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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

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.

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.

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.

Antibodies are essential components of an adaptive immune response. Immunoglobulin G (IgG) is the most common type of antibody found in circulation and extracellular fluids. Although IgG alone can directly protect the body from infection through the activities of its antigen binding region, the majority of IgG immune functions are mediated via proteins and receptors expressed by specialized cell subsets that bind to the fragment crystallizable (Fc) region of IgG. Fc gamma (γ) receptors (FcγR) belong to a broad family of proteins that presently include classical membrane-bound surface receptors as well as atypical intracellular receptors and cytoplasmic glycoproteins. Among the atypical FcγRs, the neonatal Fc receptor (FcRn) has increasingly gained notoriety given its intimate influence on IgG biology and its ability to also bind to albumin. FcRn functions as a recycling or transcytosis receptor that is responsible for maintaining IgG and albumin in the circulation, and bidirectionally transporting these two ligands across polarized cellular barriers. More recently, it has been appreciated that FcRn acts as an immune receptor by interacting with and facilitating antigen presentation of peptides derived from IgG immune complexes (IC). Here we review FcRn biology and focus on newer advances including how emerging FcRn-targeted therapies may affect the immune responses to IgG and IgG IC.

Tripartite motif containing-21 (TRIM21) is a cytosolic ubiquitin ligase and antibody receptor that provides a last line of defense against invading viruses. It does so by acting as a sensor that intercepts antibody-coated viruses that have evaded extracellular neutralization and breached the cell membrane. Upon engagement of the Fc of antibodies bound to viruses, TRIM21 triggers a coordinated effector and signaling response that prevents viral replication while at the same time inducing an anti-viral cellular state. This dual effector function is tightly regulated by auto-ubiquitination and phosphorylation. Therapeutically, TRIM21 has been shown to be detrimental in adenovirus based gene therapy, while it may be favorably utilized to prevent tau aggregation in neurodegenerative disorders. In addition, TRIM21 may synergize with the complement system to block viral replication as well as transgene expression. TRIM21 can also be utilized as a research tool to deplete specific proteins in cells and zebrafish embryos. Here, we review our current biological understanding of TRIM21 in light of its versatile 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.

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.

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.

It is becoming increasingly clear that T cell responses against many antigens are dominated by public α/β T cell receptors (TCRs) with restricted heterogeneity. Because expression of public TCRs may be related to resistance, or predisposition to diseases, it is relevant to measure their frequencies. Although staining with tetrameric peptide/major histocompatibility complex (pMHC) molecules gives information about specificity, it does not give information about the TCR composition of the individual T cells that stain. Moreover, next-generation sequencing of TCR does not yield information on pairing of α- and β-chains in single T cells. In an effort to overcome these limitations, we have here investigated the possibility of raising a monoclonal antibody (moAb) that recognizes a public TCR. As a model system, we have used T cells responding to the 91-101 CDR3 peptide of an Ig L-chain (λ2³¹⁵), presented by the MHC class II molecule I-E(d). The CD4⁺ T cell responses against this pMHC are dominated by a receptor composed of Vα3Jα1;Vβ6DβJβ1.1. Even the V(D)J junctions are to a large extent shared between T cell clones derived from different BALB/c mice. We here describe a murine moAb (AB10) of B10.D2 origin that recognizes this public TCR, while binding to peripheral T cells is negligible. Binding of the moAb is abrogated by introduction of two Gly residues in the D-J junction of the CDR3 of the β-chain. A model for the public TCR determinant is presented.

Albumin has a serum half-life of 3 weeks in humans. This has been utilized to extend the serum persistence of biopharmaceuticals that are fused to albumin. In light of the fact that the neonatal Fc receptor (FcRn) is a key regulator of albumin homeostasis, it is crucial to address how fusion of therapeutics to albumin impacts binding to FcRn. Here, we report on a detailed molecular investigation on how genetic fusion of a short peptide or an single-chain variable fragment (scFv) fragment to human serum albumin (HSA) influences pH-dependent binding to FcRn from mouse, rat, monkey, and human. We have found that fusion to the N- or C-terminal end of HSA only slightly reduces receptor binding, where the most noticeable effect is seen after fusion to the C-terminal end. Furthermore, in contrast to the observed strong binding to human and monkey FcRn, HSA and all HSA fusions bound very poorly to mouse and rat versions of the receptor. Thus, we demonstrate that conventional rodents are limited as preclinical models for analysis of serum half-life of HSA-based biopharmaceuticals. This finding is explained by cross-species differences mainly found within domain III (DIII) of albumin. Our data demonstrate that although fusion, particularly to the C-terminal end, may slightly reduce the affinity for FcRn, HSA is versatile as a carrier of biopharmaceuticals.

