Today it is generally accepted that B cells require cognate interactions with CD4 IntroductionThe most serious complication in replacement therapy with FVIII products is the development of neutralizing antibodies against FVIII (FVIII inhibitors), which is observed in approximately 25% to 30% of patients with severe hemophilia A. 1 Although several genetic 2 and nongenetic 3 factors that contribute to the risk for patients to develop these antibodies have been identified, why some patients develop antibodies while others do not remains largely unknown.Today it is generally accepted that B cells require cognate interactions with CD4 ϩ T cells to develop high-affinity antibodies against protein antigens. 4,5 In line with this perception, several lines of evidence have supported the involvement of CD4 ϩ T cells in the generation of antibody responses against FVIII in patients with hemophilia A and in murine hemophilia models. 6,7 CD4 ϩ T cells express T-cell receptors that recognize antigen-derived peptides (CD4 ϩ T-cell epitopes) presented by MHC class II molecules, which are expressed on specialized antigen-presenting cells. 8 Structural features of both the MHC class II molecule and the peptide determine the specificity of CD4 ϩ T cells that can bind to the MHC class II-peptide complex. 8,9 The conditions under which CD4 ϩ T cells interact with this complex determine whether the immune system reacts with nonresponsiveness, is activated to develop specific antibodies, or is tolerized to suppress antibody responses. 9,10 Therefore, it is crucial to understand which FVIII peptides are presented by MHC class II complexes under conditions of FVIII replacement therapy and how CD4 ϩ T cells interact with MHC class II-FVIII peptide complexes expressed by antigenpresenting cells. The available information on FVIII peptides presented in the context of specific human MHC class II molecules is limited. Several studies used peripheral blood cells of patients and healthy controls 11 to identify CD4 ϩ T-cell epitopes in the A2 domain, 12 A3 domain, 13 and C2 domain of FVIII. 14 However, these studies lack information on the specific MHC class II molecules associated with the FVIII peptides identified. Jacquemin et al identified T-cell epitopes of FVIII using CD4 ϩ T-cell clones isolated from a mild hemophilia A patient carrying an Arg2150His mutation in the C1 domain of FVIII. 15 All clones recognized FVIII peptides encompassing residue Arg2150. Peptides were presented by HLA-DRB1*0401/HLA-DRB4*01 or HLA-DRDRB1*1501/ HLA-DRB5*0101. One of the peptides identified was a promiscuous epitope that bound to several different HLA-DR proteins. James et al used MHC class II tetramers to analyze FVIII-specific CD4 ϩ T cells obtained from a mild hemophilia A patient carrying an Ala2201Pro mutation in the C2 domain of FVIII. 16 Responses of CD4 ϩ T cells to sequences containing Ala2201 (wild-type), Pro2201 (hemophilic), and other predicted T-cell epitopes were evaluated and resulted in the identification of an HLA-DRB1*0101 restricted T-ce...
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...
Summary. MHC class II molecules are essential for shaping the CD4+ T-cell repertoire in the thymus and for selecting antigenic peptides that are presented to CD4+ T cells in the periphery. A range of different mouse models humanized for HLA class II antigens have been developed to study the regulation of MHC-class II restricted immune responses. These mouse models have been used to identify immunodominant peptides that trigger diseases and to characterize the interactions of T-cell receptors with disease-associated peptides and MHC class II molecules. Peptides presented to CD4+ T cells in these mouse models were shown to be similar to peptides presented to CD4+ T cells in patients who carry the same MHC class II haplotype. Opportunities and limitations associated with these mouse models will be discussed and the potential application of these models for understanding the regulation of antibody responses against factor VIII in hemophilia A will be indicated.
The new mouse model is suitable to study the influence of the innate immune system on maintenance and break of immune tolerance against FVIIa and could be used to assess the immunogenicity of new FVIIa products during pre-clinical development.
Therapy of hemophilia A has greatly benefited from the development of safe recombinant and plasmatic factor VIII (FVIII) concentrates. Current efforts to improve products focus on the extension of half-life by chemical and/or molecular modifications of FVIII. However, any modification of the FVIII protein poses the risk of creating neo-antigens that might cause FVIII inhibitors to be induced in patients. Therefore it is important to monitor the potential creation of neo-antigens during preclinical and clinical phases of drug development. Currently available animal models for hemophilia A develop high titers of anti-FVIII antibodies when treated with human FVIII. Using these models, it is difficult to differentiate between immune responses against native human FVIII and immune responses against human FVIII that carries neo-antigens. Considering these limitations, our aim is to develop a new model for hemophilia A that does not respond with antibodies to native human FVIII but develops antibodies against human FVIII that carries neo-antigens. We created a series of hemophilic mouse lines that carry a transgene for human FVIII that was placed under the control of an albumin promoter to direct liver-specific expression. Transgenic founder mice were generated by direct microinjection of the vector into the male pronucleus of fertilized oocytes obtained from mated female C57BL/6J mice after superovulation. Transgenic mice were crossed with hemophilic mice and bred to homozygousity for the expression of the human FVIII transgene. We analyzed the expression of human FVIII by real time PCR in lung, kidney, liver, heart, muscle, spleen, lymph nodes and reproductive organs. Gene expression analysis of bone marrow and thymus are currently ongoing. We selected three sublines (E, G and I) that show different levels of liver-specific expression of human FVIII for further analysis. We did not detect any FVIII antigen in the circulation in any of these three sublines when we used two different ELISA systems with detection limits around 1 ng/ml. We treated mice of sublines E, G and I intravenously with eight weekly doses of 200 ng of human FVIII (Advate) and analyzed the potential development of antibodies against native human FVIII. Our results indicate that transgenic mice of sublines E and I are immunologically tolerant to native human FVIII. They do not develop anti-FVIII antibodies (about 90% of all mice tested) or develop low titers (below 1:80 in 10% of mice tested) only. In contrast, mice of subline G develop high titers of anti-FVIII antibodies indicating that they are not immunologically tolerant to human FVIII. Preliminary data suggest that the degree of immunological tolerance against human FVIII correlates to a certain extent with the expression levels of the human FVIII transgen in liver and/or thymus. We are in the process of verifying these preliminary data. Furthermore, we have started to analyze FVIII-specific T-cell responses to define potential differences in the repertoire of FVIIIspecific T cells between the three sublines. We conclude that transgenic expression of human FVIII under the control of an albumin promoter is able to induce immune tolerance to human native FVIII in hemophilic mice. However, a certain threshold level of gene expression might be required for the induction of immune tolerance.
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