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While the established route for vaccines against the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is intramuscular, it may be preferable to deliver vaccines intranasally to secure mucosal protection at the site of infection. This will limit the spread of the virus, ease administration and likely improve vaccine acceptance. Here, we report on a subunit vaccine platform, where the antigen is genetically fused to engineered human albumin. Upon intranasal delivery the subunit vaccines target the neonatal Fc receptor (FcRn) and induce both local and systemic antigen-specific antibody responses at magnitudes higher than after intramuscular delivery. We provide evidence that such needle-free vaccination induces production of antibodies with neutralizing capacity against SARS-CoV-2 or influenza A. Thus, the vaccine platform is particularly well suited for design of subunit vaccines against these and other infectious respiratory diseases.

Humans have four IgG antibody subclasses that selectively or differentially engage immune effector molecules to protect against infections. Although IgG1 has been studied in detail and is the subclass of most approved antibody therapeutics, increasing evidence indicates that IgG3 is associated with enhanced protection against pathogens. Here, we report that IgG3 has superior capacity to mediate intracellular antiviral immunity compared with the other subclasses due to its uniquely extended and flexible hinge region, which facilitates improved recruitment of the cytosolic Fc receptor TRIM21, independently of Fc binding affinity. TRIM21 may also synergize with complement C1/C4-mediated lysosomal degradation via capsid inactivation. We demonstrate that this process is potentiated by IgG3 in a hinge-dependent manner. Our findings reveal differences in how the four IgG subclasses mediate intracellular immunity, knowledge that may guide IgG subclass selection and engineering of antiviral antibodies for prophylaxis and therapy.

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., Communications Biology, 3 (1), ISSN:2399-3642

Introduction: The acquisition of humoral immunity in utero is essential for the fetus. The crucial protein, which is responsible for this part of immunity, is immunoglobulin-G (IgG). Immune functions of IgGs are mediated via the interaction of the crystallizable fragment (Fc) region of IgG with specific Fc gamma receptors (Fc gamma Rs). However, an atypical Fc gamma R, the neonatal Fc receptor (FcRn), is a key regulator of IgG transfer across the human placenta. During the last four decades ex vivo placental perfusion studies have contributed significantly to the study of mechanisms of IgG transfer across the multicellular placental barrier. Method: A PubMed search was conducted by using specific keywords: placenta, perfusion and IgG to review manuscripts using human placental perfusion to study the transplacental transfer of IgG. Relevant studies found in reference lists of these manuscripts were also added to the review, and references were included that supported or gave nuance to the discussion of the mechanisms of IgG kinetics in the placenta. Results and Discussion: We found twenty publications on the study of transplacental transfer of IgG using human ex vivo placental perfusion, by research groups with partly different settings. This review summarizes knowledge about placental IgG transfer, with a strong focus on the contributions from ex vivo placental perfusion studies.Superscript/Subscript Available

TCR-like antibodies represent a unique type of engineered antibodies with specificity toward pHLA, a ligand normally restricted to the sensitive recognition by T cells. Here, we report a phage display-based sequential development path of such antibodies. The strategy goes from initial lead identification through in silico informed CDR engineering in combination with framework engineering for affinity and thermostability optimization, respectively. The strategy allowed the identification of HLA-DQ2.5 gluten peptide-specific TCR-like antibodies with low picomolar affinity. Our method outlines an efficient and general method for development of this promising class of antibodies, which should facilitate their utility including translation to human therapy.

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. The ordered display of antigen patterns on DNA origami platforms combined with surface plasmon resonance chips allows the investigation of the affinity and kinetics of the antigen–antibody bivalent binding in relation to the antigen distance and antibody flexibility.

