeutic properties of activated factor VII. Our findings highlight QMP as a suitable carrier of protein-based biologics that may enhance plasma half-life and delivery across mucosal barriers.

Engineered albumin-functionalized nanoparticles for improved FcRn binding enhance oral delivery of insulin.

Oral delivery of biopharmaceuticals, as insulin, is hampered by rapid degradation and inefficient absorption in the gastrointestinal tract (GIT). To solve this, a new class of biodegradable poly(lactic-co-glycolic)-poly(ethylene glycol) (PLGA-PEG) mucodiffusive nanoparticles (NPs) was designed. Specifically, these were decorated with site-specific conjugated human albumin, engineered for improved pH dependent binding to the neonatal Fc receptor (FcRn), which naturally mediates transport of albumin across the intestinal epithelium. The designed NPs of monodisperse 150 nm in size were 10% loaded with insulin and their surface was successfully functionalized with human albumin. Importantly, the engineered albumin-functionalized NPs bound human FcRn favorably in a pH dependent manner and showed enhanced transport across polarized cell layers. When orally administered to human FcRn expressing mice induced with diabetes, a reduction of glycemia was measured as a function of receptor targeting, with up to around 40% reduction after 1 h post-delivery. Thus, biodegradable PLGA-PEG NPs decorated with human albumin for improved FcRn-dependent transport offer a novel attractive strategy for delivery of encapsulated biopharmaceuticals across intestinal barriers.

FcRn is a CD32a coreceptor that determines susceptibility to IgG immune complex-driven autoimmunity.

IgG immune complexes (ICs) promote autoimmunity through binding fragment crystallizable (Fc) γ-receptors (FcγRs). Of these, the highly prevalent FcγRIIa (CD32a) histidine (H)-131 variant (CD32aH) is strongly linked to human autoimmune diseases through unclear mechanisms. We show that, relative to the CD32a arginine (R)-131 (CD32aR) variant, CD32aH more avidly bound human (h) IgG1 IC and formed a ternary complex with the neonatal Fc receptor (FcRn) under acidic conditions. In primary human and mouse cells, both CD32a variants required FcRn to induce innate and adaptive immune responses to hIgG1 ICs, which were augmented in the setting of CD32aH. Conversely, FcRn induced responses to IgG IC independently of classical FcγR, but optimal responses required FcRn and FcγR. Finally, FcRn blockade decreased inflammation in a rheumatoid arthritis model without reducing circulating autoantibody levels, providing support for FcRn's direct role in IgG IC-associated inflammation. Thus, CD32a and FcRn coregulate IgG IC-mediated immunity in a manner favoring the CD32aH variant, providing a novel mechanism for its disease association.

An intact C-terminal end of albumin is required for its long half-life in humans.

Albumin has an average plasma half-life of three weeks and is thus an attractive carrier to improve the pharmacokinetics of fused therapeutics. The half-life is regulated by FcRn, a cellular receptor that protects against intracellular degradation. To tailor-design the therapeutic use of albumin, it is crucial to understand how structural alterations in albumin affect FcRn binding and transport properties. In the blood, the last C-terminal residue (L585) of albumin may be enzymatically cleaved. Here we demonstrate that removal of the L585 residue causes structural stabilization in regions of the principal FcRn binding domain and reduces receptor binding. In line with this, a short half-life of only 3.5 days was measured for cleaved albumin lacking L585 in a patient with acute pancreatitis. Thus, we reveal the structural requirement of an intact C-terminal end of albumin for a long plasma half-life, which has implications for design of albumin-based therapeutics.

NHDL, a recombinant VL/VH hybrid antibody control for IgG2/4 antibodies.

The mechanism of action of recombinant IgG2/4 antibodies involves blocking of their target without the induction of effector functions. Examples are eculizumab (Soliris®), which is used clinically to block complement factor C5, as well as anti-human CD14 (r18D11) and anti-porcine CD14 (rMIL2) produced in our laboratory. So far, no proper IgG2/4 control antibody has been available for controlled validation of IgG2/4 antibody functions. Here, we describe the design of a recombinant control antibody (NHDL), which was generated by combining the variable light (VL) and heavy (VH) chains from two unrelated specificities. NHDL was readily expressed and purified as a stable IgG2/4 antibody, and showed no detectable specificity toward any putative antigen present in human or porcine blood. The approach of artificial VL/VH combination may be adopted for the design of other recombinant control antibodies.

Publisher Correction: Binding to nanopatterned antigens is dominated by the spatial tolerance of antibodies.

In the Supplementary Information file originally published with this Article, the Supplementary references 48-62 were missing; the amended file has now been uploaded.

Binding to nanopatterned antigens is dominated by the spatial tolerance of antibodies.

Although repetitive patterns of antigens are crucial for certain immune responses, an understanding of how antibodies bind and dynamically interact with various spatial arrangements of molecules is lacking. Hence, we introduced a new method in which molecularly precise nanoscale patterns of antigens are displayed using DNA origami and immobilized in a surface plasmon resonance set-up. Using antibodies with identical antigen-binding domains, we found that all the subclasses and isotypes studied bind bivalently to two antigens separated at distances that range from 3 to 17 nm. The binding affinities of these antibodies change with the antigen distances, with a distinct preference for antigens separated by approximately 16 nm, and considerable differences in spatial tolerance exist between IgM and IgG and between low- and high-affinity antibodies.

