Long-term Persistence of Anti-factor VIII Antibody-secreting Cells in Hemophilic Mice after Treatment with Human Factor VIII

Hausl, Christina; Maier, Elisabeth; Schwarz, Hans; Ahmad, Rafi; Turecek, Peter L.; Dorner, Friedrich; Reipert, Birgit M.

doi:10.1055/s-0037-1613094

Cited by 46 publications

(57 citation statements)

References 20 publications

(42 reference statements)

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“…32 ELISA plates were coated with 1 g/mL of one of the following proteins: recombinant human FVIII, PEGylated human FVIII (mFVIII1 or mFVIII2), or recombinant human VWF.…”

Section: Analysis Of Antibodies and Antibody-secreting Cellsmentioning

confidence: 99%

Maintenance and break of immune tolerance against human factor VIII in a new transgenic hemophilic mouse model

Helden

Unterthurner

Hermann

et al. 2011

Blood

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Replacement of the missing factor VIII (FVIII) is the current standard of care for patients with hemophilia A. However, the short half-life of FVIII makes frequent treatment necessary. Current efforts focus on the development of longer-acting FVIII concentrates by introducing chemical and genetic modifications to the protein. Any modification of the FVIII protein, however, risks increasing its immunogenic potential to induce neutralizing antibodies (FVIII inhibitors), and this is one of the major complications in current therapy. It would be highly desirable to identify candidates with a high risk for increased immunogenicity before entering clinical development to minimize the risk of exposing patients to such altered FVIII proteins. In the present study, we describe a transgenic mouse line that expresses a human F8 cDNA. This mouse is immunologically tolerant to therapeutic doses of native human FVIII but is able to mount an antibody response when challenged with a modified FVIII protein that possesses altered immunogenic properties. In this situation, immunologic tolerance breaks down and antibodies develop that recognize both the modified and the native human FVIII. The applicability of this new model for preclinical immunogenicity assessment of new FVIII molecules and its potential use for basic research are discussed. (Blood. 2011; 118(13):3698-3707) IntroductionHemophilia A is an X-linked bleeding disorder that is caused by reduced function or lack of clotting factor VIII (FVIII). 1 Replacement of the missing protein is the current standard of care for patients. However, the short half-life of FVIII, ϳ 7-17 hours, 2 makes frequent treatment necessary. Current efforts focus on the development of longer-acting FVIII concentrates, which should decrease the required treatment frequency and therefore improve the quality of life for patients. Recently described approaches in the development of longer-acting concentrates are based on strategies that have been successfully applied to other therapeutic proteins: chemical modifications such as the addition of polyethylene glycol (PEG) polymers, polysialic acids, or hydroxyethyl starch 3,4 ; alternative formulations with PEG-modified liposomes 3 ; and fusion to the Fc part of human IgG. 5 In addition, molecular modifications of the FVIII protein aimed at increasing the duration of its cofactor activity or reducing its clearance in vivo have been reported. 3 Any chemical or molecular modification of the FVIII protein can potentially increase its immunogenic potential. Modifications could generate neo-epitopes for both B and T cells or may induce altered structures that could bind and trigger receptors expressed on cells of the innate immune system, thereby amplifying potential anti-FVIII antibody responses. 6 Finally, modifications could generate repetitive epitopes for B cells that might cause the activation and differentiation of B cells and the subsequent production of antibodies without the requirement for T-cell help. 7,8 Therefore, the potential impact that any...

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“…32 ELISA plates were coated with 1 g/mL of one of the following proteins: recombinant human FVIII, PEGylated human FVIII (mFVIII1 or mFVIII2), or recombinant human VWF.…”

Section: Analysis Of Antibodies and Antibody-secreting Cellsmentioning

confidence: 99%

Maintenance and break of immune tolerance against human factor VIII in a new transgenic hemophilic mouse model

Helden

Unterthurner

Hermann

et al. 2011

Blood

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“…The latter could lead to lymphocyte activation in the spleen as described for intravenous protein infusion. 21 We wanted to investigate whether systemic or local hF.IX antigen is the substrate for induction of an anti-hF.IX response. In order to study the activation of hF.IX-specific B cells in lymphoid organs, we adapted an ELISPOT technique to quantitate hF.IX-specific antibody secreting cells (ASCs) isolated from draining LN of the injected muscle (inguinal and popliteal LN) or from the spleen.…”