Summary 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., Graphical abstract, Highlights • IgG variable region sequences greatly affect cellular uptake and recycling • Variable region charge patches affect FcRn binding and transport • The presence of cognate antigen modulates cellular transport and FcRn binding • Fc-engineering for improved FcRn binding can overcome unfavorable charge patches, Biological sciences; Immunology; Biophysics

The neonatal Fc receptor (FcRn) was first recognized for its role in transfer of maternal IgG to the fetus or newborn, providing passive immunity early in life. However, it has become clear that the receptor is versatile, widely expressed and plays an indispensable role in both immunological and non-immunological processes throughout life. The receptor rescues immunoglobulin G (IgG) and albumin from intracellular degradation and shuttles the ligands across polarized cell barriers, in all cases via a pH dependent binding-and-release mechanism. These processes secure distribution and high levels of both IgG and albumin throughout the body. At mucosal sites, FcRn transports IgG across polarized epithelial cells where it retrieves IgG in complex with luminal antigens that is delivered to tissue-localized immune cells. In dendritic cells (DCs), FcRn orchestrates processing of IgG-opsonized immune complexes (ICs) in concert with classical Fcγ receptors, which results in antigen presentation and cross-presentation of antigenic peptides on MHC class II and I to CD4+ and CD8+ T cells, respectively. Hence, FcRn regulates transport of the ligands within and across different types of cells, but also processing of IgG-ICs by immune cells. As such, the receptor is involved in immune surveillance and protection against infections. In this brief review, we highlight how FcRn expressed by hematopoietic and non-hematopoietic cells contributes to immune regulation at mucosal barriers - biology that can be utilized in development of biologics and subunit vaccines for non-invasive delivery.

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 favourable 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 behaviour 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 unfavourable charge patches, may partly be overcome by Fc-engineering for improved FcRn binding.

Antibodies specific for peptides bound to human leukocyte antigen (HLA) molecules are valuable tools for studies of antigen presentation and may have therapeutic potential. Here, we generated human T-cell receptor (TCR)-like antibodies towards the immunodominant signature gluten epitope DQ2.5-glia-α2 in celiac disease (CeD). Phage display selection combined with secondary targeted engineering was used to obtain highly specific antibodies with picomolar affinity. The crystal structure of a Fab fragment of the lead antibody 3.C11 in complex with HLA-DQ2.5:DQ2.5-glia-α2 revealed a binding geometry and interaction mode highly similar to prototypic TCRs specific for the same complex. Assessment of CeD biopsy material confirmed disease specificity and reinforced the notion that abundant plasma cells present antigen in the inflamed CeD gut. Further, 3.C11 specifically inhibited activation and proliferation of gluten-specific CD4(+) T cells in vitro and in HLA-DQ2.5 humanized mice, suggesting a potential for targeted intervention without compromising systemic immunity. ONE SENTENCE SUMMARY: A human TCR-like antibody blocks gluten-dependent activation of celiac disease T-cells in vitro and in HLA-DQ2.5 humanized mice.

Targeted delivery of antigen to antigen-presenting cells (APCs) enhances antigen presentation and thus, is a potent strategy for making more efficacious vaccines. This can be achieved by use of antibodies with specificity for endocytic surface molecules expressed on the APC. We aimed to compare two different antibody-antigen fusion modes in their ability to induce T-cell responses; first, exchange of immunoglobulin (Ig) constant domain loops with a T-cell epitope (Troybody), and second, fusion of T-cell epitope or whole antigen to the antibody C-terminus. Although both strategies are well-established, they have not previously been compared using the same system. We found that both antibody-antigen fusion modes led to presentation of the T-cell epitope. The strength of the T-cell responses varied, however, with the most efficient Troybody inducing CD4 T-cell proliferation and cytokine secretion at 10-100-fold lower concentration than the antibodies carrying antigen fused to the C-terminus, both in vitro and after intravenous injection in mice. Furthermore, we exchanged this loop with an MHCI-restricted T-cell epitope, and the resulting antibody enabled efficient cross-presentation to CD8 T cells in vivo. Targeting of antigen to APCs by use of such antibody-antigen fusions is thus an attractive vaccination strategy for increased activation of both CD4 and CD8 peptide-specific T cells.

