Sequence features of variable region determining physicochemical properties and polyreactivity of therapeutic antibodies

International audienceTherapeutic antibodies have transformed the clinical practice. Not surprisingly, development of antibody thera-peutics is currently the main focus of the biotechnology industry. Nonetheless, the development process is complex , and many antibodies do not reach the clinic. Reasons for the failures include, undesired binding behavior (polyreactivity), low stability, poor expression yields, unfavorable pharmacokinetics etc. Numerous studies have proposed different analytical methods for assessment of physicochemical parameters of antibodies and identification of problematic molecules at early stages of the development process. These studies, however, have not addressed the basic mechanistic question of how sequence features of variable regions determinate the different biophysical characteristics and the binding behavior of the antibodies. In a recent study, Jain et al assessed 12 biophysical qualities of 137 monoclonal therapeutic antibodies. We used the raw data from this comprehensive study to perform correlation analyses of different biophysical measurables with various sequence features of variable regions of the antibodies-number of mutations, length of hypervariable loops, and frequency of amino acid residue types. The obtained data reveled significant relationships between the sequence characteristics of the variable domains and different physiochemical properties of antibodies. The data from this study can assist in design of a set of criteria for early identification of antibodies with developability issues. Moreover, our findings provide novel fundamental insights into the sequence-function relationship of antibodies

Rôle physiopathologique des anticorps catalytiques et des anticorps polyréactifs

Les anticorps sont les molécules effectrices de l immunité adaptatrice humorale. Ils se lient spécifiquement et neutralisent une large panoplie d antigènes. Au-delà de leurs fonctions classiques, les anticorps possèdent les propriétés moins explorées que sont l activité catalytique, qui permet aux anticorps de se comporter comme des enzymes, et la polyréactivité, qui représente la capacité d une molécule d anticorps à se lier à plusieurs antigènes structurellement différents. Les anticorps catalytiques sont retrouvés dans plusieurs pathologies chez l homme, telle que l hémophilie acquise, une maladie caractérisée par la survenue d autoanticorps anti-facteur VIII. Dans ce travail, nous décrivons des IgG hydrolysant et activant le facteur IX de la coagulation chez les patients avec hémophilie acquise. Par ailleurs, nous avons effectué une étude longitudinale de deux ans des IgG catalytiques chez les patients subissant une transplantation rénale. Les anticorps polyréactifs représentent une proportion importante du répertoire des immunoglobulines circulantes. De plus, les sites inflammatoires sont abondants en molécules, telles que l hème libre, capables de rendre polyréactives certaines IgG monoréactives. Nous avons étudié l influence de la nature des régions constantes de la chaîne lourde des anticorps sur leur susceptibilité à devenir polyréactifs. Ce travail apporte un nouvel éclairage sur l importance physiopathologique des anticorps catalytiques et polyréactifs.Antibodies are effector molecules of the humoral arm of the adaptive immune system that bind specifically and neutralize diverse array of antigens. Beyond the classical function of antibodies exist the relatively less explored properties, of catalytic activity that enable antibodies to act as enzymes and polyreactivity that confers the ability to bind to several structurally unrelated antigens. Catalytic antibodies have been associated with several autoimmune, inflammatory and infectious diseases. Acquired hemophilia is an autoimmune disease, reported with the presence of catalytic antibodies against coagulation factor FVIII. In the present work, we have investigated the presence of factor IX (FIX) hydrolyzing IgG in patients with acquired hemophilia. We investigated the molecular mechanism and the physiological relevance of FIX activation upon hydrolysis by patients IgG. In addition, a longitudinal follow-up for 2 years was done in patients undergone renal transplant to investigate the evolution of catalytic antibodies in the course of disease. Polyreactive antibodies constitute a major portion of the natural antibody repertoire. Additionally, sites of inflammation are abundant in protein destabilizing agents like free heme that can induce polyreactivity in monoreactive antibodies. We have investigated the effect of the antibody constant domain on heme-induced polyreactivity. The present work has allowed us a better understanding of the physiopathological relevance of catalytic and polyreactive antibodies.COMPIEGNE-BU (601592101) / SudocSudocFranceF

Immunogenicity of long-lasting recombinant factor VIII products

International audienceReplacement therapy for patients with hemophilia A using plasma-derived or recombinant factor VIII (FVIII) is complicated by the short half-life of the FVIII products and by the occurrence of neutralizing antibodies in a substantial number of patients. In the recent years, enormous efforts have been invested to develop new generations of coagulation factors with extended half-lives. Presumably, the use of long-lasting FVIII products should reduce the frequency of administration to the patients and drastically improve their quality of life. The question of their immunogenicity remains however unanswered as yet. The present review proposes a summary of the different strategies developed to enhance the half-life of FVIII, including fusion of FVIII to the Fc fragment of the human IgG1 or to human serum albumin, or attachment of polyethylene glycol. Based on the available literature, we hypothesize on the potential benefits or risks associated with each of the latter strategies in terms of immunogenicity of the newly derived hemostatic drugs

Correction of bleeding in experimental severe hemophilia A by systemic delivery of factor VIII- encoding mRNA Short title: mRNA-based therapy for hemophilia A Scientific category: Thrombosis and Hemostasis

