Host Institution: National Institute of Health and Medical Research (INSERM), Paris, France
Primary Supervisor: Dr. LACROIX-DESMAZES Sebastien
Introduction: A substantial number of Hemophilia A (HA) patients develop inhibitory anti-factor VIII (FVIII) antibodies after replacement therapy with FVIII. Ergo, immuno-intervention to tackle FVIII immunogenicity has taken great importance. The injection to pregnant mice of Fc-fused A2 and C2 FVIII domains leads to (1) the transplacental delivery of A2Fc/C2Fc, mediated by the neonatal Fc receptor, (2) induction of FVIII-specific regulatory T cells in the offspring and (3) partial tolerance to exogenous therapeutic FVIII later in life. Using the A2 and C2 domain of FVIII that represent 20% of the protein, the anti-FVIII immune response was reduced by 10-fold, hence, we hypothesize that the transplacental delivery of the entire FVIII protein should confer complete tolerance. Nonetheless, preliminary data show that rFVIIIFc is delivered through the placenta to lesser extents than human IgG and C2Fc. Moreover, this phenomenon it is not due to the binding of FVIII to its carrier-protein, von Willebrand factor (VWF), but, possibly because of other extracellular or intracellular interactions of FVIIIFc.
Aims: The main objective is to develop strategies to induce immune tolerance to therapeutic FVIII as early as during fetal life, through the transplacental delivery of FVIII. A1: I will develop different rFVIIIFc mutants to modify their interactions with extra/intracellular molecules, modify their half-life and biodistribution in the organism; A2: I will Investigate the capacity of the rFVIIIFc mutants to cross the placenta in vitro and in pregnant mice. A3: In FVIII-KO pregnant mice, I will evaluate the induction of FVIII-specific tolerance at the T and B-cell levels in offspring of mothers treated with rFVIIIFc mutants.
Expected Results: R1: The injection to pregnant mice of rFVIIIFc mutants will lead to the transplacental delivery of FVIII, induction of FVIII-specific Tregs in the offspring and complete long-lasting tolerance to exogenous therapeutic FVIII. R2: Our results will pave the way towards the design of novel strategies for immuno-intervention as early as during fetal life.
Planned secondment: A international 5-month secondment at SOBI, Geneva, Switzerland, is planned to support the expression, purification and biochemical validation of rFVIIIFc mutants.
Enrolment in Doctoral degree(s): Sorbonne University
Host Institution: Stichting Sanquin Bloedvoorziening-Sanquin (Amsterdam, The Netherlands)
Primary Supervisor: Prof. Jan Voorberg
Introduction: Major histocompatibility complex class II (MHCII)-restricted peptide presentation is crucial for the selection and subsequent proliferation of antigen-specific CD4+T cells. We have recently identified the repertoire of FVIII peptides presented by HLA-DQ and HLA-DR. The goal of my research project is to explore the contribution of HLA-DP to the presentation of FVIII-derived peptides to FVIII-specific T cells. Further, the development of a unique MHC class II dextramer technology by Immudex will assess whether HLA-DP restricted CD4+ T cells contribute to the immune response to FVIII.
Aims: A1: To delineate the HLA-DP-restricted FVIII peptidome. A2: To validate the binding of the identified peptides to purified HLA-DP4 and assess their immunogenic nature. A3: To generate a revolutionary family of DNA barcoded DP4 Dextramer reagents.
Expected Results: E1: the exhaustive delineation of HLA-DP and HLA-DP4-restricted naturally-processed peptides from FVIII. E2: the validation of DP4-associated immunodominant T-cell epitopes and the validation of FVIII-specific DP4 Dextramer for the detection of FVIII-specific CD4+ T cells in healthy donors and patients.
Planned secondment: I will undertake a 2-month secondment at CEA under the supervision of Dr. Bernard Maillère to validate the immuno-dominant HLA-DP4-restricted FVIII epitopes and amplify FVIII-induced T-cell lines. I will undertake a 8-month secondment at Immudex under the supervision of Dr. Liselotte Brix to generate and validate DNA barcoded DP4 Dextramer reagents displaying the identified immune-dominant FVIII peptides as immune-monitoring tools for HA patients.