FcRn and CD32a form a ternary complex under acidic conditions and act together in determining responses to IgG immune complexes. The human autoimmune disease–associated CD32a-H131 variant more avidly forms this ternary complex, leading to greater FcRn-dependent immune responses to IgG., 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.

The semi-public T-cell response towards the gluten epitope DQ2.5-glia-α2 uses a prototypic TCR encoded by the germline segments TRAV26-1 and TRBV7-2. Through mutagenesis experiments, we show that a TRAV26-1encoded recognition motif contacts the MHC β-chain and the TCR CDR3β loop underpinning this conserved T-cell response restricted to the prototypic TCRs.

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.

Albumin has a long serum half-life due to its unique ability to bind the cellular neonatal Fc receptor (FcRn), which provides protection from intracellular degradation. The interaction can be capitalized to improve the efficacy of drugs by extending their serum persistence. However, species-specific binding of albumin to FcRn challenges preclinical development. The goal of this brief review is to provide insights into how FcRn and cross-species binding differences affect the pharmacokinetics of human serum albumin (HSA) in different animal models, and gives an overview of genetically modified mice that may serve as improved models for testing of albumin-based drugs.

IgM molecules circulate in serum as large polymers, mainly pentamers, which can be transported by the poly-Ig receptor (pIgR) across epithelial cells to mucosal surfaces and released as secretory IgM (SIgM). The mucosal SIgM molecules have non-covalently attached secretory component (SC), which is the extracellular part of pIgR which is cleaved from the epithelial cell membrane. Serum IgM antibodies do not contain SC, and have previously been shown to make a conformational change from “a star” to a “staple” conformation upon reaction with antigens on a cell surface, enabling them to activate complement. However, it is not clear if SIgM similarly can induce complement activation. In order to clarify this issue, we constructed recombinant chimeric (mouse/human) IgM antibodies against hapten 5-iodo-4-hydroxy-3-nitro-phenacetyl (NIP) and in addition studied polyclonal IgM formed after immunization with a meningococcal group B vaccine. The monoclonal and polyclonal IgM molecules were purified by affinity chromatography on a column containing human SC in order to isolate joining-chain (J-chain) containing IgM, followed by addition of excess amounts of soluble SC to create SIgM (IgM J+ SC+). These SIgM preparations were tested for complement activation ability and shown to be nearly as active as the parental IgM J+ molecules. Thus, SIgM may offer protection against pathogens at mucosal surface by complement-mediated cell lysis or by phagocytosis mediated by complement receptors present on effector cells on mucosa. This article is protected by copyright. All rights reserved.

Antibodies specific for antigenic peptides bound to major histocompatibility complex (MHC) molecules are valuable tools for studies of antigen presentation. Such T-cell receptor (TCR)-like antibodies may also have therapeutic potential in human disease due to their ability to target disease-associated antigens with high specificity. We previously generated celiac disease (CeD) relevant TCR-like antibodies that recognize the prevalent gluten epitope DQ2.5-glia-α1a in complex with HLA-DQ2.5. Here, we report on second-generation high-affinity antibodies towards this epitope as well as a panel of novel TCR-like antibodies to another immunodominant gliadin epitope, DQ2.5-glia-α2. The strategy for affinity engineering was based on Rosetta modeling combined with pIX phage display and is applicable to similar protein engineering efforts. We isolated picomolar affinity binders and validated them in Fab and IgG format. Flow cytometry experiments with CeD biopsy material confirm the unique disease specificity of these TCR-like antibodies and reinforce the notion that B cells and plasma cells have a dominant role in gluten antigen presentation in the inflamed CeD gut. Further, the lead candidate 3.C11 potently inhibited CD4+ T-cell activation and proliferation in vitro in an HLA and epitope specific manner, pointing to a potential for targeted disease interception without compromising systemic immunity.Significance StatementConsumption of gluten-containing food drives celiac disease in genetically predisposed individuals. The underlying disease mechanism is not fully understood, but it is strictly dependent on activation of pathogenic T cells. We have engineered high-affinity human antibodies recognizing the T-cell target HLA-DQ2.5 in complex with gluten epitopes and studied cell-specific antigen presentation in patients, which shows that plasma cells and not dendritic cells dominate the inflamed tissue. The only available treatment is lifelong adherence to a gluten-free diet, which is difficult and not effective in all cases. We show that at least one of our antibodies can specifically inhibit activation of pathogenic T-cells in vitro and therefore shows promise for therapy.