Human and mouse albumin bind their respective neonatal Fc receptors differently.

Albumin has a serum half-life of three weeks in humans and is utilized to extend the serum persistence of drugs that are genetically fused or conjugated directly to albumin or albumin-binding molecules. Responsible for the long half-life is FcRn that protects albumin from intracellular degradation. An in-depth understanding of how FcRn binds albumin across species is of importance for design and evaluation of albumin-based therapeutics. Albumin consists of three homologous domains where domain I and domain III of human albumin are crucial for binding to human FcRn. Here, we show that swapping of two loops in domain I or the whole domain with the corresponding sequence in mouse albumin results in reduced binding to human FcRn. In contrast, humanizing domain I of mouse albumin improves binding. We reveal that domain I of mouse albumin plays a minor role in the interaction with the mouse and human receptors, as domain III on its own binds with similar affinity as full-length mouse albumin. Further, we show that P573 in domain III of mouse albumin is required for strong receptor binding. Our study highlights distinct differences in structural requirements for the interactions between mouse and human albumin with their respective receptor, which should be taken into consideration in design of albumin-based drugs and evaluation in mouse models.

Pharmaceutical compounding of aflibercept in prefilled syringes does not affect structural integrity, stability or VEGF and Fc binding properties.

Macular edema due to neovascular age-related macular degeneration, diabetes or retinal vein occlusion can cause central vision loss. Intravitreal treatment with antibody-based biopharmaceutical compounds designed to neutralize vascular endothelial growth factor (VEGF) has proven to be an efficient strategy to ameliorate macular edema and restore visual acuity. At the same time, the use of anti-VEGF drugs places an economic burden on the health care system; the drugs are expensive, and repeated injections are usually required to maintain the therapeutic effect. Thus, there is an unmet need for more cost-effective procedures. We here describe how the most recently approved anti-VEGF drug, aflibercept, can be compounded into prefilled sterile syringes and stored for up to 4 weeks without compromising its quality, stability or functional properties, including VEGF and neonatal Fc receptor (FcRn) binding. The novel compounding method for repackaging of aflibercept in sterile plastic syringes can greatly reduce both cost and time spent per patient in the injection room.

A human endothelial cell-based recycling assay for screening of FcRn targeted molecules.

Albumin and IgG have remarkably long serum half-lives due to pH-dependent FcRn-mediated cellular recycling that rescues both ligands from intracellular degradation. Furthermore, increase in half-lives of IgG and albumin-based therapeutics has the potential to improve their efficacies, but there is a great need for robust methods for screening of relative FcRn-dependent recycling ability. Here, we report on a novel human endothelial cell-based recycling assay (HERA) that can be used for such pre-clinical screening. In HERA, rescue from degradation depends on FcRn, and engineered ligands are recycled in a manner that correlates with their half-lives in human FcRn transgenic mice. Thus, HERA is a novel cellular assay that can be used to predict how FcRn-binding proteins are rescued from intracellular degradation.

TRIM21 Immune Signaling Is More Sensitive to Antibody Affinity Than Its Neutralization Activity.

Ab-coated viruses can be detected in the cytosol by the FcR tripartite motif-containing 21 (TRIM21), which rapidly recruits the proteasomal machinery and triggers induction of immune signaling. As such, TRIM21 plays a key role in intracellular protection by targeting invading viruses for destruction and alerting the immune system. A hallmark of immunity is elicitation of a balanced response that is proportionate to the threat, to avoid unnecessary inflammation. In this article, we show how Ab affinity modulates TRIM21 immune function. We constructed a humanized monoclonal IgG1 against human adenovirus type 5 (AdV5) and a panel of Fc-engineered variants with a wide range of affinities for TRIM21. We found that IgG1-coated viral particles were neutralized via TRIM21, even when affinity was reduced by as much as 100-fold. In contrast, induction of NF-κB signaling was more sensitive to reduced affinity between TRIM21 and the Ab variants. Thus, TRIM21 mediates neutralization under suboptimal conditions, whereas induction of immune signaling is balanced according to the functional affinity for the incoming immune stimuli. Our findings have implications for engineering of antiviral IgG therapeutics with tailored effector functions.

Enhanced FcRn-dependent transepithelial delivery of IgG by Fc-engineering and polymerization.