Albumin escapes intracellular degradation by binding to the neonatal Fc receptor (FcRn), which results in a very long serum half-life of nearly 3 weeks in humans. The broadly expressed FcRn is unique in that it binds both its ligands, immunoglobulin G (IgG) and albumin, in a strictly pH-dependent fashion, and this has proven to be fundamental for rescue from degradation. Further, elucidation of the biology of FcRn as well as its relationship with albumin is necessary to obtain a better understanding of how albumin homeostasis is regulated. This will be of great importance for optimal applications of albumin as a therapeutic molecule. Indeed, albumin is attracting increasing interest as it is utilized to extend the serum half-life of drugs and improve pharmacokinetics. We review the current status of albumin-based therapeutics in light of FcRn biology and the prospect of a new generation of albumin molecules with improved binding to FcRn.

Background: The major histocompatibility class I-related neonatal Fc receptor, FcRn, salvages both IgG and albumin from degradation and thus contributes to maintain high serum levels of these proteins. Analbuminemia is a rare autosomal recessive disorder characterized by clinically observed allelic albumin variants that are absent or found in very low concentrations in the blood circulation. Such variants may have altered FcRn binding properties that affect their half-life, biodistribution and thereby transport ability. Methods: We established an easy cloning, expression and purification strategy to obtain recombinant GST-tagged human serum albumin (HSA) variants for evaluation of pH dependent FcRn binding properties using an enzyme-linked immunosorbent assay (ELISA) and a real time surface plasmon resonance (SPR) biosensor system. Results: The strategy yielded purified GST-tagged albumin variants. A recombinant truncated HSA variant similar to a clinically observed splice mutant denoted Bartin, here abrogated HSA Bartin , showed no detectable pH dependent FcRn binding compared to a fully functional albumin wild type variant, HSA Wt , and a truncated HSA variant consisting of only the carboxy terminal domain III (HSA DIII ). Conclusions: The approach described can be used to rapidly screen clinically observed truncated or otherwise mutant or modified HSA variants regarding their pH dependent FcRn binding properties. Here, we demonstrate that a recombinant truncated HSA variant, HSA Bartin , does not interact with FcRn, which gives a molecular explanation for the low serum levels. In addition, DIII of HSA alone was shown to retain its FcRn binding property.

The half-life of the two most abundant proteins in blood, immunoglobulin G (IgG) and serum albumin, is extraordinary (approximately 19-23 days) compared to other circulating proteins. This phenomenon secures a broad biodistribution throughout the body of both molecules. The long half-life has made IgG the natural choice for engineering of antibody based therapeutics, while albumin is used as a fusion partner for or carrier of drugs. Remarkably, the half-life of these unrelated proteins has been shown prolonged by a receptor recycling pathway mediated by a common cell bound receptor named the neonatal Fc receptor (FcRn). This review summarizes our current understanding of FcRn function and discusses its relevance for development of new IgG and albumin based therapeutics and diagnostics.

The neonatal Fc receptor (FcRn) is a major histocompatibility complex class I-related molecule that regulates the half-life of IgG and albumin. In addition, FcRn directs the transport of IgG across both mucosal epithelium and placenta and also enhances phagocytosis in neutrophils. This new knowledge gives incentives for the design of IgG and albumin-based diagnostics and therapeutics. To study FcRn in vitro and to select and characterize FcRn binders, large quantities of soluble human FcRn are needed. In this report, we explored the impact of two free cysteine residues (C48 and C251) of the FcRn heavy chain on the overall structure and function of soluble human FcRn and described an improved bacterial production strategy based on removal of these residues, yielding ∼ 70 mg·L−1 of fermentation of refolded soluble human FcRn. The structural and functional integrity was proved by CD, surface plasmon resonance and MALDI-TOF peptide mapping analyses. The strategy may generally be translated to the large-scale production of other major histocompatibility complex class I-related molecules with nonfunctional unpaired cysteine residues. Furthermore, the anti-FcRn response in goats immunized with the FcRn heavy chain alone was analyzed following affinity purification on heavy chain-coupled Sepharose. Importantly, purified antibodies blocked the binding of both ligands to soluble human FcRn and were thus directed to both binding sites. This implies that the FcRn heavy chain, without prior assembly with human β2-microglobulin, contains the relevant epitopes found in soluble human FcRn, and is therefore sufficient to obtain binders to either ligand-binding site. This finding will greatly facilitate the selection and characterization of such binders.

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.

The remarkable clinical success of Fc-fusion proteins has driven intense investigation for even more potent replacements. Using quality-by-design (QbD) approaches, we generated hexameric-Fc (hexa-Fc), a ~20 nm oligomeric Fc-based scaffold that we here show binds low-affinity inhibitory receptors (FcRL5, FcγRIIb and DC-SIGN) with high avidity and specificity, whilst eliminating significant clinical limitations of monomeric Fc-fusions for vaccine and/or cancer therapies, in particular their poor ability to activate complement. Mass spectroscopy of hexa-Fc reveals high-mannose, low-sialic acid content, suggesting that interactions with these receptors are influenced by the mannose-containing Fc. Molecular dynamics (MD) simulations provides insight into the mechanisms of hexa-Fc interaction with these receptors and reveals an unexpected orientation of high-mannose glycans on the human Fc that provides greater accessibility to potential binding partners. Finally, we show that this biosynthetic nanoparticle can be engineered to enhance interactions with the human neonatal Fc receptor (FcRn) without loss of the oligomeric structure, a crucial modification for these molecules in therapy and/or vaccine strategies where a long plasma half-life is critical.