Monoclonal antibodies (mAbs) are valuable as research reagents, in diagnosis and in therapy. Their high specificity, the ease in production, favorable biophysical properties and the opportunity to engineer different properties make mAbs a versatile class of biologics. mAbs targeting peptide−major histocompatibility molecule (pMHC) complexes are often referred to as “TCR-like” mAbs, as pMHC complexes are generally recognized by T-cell receptors (TCRs). Presentation of self- and non-self-derived peptide fragments on MHC molecules and subsequent activation of T cells dictate immune responses in health and disease. This includes responses to infectious agents or cancer but also aberrant responses against harmless self-peptides in autoimmune diseases. The ability of TCR-like mAbs to target specific peptides presented on MHC allows for their use to study peptide presentation or for diagnosis and therapy. This extends the scope of conventional mAbs, which are generally limited to cell-surface or soluble antigens. Herein, we review the strategies used to generate TCR-like mAbs and provide a structural comparison with the analogous TCR in pMHC binding. We further discuss their applications as research tools and therapeutic reagents in preclinical models as well as challenges and limitations associated with their use.

The complement system is vital for anti-microbial defense. In the classical pathway, pathogen-bound antibody recruits the C1 complex (C1qC1r2C1s2) that initiates a cleavage cascade involving C2, C3, C4, and C5 and triggering microbial clearance. We demonstrate a C4-dependent antiviral mechanism that is independent of downstream complement components. C4 inhibits human adenovirus infection by directly inactivating the virus capsid. Rapid C4 activation and capsid deposition of cleaved C4b are catalyzed by antibodies via the classical pathway. Capsid-deposited C4b neutralizes infection independent of C2 and C3 but requires C1q antibody engagement. C4b inhibits capsid disassembly, preventing endosomal escape and cytosolic access. C4-deficient mice exhibit heightened viral burdens. Additionally, complement synergizes with the Fc receptor TRIM21 to block transduction by an adenovirus gene therapy vector but is partially restored by Fab virus shielding. These results suggest that the complement system could be altered to prevent virus infection and enhance virus gene therapy efficacy.

Background & Aims Development of celiac disease is believed to involve the transglutaminase-dependent response of CD4+ T cells toward deamidated gluten peptides in the intestinal mucosa of individuals with specific HLA-DQ haplotypes. We investigated the antigen presentation process during this mucosal immune response. Methods We generated monoclonal antibodies (mAbs) specific for the peptide–MHC (pMHC) complex of HLA-DQ2.5 and the immunodominant gluten epitope DQ2.5-glia-α1a using phage display. We used these mAbs to assess gluten peptide presentation and phenotypes of presenting cells by flow cytometry and enzyme-linked immune absorbent spot (ELISPOT) in freshly prepared single-cell suspensions from intestinal biopsies from 40 patients with celiac disease (35 untreated and 5 on a gluten-free diet) as well as 18 subjects with confirmed noninflamed gut mucosa (controls, 12 presumed healthy, 5 undergoing pancreatoduodenectomy, and 1 with potential celiac disease). Results Using the mAbs, we detected MHC complexes on cells from intestinal biopsies from patients with celiac disease who consume gluten, but not from patients on gluten-free diets. We found B cells and plasma cells to be the most abundant cells that present DQ2.5-glia-α1a in the inflamed mucosa. We identified a subset of plasma cells that expresses B-cell receptors (BCR) specific for gluten peptides or the autoantigen transglutaminase 2 (TG2). Expression of MHC class II (MHCII) was not restricted to these specific plasma cells in patients with celiac disease but was observed in an average 30% of gut plasma cells from patients and controls. Conclusions A population of plasma cells from intestinal biopsies of patients with celiac disease express MHCII; this is the most abundant cell type presenting the immunodominant gluten peptide DQ2.5-glia-α1a in the tissues from these patients. These results indicate that plasma cells in the gut can function as antigen-presenting cells and might promote and maintain intestinal inflammation in patients with celiac disease or other inflammatory disorders.

