(M1330-05-27) Tolerogenic Form of FVIII Provides Durable Tolerance in Hemophilia A Mice Following 3 Months of Treatment

Purpose: Human factor VIII (FVIII) is a large multidomain glycoprotein that serves as a critical cofactor in the blood coagulation cascade. FVIII is involved in the intrinsic pathway of the coagulation cascade by anchoring activated factor IX to its substrate, factor X, to the surface of blood platelets [1]. The role of FVIII is very important in coagulation, so much so that deficiency or dysfunction of FVIII results in Hemophilia A (HA), a genetic bleeding disorder [2, 3]. Treatment for HA typically involves replacement therapy using recombinant FVIII to restore hemostatic activity. A major complication that occurs in roughly 30% of severe HA patients ( < 1% FVIII activity) is the development of inhibitory antibodies that abrogate the biological activity of the protein, leading to decreased efficacy and increased hemorrhage risk of the patients [4, 5]. After inhibitor development, treatment options become limited and expensive. Our lab has developed a tolerogenic lipid nanoparticle that uses single chain phosphatidylserine (LysoPS) to mimic the properties of an apoptotic cell. This LysoPS-based lipid nanoparticle (TOLIP) has shown the ability to “teach” the immune system to tolerate a protein, effectively converting it from an “immunogen” into a “tolerogen”[6]. We have shown that TOLIP can prevent an immunogenic response toward FVIII in a HA mouse model through IV administration, despite repeated rechallenge of free FVIII [7]. However, we do not know if pretreatment of TOLIP-FVIII would be sufficient to induce a durable tolerance, or if other dosing regimes should be investigated as well. We hypothesize that TOLIP-FVIII would be able to provide a durable tolerance following 3 months after TOLIP-FVIII treatment. In this study, we investigated the durability of tolerance in TOLIP-FVIII following 3 months of treatment.

Methods: 18:1 LysoPS lipids were mixed with 1,2-dimyristoyl-sn-glycero-3-phosphocoline (DMPC) at 30%:70% molar ratio using thin film dehydration and rehydration methods, followed by high-pressure membrane extrusion, to generate uniform, homogenous nanoparticles with a 100 nm diameter. To investigate the durability of tolerance of TOLIP-FVIII, 10 male HA mice per group were immunized with either free FVIII or TOLIP-FVIII intravenously once every 2 weeks for 8 weeks (Figure 1). Following prophylactic dosing, mice were then dosed with free FVIII subcutaneously once a month for 2 months then twice (weekly) on month 3. Following a 3-week washout period, mice were sacrificed, and plasma was collected to study development of neutralizing anti-FVIII antibody titers. Additionally, the spleen was crushed to study development of regulatory T cells, regulatory B cells (B10), and germinal center B cells. Inhibitor development against FVIII was quantified using an activated partial thromboplastin time assay (aPTT) following Nijmegen’s modified Bethesda assay and expressed in Bethesda Units (BU/mL) as described previously [8, 9].

Results: Figure 2 shows the FVIII inhibitory titers determined from the mice plasma after 3 months. As shown, there is a statistically significant difference between the FVIII and TOLIP-FVIII groups, with the TOLIP-FVIII group having significantly lower titer values compared to the FVIII group. This trend was accompanied with a significant decrease in splenic germinal center B cells in the TOLIP-FVIII group, which indicates that the reduction in inhibitory titers reflects a meaningful immunological response (Figure 3).

Conclusion: TOLIP-FVIII pretreatment is able to effectively reduce inhibitor development in a naïve setting for 3 months.

References: 1. Chavin, S.I., Factor VIII: structure and function in blood clotting. Am J Hematol, 1984. 16(3): p. 297-306.
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3. Franchini, M., The modern treatment of haemophilia: a narrative review. Blood Transfusion, 2013. 11(2): p. 178.
4. Witmer, C. and G. Young, Factor VIII inhibitors in hemophilia A: rationale and latest evidence. Ther Adv Hematol, 2013. 4(1): p. 59-72.
5. van den Berg, H.M., et al., Timing of inhibitor development in more than 1000 previously untreated patients with severe hemophilia A. Blood, 2019. 134(3): p. 317-320.
6. Schneider, J.L. and S.V. Balu-Iyer, Phosphatidylserine Converts Immunogenic Recombinant Human Acid Alpha-Glucosidase to a Tolerogenic Form in a Mouse Model of Pompe Disease. J Pharm Sci, 2016. 105(10): p. 3097-3104.
7. Nguyen, N.H., R.K. Dingman, and S.V. Balu-Iyer, Tolerogenic form of Factor VIII to prevent inhibitor development in the treatment of Hemophilia A. J Thromb Haemost, 2021. 19(11): p. 2744-2750.
8. Ramani, K., et al., Lipid binding region (2303-2332) is involved in aggregation of recombinant human FVIII (rFVIII). J Pharm Sci, 2005. 94(6): p. 1288-99.
9. Verbruggen, B., et al., The Nijmegen modification of the Bethesda assay for factor VIII:C inhibitors: improved specificity and reliability. Thromb Haemost, 1995. 73(2): p. 247-51.

Acknowledgements: Funding was provided by the National Heart Lung and Blood Institute R61 HL161818. We would like to thank Drs. Kazazian and Sarkar of the University of Pennsylvania for providing the initial HA mice model. We are grateful for the generous donation of FVIII products provided by Western New York BloodCare. The authors would also like to acknowledge the UB Optical Imaging and Analysis Facility for the use of the flow cytometer.

Figure 1: IV dosing strategy in HA Mice. Mice were injected with free FVIII or TOLIP-FVIII once every 2 weeks for 8 weeks. Following prophylactic dosing, mice were dosed with free FVIII subcutaneously once a month for 2 months then twice on month 3. Mice were sacrificed at 3 months and plasma, spleen, and bone marrow cells were collected.

Figure 2: Inhibitory titer level (Mean ± SEM) of FVIII and TOLIP-FVIII pre-treatment via IV immunotherapy followed by aggressive SC rechallenge. Statistics were performed using two-sided Welch’s T test.

Figure 3: A) Flow Cytometry gating strategy for germinal center B cells (CD19+BCL6+IgDlowCD38int). B) Flow cytometry depicting frequency splenic germinal center B cells (CD19+BCL6+IgDlowCD38int) from CD19+IgDlow cells. Statistics were performed using two-sided Welch’s T test.