The high-affinity IgG receptor, Fcgamma receptor I (FcgammaRI), is expressed exclusively on myeloid cells, and there is a great interest in the targeting of vaccine antigens to FcgammaRI using anti-human FcgammaRI antibodies or fragments derived from such molecules. In order to reduce the size and complexity of the targeting reagent, we have searched for FcgammaRI binding peptides in peptide libraries displayed on phage. The human monocytic cell line U937 was used as target. Phages that displayed the consensus peptide CLRSGXGC were selected and revealed increased binding to IFN-gamma stimulated versus non-stimulated U937 cells as well as to FcgammaRI transfected versus non-transfected IIA1.6 cells. Furthermore, they bound the extracellular domains of soluble FcgammaRI, but neither FcgammaRIIA, FcgammaRIIB nor FcgammaRIIIB. Binding was inhibited by a synthetic version of the peptide, whereas neither human IgG nor the FcgammaRI-specific monoclonal antibodies (mAb) mAb22 and 32.2 interfered. Flow-cytometry analysis and internalization studies showed that a synthetic biotin-conjugated peptide ADGACLRSGRGCGAAK-bio was able to target U937 cells and FcgammaRI transfected IIA1.6 cells, and further to promote internalization and vesicular degradation of streptavidin coupled to 1 microm magnetic beads. These peptides may have potential as FcgammaRI targeting reagents.

There is still no general vaccine for prevention of disease caused by group-B meningococcal strains. Meningococcal lipopolysaccharides (LPSs) have received attention as potential vaccine candidates, but concerns regarding their safety have been raised. Peptide mimics of LPS epitopes may represent safe alternatives to immunization with LPS. The monoclonal antibody (MoAb) 9-2-L3,7,9 specific for Neisseria meningitidis LPS immunotype L3,7,9 is bactericidal and does not cross-react with human tissue. To explore the possibility of isolating peptide mimics of the epitope recognized by MoAb 9-2-L3,7,9, we have constructed two phage display libraries of six and nine random amino acids flanked by cysteines. Furthermore, we developed a system for the easy exchange of peptide-encoding sequences from the phage-display system to a hepatitis B core (HBc) expression system. Cyclic peptides that specifically bound MoAb 9-2-L3,7,9 at a site overlapping with the LPS-binding site were selected from both libraries. Three out of four tested peptides which reacted with MoAb 9-2-L3,7,9 were successfully presented as fusions to the immunodominant loop of HBc particles expressed in Escherichia coli. However, both peptide conjugates to keyhole limpet haemocyanin and HBc particle fusions failed to give an anti-LPS response in mice.

A major objective in vaccine development is the design of reagents that give strong, specific T cell responses. We have constructed a series of rAb with specificity for MHC class II (I-E). Each has one of four different class II-restricted T cell epitopes genetically introduced into the first C domain of the H chain. These four epitopes are: 91–101 λ2315, which is presented by I-Ed; 110–120 hemagglutinin (I-Ed); 323–339 OVA (I-Ad); and 46–61 hen egg lysozyme (I-Ak). We denote such APC-specific, epitope-containing Ab “Troybodies.” When mixed with APC, all four class II-specific Troybodies were ∼1,000 times more efficient at inducing specific T cell activation in vitro compared with nontargeting peptide Ab. Furthermore, they were 1,000–10,000 times more efficient than synthetic peptide or native protein. Conventional intracellular processing of the Troybodies was required to load the epitopes onto MHC class II. Different types of professional APC, such as purified B cells, dendritic cells, and macrophages, were equally efficient at processing and presenting the Troybodies. In vivo, class II-specific Troybodies were at least 100 times more efficient at targeting APC and activating TCR-transgenic T cells than were the nontargeting peptide Ab. Furthermore, they were 100–100,000 times more efficient than synthetic peptide or native protein. The study shows that class II-specific Troybodies can deliver a variety of T cell epitopes to professional APC for efficient presentation, in vitro as well as in vivo. Thus, Troybodies may be useful as tools in vaccine development.

Type-B T cells raised against the immunodominant peptide in hen egg lysozyme (HEL48–62) do not respond to whole lysozyme, and this has been thought to indicate that peptide can bind to l-Ak in different conformations. Here we demonstrate that such T cells recognize a deamidated form of the HEL peptide and not the native peptide. The sequence of the HEL epitope facilitates rapid and spontaneous deamidation when present as a free peptide or within a flexible domain. However, this deamidated epitope is not created within intact lysozyme, most likely because it resides in a highly structured part of the protein. These findings argue against the existence of multiple conformations of the same peptide–MHC complex and have important implications for the design of peptide-based vaccines. Furthermore, as the type-B T cells are known to selectively evade induction of tolerance when HEL is expressed as a transgene, these results suggest that recognition of posttranslationally modified self-antigen may play a role in autoimmunity.