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.

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.

There is a quest for production of soluble protein of high quality for the study of T-cell receptors (TCRs), but expression often results in low yields of functional molecules. In this study, we used an E. coli chaperone-assisted periplasmic production system and compared expression of 4 different soluble TCR formats: single-chain TCR (scTCR), two different disulfide-linked TCR (dsTCR) formats, and chimeric Fab (cFab). A stabilized version of scTCR was also included. Additionally, we evaluated the influence of host (XL1-Blue or RosettaBlueTM) and the effect of IPTG induction on expression profiles. A celiac disease patient-derived TCR with specificity for gluten was used, and we achieved detectable expression for all formats and variants. We found that expression in RosettaBlueTM without IPTG induction resulted in the highest periplasmic yields. Moreover, after large-scale expression and protein purification, only the scTCR format was obtained in high yields. Importantly, stability engineering of the scTCR was a prerequisite for obtaining reliable biophysical characterization of the TCR-pMHC interaction. The scTCR format is readily compatible with high-throughput screening approaches that may enable both development of reagents allowing for defined peptide MHC (pMHC) characterization and discovery of potential novel therapeutic leads.

Antibodies are key molecules in the fight against infections. Although previously thought to mediate protection solely in the extracellular environment, recent research has revealed that antibody-mediated protection extends to the cytosolic compartment of cells. This postentry viral defense mechanism requires binding of the antibody to a cytosolic Fc receptor named tripartite motif containing 21 (TRIM21). In contrast to other Fc receptors, TRIM21 shows remarkably broad isotype specificity as it does not only bind IgG but also IgM and IgA. When viral pathogens coated with these antibody isotypes enter the cytosol, TRIM21 is rapidly recruited and efficient neutralization occurs before the virus has had the time to replicate. In addition, inflammatory signaling is induced. As such, TRIM21 acts as a cytosolic sensor that engages antibodies that have failed to protect against infection in the extracellular environment. Here, we summarize our current understanding of how TRIM21 orchestrates humoral immunity in the cytosolic environment.

Albumin is the most abundant protein in blood and acts as a molecular taxi for a plethora of small insoluble substances such as nutrients, hormones, metals and toxins. In addition, it binds a range of medical drugs. It has an unusually long serum half-life of almost 3 weeks, and although the structure and function of albumin has been studied for decades, a biological explanation for the long half-life has been lacking. Now, recent research has unravelled that albumin-binding cellular receptors play key roles in the homeostatic regulation of albumin. Here, we review our current understanding of albumin homeostasis with a particular focus on the impact of the cellular receptors, namely the neonatal Fc receptor (FcRn) and the cubilin–megalin complex, and we discuss their importance on uses of albumin in drug delivery.

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. This research was originally published in: Journal of Biological Chemistry. © the American Society for Biochemistry and Molecular Biology.

