Neutralizing Antibodies Against Factor VIII Can Occur Through a Non-Germinal Center Pathway.

Humoral immunity to factor VIII (FVIII) represents a significant challenge for the treatment of patients with hemophilia A. Current paradigms indicate that neutralizing antibodies against FVIII (inhibitors) occur through a classical CD4 T cell, germinal center (GC) dependent process. However, clinical observations suggest that the nature of the immune response to FVIII may differ between patients. While some patients produce persistent low or high inhibitor titers, others generate a transient response. Moreover, FVIII reactive memory B cells are only detectable in some patients with sustained inhibitor titers. The determinants regulating the type of immune response a patient develops, let alone how the immune response differs in these patients remains incompletely understood. One hypothesis is that polymorphisms within immunoregulatory genes alter the underlying immune response to FVIII, and thereby the inhibitor response. Consistent with this, studies report that inhibitor titers to FVIII differ in animals with the same F8 pathogenic variant but completely distinct backgrounds; though, how these genetic disparities affect the immune response to FVIII remains to be investigated. Given this, we sought to mechanistically dissect how genetics impact the underlying immune response to FVIII. In particular, as the risk of producing inhibitors is weakly associated with differences in HLA, we hypothesized that genetic factors other than HLA influence the immune response to FVIII and downstream inhibitor formation. Our data demonstrate that FVIII deficient mice encoding the same MHC and F8 variant produce disparate inhibitor titers, and that the type of inhibitor response formed associates with the ability to generate GCs. Interestingly, the formation of antibodies through a GC or non-GC pathway does not appear to be due to differences in CD4 T cell immunity, as the CD4 T cell response to an immunodominant epitope in FVIII was similar in these mice. These results indicate that genetics can impact the process by which inhibitors develop and may in part explain the apparent propensity of patients to form distinct inhibitor responses. Moreover, these data highlight an underappreciated immunological pathway of humoral immunity to FVIII and lay the groundwork for identification of biomarkers for the development of approaches to tolerize against FVIII.

Pharmacokinetic analysis identifies a factor VIII immunogenicity threshold after AAV gene therapy in hemophilia A mice.

Advances in the development of novel treatment options for hemophilia A are prevalent. However, the anti-factor VIII (FVIII) neutralizing antibody (inhibitor) response to existing FVIII products remains a major treatment challenge. Although some novel products are designed to function in the presence of inhibitors, they do not specific address the immunogenicity risk or mechanistic causes of inhibitor development, which remain unclear. Furthermore, most preclinical studies supporting clinical gene therapy programs have reported immunogenicity signals in animal models, especially at higher vector doses and sometimes using multiple vector designs. In these settings, immunogenicity risk factor determination, comparative immunogenicity of competing vector designs, and the potential for obtaining meaningful prognostic data remain relatively unexplored. Additionally, there remains the opportunity to investigate clinical gene therapy as an alternative to standard immune tolerance induction therapy. The current study was designed to address these issues through longitudinal dose-response evaluation of 4 adeno-associated viral (AAV) vector candidates encoding 2 different FVIII transgenes in a murine model of hemophilia A. Plasma FVIII activity and anti-FVIII antibody data were used to generate a pharmacokinetic model that (1) identifies initial AAV-FVIII product expression kinetics as the dominant risk factor for inhibitor development, (2) predicts a therapeutic window where immune tolerance is achieved, and (3) demonstrates evidence of gene therapy-based immune tolerance induction. Although there are known limitations to the predictive value of preclinical immunogenicity testing, these studies can uncover or support the development of design principles that can guide the development of safe and effective genetic medicines.

Non-genotoxic conditioning facilitates hematopoietic stem cell gene therapy for hemophilia A using bioengineered factor VIII.

Hematopoietic stem and progenitor cell (HSPC) lentiviral gene therapy is a promising strategy toward a lifelong cure for hemophilia A (HA). The primary risks associated with this approach center on the requirement for pre-transplantation conditioning necessary to make space for, and provide immune suppression against, stem cells and blood coagulation factor VIII, respectively. Traditional conditioning agents utilize genotoxic mechanisms of action, such as DNA alkylation, that increase risk of sterility, infection, and developing secondary malignancies. In the current study, we describe a non-genotoxic conditioning protocol using an immunotoxin targeting CD117 (c-kit) to achieve endogenous hematopoietic stem cell depletion and a cocktail of monoclonal antibodies to provide transient immune suppression against the transgene product in a murine HA gene therapy model. This strategy provides high-level engraftment of hematopoietic stem cells genetically modified ex vivo using recombinant lentiviral vector (LV) encoding a bioengineered high-expression factor VIII variant, termed ET3. Factor VIII procoagulant activity levels were durably elevated into the normal range and phenotypic correction achieved. Furthermore, no immunological rejection or development of anti-ET3 immunity was observed. These preclinical data support clinical translation of non-genotoxic antibody-based conditioning in HSPC LV gene therapy for HA.

The Immune Response to the fVIII Gene Therapy in Preclinical Models.

Neutralizing antibodies to factor VIII (fVIII), referred to as "inhibitors," remain the most challenging complication post-fVIII replacement therapy. Preclinical development of novel fVIII products involves studies incorporating hemophilia A (HA) and wild-type animal models. Though immunogenicity is a critical aspect of preclinical pharmacology studies, gene therapy studies tend to focus on fVIII expression levels without major consideration for immunogenicity. Therefore, little clarity exists on whether preclinical testing can be predictive of clinical immunogenicity risk. Despite this, but perhaps due to the potential for transformative benefits, clinical gene therapy trials have progressed rapidly. In more than two decades, no inhibitors have been observed. However, all trials are conducted in previously treated patients without a history of inhibitors. The current review thus focuses on our understanding of preclinical immunogenicity for HA gene therapy candidates and the potential indication for inhibitor treatment, with a focus on product- and platform-specific determinants, including fVIII transgene sequence composition and tissue/vector biodistribution. Currently, the two leading clinical gene therapy vectors are adeno-associated viral (AAV) and lentiviral (LV) vectors. For HA applications, AAV vectors are liver-tropic and employ synthetic, high-expressing, liver-specific promoters. Factors including vector serotype and biodistribution, transcriptional regulatory elements, transgene sequence, dosing, liver immunoprivilege, and host immune status may contribute to tipping the scale between immunogenicity and tolerance. Many of these factors can also be important in delivery of LV-fVIII gene therapy, especially when delivered intravenously for liver-directed fVIII expression. However, ex vivo LV-fVIII targeting and transplantation of hematopoietic stem and progenitor cells (HSPC) has been demonstrated to achieve durable and curative fVIII production without inhibitor development in preclinical models. A critical variable appears to be pre-transplantation conditioning regimens that suppress and/or ablate T cells. Additionally, we and others have demonstrated the potential of LV-fVIII HSPC and liver-directed AAV-fVIII gene therapy to eradicate pre-existing inhibitors in murine and canine models of HA, respectively. Future preclinical studies will be essential to elucidate immune mechanism(s) at play in the context of gene therapy for HA, as well as strategies for preventing adverse immune responses and promoting immune tolerance even in the setting of pre-existing inhibitors.