Monoclonal IgG antibodies (Abs) are used extensively in the clinic to treat cancer and autoimmune diseases. In addition, therapeutic proteins are genetically fused to the constant Fc part of IgG. In both cases, the Fc secures a long serum half-life and favourable pharmacokinetics due to its pH-dependent interaction with the neonatal Fc receptor (FcRn). FcRn also mediates transport of intact IgG across polarized epithelial barriers, a pathway that is attractive for delivery of Fc-containing therapeutics. So far, no study has thoroughly compared side-by-side how IgG and different Fc-fusion formats are transported across human polarizing epithelial cells. Here, we used an in vitro cellular transport assay based on the human polarizing epithelial cell line (T84) in which both IgG1 and Fc-fusions were transported in an FcRn-dependent manner. Furthermore, we found that the efficacy of transport was dependent on the format. We demonstrate that transepithelial delivery could be enhanced by Fc-engineering for improved FcRn binding as well as by Fc-polymerization. In both cases, transport was driven by pH-dependent binding kinetics and the pH at the luminal side. Hence, efficient transcellular delivery of IgG-based drugs across human epithelial cells requires optimal pH-dependent FcRn binding that can be manipulated by avidity and Fc-engineering, factors that should inspire the design of future therapeutics targeted for transmucosal delivery.

Fc Engineering of Human IgG1 for Altered Binding to the Neonatal Fc Receptor Affects Fc Effector Functions.

Engineering of the constant Fc part of monoclonal human IgG1 (hIgG1) Abs is an approach to improve effector functions and clinical efficacy of next-generation IgG1-based therapeutics. A main focus in such development is tailoring of in vivo half-life and transport properties by engineering the pH-dependent interaction between IgG and the neonatal Fc receptor (FcRn), as FcRn is the main homeostatic regulator of hIgG1 half-life. However, whether such engineering affects binding to other Fc-binding molecules, such as the classical FcγRs and complement factor C1q, has not been studied in detail. These effector molecules bind to IgG1 in the lower hinge-CH2 region, structurally distant from the binding site for FcRn at the CH2-CH3 elbow region. However, alterations of the structural composition of the Fc may have long-distance effects. Indeed, in this study we show that Fc engineering of hIgG1 for altered binding to FcRn also influences binding to both the classical FcγRs and complement factor C1q, which ultimately results in alterations of cellular mechanisms such as Ab-dependent cell-mediated cytotoxicity, Ab-dependent cellular phagocytosis, and Ab-dependent complement-mediated cell lysis. Thus, engineering of the FcRn-IgG1 interaction may greatly influence effector functions, which has implications for the therapeutic efficacy and use of Fc-engineered hIgG1 variants.

Interaction with both domain I and III of albumin is required for optimal pH-dependent binding to the neonatal Fc receptor (FcRn).

Albumin is an abundant blood protein that acts as a transporter of a plethora of small molecules like fatty acids, hormones, toxins, and drugs. In addition, it has an unusual long serum half-life in humans of nearly 3 weeks, which is attributed to its interaction with the neonatal Fc receptor (FcRn). FcRn protects albumin from intracellular degradation via a pH-dependent cellular recycling mechanism. To understand how FcRn impacts the role of albumin as a distributor, it is of importance to unravel the structural mechanism that determines pH-dependent binding. Here, we show that although the C-terminal domain III (DIII) of human serum albumin (HSA) contains the principal binding site, the N-terminal domain I (DI) is important for optimal FcRn binding. Specifically, structural inspection of human FcRn (hFcRn) in complex with HSA revealed that two exposed loops of DI were in proximity with the receptor. To investigate to what extent these contacts affected hFcRn binding, we targeted selected amino acid residues of the loops by mutagenesis. Screening by in vitro interaction assays revealed that several of the engineered HSA variants showed decreased binding to hFcRn, which was also the case for two missense variants with mutations within these loops. In addition, four of the variants showed improved binding. Our findings demonstrate that both DI and DIII are required for optimal binding to FcRn, which has implications for our understanding of the FcRn-albumin relationship and how albumin acts as a distributor. Such knowledge may inspire development of novel HSA-based diagnostics and therapeutics.

Maternofetal transplacental transport of recombinant IgG antibodies lacking effector functions.

The neonatal Fc receptor (FcRn) directs the transfer of maternal immunoglobulin G (IgG) antibodies across the placenta and thus provides the fetus and newborn with passive protective humoral immunity. Pathogenic maternal IgG antibodies will also be delivered via the placenta and can cause alloimmunity, which may be lethal. A novel strategy to control pathogenic antibodies would be administration of a nondestructive IgG antibody blocking antigen binding while retaining binding to FcRn. We report on 2 human IgG3 antibodies with a hinge deletion and a C131S point mutation (IgG3ΔHinge) that eliminate complement activation and binding to all classical Fcγ receptors (FcγRs) and to C1q while binding to FcRn is retained. Additionally, 1 of the antibodies has a single point mutation in the Fc (R435H) at the binding site for FcRn (IgG3ΔHinge:R435H). We compared transplacental transport with wild-type IgG1 and IgG3, and found transport across trophoblast-derived BeWo cells and ex vivo placenta perfusions with hierarchies as follows: IgG3ΔHinge:R435H>wild-type IgG1≥IgG3ΔHinge and IgG3ΔHinge:R435H=wild-type IgG1=wild-type IgG3>>>IgG3ΔHinge, respectively. Collectively, IgG3ΔHinge:R435H was transported efficiently from the maternal to the fetal placental compartment. Thus, IgG3ΔHinge:R435H may be a good candidate for transplacental delivery of a nondestructive antibody to the fetus to combat pathogenic antibodies.