Human Fc Receptor Research Articles

A preliminary study of expression of human neonatal Fc-receptor (FcRn) in Escherichia coli

IntroductionHuman neonatal Fc-receptor is a potential novel antibody binding protein for development of immunoassay. Previous studies had shown that this human protein has high affinity to Ig G either in vivo or in vitro. However, none of the studies had attempted exploring the utility of human FcRn in antibody immobilisation strategy. In this study, we are in the direction towards development of a novel antibody binding protein by using human FcRn as an immobilisation platform. The three-dimensional structure of the protein was analysed. This protein was successful expressed in bacteria E. coli BL21 (DE3) by using expression vector pET-28b. The pET-28b vector contains poly-histidine tagged which allows detection and purification of expressed proteins. Objectives(1) To study the three dimensional structure of human FcRn, (2) To clone the human FcRn into pET-28b expression vector, (3) To express human FcRn in bacteria E. coli BL21 (DE3). MethodsHuman FcRn cDNA was ligated into pET-28b vector through EcoRI restriction site. The successful clone was transformed into E. coli BL21 (DE3) for expression. Expression in bacteria was induced by IPTG. Induction was conducted under 30°C and 37°C. Expression products were analysed by performing SDS-Page and Western Blot. The 3D structure of human FcRn was studied by using ViewerLite program. Results & DiscussionHuman FcRN was expressed under two sets of temperature, 30°C and 37°C, in 1 hour, 2 hours, 3 hours, and 6 hours. The induction for 3 hours showed highest amount of expressed products, under the above mentioned temperature. The amount of protein expressed in 6 hours of induction showed almost same thickness of band in SDS-PAGE compare to the induction under 3 hours period. ConclusionExpression of human FcRn can be conducted using bacteria expression system, instead of mammalian cell expression system which requires longer time and complicated process. Further study will be conducted to determine the antibody-binding activity and stability of the expressed protein.

Polymorphisms and Interspecies Differences of the Activating and Inhibitory FcγRII of Macaca nemestrina Influence the Binding of Human IgG Subclasses

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

The FcγR of humans and non-human primates and their interaction with IgG: implications for induction of inflammation, resistance to infection and the use of therapeutic monoclonal antibodies.

Considerable effort has focused on the roles of the individual members of the FcγR receptor (FcγR) family in inflammatory diseases and humoral immunity. Recent work has revealed major roles in infection and in particular HIV pathogenesis and immunity. In addition, FcγR functions underpin the action of many of the successful therapeutic monoclonal antibodies. This emphasises the need for a greater understanding of FcγR function in humans and in the NHP which provides a key model for human immunity and preclinical testing of antibodies. We discuss recent key aspects of the human FcγR receptor biology and structure to define differences and similarities in activity between the human and macaque Fc receptors. These differences and similarities nuance the interpretation of infection and vaccine studies in the macaque. Indeed passive IgG antibody protection in lentivirus infection models in the macaque provided early evidence for the role of Fc receptors in anti-HIV immunity that have subsequently gained support from human vaccine trials. None-the-less the diverse functions and cellular contexts of FcγR receptor expression ensure there is much still to understand of the protective and deleterious effects of FcγRs in HIV infection. Careful comparative studies of human and non-human primate FcγRs will facilitate our appreciation of what attributes of HIV specific IgG antibodies, either acquired naturally or via vaccination, are most important for protection.

Enhanced production of human full-length immunoglobulin G1 in the periplasm of Escherichia coli

Monoclonal antibodies are currently the most important pharmaceutical proteins, and the economic production of functional immunoglobulin G (IgG) is an important issue in biotechnology. Recent successes in the development of aglycosylated IgG variants that do not require glycosylation for effector functions have increased the use of Escherichia coli as an alternative host for economic production of IgG, instead of traditional mammalian host expression systems. Here, we have developed a new E. coli host-vector system for the high-level production of full-length IgG1 by examining (1) E. coli strains, (2) modification of 5' untranslated region sequences, and (3) co-expression of periplasmic foldase. With the engineered host-vector system, fed-batch cultivations were conducted at two different conditions, and under optimized conditions, up to 362 mg/L of full-length IgG1 could be produced in a relatively short-time (22 h) cultivation. The fully assembled IgG1 from fed-batch cultivation was purified with high purity and yield. With the purified IgG1, the specific bindings to an antigen, anthrax toxin PA, and to human neonatal Fc receptor were successfully demonstrated.

