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1.
Haematologica ; 108(5): 1322-1334, 2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-36655430

RESUMO

Neutralizing anti-factor VIII (FVIII) antibodies, known as FVIII inhibitors, represent a major drawback of replacement therapy in persons with congenital hemophilia A (PwHA), rendering further infusions of FVIII ineffective. FVIII inhibitors can also appear in non-hemophilic individuals causing acquired hemophilia A (AHA). The use of non-FVIII bypassing agents in cases of bleeds or surgery in inhibitor-positive patients is complicated by the lack of reliable biological monitoring and increased thrombotic risk. Imlifidase (IdeS) is an endopeptidase that degrades human immunoglobulin G (IgG); it was recently approved for hyperimmune patients undergoing renal transplants. Here we investigated the ability of IdeS to eliminate FVIII inhibitors in vitro and in a model of inhibitor-positive HA mice. IdeS cleaved anti-FVIII plasma IgG from PwHA and AHA patients, and hydrolyzed recombinant human anti-FVIII IgG independently from their subclass or specificity for the A2, A3, C1 or C2 domains of FVIII. In HA mice passively immunized with recombinant human anti-FVIII IgG, IdeS restored the hemostatic efficacy of FVIII, as evidenced by the correction of the bleeding tendency. Our results provide the proof of concept for the transient removal of FVIII inhibitors by IdeS, thereby opening a therapeutic window for efficient FVIII replacement therapy in inhibitor-positive patients.


Assuntos
Hemofilia A , Hemostáticos , Humanos , Camundongos , Animais , Hemofilia A/tratamento farmacológico , Hemorragia , Imunoglobulina G , Imunossupressores/uso terapêutico
2.
J Thromb Haemost ; 22(5): 1489-1495, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38325597

RESUMO

BACKGROUND: The recruitment of activated factor VIII (FVIII) at the surface of activated platelets is a key step toward the burst of thrombin and fibrin generation during thrombus formation at the site of vascular injury. It involves binding to phosphatidylserine and, possibly, to fibrin-bound αIIbß3. Seminal work had shown the binding of FVIII to resting platelets, yet without a clear understanding of a putative physiological relevance. OBJECTIVES: To characterize the effects of FVIII-platelet interaction and its potential modulation of platelet function. METHODS: FVIII was incubated with washed platelets. The effects on platelet activation (spontaneously or triggered by collagen and thrombin) were studied by flow cytometry and light transmission aggregometry. We explored the involvement of downstream pathways by studying phosphorylation profiles (Western blot). The FVIII-glycoprotein (GP) VI interaction was investigated by ELISA, confocal microscopy, and proximity ligation assay. RESULTS: FVIII bound to the surface of resting and activated platelets in a dose-dependent manner. FVIII at supraphysiological concentrations did not induce platelet activation but rather specifically inhibited collagen-induced platelet aggregation and altered glycoprotein VI (GPVI)-dependent phosphorylation. FVIII, freed of its chaperone protein von Willebrand factor (VWF), interacted in close proximity with GPVI at the platelet surface. CONCLUSION: We showed that VWF-free FVIII binding to, or close to, GPVI modulates platelet activation in vitro. This may represent an uncharacterized negative feedback loop to control overt platelet activation. Whether locally activated FVIII concentrations achieved during platelet accumulation and thrombus formation at the site of vascular injury in vivo are compatible with such a function remains to be determined.


Assuntos
Plaquetas , Fator VIII , Ativação Plaquetária , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas , Humanos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Plaquetas/metabolismo , Fosforilação , Fator VIII/metabolismo , Colágeno/metabolismo , Ligação Proteica , Citometria de Fluxo , Trombina/metabolismo , Relação Dose-Resposta a Droga , Microscopia Confocal
3.
J Thromb Haemost ; 21(10): 2776-2783, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37473843

RESUMO

BACKGROUND: Emicizumab is a bispecific, chimeric, humanized immunoglobulin G (IgG)4 that mimics the procoagulant activity of factor (F) VIII (FVIII). Its long half-life and subcutaneous route of administration have been life-changing in treating patients with hemophilia A (HA) with or without FVIII inhibitors. However, emicizumab only partially mimics FVIII activity; it prevents but does not treat acute bleeds. Emergency management is particularly complicated in patients with FVIII inhibitors receiving emicizumab prophylaxis in whom exogenous FVIII is inefficient. We have shown recently that Imlifidase (IdeS), a bacterial IgG-degrading enzyme, efficiently eliminates human anti-FVIII IgG in a mouse model of severe HA with inhibitors and opens a therapeutic window for the administration of exogenous FVIII. OBJECTIVES: To investigate the impact of IdeS treatment in inhibitor-positive HA mice injected with emicizumab. METHODS: IdeS was injected to HA mice reconstituted with human neutralizing anti-FVIII IgG and treated with emicizumab. RESULTS: IdeS hydrolyzed emicizumab in vitro and in vivo, albeit, at slower rates than another recombinant human monoclonal IgG4. While F(ab')2 fragments were rapidly cleared from the circulation, thus leading to a rapid loss of emicizumab procoagulant activity, low amounts of single-cleaved intermediate IgG persisted for several days. Moreover, the IdeS-mediated elimination of the neutralizing anti-FVIII IgG and restoration of the hemostatic efficacy of exogenous FVIII were not impaired by the presence of emicizumab and polyclonal human IgG in inhibitor-positive HA mice. CONCLUSION: Our results suggest that IdeS could be administered to inhibitor-positive patients with HA receiving emicizumab prophylaxis to improve and ease the management of breakthrough bleeds or programmed major surgeries.


