Effect of an Active Phosphatidylserine Species on Antigen‐Specific Tolerance Induction Towards Factor VIII in Hemophilia A Mice
Authors: Yau Fiona C , Balu‐Iyer Sathy V 
Published: November 2023

Purpose

The development of antibodies towards Factor VIII (FVIII) in Hemophilia A (HA) patients is a clinical complication that could lead to the loss of efficacy of treatment and increased bleeding episodes. Therefore, a reduction in immunogenicity to FVIII is an unmet clinical need. Our lab has previously shown that pre‐exposure of Factor VIII (FVIII) in the presence of phosphatidylserine (PS) is able to induce immunological tolerance or hypo‐responsiveness to FVIII in Hemophilia A mice by converting an immunogen to a tolerogen (Gaitonde et al, 2013). The PS that was previously used was derived from an animal source and was heterogeneous in its structure. The identification of an active synthetically‐derived PS species with optimal biophysical and structural requirements for an effective tolerance is critical. This work investigated the ability of a synthetic PS species (s‐PS) with acyl chain oxidation and unsaturation in antigen‐specific tolerance induction towards FVIII in HA mice.

Methods

In vivo studies were carried out in FVIII KO mice and all protein doses were 0.4 μg. Mice received weekly subcutaneous (SC) injections of FVIII in the absence and presence of PS and s‐PS liposomes. On week 6, mice received four weekly intravenous re‐challenge injections of FVIII 24 hours after SC liposome treatment. Two weeks after the last re‐challenge, all animals were sacrificed and plasma collected for titer analysis. To investigate the antigen specificity, mice received four weekly SC injections of FVIII in the presence and absence of s‐PS liposomes. Two weeks after the last injection, half the animals were re‐challenged with four weekly SC injections of FVIII alone while the other half received OVA, an irrelevant antigen. Two weeks after the last re‐challenge, all animals were sacrificed and plasma analyzed for anti‐FVIII and anti‐OVA antibody development.

Results

Animals that received the s‐PS treatment displayed a significant reduction in both the total anti‐FVIII titers and neutralizing titers compared to animals that received free FVIII alone. Animals that received PS liposomes displayed a significant reduction in only the neutralizing titers compared to animals that received free FVIII alone. In the antigen specificity study, mice treated with s‐PS liposomes displayed a significant reduction in anti‐FVIII titers compared to those treated with free FVIII alone at baseline. Mice treated with s‐PS liposomes displayed a trend towards lower antibody development towards FVIII at the end of the study compared to mice treated with free FVIII alone. All mice, irrespective of treatment with s‐PS liposomes or free FVIII, displayed a robust immune response to the irrelevant antigen, OVA.

Conclusion

Reduction in immunogenicity and tolerance induction towards FVIII was observed when mice were treated with s‐PS liposomes. This tolerance induction strategy using s‐PS liposomes was shown to be antigen‐specific and not general immunosuppression. The identification of a single PS species that could be responsible for the PS‐mediated effects observed is important to allow for further optimization and clinical translation.

Support or Funding Information

This work was supported by the National Institute of Health grant (R01 HL‐70227) to Dr. Sathy V. Balu‐Iyer.