Section: Elispot Assays For Antibody and Cytokine Secreting Cells Shomentioning

confidence: 99%

Major role of local immune responses in antibody formation to factor IX in AAV gene transfer

et al. 2005

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“…After 3 or 6 days of culture, newly formed ASCs were detected by enzyme-linked immunospot (ELISPOT) assays as described. 16,20 To test for the effect of high concentrations of FVIII on T-cell function, CD138 Ϫ spleen cells were cultured for 3 days as indicated, washed twice with culture medium, and subsequently used for the isolation of T cells by negative selection as described in "Spleen-cell preparation." T cells were then mixed in a ratio of 1:2 with freshly isolated CD138 Ϫ spleen cells depleted of T cells, which were obtained from the spleens of hemophilic mice treated with 4 doses of FVIII.…”

mentioning

confidence: 99%

“…Blood samples were taken from each mouse by tail snipping at 7 and 14 days after treatment with FVIII for measurement of anti-FVIII antibodies by enzyme-linked immunosorbent assay (ELISA) as described. 16,20 …”

mentioning

confidence: 99%

High-dose factor VIII inhibits factor VIII–specific memory B cells in hemophilia A with factor VIII inhibitors

Hausl

Ahmad

Sasgary

et al. 2005

Blood

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108

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IntroductionThe development of neutralizing anti-factor VIII (FVIII) antibodies is the major complication in the treatment of patients with hemophilia A with FVIII products. 1,2 Long-term application of high doses of FVIII has evolved as an effective therapy to eradicate the antibodies and to induce long-lasting immune tolerance. [3][4][5][6] Despite clinical experience with the therapy, little is known about the immunologic mechanisms that cause the down-modulation of FVIII-specific immune responses and the induction of long-lasting immune tolerance against FVIII. We asked the question whether the restimulation of FVIII-specific memory B cells is affected by high concentrations of FVIII in vitro or high doses of FVIII in vivo. Memory B cells play an essential role in the maintenance of established antibody responses. On re-exposure to the same antigen, they are rapidly restimulated to proliferate and differentiate into antibody-secreting plasma cells (ASCs) that secrete high-affinity antibodies. 7,8 Furthermore, memory B cells have the potential to act as very efficient antigen-presenting cells and stimulators of CD4 ϩ T cells because of the expression of highaffinity antigen receptors, major histocompatibility complex (MHC) class II and costimulatory molecules. 9 It is, therefore, reasonable to believe that memory B cells have to be eradicated or functionally inactivated during a successful immune tolerance induction therapy with FVIII inhibitors in patients with hemophilia A.We used a murine model of hemophilia A that is characterized by complete deficiency of functionally active FVIII because of a targeted disruption of exon 17 of the F8 gene. 10,11 Intravenous injection of human FVIII into these mice results in high titers of anti-FVIII antibodies that have similar characteristics to those of FVIII inhibitors in patients. [12][13][14][15] Using this model, we demonstrated previously that the differentiation of FVIII-specific memory B cells into ASCs depends on the presence of activated T cells and requires CD40-CD40 ligand and CD80/CD86-CD28 costimulatory interactions. 16 Here, we show that concentrations of FVIII below the physiologic plasma concentration of 0.1 g/mL (1 U/mL) restimulate FVIII-specific memory B cells and induce their differentiation into ASCs. Concentrations above 0.1 g/mL (1 U/mL), however, inhibit memory B-cell restimulation and prevent the formation of ASCs. This inhibition is irreversible and involves the activation of caspases. Materials and methods Hemophilic E-17 miceOur colony of fully inbred hemophilic E-17 mice (characterized by a targeted disruption of exon 17 of the F8 gene) was established with a breeding pair from the original colony 10,11 and crossed into the C57BL/6J background as described. 17 All mice were male and aged 8 to 10 weeks at the beginning of the experiments. All studies were carried out in accordance with Austrian federal law (Act BG 501/1989) regulating animal experimentation and approved by the local authority in Vienna, Austria. Immunization of mice with FV...

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Long-term Persistence of Anti-factor VIII Antibody-secreting Cells in Hemophilic Mice after Treatment with Human Factor VIII

Cited by 46 publications

References 20 publications

Maintenance and break of immune tolerance against human factor VIII in a new transgenic hemophilic mouse model

Maintenance and break of immune tolerance against human factor VIII in a new transgenic hemophilic mouse model

Major role of local immune responses in antibody formation to factor IX in AAV gene transfer

High-dose factor VIII inhibits factor VIII–specific memory B cells in hemophilia A with factor VIII inhibitors

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