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Contrôle de la réponse immunitaire dirigée contre le facteur VIII thérapeutique

L administration de FVIII thérapeutique aux patients hémophiles A induit, chez 10 à 30 % d entre eux, une réponse alloimmune et l apparition d IgG anti-FVIII qui inhibent l activité procoagulante du FVIII. L apparition des inhibiteurs du FVIII rend tout traitement ultérieur inefficace, a des conséquences dramatiques sur la qualité de vie des patients, et entraîne une augmentation considérable du coût des traitements. Des stratégies existent en clinique pour éliminer les inhibiteurs chez les patients hémophiles A. Bien qu efficaces, ces différentes stratégies d éradication des inhibiteurs sont coûteuses, peu spécifiques du FVIII, et leur application contraignante pour les patients. Durant ma thèse, j ai exploré de nouvelles approches d immunointervention dans le but de contrôler la réponse immunitaire anti-FVIII dans un modèle murin d hémophilie A sévère. La première approche consistait à éliminer les plasmocytes spécifiques du FVIII à l aide d un inhibiteur du protéasome, le bortezomib. Son utilisation prévenait le développement de la réponse anti-FVIII chez les souris naïves, mais ne réduisait pas le titre d inhibiteur une fois la réponse anti-FVIII déclarée. La seconde approche montrait que le transfert d IgG anti-FVIII maternelles, pendant la gestation et la lactation, retardait l apparition des inhibiteurs chez la progéniture à l âge adulte. La dernière approche, qui consistait à induire une tolérance centrale au FVIII par transfert materno-fœtal de constructions FVIII-Ig chez la souris hémophile A, reste à être validée. Des résultats positifs permettraient d envisager une application clinique dans la prévention des réponses contre des protéines thérapeutiquesPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Influence de la polyréactivité naturelle et induite des immunoglobulines solubles et des récepteurs de lymphocytes B sur la modulation de la réponse immunitaire

Afin de faire face à l extraordinaire diversité des antigènes auxquels l organisme est exposé, des mécanismes élaborés de génération de diversité des récepteurs des lymphocytes T et B sont apparus au cours de l évolution. Toutefois, ces mécanismes aléatoires permettent l émergence de récepteurs potentiellement délétères dirigés contre des antigènes du Soi. Aussi les effecteurs cellulaires subissent-ils une éducation leur permettant de discriminer le Soi du non-Soi. Pourtant, une fraction non-négligeable des anticorps circulants est capable de reconnaître plusieurs antigènes structurellement différents et notamment des antigènes du Soi. En outre, des travaux récents ont démontré que certaines immunoglobulines monoréactives deviennent polyréactives après exposition à différents agents pro-oxydants. Lors de mon travail de thèse, j ai étudié la polyréactivité des lymphocytes B naïfs murins et j ai observé que la faible stimulation du BcR confère un phénotype régulateur caractérisé par la production d IL-10 et la polarisation des lymphocytes T CD4+ vers un profil Tr1/Th2. En parallèle, j ai observé différents degrés de sensibilité des immunoglobulines solubles à l induction de polyréactivité par différents agents chaotropiques et pro-oxydants. Les résultats de mon travail soulèvent la question de l importance des lymphocytes B poly/autoréactifs naturels comme source de cellules régulatrices dans les conditions physiologiques. Par ailleurs, mes résultats mettent en exergue l importance des molécules libérées dans le microenvironnements inflammatoires sur les fonctions des immunoglobulines et des lymphocytes BIn order to counter the enormous antigen diversity, elaborated mechanisms for the generation of diversity of the T and B-cell receptors appeared along the evolution. However, such random mechanisms enable the appearance of broadly-reactive receptors that could be potentially deleterious. Thus, the cellular effectors are educated along their development in order to discriminate self from non-self. The education of the immune system implies several mechanisms of negative selection of self-reactive T and B cells during their ontogeny. However, a non-negligible fraction of circulating immunoglobulins is able to bind to various structurally unrelated antigens including self-antigens. Besides, recent studies have demonstrated that certain monoreactive immunoglobulins become polyreactive following exposure to various chaotropic agents and pro-oxidative molecules. During my PhD work, I studied the natural polyreactivity of B lymphocytes from naïve mice and I observed that a weak BcR-stimulation confers them regulatory properties characterised by the production of IL-10 and the polarization of CD4+ T cells into Tr1/Th2 cells. I also observed different degrees of sensibility of soluble immunoglobulins to the induction of polyreactivity depending on the nature of the pro-oxidative molecules. The results of my work emphasize the question of the importance of the naturally poly/self-reactive B cells as a source of regulatory cells under physiological conditions. Besides, my results highlight the importance of molecules present at the site of inflammation and their functions on soluble immunoglobulins and circulating B cellsPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Noncanonical Functions of Antibodies

International audienceThe canonical functions of antibodies are based on joining the process of antigen recognition with initiation of innate immune reactions. With the introduction of modern research approaches, and the use of sophisticated model systems, the recent years have witnessed the discovery of numbers of non-canonical functions of antibodies. These functions encompass either untypical strategies for neutralization of pathogens or exertion of activities by antibodies that are typically characteristic for other proteins (cytokines, chaperons or enzymes). Here we provide an overview of non-canonical functions of antibodies and discuss their mechanisms and implications for immune regulation and immune defence. A better comprehension of these functions will enrich our knowledge about the adaptive immune response and shall inspire the development of novel therapeutics

A molecular jewel for hemophilia A treatment

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Pathogenic immune response to therapeutic factor VIII: exacerbated response or failed induction of tolerance?