Robust Expression of the Human Neonatal Fc Receptor in a Truncated Soluble Form and as a Full-Length Membrane-Bound Protein in Fusion with eGFP

Studies on the neonatal Fc receptor (FcRn) have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA) from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding.

Delayed Anti-CD3 Therapy in a Mouse Heart Transplant Model Induced Tolerance and Long-Term Survival of Allograft: Achieving Tolerance

Goto R, You S, Zaitsu M, Chatenoud L, Wood KJ. Delayed anti-CD3 therapy results in depletion of alloreactive T cells and the dominance of Foxp3(+) CD4(+) graft infiltrating cells. Am. J. Transplant. 13(7), 1655-1664 (2013). Humanized Fc receptor nonbinding anti-CD3 monoclonal antibodies have been tested in patients with autoimmune diseases with the goal of inducing immune tolerance. However, the timing of drug administration may be an important determinant of the biologic effects, since not all T cells are equally affected, and there may be different subsets of cells involved during the evolution of immune responses. The study by Goto et al. showed that delayed administration of anti-CD3 therapy was more effective in depleting alloreactive T cells than administration at the time of transplant, and resulted in long-term survival of the graft by promoting infiltration of CD4 Tregs into the graft.

Genetically Engineered Humanized Mouse Models for Preclinical Antibody Studies

The use of genetic engineering has vastly improved our capabilities to create animal models relevant in preclinical research. With the recent advances in gene-editing technologies, it is now possible to very rapidly create highly tunable mouse models as needs arise. Here, we provide an overview of genetic engineering methods, as well as the development of humanized neonatal Fc receptor (FcRn) models and their use for monoclonal antibody in vivo studies.

Cloning, Expression and Characterization of Recombinant Human Fc Receptor Like 1, 2 and 4 Molecules

Background: The Fc receptor like (FCRL) molecules belong to the immunoglobulin (Ig) superfamily with potentially immunoregulatory function. Among the FCRL family FCRL2 and 4 are predominantly expressed on memory B cells and FCRL1 is a pan- B cell marker. To date, no ligand has been identified for the human FCRL1, 2 and 4 molecules. Objectives: Cloning, expression, purification and structural analysis of the extracellular domain of human FCRL1, 2 and 4 proteins. Materials and Methods: In this study, the extracellular part of human FCRL1, 2 and 4 were subcloned into prokaryotic expression vectors pET-28b (+) and transformed into BL21-DE3 E.coli strain. Protein expression was optimized by fine adjustments such as induction time, incubation temperature and expression hosts. Recombinant FCRL proteins were purified by metal affinity chromatography using Ni-NTA resin. Purified FCRL proteins were further characterized by SDS-PAGE and immunoblotting using His-tag and FCRL specific polyclonal antibodies. Results: Our results demonstrated that FCRL1, 2 and 4 were successfully expressed in pET-28b (+) vector. Optimization of the expression procedure showed that IPTG induction at OD600 = 0.9 and overnight incubation at 37˚C resulted in the highest expression levels of FCRL proteins ranging from approximately 15% (FCRL1) to 25% (FCRL2 and 4) of the total bacterial lysate proteins. Conclusions: These purified recombinant proteins are potentially a valuable tool for investigating the immunoregulatory function of FCRL molecules and the production of specific mAbs for immunotherapeutic interventions.