The classical complement activation cascade of the immune system is initiated by multivalent binding of its first component, C1q, to the Fc region of immunoglobulins in immune complexes. The C1q binding site on mouse IgG2b has been shown to contain the amino acids Glu 318, Lys 320 and Lys 322 in the C(H)2 domain (Duncan, A.R., Winter, G.,1988. The binding site for C1q on IgG. Nature 322 738-740). Identical or closely related motifs are found on all IgGs in all species, and the binding site has therefore been thought to be universal. However, the results from another study indicate that the site is different in human IgG1 molecules (Morgan, A., Jones, N.D., Nesbitt, A.M., et al., 1995. The N-terminal end of the C(H)2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, Fc gamma RI and Fc gamma RIII binding. Immunology 86 319-324). To determine the site(s) responsible for complement activation in anti-NIP-mouse/human IgG3 antibodies, we have mutated amino acids Lys 276, Tyr 278, Asp 280, Glu 318, Lys 320 and Lys 322 in two beta-strands in the C(H)2 domains of human IgG3. In addition, we mutated the Glu 333, which resides in close proximity to the postulated C1q-binding site of mouse IgG2b, as well as Leu 235 in the lower hinge region. All mutants were tested in Antibody Dependent Complement Mediated Lysis (ADCML)(4) assays, where the antigen concentration on target cells was varied and human serum was complement source. Only the mutants that lacked the positively charged side chain of lysine in position 322 showed strong reduction in ADCML, particularly at low antigen density on target cells. Alanine scanning of positions 318 and 320 did not affect ADCML, contrary to what was observed for mouse IgG2b. Neither did a leucine to glutamic acid mutation in position 235 have the effect that has been reported for human IgG1. These results suggest that the complement binding site on human IgG3 molecules is different from that found on mouse IgG2b, and possibly on human IgG1 as well. Thus the contact site may not be conserved.

Interactions between human IgG with human FcgammaRI and FcgammaRIIa (R131) were studied to investigate the role of the hinge region of IgG3 and IgG1 in the binding of the antibodies to FcgammaR. It was found that a hinge deletion mutant of IgG3 (IgG3 m15) was reduced in its ability to bind to FcgammaRI and FcgammaRIIa but was more potent at activating ADCC by activated lymphocytes (FcgammaRIIIa-mediated), compared to the wild-type version of IgG3. The human IgG1 allotype G1m(a,z) was more efficient at binding to FcgammaRI than the two IgG3 antibodies tested. The IgG1 and IgG3 wild type antibodies were better able to bind to FcgammaRII than the hinge deletion mutant version of IgG3. The data suggest a role for the hinge region in influencing FcgammaR mediated effector functions in IgG3.

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.

A series of anti-tumor/anti-chelate bispecific antibody formats were developed for pre-targeted radioimmunotherapy. Based on the anti-carcinoembryonic antigen humanized hT84.66-M5A monoclonal antibody and the anti-DOTA C8.2.5 scFv antibody fragment, this cognate series of bispecific antibodies were radioiodinated to determine their tumor targeting, biodistribution and pharmacokinetic properties in a mouse xenograft tumor model. The in vivo biodistribution studies showed that all the bispecific antibodies exhibited specific high tumor uptake but the tumor targeting was approximately one-half of the parental anti-CEA mAb due to faster blood clearance. Serum stability and FcRn studies showed no apparent reason for the faster blood clearance. A dual radiolabel biodistribution study revealed that the (111)In-DOTA bispecific antibody had increased liver and spleen uptake, not seen for the (125)I-version due to metabolism and release of the radioiodine from the cells. These data suggest increased clearance of the antibody fusion formats by the mononuclear phagocyte system. Importantly, a pre-targeted study showed specific tumor uptake of (177)Lu-DOTA and a tumor : blood ratio of 199 : 1. This pre-targeted radiotherapeutic and substantial reduction in the radioactive exposure to the bone marrow should enhance the therapeutic potential of RIT.