Enrolment in Doctoral degree(s): AMC Graduate School of the University of Amsterdam
Host Institution: University of Milan
Primary Supervisor: Prof. Flora Peyvandi
Introduction: The anti-FVIII immune response is characterized by the production of polyclonal IgG directed against multiple epitopes located on the A2, A3-C1 and C2 domains of FVIII. UMIL documented that pre-existing anti-FVIII antibodies are an important risk factor for the development of FVIII inhibitors. A controversy has prevailed over the last two decades on the higher immunogenicity of therapeutic recombinant FVIII (rFVIII) as compared to plasma-drive FVIII (pdFVIII). The prospective randomized SIPPET study, substantiated by a recent observational open cohort, has shown that rFVIII concentrates are indeed associated with an 87% higher incidence of inhibitor development than pdFVIII products. Nonetheless, some cohort studies report contradictory results and the mechanisms underlying the higher immunogenicity of rFVIII over pdFVIII remain unknown.
Aims: A1: This task will investigate differences in epitope profiles between patients who have developed FVIII inhibitors under pdFVIII or rFVIII. Our preliminary data reveal distinct immuno-profiles in plasma obtained pre- and post-treatment from patients with or without inhibitors. A2 I will perform analysis from the plasma of 138 severe HA patients with and without inhibitors who received either rFVIII (n=70) or pdFVIII (n=68). A3 I will then perform DNA extraction of the selected phages and sequencing of 50bp single-end reads on Illumina HiSeq platform.
Expected Results: R1: The project will allow a fine mapping of the existing FVIII epitopes previously identified as targets for anti-FVIII alloantibodies as well as identification of new FVIII epitopes. R2 It will also provide the first detailed comparison of the base-line differences in IgG reactivities between patients that will or will not develop FVIII inhibitors, prior to exposure to FVIII. R3 The results will identify immunogens other than FVIII that predispose patients to inhibitor development.
Planned secondment: There will be a secondment allocated to INGM (National Institute of Molecular Genetics, Milan) to get training and perform analysis for precision immunotherapy.
Enrolment in Doctoral degree(s): University of Milan
Host Institution: University Hospital Frankfurt, Goethe University, Frankfurt am Main
Primary Supervisor: Dr. Christoph Königs
Introduction: Hemophilia A (HA) is a severe bleeding disorder that affects 1 in 5000 males and very few females. HA patients have a deficiency in coagulation factor VIII (FVIII) protein and so their plasma clotting time is delayed compared to healthy individuals. Recombinant or plasma-derived FVIII is used for prophylaxis and treatment in HA patients, but almost 30% of patients with the severe form of the disease produce inhibitory anti-FVIII antibodies, because of a lack of tolerance for FVIII. Thus, it remains important to develop an innovative, effective and tolerogenic strategy to allow a safe and effective therapy for inhibitor patients. Recently, we and others have shown that CAR-transduced T regulatory cells are able to inhibit the activation of FVIII-specific helper T cells in vitro, resulting in the inhibition of differentiation of murine FVIII-specific memory B cells into antibody-secreting cells. The main goal of the current project is to develop third generation chimeric antigen receptor (CAR) regulatory T cells able to potently prevent and/or reverse anti-FVIII antibody formation.