Call to Action for Dengue Vaccine Failure

Call to Action for Dengue Vaccine Failure

N-Terminal Truncation of an Isolated Human IgG1 CH2 Domain Significantly Increases Its Stability and Aggregation Resistance

Isolated human immunoglobulin G (IgG) CH2 domains are promising scaffolds for novel candidate therapeutics. Unlike other human IgG domains, CH2 is not involved in strong interchain interactions, and isolated CH2 is relatively stable. However, isolated single CH2 is prone to aggregation. In native IgG and Fc molecules, the N-terminal residues of CH2 from the two heavy chains interact with each other and form hinge regions. By contrast, the N-terminal residues are highly disordered in isolated CH2. We have hypothesized that the removal of the CH2 N-terminal residues may not only increase its stability but also its aggregation resistance. To test this hypothesis we constructed a shortened variant of IgG1 CH2 (CH2s) where the first seven residues of the N-terminus were deleted. We found that the thermal stability of CH2s was increased by 5 °C compared to CH2. Importantly, we demonstrated that CH2s is significantly less prone to aggregation than CH2 as measured by Thioflavin T (ThT) fluorescence, turbidity, and light scattering. We also found that the CH2s exhibited pH-dependent binding to a soluble single-chain human neonatal Fc receptor (shFcRn) which was significantly stronger than the very weak binding of CH2 to shFcRn as measured by flow cytometry. Computer modeling suggested a possible mode of CH2 aggregation involving its N-terminal residues. Therefore, deletion of the N-terminal residues could increase drugability of CH2-based therapeutic candidates. This strategy to increase stability and aggregation resistance could also be applicable to other Ig-related proteins.

Correlations between pharmacokinetics of IgG antibodies in primates vs. FcRn-transgenic mice reveal a rodent model with predictive capabilities

Transgenic mice expressing human neonatal Fc receptor (FcRn) instead of mouse FcRn are available for IgG antibody pharmacokinetic (PK) studies. Given the interest in a rodent model that offers reliable predictions of antibody PK in monkeys and humans, we set out to test whether the PK of IgG antibodies in such mice correlated with the PK of the same antibodies in primates. We began by using a single research antibody to study the influence of: (1) different transgenic mouse lines that differ in FcRn transgene expression; (2) homozygous vs. hemizygous FcRn transgenic mice; (3) the presence vs. absence of coinjected high-dose human intravenous immunoglobulin (IVIG), and (4) the presence vs. absence of coinjected high-dose human serum albumin (HSA). Results of those studies suggested that use of hemizygous Tg32 mice (Tg32 hemi) not treated with IVIG or HSA offered potential as a predictive model for PK in humans. Mouse PK studies were then done under those conditions with a panel of test antibodies whose PK in mice and primates is not significantly affected by target binding, and for which monkey or human PK data were readily available. Results from the studies revealed significant correlations between terminal half-life or clearance values observed in the mice and the corresponding values reported in humans. A significant relationship in clearance values between mice and monkeys was also observed. These correlations suggest that the Tg32 hemi mouse model, which is both convenient and cost-effective, can offer value in predicting antibody half-life and clearance in primates.

Intracellular Trafficking of an Antibody Bipolar Bridged Complex of HSV‐1 gE‐gI, IgG, and a Viral Antigen

Herpes Simplex Virus 1 (HSV‐1) glycoproteins, gE and gI, mimic a human Fc‐receptor to hinder host immune responses. gE‐gI binds the Fc region of human IgG (hIgG), which can be internalized and released into endocytic organelles. gE‐gI also mediates antibody bipolar bridging (ABB), a process whereby IgG Fabs bind an antigen while its Fc region binds to gE‐gI. The cellular fate of the ABB complex is unknown. Here, we engineered an in vitro system to determine whether ABB complexes are internalized and degraded in host cells. Human cell lines transiently expressing gE‐gI and an HSV‐1 antigen, gD, normally localized at the cell surface of HSV‐1 virions and infected cells, were incubated with two forms of a monoclonal IgG anti‐gD antibody: one with human Fc to allow ABB and one with mouse Fc, a control which binds gD but not gE‐gI and cannot enable ABB. A hIgG against an irrelevant antigen was used as another non ABB control. 4D time‐lapse confocal fluorescence data indicate that gD is internalized in an ABB specific fashion and internalization is dependent upon gE‐gI. Internalized hIgGs bound to a surface viral antigen were targeted to lysosomes and biochemical analysis of enriched lysosomal compartments revealed degraded gD. These results suggest that gE‐gI plays an active role in clearing the infected cell surface of both host IgG and viral antigens, providing HSV‐1 with a mechanism to evade immune responses.