Assuntos
Anticorpos Biespecíficos , Hemofilia A , Humanos , Animais , Camundongos , Hemofilia A/tratamento farmacológico , Fator VIII/uso terapêutico , Anticorpos Biespecíficos/uso terapêutico , Hemorragia/tratamento farmacológico , Imunossupressores/uso terapêutico , Imunoglobulina G
4.
J Thromb Haemost ; 21(9): 2405-2417, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37271431

RESUMO

BACKGROUND: Transplacental delivery of maternal immunoglobulin G (IgG) provides humoral protection during the first months of life until the newborn's immune system reaches maturity. The maternofetal interface has been exploited therapeutically to replace missing enzymes in the fetus, as shown in experimental mucopolysaccharidoses, or to shape adaptive immune repertoires during fetal development and induce tolerance to self-antigens or immunogenic therapeutic molecules. OBJECTIVES: To investigate whether proteins that are administered to pregnant mice or endogenously present in their circulation may be delivered through the placenta. METHODS: We engineered monovalent immunoglobulin G (FabFc) specific for different domains of human factor VIII (FVIII), a therapeutically relevant model antigen. FabFc was injected with exogenous FVIII into pregnant severe hemophilia A mice or pregnant mice expressing human FVIII following AAV8-mediated gene therapy. FabFc and FVIII were detected in the pregnant mice and/or fetuses by enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS: Administration of FabFc to pregnant mice allowed the maternofetal delivery of FVIII in a FcRn-dependent manner. FVIII antigen levels achieved in the fetuses represented 10% of normal plasma levels in the human. We identified antigen/FabFc complex stability, antigen size, and shielding of promiscuous protein patches as key parameters to foster optimal antigen delivery. CONCLUSION: Our results pave the way toward the development of novel strategies for the in utero delivery of endogenous maternal proteins to replace genetically deficient fetal proteins or to educate the immune system and favor active immune tolerance upon protein encounter later in life.


Assuntos
Hemofilia A , Imunoglobulina G , Gravidez , Feminino , Camundongos , Humanos , Animais , Fator VIII , Hemofilia A/genética , Hemofilia A/terapia , Placenta , Terapia Genética , Tolerância Imunológica
5.
Commun Biol ; 3(1): 96, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132640

RESUMO

Therapeutic normal IgG intravenous immunoglobulin (IVIG) is a well-established first-line immunotherapy for many autoimmune and inflammatory diseases. Though several mechanisms have been proposed for the anti-inflammatory actions of IVIG, associated signaling pathways are not well studied. As ß-catenin, the central component of the canonical Wnt pathway, plays an important role in imparting tolerogenic properties to dendritic cells (DCs) and in reducing inflammation, we explored whether IVIG induces the ß-catenin pathway to exert anti-inflammatory effects. We show that IVIG in an IgG-sialylation independent manner activates ß-catenin in human DCs along with upregulation of Wnt5a secretion. Mechanistically, ß-catenin activation by IVIG requires intact IgG and LRP5/6 co-receptors, but FcγRIIA and Syk are not implicated. Despite induction of ß-catenin, this pathway is dispensable for anti-inflammatory actions of IVIG in vitro and for mediating the protection against experimental autoimmune encephalomyelitis in vivo in mice, and reciprocal regulation of effector Th17/Th1 and regulatory T cells.


Assuntos
Células Dendríticas/efeitos dos fármacos , Imunoglobulinas Intravenosas/farmacologia , beta Catenina/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/efeitos dos fármacos
6.
Med Sci (Paris) ; 36(4): 341-347, 2020 Apr.
Artigo em Francês | MEDLINE | ID: mdl-32356710

RESUMO

The use of therapeutic proteins induces in some patients the appearance of neutralizing antibodies. This is the case of pro-coagulant factor VIII (FVIII) used in patients with hemophilia A. Several parameters related to the protein itself, to the type of pathology or to the patients, condition the immunogenicity of a therapeutic protein. Understanding these parameters would help to anticipate or prevent the development of neutralizing antibodies. In the case of FVIII, we propose that the development of neutralizing antibodies does not result from an unpredicted immune response but rather from the inability of the patient's organism to develop an anti-inflammatory or regulatory response.


TITLE: Origine et nature de la réponse immunitaire neutralisante contre le facteur VIII thérapeutique. ABSTRACT: L'utilisation de protéines thérapeutiques se heurte, chez certains patients, à l'apparition d'anticorps neutralisants. C'est le cas, par exemple, du facteur VIII pro-coagulant qui est utilisé pour traiter les patients atteints d'hémophilie A. Plusieurs paramètres, liés à la protéine elle-même, au type de pathologie ou aux patients, conditionnent l'immunogénicité d'une protéine thérapeutique. Les comprendre permettrait d'anticiper ou de prévenir la survenue d'anticorps neutralisants. Nous proposons dans cette revue de montrer que, dans le cas du facteur VIII, la survenue de ces anticorps neutralisants ne résulte pas d'une réponse immunitaire inopinée, mais plutôt de l'incapacité de l'organisme des patients à développer une réponse anti-inflammatoire ou régulatrice.


Assuntos
Anticorpos Neutralizantes/metabolismo , Fator VIII/imunologia , Fator VIII/uso terapêutico , Hemofilia A/tratamento farmacológico , Hemofilia A/terapia , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos/fisiologia , Hemofilia A/imunologia , Humanos , Tolerância Imunológica/fisiologia
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