Aims: A1: Deciphering essential parameters for effective FVIII-specific CAR-Tregs by design and generation. FVIII-specific scFvs isolated from phage display libraries will be combined with relevant intracellular and transmembrane domains of CARs. The final constructs will be ligated into lentiviral vectors used to transduce T regulatory cells (Tregs). Experiments will be performed using both murine and human cells, indeed Tregs will be isolated from human peripheral blood cells and from the spleen of naïve FVIII-KO mice. A2: Analysis of transduced Tregs phenotype (in presence and absence of FVIII stimuli) and their ability to suppress humoral and cellular responses in vitro and in vivo, focusing on cytokine production, expression of costimulatory receptors and markers of T-cell activation and exhaustion. The suppressive effect of the most potent CAR-Treg candidates will be validated in vitro using human peripheral blood cells and splenic B cells from FVIII-challenged FVIII-KO mice. Then, the most potent human CAR-Tregs will be validated in vivo by adoptive transfer to RAG-KO mice reconstituted with splenocytes from FVIII-challenged mice and ex vivo using peripheral blood cells from inhibitor-positive hemophilia A patients. In parallel, the most potent mouse CAR-Tregs will be validated in vivo in a preventive approach (adoptive transfer to naïve mice followed by FVIII replacement therapy) as well as in a therapeutic approach (adoptive transfer to FVIII-challenged FVIII-KO mice). A3: Translation of the best FVIII-specific CAR-Treg candidate in terms of tolerance induction from the laboratory to a clinical application to efficiently control FVIII inhibitor formation.
Expected Results: R1: Deciphering essential criteria for FVIII-specific CAR-Treg design. R2: Generation of FVIII-specific CAR-Tregs able to suppress humoral and cellular response, inducing tolerance towards FVIII. R3: Analyzing FVIII- specific CAR-T regs potential in vitro and in vivo.
Planned secondment: The ESR4 will be seconded at Biotest, under the supervision of PD Dr. Jörg Schüttrumpf. Plans for secondments are currently redeveloped to achieve a maximal benefit for the ESRs under pandemic conditions.
Enrolment in Doctoral degree(s): Goethe University, Frankfurt am Main
Introduction: A large variety of mutations causing hemophilia A (HA) has been identified. Large deletions, intron inversions and nonsense mutations lead to the absence of FVIII production or production of truncated molecules. Importantly, the prevalence of FVIII inhibitor development shows great variation depending on the type of mutation: 88% and 25% for multi-domain and single domain large deletions, respectively, 31% for nonsense mutations and 22% for intron 22 inversions (INV22) In particular, preterminal stop codons (PTCs) located in the FVIII light chain (A3-C1-C2 domains) are associated with a higher risk to develop FVIII inhibitors as compared to PTCs in the FVIII heavy chain (A1-A2-B domains). Our preliminary data document the presence of significant amounts of intracellular FVIII in iPS-derived vascular endothelial cells (vECs) from HA patients with INV22 (P-I22I) and light chain-located PTCs (R1941X & R2209X). Our working hypotheses is that, under physiological conditions, MHC expressed at the surface of ECs, which favors the establishment of peripheral tolerance, presents endogenous FVIII. Conversely, in some HA patients with nonsense mutations, part of the FVIII molecule is not presented which alters the acquisition of peripheral tolerance and exposes the patients to an increased risk of alloimmunization to therapeutic FVIII.
Aims: A1: Decipher the mutation-related mechanism at the basis of a variation in inhibitor development risk. Patient-derived induced pluripotent stem (IPS) cells will be differentiated into vECs to analyze the intracellular fate of endogenous FVIII harboring different non-sense mutations; A2: Compare the amount of endogenous FVIII using immunoprecipitation and immunostaining approaches; A3: Co-stain FVIII with proteins involved in the classical secretory pathway, in the ubiquitin-proteasomal or lysosomal degradation pathways, or in the autophagosome pathway; A4: ECs in the microvasculature basally express MHC-I and low levels of MHC-II; MHC-II expression can be induced to present endothelial antigens to immune cells. I will analyse the FVIII peptidome presented by MHC-I and MHC-II of vECs derived from healthy donors and HA patients with different mutations; A5: vECs will be incubated with FVIII-specific CD4+ T-cell lines generated by ESR8 (Maillère lab, CEA) to confirm a link between the type of non-sense mutation and the presentation of endogenous FVIII to CD4+ T cells.
Expected Results: R1: The results will indicate whether different non-sense mutations differentially affect the intracellular trafficking of endogenously produced FVIII, targeting it differentially to the ubiquitin-proteasome, autophagosome or lysosome pathways. R2: A link will be established between the altered intracellular trafficking of endogenous FVIII, FVIII presentation to T cells and induction of T- cell mediated tolerance/occurrence of allo-immunisation to therapeutic FVIII. R3: The CD4+ T-cell lines will match the HLA-DR haplotype of the vEC donor and will recognize a FVIII epitope different from the peptide carrying the non-sense mutation. R4: The successful implementation of the approaches will give new insights into the mechanism of establishment of peripheral tolerance to FVIII.