CD4+ T lymphocytes play a central role in the orchestration and maintenance of the adaptive immune response. Targeting of antigen to antigen presenting cells (APCs) increases peptide loading of major histocompatibility complex (MHC) class II molecules and CD4+ T-cell activation. APCs have been targeted by APC-specific recombinant antibodies (rAbs) with single T-cell epitopes integrated in the constant region of the heavy chain (C(H)). However, the strategy may be improved if several T-cell epitopes could be delivered simultaneously by one rAb. We here demonstrate that a single rAb can be loaded with multiple identical or different T-cell epitopes, integrated as loops between β-strands in C(H) domains. One epitope was inserted in C(H)1, while two were placed in C(H)2 of IgG. T-cell proliferation assays showed that all three peptides were excised from loops and presented on MHC class II to T-cells. Induction of T-cell activation by each epitope in the multi-peptide rAb was as good, or even better, than that elicited by corresponding single-peptide rAbs. Furthermore, following DNA vaccination of mice with plasmids that encode CD40-specific rAbs loaded with either one or three peptides, T-cell responses were induced. Thus, integration of multiple epitopes in C(H) region loops of APC-specific rAbs is feasible and may be utilized in design of multi-vaccines.

Posttranslational modification of Ag is implicated in several autoimmune diseases. In celiac disease, a cereal gluten-induced enteropathy with several autoimmune features, T cell recognition of the gluten Ag is heavily dependent on the posttranslational conversion of Gln to Glu residues. Evidence suggests that the enhanced recognition of deamidated gluten peptides results from improved peptide binding to the MHC and TCR interaction with the peptide–MHC complex. In this study, we report that there is a biased usage of TCR Vβ6.7 chain among TCRs reactive to the immunodominant DQ2-α-II gliadin epitope. We isolated Vβ6.7 and DQ2-αII tetramer-positive CD4+ T cells from peripheral blood of gluten-challenged celiac patients and sequenced the TCRs of a large number of single T cells. TCR sequence analysis revealed in vivo clonal expansion, convergent recombination, semipublic response, and the notable conservation of a non-germline-encoded Arg residue in the CDR3β loop. Functional testing of a prototype DQ2-α-II–reactive TCR by analysis of TCR transfectants and soluble single-chain TCRs indicate that the deamidated residue in the DQ2-α-II peptide poses constraints on the TCR structure in which the conserved Arg residue is a critical element. The findings have implications for understanding T cell responses to posttranslationally modified Ags.

Albumin is the most abundant protein in blood where it has a pivotal role as a transporter of fatty acids and drugs. Like IgG, albumin has long serum half-life, protected from degradation by pH-dependent recycling mediated by interaction with the neonatal Fc receptor, FcRn. Although the FcRn interaction with IgG is well characterized at the atomic level, its interaction with albumin is not. Here we present structure-based modelling of the FcRn–albumin complex, supported by binding analysis of site-specific mutants, providing mechanistic evidence for the presence of pH-sensitive ionic networks at the interaction interface. These networks involve conserved histidines in both FcRn and albumin domain III. Histidines also contribute to intramolecular interactions that stabilize the otherwise flexible loops at both the interacting surfaces. Molecular details of the FcRn–albumin complex may guide the development of novel albumin variants with altered serum half-life as carriers of drugs., Albumin transport proteins circulate in the blood and are protected from degradation by interaction with the neonatal Fc receptor. Andersen et al. investigate the albumin binding site of the neonatal Fc receptor and find pH sensitive ionic networks at the binding interface.

Background: Phage display is a platform for selection of specific binding molecules and this is a clear-cut motivation for increasing its performance. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII), or the minor coat protein III (pIII). Display on other coat proteins such as pVII allows for display of heterologous peptide sequences on the virions in addition to those displayed on pIII and pVIII. In addition, pVII display is an alternative to pIII or pVIII display. Methodology/Principal Findings: Here we demonstrate how standard pIII or pVIII display phagemids are complemented with a helper phage which supports production of virions that are tagged with octa FLAG, HIS6 or AviTag on pVII. The periplasmic signal sequence required for pIII and pVIII display, and which has been added to pVII in earlier studies, is omitted altogether. Conclusions/Significance: Tagging on pVII is an important and very useful add-on feature to standard pIII and pVII display. Any phagemid bearing a protein of interest on either pIII or pVIII can be tagged with any of the tags depending simply on choice of helper phage. We show in this paper how such tags may be utilized for immobilization and separation as well as purification and detection of monoclonal and polyclonal phage populations. © 2011 Wälchli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The success of Fc-fusion bio-therapeutics has spurred the development of other Fc-fusion products for treating and/or vaccinating against a range of diseases. We describe a method to modulate their function by converting them into well-defined stable polymers. This strategy resulted in cylindrical hexameric structures revealed by tapping mode atomic force microscopy (AFM). Polymeric Fc-fusions were significantly less immunogenic than their dimeric or monomeric counterparts, a result partly owing to their reduced ability to interact with critical Fc-receptors. However, in the absence of the fusion partner, polymeric IgG1-Fc molecules were capable of binding selectively to FcγRs, with significantly increased affinity owing to their increased valency, suggesting that these reagents may prove of immediate utility in the development of well-defined replacements for intravenous immunoglobulin (IVIG) therapy. Overall, these findings establish an effective IgG Fc-fusion based polymeric platform with which the therapeutic and vaccination applications of Fc-fusion immune-complexes can now be explored.