IGG SUBCLASS VARIATION OF A MONOCLONAL ANTIBODY BINDING TO HUMAN FC-GAMMA RECEPTORS

The importance of human Fc receptors in immune regulation is well known. Their role is critical not on ly in the recruitment of cellular effector functions but also in regulating the balance in the periphery bet ween autoimmunity and tolerance. Despite their central i mportance, there is a dearth of literature on contr olled numeric comparisons in affinities of antibody subcl asses for gamma receptors. To date, no studies have directly compared humanized antibodies with the same variable region and differing Fc region subclasse s which would rule out any differences that may be at tributed to variations in the variable region. In t his study we characterized the interaction between four humanized monoclonal antibodies; IgG 1, G 2, G 3 and G4, each possessing an identical variable region and the repertoire of human Fc-gamma (Fc γ) receptors (Fc γRI, Fc γRIIA, Fc γRIIB, Fc γRIIIA and Fc γRIIIB). The studies were performed using both Surfa ce Plasmon Resonance (SPR) and Enzyme-Linked Immunosorbent-Assay (ELISA) formats. The affinities of the antibodies for their antigen molecule, an en dogenous human protein, were also analyzed by SPR. While the identity of the Fc-region had no signific ant effect on the binding to antigen, substantially different affinities for each of the Fc γ receptors, Fc γRI, Fc γRIIA, Fc γRIIB, Fc γRIIIA and Fc γRIIIB were observed across the various Fc-subclasses.

Selection of Nanobodies that Target Human Neonatal Fc Receptor

FcRn is a key player in several immunological and non-immunological processes, as it mediates maternal-fetal transfer of IgG, regulates the serum persistence of IgG and albumin, and transports both ligands between different cellular compartments. In addition, FcRn enhances antigen presentation. Thus, there is an intense interest in studies of how FcRn binds and transports its cargo within and across several types of cells, and FcRn detection reagents are in high demand. Here we report on phage display-selected Nanobodies that target human FcRn. The Nanobodies were obtained from a variable-domain repertoire library isolated from a llama immunized with recombinant human FcRn. One candidate, Nb218-H4, was shown to bind FcRn with high affinity at both acidic and neutral pH, without competing ligand binding and interfering with FcRn functions, such as transcytosis of IgG. Thus, Nb218-H4 can be used as a detection probe and as a tracker for visualization of FcRn-mediated cellular transport.

Long-term Etanercept Therapy Favors Weight Gain and Ameliorates Cachexia in Rheumatoid Arthritis Patients: Roles of Gut Hormones and Leptin

Rheumatoid arthritis (RA) is a chronic inflammatory disease that damages the synovial joints, and patients with it are often anorexic and cachectic with high morbidity and mortality. Biological therapy with anti-tumor necrosis factor (TNF)-α has been proven effective as a treatment for RA. However, the long-term effects of anti-TNF-α therapy on body weight, appetite, plasma gut hormones and leptin have not been investigated. Twenty RA patients received subcutaneous injections of etanercept, a chimeric protein of human IgG1 Fc and TNF receptor p75, twice weekly for 12 consecutive months. Sequential changes in body weight, body fat, appetite rating, lipid profiles, gut hormones and leptin were measured at baseline and at 3 and 12 months after treatment. Ten RA patients who received non-biological disease modifying anti-rheumatic drugs were enrolled as the controls and were appraised at baseline and at 12 months after treatment (a nonrandomized study). Significant weight gain, hyperuricemia, decreased fasting plasma glucose-dependent insulinotropic polypeptide (GIP) levels, and loss of post-oral glucose suppression of plasma leptin concentration were found in the patients after the 12-month course of etanercept therapy, but not in the controls. A transient decrease in fasting plasma acyl ghrelin occurred at 3 months during etanercept treatment. Appetite score and serum lipid profiles did not change in either group. Long-term therapy with anti-TNF-α is promising in ameliorating body mass decrease in patients with active RA. Plasma levels of ghrelin, GIP and leptin may play significant roles in maintaining energy homeostasis in the anti-inflammatory responses during RA remission.

Discovery and structure–activity relationships of small molecules that block the human immunoglobulin G–human neonatal Fc receptor (hIgG–hFcRn) protein–protein interaction

The neonatal Fc receptor, FcRn, prolongs the half-life of IgG in the serum and represents a potential therapeutic target for the treatment of autoimmune disease. Small molecules that block the protein–protein interactions of human IgG–human FcRn may lower pathogenic autoantibodies and provide effective treatment. A novel class of quinoxalines has been discovered as antagonists of the IgG:FcRn protein–protein interaction through optimization of a hit derived from a virtual ligand-based screen.

Binding structures and energies of the human neonatal Fc receptor with human Fc and its mutants by molecular modeling and dynamics simulations

Homology modeling and molecular dynamics simulations have been carried out to model the detailed structures of the human neonatal Fc receptor (FcRn) binding with the wild-type Fc of human immunoglobulin G1 (IgG1) and its various mutants. Based on the modeled human FcRn-Fc binding structures, it has been proposed that the protein-protein binding interface is composed of three subsites. The first subsite is a hydrophobic core where residue I39 of human Fc can be accommodated very well, and the other two subsites are all composed of critical salt bridges between human FcRn and human Fc. All of the modeled structures and the calculated binding energies are qualitatively consistent with the available experimental data, suggesting that the modeled human FcRn-Fc binding structures are reasonable. The modeled human FcRn-Fc binding structure may be valuable for future rational design of novel mutants of human Fc and Fc-fused therapeutic proteins with a potentially higher binding affinity for human FcRn and, thus, a longer in vivo half-life in humans.

Ligation of human Fc receptor like-2 (FCRL2) by specific monoclonal antibodies downregulates B cell receptor mediated signaling

Event Abstract Back to Event Ligation of human Fc receptor like-2 (FCRL2) by specific monoclonal antibodies downregulates B cell receptor mediated signaling Mahdi Shabani1, 2, Ali Ahmad Bayat1, Mahmood Jeddi-Tehrani1, Hodjatallah Rabbani1, Cristina Ulivieri3, Zahra Amirghofran2, Cosima T. Baldari3 and Fazel Shokri1, 4* 1 Avicenna Research Institute,, Department of Antibody and Antigen Engendering, Iran 2 Shiraz University of Medical Sciences, Department of Immunology, Medical School, Iran 3 University of Siena, Department of Evolutionary Biology, Italy 4 Tehran University of Medical Sciences, Department of Immunology, School of Public Health, Iran Introduction: B cell antigen receptor (BCR) signaling and its regulation are critical for balancing B cell response and function. Human Fc receptor like-2 (FCRL2) could influence B cell signaling due to possession of both activating and inhibitory tyrosine-based immunoregulatory motifs. Since the natural ligand of FCRL2 has not been identified yet, we generated FCRL2-specific monoclonal antibodies (mAbs) and employed them to investigate the influence of FCRL2 stimulation on BCR signaling in FCRL2 expressing B cell line. Methods: Anti-FCRL2 secreting hybridomas were produced using hybridoma technology and screened with the immunizing protein by ELISA and stable CHO cell line expressing FCRL2 protein by FACS. The CA46 cells (FCRL2 positive) were stimulated in different condition using different ‎concentration of anti-IgM Ab and mAbs to FCRL2 followed by ‎BCR mediators analysis by FACS and Immunoblotting. Results: Two hybridoma clones producing mAbs specific for FCRL2 were selected (5A7-E7 and 3D8-G8). Both mAbs displayed no cross-reactivity with the other members of the FCRL family as tested by FACS and ELISA techniques. Engagement of FCRL2 by these mAbs resulted in significant inhibition of BCR signaling mediators such as calcium mobilization, phosphorylation of Erk, p38 and Jnk MAP kinases. A minor or no inhibition of the phosphorylation of whole protein tyrosine, Syk, Vav and Akt observed in B cells treated with a cocktail of anti-FCRL2 and anti-BCR Abs. Conclusion: These findings indicated that the FCRL2 ITIMs motifs are functional and the anti-FCRL2 mAbs may mimic the natural ligand of FCRL2 by induction of inhibitory signals in B cells. Keywords: FCRL2, Monoclonal antibody, BCR signaling, BL cell line, MAP Kinase Signaling System Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune receptors and signaling Citation: Shabani M, Bayat A, Jeddi-Tehrani M, Rabbani H, Ulivieri C, Amirghofran Z, Baldari CT and Shokri F (2013). Ligation of human Fc receptor like-2 (FCRL2) by specific monoclonal antibodies downregulates B cell receptor mediated signaling. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00778 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 17 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Fazel Shokri, Tehran University of Medical Sciences, Department of Immunology, School of Public Health, Tehran, Iran, fazshok@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Mahdi Shabani Ali Ahmad Bayat Mahmood Jeddi-Tehrani Hodjatallah Rabbani Cristina Ulivieri Zahra Amirghofran Cosima T Baldari Fazel Shokri Google Mahdi Shabani Ali Ahmad Bayat Mahmood Jeddi-Tehrani Hodjatallah Rabbani Cristina Ulivieri Zahra Amirghofran Cosima T Baldari Fazel Shokri Google Scholar Mahdi Shabani Ali Ahmad Bayat Mahmood Jeddi-Tehrani Hodjatallah Rabbani Cristina Ulivieri Zahra Amirghofran Cosima T Baldari Fazel Shokri PubMed Mahdi Shabani Ali Ahmad Bayat Mahmood Jeddi-Tehrani Hodjatallah Rabbani Cristina Ulivieri Zahra Amirghofran Cosima T Baldari Fazel Shokri Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Engineering neonatal Fc receptor-mediated recycling and transcytosis in recombinant proteins by short terminal peptide extensions

The importance of therapeutic recombinant proteins in medicine has led to a variety of tactics to increase their circulation time or to enable routes of administration other than injection. One clinically successful tactic to improve both protein circulation and delivery is to fuse the Fc domain of IgG to therapeutic proteins so that the resulting fusion proteins interact with the human neonatal Fc receptor (FcRn). As an alternative to grafting the high molecular weight Fc domain to therapeutic proteins, we have modified their N and/or C termini with a short peptide sequence that interacts with FcRn. Our strategy was motivated by results [Mezo AR, et al. (2008) Proc Natl Acad Sci USA 105:2337-2342] that identified peptides that compete with human IgG for FcRn. The small size and simple structure of the FcRn-binding peptide (FcBP) allows for expression of FcBP fusion proteins in Escherichia coli and results in their pH-dependent binding to FcRn with an affinity comparable to that of IgG. The FcBP fusion proteins are internalized, recycled, and transcytosed across cell monolayers that express FcRn. This strategy has the potential to improve protein transport across epithelial barriers, which could lead to noninvasive administration and also enable longer half-lives of therapeutic proteins.

Engineering of recombinant human Fc receptor I by directed evolution

Human FcγRI is a high-affinity receptor for human IgG. On the basis of its binding activity, recombinant human FcγRI (rhFcγRI) has several possible applications, including as a therapeutic reagent to treat immune complex-mediated disease and as a ligand in affinity chromatography for purification of human IgG. As the stability and production rate of rhFcγRI are low, it would need to be engineered for use in such applications. In this study, we demonstrated engineering of rhFcγRI by directed evolution through random mutagenesis and integration of mutations. Engineered rhFcγRI was expressed by Escherichia coli. Screening identified 19 amino acid mutations contributing to the thermal stability and production rate of rhFcγRI. By integration of these mutations, engineered rhFcγRI containing all 19 amino acid mutations (enFcRd) was constructed and showed markedly enhanced thermal stability (transition midpoint temperature [Tm] = 65.6°C) and production rate (3.27 mg L-medium(-1) OD(600)(-1)) compared with wild-type rhFcγRI (Tm = 48.5°C; production rate, 0.07 mg L-medium(-1) OD(600)(-1)) without a change in the specificities of binding to human IgG subclasses. Moreover, the binding affinity of enFcRd for human IgG1/к (equilibrium dissociation constant [K(D)] = 0.80 × 10(-10) M) was higher than that of wild-type rhFcγRI (K(D) = 1.23 × 10(-10) M). Our study showed that substantial engineering of rhFcγRI is possible.