CE17 - Recherche translationnelle en santé

Induction of neonatal Fc receptor-mediated tolerance to autologous therapeutic proteins – Exfiltrins

Induction of neonatal Fc receptor-mediated tolerance to autologous therapeutic proteins

The apparition of neutralizing antibodies to therapeutic factor VIII (FVIII) is the major complication of bleed management in haemophilia A patients. The aim of this project is to introduce proteins during foetal life in order to confer specific immune tolerance. To do so, we aim to induce specific FVIII tolerance in the haemophilic foetus by the exposition to maternal FVIII that will be ferried though the placenta by neonatal Fc.

Hemophilia treatment by injection of FVIII leads to anti-FVIII inhibitory antibodies in 30% of patients. This is a major medical and societal challenge.

Several pathological situations result from the inability of the organism to mount immune tolerance towards exogenously administered molecules, as observed in alloimmune responses to protein therapeutics. This is particularly true of hemophilia A, a rare X-linked recessive life-threatening, crippling hemorrhagic disorder consecutive to the absence of endogenous pro-coagulant factor VIII (FVIII). Treatment of bleeding episodes by administration of exogenous FVIII results in up to 30% of cases, in the emergence of anti-FVIII IgG, that inhibit the pro-coagulant activity of therapeutic FVIII. FVIII inhibitors represent a major medical hurdle and a critical societal concern since the treatment of inhibitor-positive patients exceeds 0.3 million euros/patient/year in developed countries. <br />We have recently validated an innovative strategy wherein the antigen of interest is introduced during the fetal life in the circulation of an individual, in order to shape its immune repertoire and to let the immune system develop specific tolerance throughout its ontogeny. <br />In this project, we will develop innovative recombinant FVIII-binding molecules, referred to as «exfiltrins«: upon administration to pregnant mothers, the FVIII-binding exfiltrins, will capture some of the mother's endogenous FVIII and will deliver it to the fetuses through the placenta, where immune tolerance to the entire FVIII molecule will be induced. <br />The therapeutic potential of the exfiltrins will be validated in a novel model of transgenic mice expressing a human FVIII molecule, developed for this project. <br />Positive results in our study would have dramatic repercussions for the quality of life of the patients and, in the long range, reduce societal costs. At the same time, this selective antigen-specific approach would avoid generalized immunosuppression, which frequently carries unacceptable risk/benefit ratios.

Exfiltrins are molecules with the capacity to interact both with FVIII and with the FcRn receptor.
Our working plan is as follows:
Cloning and production of 9 different exfiltrins using phage display or recombinant technologies
Generation of a model of mice deficient for mouse FVIII and expressing human FVIII (outsourced)
Validation of the exfiltrin-mediated transplacental delivery of maternal FVIII using:
1) Biochemical assays to measure binding of exfiltrins to FVIII and to FcRn
2) Cellular assays to measure the capacity of the exfiltrins to mediate FVIII transcytosis
3) In vivo assays to determine exfiltrins half-life and test their capacity to mediate transplacental FVIII transfer. For this part, we will use imaging techniques
4) Induction of immune tolerance to FVIII by transplacental delivery of maternal FVIII in FVIII-transgenic pregnant mice

So far, we have generated 3 exfiltrines based on anti-FVIII antibodies or the D’D3 region of von Willebrand factor. These three candidates have been fully characterized in terms of binding to FVIII, binding to FcRn receptor and capacity to compete for the FVIII-von Willebrand factor interaction.
Concerning the single domain (VHH) based exfiltrins, we have already generated 14 VHH by phage display. One specific candidate (KB-FVIII-05) displays a number of characteristics compatible with a potential exfiltrin: it can compete with FVIII-von Willebrand factor binding and the interaction between this VHH and FVIII remains relatively stable at an acidic pH. The coupling of the VHH to a FcRn moiety has now been achieved and its characterization is ongoing.
We also have been able to show transplacental delivery of exfiltrins and for some of them, their ability to deliver FVIII from the maternal side to the foetal side.
Finally, the transgenic mice expressing human FVIII under the control of the murine endogenous FVIII promoter have been engineered and are currently being characterized.

From a scientific point of view, the project is proceeding as originally planned. The humanized mice have been delivered to our consortium. A number of exfiltrins have been generated. From a timing point of view, the covid-19 pandemic has obviously led to a serious delay in our work. Between the lockdown period and the unavoidable inertia, we estimate a 6-month delay in our original calendar.
Our first priority is to finalize the construction and characterization of the different exfiltrins.
The most important characteristics for an ideal exfiltrin are as follows:
1) Binding to FVIII and to FcRn and stability of these interactions at acidic pH
2) Capacity to inhibit the FVIII-von Willebrand factor interaction
3) No inhibition of FVIII procoagulant activity
The exfiltrins with the best properties will be selected for the follow-up of the project which involves mostly in vivo work in order to:
1) Determine exfiltrins half-life and test their capacity to mediate transplacental FVIII transfer. For this part, we will use imaging techniques
2) Induce immune tolerance to FVIII by exfiltrin-mediated transplacental delivery of maternal FVIII in FVIII-transgenic pregnant mice

Review article in a peer-reviewed international journal
Mimoun A, Delignat S, Peyron I, Daventure V, Lecerf M, Dimitrov JD, Kaveri SV, Bayry J and Lacroix-Desmazes S (2020) Relevance of the Materno-Fetal Interface for the Induction of Antigen-Specific Immune Tolerance. Front. Immunol. 11:810. doi: 10.3389/fimmu.2020.00810