Background Whereas T cell receptors (TCRs) detect peptide/major histocompatibility complexes (pMHCs) with exquisite specificity, there are challenges regarding their expression and use as soluble detection molecules due to molecular instability. We have investigated strategies for the production of TCR-immunoglobulin (Ig) fusion proteins. Two different TCRs that are characteristic of a mouse model for idiotype (Id) dependent immune regulation were engineered. They are structurally unrelated with different variable (V), diversity (D) and joining (J) segments, but each share one V gene segment, either Vα or Vβ, with the well characterized murine TCR, 2C. Results Several TCR-Ig formats were assessed. In one, the TCR V domains were fused to Ig constant (C) regions. In others, the complete extracellular part of the TCR was fused either to a complete Ig or an Ig Fc region. All molecules were initially poorly secreted from eukaryotic cells, but replacement of unfavourable amino acids in the V regions improved secretion, as did the introduction of a disulfide bridge between the TCR C domains and the removal of an unpaired cysteine. A screening strategy for selection of mutations that stabilize the actual fusion molecules was developed and used successfully. Molecules that included the complete heterodimeric TCR, with a stabilizing disulfide bridge, were correctly folded as they bound TCR-specific antibodies (Abs) and detected pMHC on cells after specific peptide loading. Conclusions We show that fully functional TCR-Ig fusion proteins can be made in good yields following stabilizing engineering of TCR V and C region genes. This is important since TCR-Ig fusions will be important probes for the presence of specific pMHCs in vitro and in vivo. In the absence of further affinity maturation, the reagents will be very useful for the detection of kinetic stability of complexes of peptide and MHC.

The neonatal Fc receptor (FcRn) regulates the serum half-life of both IgG and albumin through a pH-dependent mechanism that involves salvage from intracellular degradation. Therapeutics and diagnostics built on IgG, Fc, and albumin fusions are frequently evaluated in rodents regarding biodistribution and pharmacokinetics. Thus, it is important to address cross-species ligand reactivity with FcRn, because in vivo testing of such molecules is done in the presence of competing murine ligands, both in wild type (WT) and human FcRn (hFcRn) transgenic mice. Here, binding studies were performed in vitro using enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant soluble forms of human (shFcRnWT) and mouse (smFcRnWT) receptors. No binding of albumin from either species was observed at physiological pH to either receptor. At acidic pH, a 100-fold difference in binding affinity was observed. Specifically, smFcRnWT bound human serum albumin with a KD of ∼90 μm, whereas shFcRnWT bound mouse serum albumin with a KD of 0.8 μm. shFcRnWT ignored mouse IgG1, and smFcRnWT bound strongly to human IgG1. The latter pair also interacted at physiological pH with calculated affinity in the micromolar range. In all cases, binding of albumin and IgG from either species to both receptors were additive. Cross-species albumin binding differences could partly be explained by non-conserved amino acids found within the α2-domain of the receptor. Such distinct cross-species FcRn binding differences must be taken into consideration when IgG- and albumin-based therapeutics and diagnostics are evaluated in rodents for their pharmacokinetics. This research was originally published in: Journal of Biological Chemistry. © the American Society for Biochemistry and Molecular Biology.

The C1q binding epicentre on IgG molecules involves residues Asp(270), Lys(322), Pro(329) and Pro(331) in the C(H)2 domain. IgG1 and IgG3 are usually the most efficient of the four human IgG subclasses in activating complement and they both share all these residues. To reveal possible differences in the structural requirement for complement activation, we created a number of NIP (5-iodo-4-hydroxy-3-nitro-phenacetyl) specific IgG1 and IgG3 antibodies with parallel mutations in or near the putative C1q binding site. The mutants were tested simultaneously for antibody induced, antibody-dependent complement-mediated lysis (ADCML) at high and low antigen concentration on the target cells using sera of human, rabbit and guinea pig as complement source. In addition, we tested the antibodies against target cells decorated with the NP hapten, which has 10-fold lower affinity for the antibodies compared to the NIP hapten. We also used ELISA methods to measure complement activation. We observed a clear difference between IgG1 and IgG3 localized to residues Asp(270), Leu(334), Leu(335). For all these residues, and especially for Asp(270), IgG1 was heavily reduced in complement activation, while IgG3 was only moderated reduced, by alanine substitution. This difference was independent of the long hinge region of IgG3, demonstrated by hinge region truncation of this isotype such that it resembles that of IgG1. This report indicates the presence of structural differences between human IgG1 and IgG3 in the C1q binding site, and points to a specialization of the two isotypes with respect to complement activation.

Phage display holds a key position in the use of combinatorial library approaches for the purpose of protein engineering and discovery. However, modifying the pIII protein of the phage can severely and negatively influence the infectiousness of the phage particle. This concern is particularly relevant when large pIII fusions in combination with multivalent display systems are in use. We here describe the use of trypsin to restore wild-type pIII phenotype as a small modification to the standard titration protocol. The results show that the trypsin treatment has a very large but heterogeneous effect on the phage infection efficiency, depending on the pIII fusion domain and the valence of display.