Selection of biased T cell receptor (TCR) repertoires across individuals is seen in both infectious diseases and autoimmunity, but the underlying molecular basis leading to these shared repertoires remains unclear. Celiac disease (CD) occurs primarily in HLA-DQ2.5+ individuals and is characterized by a CD4+ T cell response against gluten epitopes dominated by DQ2.5-glia-α1a and DQ2.5-glia-α2. The DQ2.5-glia-α2 response recruits a highly biased TCR repertoire composed of TRAV26-1 paired with TRBV7-2 harboring a semipublic CDR3β loop. We aimed to unravel the molecular basis for this signature. By variable gene segment exchange, directed mutagenesis, and cellular T cell activation studies, we found that TRBV7-3 can substitute for TRBV7-2, as both can contain the canonical CDR3β loop. Furthermore, we identified a pivotal germline-encoded MHC recognition motif centered on framework residue Y40 in TRAV26-1 engaging both DQB1*02 and the canonical CDR3β. This allowed prediction of expanded DQ2.5-glia-α2–reactive TCR repertoires, which were confirmed by single-cell sorting and TCR sequencing from CD patient samples. Our data refine our understanding of how HLA-dependent biased TCR repertoires are selected in the periphery due to germline-encoded residues. This research was originally published in JCI Insight. © 2018 American Society for Clinical Investigation

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., The development of IgG and albumin-based therapeutics with increased half-lives needs more efficient screening procedures. Here the authors report a human endothelial cell-based recycling assay enabling screening of IgG and albumin variants without chemical labelling and prior to animal testing.

Scientific Reports 6: Article number: 37457; published online: 24 November 2016; updated: 06 April 2017 This Article contains a typographical error in the Methods section under the subheading ‘Crystallization’, where the Protein Data Bank accession code ‘5LDN’ was incorrectly given as ‘5LDV’.

During infection with non-enveloped viruses, antibodies stimulate immunity from inside cells by activating the cytosolic Fc receptor TRIM21. This intracellular humoral response relies on opsonized viral particles reaching the cytosol intact but the antigenic and kinetic constraints involved are unknown. We have solved the structure of a potent TRIM21-dependent neutralizing antibody in complex with human adenovirus 5 hexon and show how these properties influence immune activity. Structure-guided mutagenesis was used to generate antibodies with 20,000-fold variation in affinity, on-rates that differ by ~50-fold and off-rates by >175-fold. Characterization of these variants during infection revealed that TRIM21-dependent neutralization and NFκB activation was largely unaffected by on-rate kinetics. In contrast, TRIM21 antiviral activity was exquisitely dependent upon off-rate, with sub-μM affinity antibodies nevertheless unable to stimulate signaling because of fast dissociation kinetics. These results define the antibody properties required to elicit an efficient intracellular immune response during viral infection.

The neonatal crystallizable fragment receptor (FcRn) is responsible for maintaining the long half-life and high levels of the two most abundant circulating proteins, albumin and IgG. In the latter case, the protective mechanism derives from FcRn binding to IgG in the weakly acidic environment contained within endosomes of hematopoietic and parenchymal cells, whereupon IgG is diverted from degradation in lysosomes and is recycled. The cellular location and mechanism by which FcRn protects albumin are partially understood. Here we demonstrate that mice with global or liver-specific FcRn deletion exhibit hypoalbuminemia, albumin loss into the bile, and increased albumin levels in the hepatocyte. In vitro models with polarized cells illustrate that FcRn mediates basal recycling and bidirectional transcytosis of albumin and uniquely determines the physiologic release of newly synthesized albumin into the basal milieu. These properties allow hepatic FcRn to mediate albumin delivery and maintenance in the circulation, but they also enhance sensitivity to the albumin-bound hepatotoxin, acetaminophen (APAP). As such, global or liver-specific deletion of FcRn results in resistance to APAP-induced liver injury through increased albumin loss into the bile and increased intracellular albumin scavenging of reactive oxygen species. Further, protection from injury is achieved by pharmacologic blockade of FcRn–albumin interactions with monoclonal antibodies or peptide mimetics, which cause hypoalbuminemia, biliary loss of albumin, and increased intracellular accumulation of albumin in the hepatocyte. Together, these studies demonstrate that the main function of hepatic FcRn is to direct albumin into the circulation, thereby also increasing hepatocyte sensitivity to toxicity.