Invited Conference
Sébastien Lacroix-Desmazes. Transplacental Immune Tolerance. NHF's 15th Workshop on Novel Technologies and Gene Transfer for Hemophilia, Washington, USA (12 septembre 2019)

Oral presentations
Angelina Mimoun, Victoria Daventure, Sandrine Delignat, Melissa Bou Jaoudeh, Olivier Christophe, Peter Lenting, Cécile Denis, Sébastien lacroix-Desmazes. Towards the transplacental delivery of maternal FVIII to FVIII-deficient progeny for induction of active immune tolerance to therapeutic FVIII. 10th BIC International Conference/3rd international conference on inhibitors in hemophilia. Genoa, Italy (6-8 septembre 2019)

Angelina Mimoun, Victoria Daventure, Sandrine Delignat, Melissa Bou Jaoudeh, Olivier Christophe, Peter Lenting, Cécile Denis, Sébastien Lacroix-Desmazes. Towards the transplacental delivery of maternal FVIII to FVIII-deficient progeny for induction of active immune tolerance to therapeutic FVIII. 2e`mes Journe´es Club Anticorps SFI. Paris, France (8 octobre 2019)

Posters
Angelina Mimoun, Melissa Bou Jaoudeh, Ivan Peyron, Victoria Daventure, Sandrine Delignat, Olivier Christophe, Peter Lenting, Cécile Denis, Sébastien Lacroix-Desmazes. Transplacental delivery of maternal factor VIII for induction of immune tolerance to therapeutic factor VIII. ISTH 2020 Virtual congress (12-14 juillet 2020)

Reports
Angelina Mimoun. Induction de tolérance materno-fœtale envers le FVIII thérapeutique. Rapport de mi-thèse – 1ère année. ED394, Sorbonne-Université.

Several pathological situations result from the inability of the organism to mount immune tolerance towards exogenously administered molecules, as observed in alloimmune responses to protein therapeutics. This is particularly true of hemophilia A, a rare X-linked recessive life-threatening, crippling hemorrhagic disorder consecutive to the absence of endogenous pro-coagulant factor VIII (FVIII). Treatment of bleeding episodes by administration of exogenous FVIII results in up to 30% of cases, in the emergence of anti-FVIII IgG, that inhibit the pro-coagulant activity of therapeutic FVIII. FVIII inhibitors represent a major medical hurdle and a critical societal concern since the treatment of inhibitor-positive patients exceeds 0.3 million euros/patient/year in developed countries. To date, no efficient strategy exists to induce tolerance to therapeutic FVIII in patients with hemophilia A and to prevent the onset of FVIII inhibitors.
We have recently validated an innovative strategy wherein the antigen of interest is introduced during the fetal life in the circulation of an individual, in order to shape its immune repertoire and to let the immune system develop specific tolerance throughout its ontogeny. In this strategy, the target antigen is coupled to the Fc portion of an immunoglobulin G (IgG) and is injected into the blood of pregnant mothers. The antigen-Fc chimeric protein is then targeted to the offspring upon binding to the neonatal Fc receptor (FcRn) expressed on the syncytiotrophoblast and is transferred into the fetuses' circulation. Indeed, the treatment of pregnant FVIII-deficient mice with FVIII A2 and C2 domains fused to the Fc fragment of IgG allowed the transplacental delivery of the Fc-fused FVIII domains during fetal development. The transplacentally delivered A2-Fc and C2-Fc induced long-lived central and peripheral regulatory T cells in the fetuses, that conferred partial immune tolerance to therapeutic FVIII later in life. The use of the entire Fc-fused FVIII molecule in this approach should confer complete immune tolerance to FVIII. It is however hampered by the formation of large complexes formed between FVIII-Fc and endogenous von Willebrand factor in the mother's circulation, that prevent crossing of the placenta.
In this project, rather than producing expensive and labile FVIII-derived fusion proteins, we will develop innovative recombinant FVIII-binding molecules, referred to as "exfiltrins": upon administration to pregnant mothers, the FVIII-binding exfiltrins, will capture some of the mother's endogenous FVIII and will deliver it to the fetuses through the placenta, where immune tolerance to the entire FVIII molecule will be induced. Moreover, in order to avoid formation of large molecular complexes and ensure optimal placental transfer, the FVIII-binding exfiltrins will be monovalent and will dissociate maternal FVIII from endogenous von Willebrand factor. The therapeutic potential of the exfiltrins will be validated in a novel model of transgenic mice expressing a human FVIII molecule, developed for this project. If successful, our preventive approach could be translated into clinical trials. Interestingly, hemophilia in humans can be anticipated based on the familial history of the disease in 2/3 of the cases and can be diagnosed during pregnancy. In addition, the risks for a patient-to-be-born to develop FVIII inhibitors after initiation of replacement therapy can also be estimated before birth, according to the type of mutation in the F8 gene, on the history of an inhibitor among hemophiliac brothers or uncles, and on polymorphisms in the IL-10, TNF-a, CTLA-4 and HMOX-1 encoding genes. Positive results in our study would have dramatic repercussions for the quality of life of the patients and, in the long range, reduce societal costs. At the same time, this selective antigen-specific approach would avoid generalized immunosuppression, which frequently carries unacceptable risk/benefit ratios.

Project coordination

Cecile DENIS (Hémostase, Inflammation, Thrombose)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

HITh Hémostase, Inflammation, Thrombose
CRC CENTRE DE RECHERCHE DES CORDELIERS

Help of the ANR 453,600 euros
Beginning and duration of the scientific project: - 36 Months

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