Periodic Reporting for period 2 - EDUC8 (Early Stage Researchers EDUCational Program on Factor VIII Immunogenicity)
Reporting period: 2022-04-01 to 2024-09-30
The development of anti-drug antibodies is the major complication of treatment with biotherapeutics. This is particularly true of patients with haemophilia A (HA) who develop neutralizing antibodies to therapeutic factor VIII (FVIII) following replacement therapy with exogenous pro-coagulant FVIII. The onset of neutralizing antibodies to FVIII, referred to as FVIII inhibitors, represents a major societal concern owing to the enormous cost associated to the clinical management of inhibitor-positive patients, that exceeds 200,000€/patient/year. The EDUC8 program is a multidisciplinary training program with exposure of the enrolled ESRs to a core common educational package and development of individual PhD research projects dedicated to decreasing the societal burden associated with the development of anti-FVIII antibodies in Europe. The core educational program included exposure to basic science in the fields of immunology and hemostasis, to the most advanced “omic” technologies in order to anticipate tomorrow’s scientific discoveries, and to the importance of ethical considerations and public awareness in today’s research. A key component of the training program was provided by immersion of the ESRs in a clinical center for HA, which provided them with a unique experience and foster the conception of novel and realistic therapeutic approaches. The ESRs exploited the accumulated theoretic and practical experience in an entrepreneurship training on key strategic, technical, financial and human resource issues required for the creation of startups. In a tribute to the concept of the fully developed mind coined by the French philosopher Michel de Montaigne on education, our ambition has been to form a novel generation of scientists with deep scientific knowledge, multicultural exposure, imagination, pragmatism and understanding of both the academic and private sectors. This shall foster translational research in European on the immunogenicity of therapeutic proteins.
During this reporting period, the research has focused on different targets. A thorough analysis of the repertoires of HLADP4 and HLADR-restricted FVIII peptides presented by antigen presenting cells such as dendritic cells and endothelial cells has been performed. Further, the protocol to derive endothelial cells (liver sinusoidal endothelial-like) from healthy donors and haemophilia A patients with different haemophilia-causing gene abnormalities has been refined and validated. Work is now focusing on confirming the presence of endogenous of FVIII, its localization into different cellular compartment, the expression of HLADR molecules, as a pre-requisite to perform peptide repertoire analyses. Likewise, the protocols to study HLADP4-restrcited FVIII peptides have been validated and the results are summarized in an article under submission for publication. The goal here was to deliver a list of 5 most immunogenic HALDP4-restricted FVIII peptides. Another aspect has been to delineate the repertoire of FVIII-specific CD4+ T cells. More than 800 FVIII-specific T cells lines were successfully generated from more than 22 healthy donors altogether. The specific T cell lines have been identified and tested for the recognition of 63 predicted HLADR-restricted T cell epitopes. The analysis of the repertoires of the the Vbeta CDR3 regions of the T cell receptors was performed which provided a full picture of the heterogeneity of this peculiar cell population. In parallel, several recombinant proteins have been cloned and produced including Fc-fused FVIII domains, immunogenic FVIII A2 or C2 domains linked to tolerogenic molecules. Immunodominant FVIII peptides were selected and synthesized in collaboration with other Beneficiaries and the protocols were delineated for the conjugation of synthetic immuno-dominant FVIII peptides to DC4U’s GlycoDCTM platform dedicated to the induction of T cell mediated immune tolerance in constant interaction with the partner organization DC4U. Likewise, second and third generation FVIII-specific CAR constructs with different intracellular domain combinations have been cloned into lentiviral vectors and used to transduce T cells for further in vitro characterization. In addition, several in vitro culture assays have been validated including the characterization of liver sinusoidal endothelial cells from different from commercial sources, or the preliminary cell culture to study the response to FVIII of transduced CAR CD4+ T cells from healthy donors. Likewise, the experimental protocols to study transcytosis in vitro by human placental cells have been performed; the results are now summarized in an article under submission for publication. In conclusion, most of the tools (peptides, recombinant proteins, primary cell preparation, establishment of cell lines, in vivo experimentation) have been validated and the fundamental experimental protocols for our research have been established. For most of the ESRs, sufficiently original and numerous results have been accumulated to allow the redaction of several research articles, some have been published and some are under submission.
Biotherapeutic pharmaceuticals developed in the last decades have provided life-saving therapies for many diseases thus remarkably improving patients’ quality of life in Europe and in the world. In 2014, over 200 biopharmaceuticals were marketed for clinical use in Europe or the US. This progress comes at a price since many biotherapeutics give rise to unwanted immune responses resulting in anti-drug antibodies (ADA), which greatly reduce treatment efficacy. Our current understanding of the immunogenicity of biotherapeutics is incomplete due to the lack of studies wherein patient- and product-related factors are investigated in an integrated fashion. In this project, we will “educ8” and train a novel generation of ESRs on innovative and integrated approaches to study biotherapeutics immunogenicity and to design novel strategies to mitigate undesirable immune responses to current and future generations of biotherapeutics. To be able to adequately address the complex interplay between patient- and product-related factors, we have focused our project on the immunogenicity of therapeutic FVIII for which a large variety of biotherapeutic products is currently available. Incorporating large existing patients’ cohorts that have been specifically designed to identify risk factors for the development of ADA provides a unique and excellent opportunity to design a roadmap for systematically reducing immunogenicity as well as to provide personal medicine based-approaches for ADA eradication. At term, our results will be translated to other disorders in which the development of ADA prohibits optimal and economically sustainable patient treatment with biotherapeutics.