International audienceTherapeutic factor VIII is highly immunogenic. Despite intensive research in the last decades, the reasons why 5-30% of patients with hemophilia A (of all severities) develop inhibitory anti-factor VIII antibodies (inhibitors) following replacement therapy remain an enigma. Under physiological conditions, endogenous factor VIII is recognized by the immune system. Likewise, numerous observations indicate that, in hemophilia A patients without inhibitors, exogenous therapeutic factor VIII is immunologically assessed and tolerated. A large part of the research on the immunogenicity of therapeutic factor VIII is attempting to identify the ‘danger signals’ that act as adjuvants to the deleterious anti-factor VIII immune responses. However, several of the inflammatory assaults concomitant to factor VIII administration initially hypothesized as potential sources of danger signals (e.g., bleeding, infection, and vaccination) have been disproved to be such signals. Conversely, recent evidence suggests that cells from inhibitor-negative patients are able to activate anti-inflammatory and tolerogenic mechanisms required to suppress deleterious immune responses, while cells from inhibitor-positive patients are not. Based on the available observations, we propose a model in which all hemophilia A patients develop anti-factor VIII immune responses during replacement therapy irrespective of associated danger signals. We further postulate that the onset of clinically relevant factor VIII inhibitors results from an inability to develop counteractive tolerogenic responses to exogenous factor VIII rather than from an exacerbated activation of the immune system at the time of factor VIII administration. A better understanding of the pathogenesis of neutralizing anti-factor VIII antibodies will have repercussions on the clinical management of patients and highlight new strategies to achieve active immune tolerance to therapeutic factor VIII

Les signaux de danger dans l'initiation de la réponse immunitaire contre le facteur VIII thérapeutique

L hémophilie A (HA) est une maladie hémorragique congénitale qui se traduit par un défaut en facteur VIII (FVIII) de la coagulation. Le traitement privilégié des saignements est l administration de FVIII exogène cependant, 30% des patients développent une réponse anticorps alloimmune contre le FVIII thérapeutique qui inhibe son activité pro-coagulante. L'endocytose du FVIII par les cellules dendritiques (DC) et sa présentation aux lymphocytes T ont été documentées. Cependant, la nature des signaux de danger (SD) responsables de la maturation des DC indispensable à l initiation de la réponse immunitaire anti-FVIII, est inconnue. Au cours de ma thèse, j ai cherché à identifier l origine de ces SD et envisagé 3 possibilités: Une origine liée à la structure intrinsèque du FVIII, au contexte inflammatoire avant l administration de FVIII, ou au contexte inflammatoire généré par l injection de FVIII. Mes résultats ont montré que le FVIII n était pas capable d induire la maturation de macrophages ou d activer directement le TLR2. J ai également écarté l hypothèse d un état d activation compensatoire des plaquettes (PLT) dans un organisme privé de FVIII. En revanche, mes travaux ont mis en évidence un rôle des PLT dans l initiation de la réponse immunitaire anti-FVIII dans le modèle murin d HA. Mes résultats suggèrent que l implication des PLT passe par leur activation par la thrombine générée lors de l administration de FVIII. L identification des médiateurs de l inflammation issus de l activation plaquettaire devrait ouvrir des perspectives thérapeutiques intéressantes dans le contrôle de l inflammation au moment de l administration de FVIII, afin de réduire son immunogénicitéHemophilia A (HA) is a rare congenital hemorrhagic disorder resulting from a defective production of coagulation factor VIII (FVIII). Treatment of HA patients with therapeutic FVIII results, in up to 30% of the cases, in the emergence of anti-FVIII antibodies that inhibit the pro-coagulant activity of the therapeutically administered FVIII. FVIII endocytosis by dendritic cells (DCs) and its presentation to T cells have been well established. However, the nature of the danger signals responsible for the maturation of DCs that is mandatory to the initiation of anti-FVIII immune response, remains poorly understood. During my thesis, I investigated the origin of these danger signals, and explored 3 possibilities: an origin linked to the intrinsic structure of FVIII, to the inflammatory environment that prevails before FVIII administration, or to the inflammatory environment generated upon injection of exogenous FVIII. My work demonstrates that FVIII is not able to trigger macrophages maturation or to induce direct TLR2 activation. I also rejected the hypothesis of a role of a compensatory activation state of HA patients platelets (PLT) in the initiation of the immune response against FVIII in a murine model of HA. My results suggest that PLT involvement depends on their activation by thrombin which is generated by FVIII administration. The identification of the inflammatory mediators released by activated PLT should open attractive therapeutic perspectives in the control of inflammation at the time of FVIII administration, in order to reduce FVIII immunogenicityPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF