Winterling, Karina (2018)
Immunogenic Determinants of Coagulation Factor VIII.
Technische Universität Darmstadt
Dissertation, Erstveröffentlichung

Kurzbeschreibung (Abstract)

Hemophilia A is a bleeding disorder, affecting about 400 000 people worldwide. In addition to prolonged bleeding times, patients suffer from spontaneous and internal bleedings, affecting their everyday life. Patients can be treated by the application of therapeutic FVIII, either derived from plasma or recombinantly produced. Whether the treatment is prophylactic or on-demand depends on the severity of the disease and on the living conditions of the patients. Although treatment allows most of the patients to live a life as normal as possible, patients can develop inhibitory antibodies against the therapeutic FVIII, rendering the molecule inactive. In most cases, this situation can be overcome by an immune-tolerance-induction therapy. However, this therapy is long, strenuous and associated with very high costs. Different recombinant FVIII products evolved within the last years, containing modifications, in order to prolong the in vivo half-life of FVIII. However, a FVIII product with a reduced immunogenicity was not yet published. A deimmunized FVIII could reduce the probability of inhibitor development, providing a safer therapy for the patients and lower the costs for therapy. In this thesis, a deimmunized FVIII molecule is generated. The work comprises the full development, starting with in silico calculations of the FVIII sequence, through the production and purification, leading to final analyses in vitro and in vivo. The deimmunization approach is based on the reduction of FVIII-peptide presentation on antigen-presenting immune cells. This is supposed to lead to a prevention of naïve T cell activation. Without FVIII-specific T helper cells, naïve B cells cannot be activated, leading to the absence of high-affinity inhibitors. The in silico analysis of FVIII was based on the identification of peptides which bind with a high affinity to the major histocompatibility complex class II, which is required for cell surface-presentation of peptides. Amino acid exchanges were proposed, in order to reduce this binding. These mutations were incorporated into the FVIII sequence. This was performed in three rounds, starting with the incorporation of single mutations, followed by the combination of several mutations, to the final combination of all mutations in one molecule. Only mutations still leading to functional FVIII were finally incorporated. Based on this, 19 mutations could be incorporated into the molecule. This led to a reduction of the immunogenicity score of FVIII from 7 to -11, indicating a reduced immunogenicity based on in silico calculations. However, a total deimmunization of all epitopes was not possible, as some regions of FVIII, especially those important for activity, folding and binding, could not be altered. Functional and structural analyses of this mutated FVIII variant revealed similarity to a control FVIII as well as to commercially available recombinant FVIII products. Additionally, an in vitro DC-T cell Assay was developed, in order to determine a reduced immunogenicity of the deimmunized FVIII variant, as the available mouse models were limited. The DCs were primed with different FVIII products and the proliferative response of regulatory T cell-depleted CD4+ T cells to these DCs was measured. The regulatory T cells were depleted, in order to be able to perform this assay with cells derived from healthy donors as well as Hemophilia A patients. Using this DC-T cell Assay, cells from healthy donors proved the significantly reduced immunogenicity of the mutated FVIII variant by revealing less proliferation of T helper cells to this variant than to the control FVIII. These results have further to be proven with cells derived from Hemophilia A patients. Unfortunately, the detection of a reduced immunogenicity in vivo was limited. Two mouse models were tested but the models had either a FVIII knockout or a human immune system. However, a combination of these two models would have been optimal but was not available. Finally, this thesis presents the first deimmunized FVIII, containing 19 amino acid mutations, which is still active and proves to be less immunogenic in an in vitro assay with cells from healthy donors. Upon confirmation of these results in vivo, this molecule can highly improve the therapy of Hemophilia A patients by reducing the risk of inhibitor development in previously treated as well as in previously untreated patients. This reduced inhibitor incidence would also reduce the costs of therapy, as inhibitor-negative patients would not require an expensive immune-tolerance-induction therapy.

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