Planned secondment: Secondment at Biotest, Germany for 8 months is planned. This will give me an opportunity to establish the methods allowing isolation of FVIII from vECs and develop protein-based analysis of FVIII via immunoprecipitation, affinity chromatography and mass spectrometry.
Enrolment in Doctoral degree(s): Bonn International Graduate School, PhD program: Immunology (ImmunSensation and infection cluster)
Host Institution: Amsterdam UMC – location VUmc
Supervisors: Prof. Yvette van Kooyk
Introduction: Hemophilia A patients carry a genetic deficiency in procoagulant protein factor VIII (FVIII). Due to the inexplicably high immunogenicity of FVIII compared to other therapeutic proteins, 1 in 4 patients develop antidrug antibodies (Scott et al. 2020, Frontiers in immunology). In patients receiving treatment in the form of concentrated FVIII, the formation of anti-FVIII antibodies is a major concern. For these reasons, studying FVIII and the reversal of its immunogenicity is of interest. It has been demonstrated that sialylation of models antigens directs the immune response towards an antigen – specific tolerogenic programme. Specifically, evidence showed that α2,3-sialic acid-conjugated antigens (Sia-Ag) are taken up by dendritic cells (DCs) via inhibitory sialic acid-binding Ig-type lectin (Siglecs) receptor, which endows them with the ability to both promote naïve CD4+ T cell differentiation to regulatory T cells (Tregs) in vitro and in vivo, at the expenses of CD4+ and CD8+ effector T cells and to dampen the functioning of established effector T cells (Perdicchio et al. 2016, PNAS). Thus, antigen sialylation is a highly attractive strategy to inhibit ongoing inflammatory immune responses. We therefore hypothesize that modification of therapeutic FVIII with α2,3 sialic acid will similarly induce tolerance and reduce its immunogenicity.
Aims: The overall objective is to exploit the GlycoDCTM technology developed by DC4U to modify therapeutic FVIII with an α2,3-sialic acid to induce preventive or therapeutic antigen-specific tolerance. A1. Firstly, we will generate several Sia – FVIII peptides candidates via conjugation of either recombinant human B domain-deleted FVIII or immuno-dominant FVIII peptides to α2,3-sialic acid. A2. Secondly, we will confirm the binding to and efficient uptake by Siglecs of Sia-FVIII/peptides candidates using mouse splenic DCs and immature human moDCs as models. Then, we will study the maturation profile of DCs by flow cytometry. A3. Thirdly, we will assess the ability of Sia-FVIII/peptides-loaded DCs to generate de novo functionally proficient CD4+Foxp3+ Tregs both using mouse and human cells. A4. Lastly, we will investigate the in vivo tolerogenic effect of the most promising Sia-FVIII/peptides candidates using two different strains of FVIII-KO mice.
Expected Results: R1: Our results will validate the hypothesis that glycosylation of therapeutic FVIII induces tolerance to the exogenous protein via internalization of Sia – FVIII by DCs and the consequent generation of both a tolerogenic cytokine profile and FVIII-specific Tregs.
Planned secondment: 5 months at INSERM and 4 months at GUF are the planned secondments to investigate the in vivo response to the Sia-FVIII/peptides candidates, under the supervisions of Dr. Sébastien Lacroix-Desmazes and Dr. Christoph Königs respectively. Injections of the FVIII to relevant FVIII-KO mouse models will validate the induction of tolerance by (1) analysing the ADA levels in plasma, (2) studying the restoration of the coagulation cascade, (3) T and B cell phenotyping and functional assays.
Enrolment in Doctoral degree(s): VU University, Amsterdam (PX), The Netherlands
More info will be available soon.
Host Institution: Topas Therapeutics GmbH, Hamburg, Germany
Supervisors: Dr. Sabine Fleischer
Introduction: Liver sinusoidal endothelial cells (LSEC) have the potential to regulate CD4+ and CD8+ T cell responses. LSECs can be targeted by intravenous injection of specialized peptide-coupled nanoparticles. After uptake by LSECs, the peptides are released from the nanoparticles and presented by MHCII/MHCI, resulting in peptide-speciﬁc tolerization of CD4+ and CD8+ T cells. We hypothesize that immunogenic FVIII peptides coupled with Topas-developed proprietary tolerizing particles (TP) will foster antigen-specific tolerance to therapeutic FVIII. My work suggests innovative approaches to induce preventive or therapeutic tolerance to therapeutic FVIII by harnessing the tolerogenic potency of the liver.
Aims: A1: To generate and characterize several types of FVIII Topas Particles Conjugates (TPCs) by chemically coupling immunogenic FVIII peptides to nanoparticles (Topas Particles (TPs)), followed by physical and chemical characterization. A2: To perform in vitro tests to analyze the presentation of the released peptide(s) after TPC uptake by APCs and the subsequent stimulation of T cells. A3: To test the tolerogenic eﬀect of FVIII-TPCs in pre-clinical models of severe HA, using either FVIII-KO mice or HLA-humanized transgenic mice immunized with human FVIII and treated with respective FVIII-TPCs.
Expected Results: R1: Implementation of the innovative TPC technology for tolerance induction towards therapeutic FVIII by developing functional Topas Particles (TPs) coupled to murine and human FVIII-peptides (TPCs). R2: Establishment and validation of both in-vitro and in-vivo assay to measure tolerogenic effect of FVIII-TPCs in several models. R3: Preclinical proof of concept for tackling anti-FVIII immune responses by Topas approach for prospective translation into clinic.
Planned secondment: An international, intersectoral 8-10 months secondment to INSERM is planned to conduct preclinical animal experiments to testify tolerogenic effect of FVIII-TPCs.
Enrolment in Doctoral degree(s): Universität Hamburg, Hamburg, Germany
Host Institution: CEA, Département Medicaments et Technologies pour la Santé (DMTS)
Primary Supervisor: MAILLERE Bernard
Introduction: T cells are a key component of the regulation of the immune response raised against factor VIII (FVIII) but the peptide specificity of FVIII-specific T cells remains unknown. T cell epitopes might vary from one donor to another owing to HLA polymorphism but also the severity of the disease. FVIII is a complete foreign molecule for severe hemophilia A patients, while it is a fully self-molecule for healthy donors. It has been recently revealed that a large T cell repertoire specific for FVIII exists in healthy donors demonstrating that many FVIII-specific T cells escape from central tolerance (Meunier et al, Blood Adv., 2017). The question arises to decipher and compare the fine specificity of the T cell response to FVIII in healthy donors and Hemophilia A patients in the perspective to anticipate the onset of FVIII inhibitors.
Aims: A1: Analyze the specificity and the immunogenicity level of biotherapeutic FVII in the T cell population of healthy individuals;
Expected Results: R1: identify the most frequent FVIII T-cell epitopes among the identified sets of naturally processed HLA-DR-restricted peptides, using in-silico prediction software to identify the eligible peptides and estimate the pro-inflammatory response using an ELISPOT assay. R2: evaluate the diversity of the specific T-cell response using cell sorting and next-generation sequencing (NGS) of the CDR3 ß TCR of the FVIII-specific T cells. R3: The minimal frequency of the FVIII-specific clones leading to in vitro anti-FVIII T-cell responses will be determined by CDR3 ß TcR NGS of whole blood CD4+ T-cells. R4: In collaboration with Immudex, generate a revolutionary family of DNA barcoded DR Dextramer reagents with the identified immuno-dominant peptides. The FVIII-specific Dextramer reagents will allow identification of FVIII-specific CD4+ TCR specificity at the single-cell level.
Planned secondment: an international 5-months secondment to the company Immudex, Copenhagen (DK), is planned to support the development of specific FVIII-specific Dextramer reagents.
Enrolment in Doctoral degree(s): Université Paris-Saclay