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

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

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

Albumin and IgG have remarkably long serum half-lives due to pH-dependent FcRn-mediated cellular recycling that rescues both ligands from intracellular degradation. Furthermore, increase in half-lives of IgG and albumin-based therapeutics has the potential to improve their efficacies, but there is a great need for robust methods for screening of relative FcRn-dependent recycling ability. Here, we report on a novel human endothelial cell-based recycling assay (HERA) that can be used for such pre-clinical screening. In HERA, rescue from degradation depends on FcRn, and engineered ligands are recycled in a manner that correlates with their half-lives in human FcRn transgenic mice. Thus, HERA is a novel cellular assay that can be used to predict how FcRn-binding proteins are rescued from intracellular degradation. Nat Commun 2018 Feb 12; 9(1):621

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

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

Human and mouse albumin bind their respective neonatal Fc receptors differently

Abstract 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

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

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

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

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

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

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

Foreign Direct Investment Law : the case of China

Cette étude tente de donner un aperçu général du développement historique, des particularismes et des principales problématiques du droit positif des investissements directs étrangers. En effet, une étude exégétique des textes législatifs et règlements portant sur des investissements étrangers est, certes, nécessaire pour la compréhension du régime juridique des investissements directs étrangers, mais sa connaissance ne suffit pas pour qu'un investisseur puisse réussir son investissement sur le territoire chinois. Le régime juridique des investissements directs étrangers est le fruit d'un travail d'imitation législative de systèmes de droits étrangers. Son développement est indéniablement lié aux éléments de la vie politique, culturelle, sociale et économique du pays. De la politique de l'économie planifiée à l'économie de marché, en quarante ans, le gouvernement a pu créer un droit quasiment exhaustif à partir du vide. Cependant, la rapidité du travail législatif n'est nullement une preuve de sa qualité. En effet, dans le but de rattraper son retard sur les systèmes juridiques des pays économiquement développés, le gouvernement a dû implanter les solutions étrangères, sans pour autant effectuer un travail approfondi d'adaptation ou d'harmonisation, diminuant ainsi la qualité et l'efficacité des lois.Initialement, le droit des investissements directs étrangers était uniquement constitué de trois lois distinctes et des règlements d'application desdites lois. Il s'agissait de la Loi sur les EJV sino-étrangères et son règlement d'application, la Loi sur les WFOE et son règlement d'application, et CJV sino-étrangères et son règlement d'application. Ce régime est ensuite complété par d'autres textes, chacun régissant qu'une seule forme spécifique d'entreprise à participation étrangère.Les litiges font partie de la vie courante non seulement des personnes morales, mais, également des personnes physiques, et permettent de mesurer l'efficacité de la justice au sein d'un État. Malgré le fait que les litiges constituent le moyen ultime de tester l'efficacité de la loi ou d'un système de droit, ils ne surviennent qu'en dernier ressort, surtout dans les États tels que la Chine, où l'histoire démontre une préférence constante envers les modes informelles de résolution des différends au détriment de la procédure étatique contentieuse.Après trois décennies de réforme, la Chine fait désormais partie intégrante de la scène internationale. Par conséquent, ses pratiques portant sur la résolution des différends sont de plus en plus en harmonie avec les standards internationaux. En d'autres termes, la recherche d'une résolution efficace des différends est similaire de celle des autres États occidentaux : idéalement un différend doit être résolu dans le délai court, à un coût bas et avec moins de stress que possible, mais, arrivant tout de même à un résultat acceptable.Le droit chinois des investissements directs étrangers a subi le plus de réformes législatives en Chine. Il est donc primordial de suivre constamment l'actualité juridique. En effet, les deux catalogues récent (2015 et de 2017) illustrent une tendance libéralisatrice du marché national en ouvrant de plus en plus les secteurs d'industrie aux investissements étrangers, et en introduisant un système proche de la " liste négative ". Puis la Réforme de 2016 est venue remplacer la procédure d'autorisation des projets d'investissements directs étrangers par un simple système d'enregistrement. Certes, ces éléments récents ont su apporter des solutions à des difficultés existantes, mais en raison du nombre de vices du droit positif, une réforme en profondeur est nécessaire. Le Projet de loi des investissements étrangers, publié par le Ministère du Commerce en 2015, a le potentiel d'effacer tous les défauts du régime actuel. Cependant, malgré les promesses du gouvernement, son futur demeure très incertain à l'heure actuelle.This study aims to give a general overview of the historical development, particularisms and main issues of the positive law of foreign direct investment, by looking at different areas of law, ranging from the history of law to the very content of foreign investment law, and through dispute settlement mechanisms for foreign investment. An exegetical study of laws and regulations on foreign investments is a necessary step for the understanding of the legal regime of foreign direct investment, but its knowledge of the law is not sufficient for an investor to succeed in his investment.Its development is undeniably linked to the elements of the political, cultural, social and economic aspects of the country. From the promotion of Marxist values of the Soviet system under the Mao Zedong period, to the adoption of legal pragmatism under the direction of Deng Xiaoping; from the policy of the planned economy to the market economy. In forty years, the government was able to create an entire legal system from almost nothing. However, the speed of legislative work is by no means a proof of its quality. Indeed, in order to catch up with the legal systems of the economically developed countries, the government had to implement foreign solutions, without carrying out any in-depth adaptation or harmonization work, thus reducing the value and the effectiveness of the law.Initially, the foreign direct investment law consisted only of three separate laws and their implementing regulations : Sino-Foreign EJV Law and its Implementing Regulation, the WFOE Law and its Implementing Regulation, and Sino-Foreign CJV and its Implementing Regulation. This was then competed by other laws and regulations, each governing only one specific form of foreign-invested enterprise.Litigation is part of everyday life which allows the legislator to measure the efficiency of justice within a State. Despite the fact that litigation is the ultimate means of testing the effectiveness a legal system, it is only a last resort, especially in states such as China, in which its history demonstrates a constant preference for alternative modes of dispute resolution to the detriment of contentious proceedings.After three decades of reform, China has become an integral part of the international scene. As a result, its practice of resolving disputes is increasingly aligned with international standards. The quest for effective dispute resolution is now similar to that of other Western states: ideally a dispute must be resolved in the short time frame, at a low cost and with as little stress as possible, but with an acceptable result.It is therefore essential to constantly follow legal news. Indeed, the two recent catalogs (2015 and 2017) illustrate a liberalizing trend of the national market by opening more sectors to foreign investments, and by introducing a system close to the "negative list". And the 2016 Reform replaced the authorization procedure for foreign direct investment projects with a simple registration system. While these recent developments have provided solutions to existing difficulties, due to the number of flaws in substantive law, a thorough reform is needed. The Draft Law on Foreign Investment, published by the Ministry of Commerce in 2015 for public commentary, has the potential to erase all the flaws of the current regime. However, despite the promises made by the government, its future remains very uncertain at the moment