The proposed study will elucidate mechanisms of immune responses to factor VIII (FVIII) in hemophilia A patients and explore FVIII sequence modifications that could be translated to make less immunogenic products for replacement therapy. A primary goal is to reduce the incidence and burden of neutralizing anti-factor VIII (FVIII) antibodies, referred to as inhibitors, which can lead to life-threatening bleeds that are extremely expensive and difficult to manage. This project seeks to better characterize the mechanisms of these pathological immune responses by mapping T-cell and B-cell epitopes on FVIII, and then applying this information to modify its amino acid sequence to render it less immunogenic (stimulatory to helper T cells) and antigenic (stimulatory to memory B cells and recognized by antibodies). The availability of less immunogenic/antigenic FVIII proteins would significantly reduce mortality, morbidity and costs of effective treatment. The novel methodologies under development in our laboratory are also broadly applicable to investigations of other pathological immune responses to therapeutic proteins, which are a concern that accompanies the development of any protein drug and which are generally not predicted accurately using animal models for preclinical testing. Several studies have indicated that African American patients have a higher incidence of inhibitors than Caucasians. Approximately half of our subject population is African American, ensuring that this patient subgroup is well represented in our studies. The related goals of this project are: 1. To map and then neutralize immunodominant T-cell epitopes in FVIII, generating FVIII proteins that are less likely to stimulate the immune system of patients with susceptible HLA types. T-cell responses to FVIII will be detected and characterized using ELISpot assays and MHC Class II tetramers loaded with synthetic FVIII peptides. The immunogenicity of FVIII peptides and proteins with modified amino acid sequences will then be evaluated utilizing primary and cloned human T cells from hemophilia A subjects. 2. To map and then neutralize B-cell epitopes, generating FVIII proteins capable of evading inhibitory antibodies. We are developing novel approaches to evaluate antibody- antigen interactions using surface plasmon resonance (SPR), in which plasma/serum samples are analyzed to determine antibody titers, apparent antibody affinities for immobilized FVIII, and B-cell epitopes. 3. To generate novel FVIII proteins with translational potential, in which T-cell and B-cell epitopes have been neutralized while retaining procoagulant activity comparable to that of wild-type FVIII. Immunogenicity of rationally designed, sequence-modified FVIII proteins will be tested using T-cell stimulation assays. Antigenicity of the proteins will be evaluated using Bethesda, ELISA and novel SPR assays.
The bleeding disorder hemophilia A, which affects 1 in 5,000 males worldwide, is caused by genetic mutations leading to a deficiency in the clotting protein factor VIII. The development of pathological anti-drug antibodies is the most serious complication of therapeutic factor VIIII infusions, affecting approximately one quarter of hemophilia A patients. The incidence of these immune responses is increasing as access to protein replacement therapy improves. The related goals of this project are to better understand the mechanisms of these immune responses and to develop novel versions of the factor VIII protein that are less likely to provoke neutralizing antibody responses.