Dolcini et al.(1) recently reported in this journal a novel splice-site mutation (denoted Bartin) that causes deletion of exon 11 in the human serum albumin (HSA) gene, ALB . In spite of their uncertain relevance to pathophysiology of diseases, differences in HSA sequence have interesting correlations with functional properties and stability. Bisalbuminemia (or alloalbuminemia) is a rare inherited or acquired condition characterized by the occurrence of 2 circulating components that are observed, typically, during routine clinical electrophoresis or in genetic surveys. Over the past decades several cases of genetic polymorphisms, described in peer-reviewed papers and listed in the analbuminemia register(2), have been characterized as representing site-specific mutations, splice-site mutations, or frame-shift mutations. The deletion reported by Dolcini et al.(1) would give rise to a C-terminally truncated HSA variant of 410 amino acids rather than the 585 amino acids …

We have employed the recently described crystallohydrodynamic approach to compare the time-averaged domain orientation of human chimeric IgG3wt (wild-type) and IgG4wt as well as two hinge mutants of IgG3 and an IgG4S331P (mutation from serine to proline at position 331, EU numbering) mutant of IgG4. The approach involves combination of the known shape of the Fab and Fc regions from crystallography with hydrodynamic data for the Fab and Fc fragments and hydrodynamic and small angle x-ray scattering data for the intact IgG structures. In this way, ad hoc assumptions over hydration can be avoided and model degeneracy (uniqueness problems) can be minimized. The best fit model for the solution structure of IgG3wt demonstrated that the Fab regions are directed away from the plane of the Fc region and with a long extended hinge region in between. The best fit model of the IgG3m15 mutant with a short hinge (and enhanced complement activation activity) showed a more open, but asymmetric structure. The IgG3HM5 mutant devoid of a hinge region (and also devoid of complement-activation activity) could not be distinguished at the low-resolution level from the structure of the enhanced complement-activating mutant IgG3m15. The lack of inter-heavy-chain disulphide bond rather than a significantly different domain orientation may be the reason for the lack of complement-activating activity of the IgG3HM5 mutant. With IgG4, there are significant and interesting conformational differences between the wild-type IgG4, which shows a symmetric structure, and the IgG4S331P mutant, which shows a highly asymmetric structure. This structural difference may explain the ability of the IgG4S331P mutant to activate complement in stark contrast to the wild-type IgG4 molecule which is devoid of this activity.

The MHC class I-related neonatal Fc receptor (FcRn) serves in the homeostatic regulation of IgG and albumin by increasing their half-lives. FcRn may bind IgG and albumin simultaneously, and in a pH-dependent manner, with ligand binding at pH 6.0-6.5 and release at pH 7.0-7.4. The FcRn-IgG interaction has been extensively characterized at the amino acid level and shown to depend on conserved histidine residues in the IgG-Fc part that interact with negatively charged residues in the alpha-2 domain of FcRn. The recently discovered FcRn-albumin interaction remains to be elucidated. Guided by the pH dependence of the FcRn-albumin interaction, we compared the sequence of the FcRn alpha-2 domain from eleven different species, and identified histidine residues that were conserved in all (H166) or seven (H161) of these. Both residues are located directly opposite to the IgG interaction site in the folded molecule. We did in vitro mutagenesis (H161A or H166A) in combination with interaction studies (ELISA and surface plasmon resonance) with recombinant, soluble, purified receptors and IgG and albumin to investigate the role of the two histidine residues. Our results show clear evidence that the conserved H166 is a key player in the FcRn-albumin interaction.

The polymeric Ig receptor (pIgR), also called membrane secretory component (SC), mediates epithelial transcytosis of polymeric immunoglobulins (pIgs). J Chain-containing polymeric IgA (pIgA) and pentameric IgM bind pIgR at the basolateral epithelial surface. After transcytosis, the extracellular portion of the pIgR is cleaved at the apical side, either complexed with pIgs as bound SC or unoccupied as free SC. This transport pathway may be exploited to target bioactive molecules to the mucosal surface. To identify small peptide motifs with specific affinity to human pIgR, we used purified free SC and selection from randomized, cysteine-flanked 6- and 9-mer phage-display libraries. One of the selected phages, called C9A, displaying the peptide CVVWMGFQQVC, showed binding both to human free SC and SC complexed with pIgs. However, the pneumococcal surface protein SpsA (Streptococcus pneumoniae secretory IgA-binding protein), which binds human SC at a site distinct from the pIg binding site, competed with the C9A phage for binding to SC. The C9A phage showed greatly increased transport through polarized Madin-Darby canine kidney cells transfected with human pIgR. This transport was not affected by pIgA nor did it inhibit pIgR-mediated pIgA transcytosis. A free peptide of identical amino acid sequence as that displayed by the C9A phage inhibited phage interaction with SC. This implied that the C9A peptide sequence may be exploited for pIgR-mediated epithelial transport without interfering with secretory immunity. This research was originally published in: Journal of Biological Chemistry. © the American Society for Biochemistry and Molecular Biology.

There are potentially two binding sites for C1q on IgG, one on each C(H)2 domain of the gamma heavy chains, close to the lower hinge region. It is not clear whether the presence and involvement of both the C1q binding sites is necessary to induce the activation signal of human IgG. In order to clarify this issue, we made a hybrid mutant IgG1/IgG3 molecule where the IgG1 half of the molecule was made unable to activate complement through the introduction of a P329A mutation. The IgG3 half of the molecule was mutated to harbor a hinge region identical to that of IgG1, and for detection a peptide tag derived from p21ras was introduced into the FG loop of the C(H)1 domain. The hybrid IgG1P329A/IgG3h1-ras molecules were isolated by Protein A affinity chromatography and shown to activate complement and induce complement-mediated lysis at the same levels as wild-type IgG1 and IgG3h1-ras molecules. Thus, one C1q binding site per IgG is sufficient to induce activation. Wild-type human IgG molecules might also normally expose only one C1q binding site as already shown for interaction with FcgammaR, were IgG expose one binding site per molecule.

Mature, naive B cells coexpress IgD and IgM with identical binding sites. In this study, the binding properties of such IgM and IgD are compared to determine how size and shape may influence their ability to bind Ag and thus function as receptors. To dissect their intrinsic binding properties, recombinant IgM and IgD were produced in soluble form as monomers of the basic H2L2 Ab architecture, each with two Ag binding sites. Since these sites are connected with a hinge region in IgD and structural Ig domains in IgM, the two molecules differ significantly in this region. The results show that IgD exhibited the larger angle and longer distance between its binding sites, as well as having the greater flexibility. Relative functional affinity was assessed on two antigenic surfaces with high or low epitope density, respectively. At high epitope density, IgM had a higher functional affinity for the Ag compared with IgD. The order was reversed at low epitope density due to a decrease in the functional affinity of IgM. Studies of binding kinetics showed similar association rates for both molecules. The dissociation rate, however, was slower for IgM at high epitope density and for IgD at low epitope density. Taken together, the results show that IgM and IgD with identical Ag binding regions have different Ag binding properties.

Mucosal surfaces are protected by polymeric immunoglobulins that are transported across the epithelium by the polymeric immunoglobulin receptor (pIgR). Only polymeric IgA and IgM containing a small polypeptide called the "joining" (J) chain can bind to the pIgR. J chain-positive IgA consists of dimers, and some larger polymers, whereas only IgM pentamers incorporate the J chain. We made domain-swap chimeras between human IgA1 and IgM and found that the COOH-terminal domains of the heavy chains (Ca3 and Cm4, respectively) dictated the size of the polymers formed and also which polymers incorporated the J chain. We also showed that chimeric IgM molecules engineered to contain Ca3 were able to bind the rabbit pIgR. Since the rabbit pIgR normally does not bind IgM, these results suggest that the COOH-terminal domain of the polymeric immunoglobulins is primarily responsible for interaction with the pIgR. Finally, we made a novel chimeric IgA immunoglobulin, containing the terminal domain from IgM. This recombinant molecule formed J chain-containing pentamers that could, like IgA, efficiently form covalent complexes with the human pIgR ectodomain, known as secretory component. This research was originally published in: Journal of Biological Chemistry. © the American Society for Biochemistry and Molecular Biology.

J chain is associated with pentameric IgM and dimeric IgA via disulfide bonds involving the penultimate cysteine residue in the secretory tailpiece of the mu or the alpha heavy chain. We have investigated the structural basis for incorporation of J chain by analyzing several IgM mutants, IgA mutants and IgG/IgM hybrid molecules. IgM mutants with the mu secretory tailpiece replaced by the alpha secretory tailpiece and/or Cys414 replaced by serine incorporated J chain, although in reduced amounts correlating with reduced pentamer/polymer formation. In addition to pentamers, tetramers of IgMC414S contained J chain, while no J chain was associated with smaller polymers or hexamers of IgM. An IgA/IgM hybrid tailpiece abolished J chain incorporation to pentameric IgM. Analysis of IgG molecules that have added a secretory tailpiece and/or have IgM domain replacements showed that J chain incorporation depends on regions of the C(mu)4 domain in addition to the tailpiece. Features of the C(mu)3 domain other than Cys414 also play a role in efficient formation of pentamers and J chain incorporation, while the C(mu)2 domain is not specifically required. By analysis of two IgA mutants that formed larger polymers than IgAwt, we found J chain equally incorporated into dimers, trimers, tetramers and pentamers. Thus, the results show that J chain incorporation into IgA does not depend on the polymeric structure, while J chain incorporation into IgM is restricted to certain polymeric conformations.