Subcutaneous administration of Lyso-phosphatidylserine nanoparticles induces immunological tolerance towards Factor VIII in a Hemophilia A mouse model

Abstract

A major complication with enzyme replacement therapy of Factor VIII (FVIII) in Hemophilia A (HA) is the development of anti-drug antibodies. Recently, we have shown that FVIII administration in the presence of heterogeneous phosphatidylserine (PS) nanoparticles derived from a natural source induces tolerance to FVIII, suggesting that PS converts an immunogen to a tolerogen. However, the specific structural features responsible for the immune-regulatory properties of PS is unclear. Identifying a specific PS species that is responsible is critical in order to further develop and optimize this nanoparticle. Further, clinical development of this lipid-based strategy requires optimization of the lipid particle that is homogeneous and synthetic. Here, we investigate the ability of mono-acylated Lyso-PS to induce hypo-responsiveness towards FVIII in HA mice. Administration of both PS and Lyso-PS FVIII significantly reduced anti-FVIII antibody responses despite rechallenge with FVIII. Additionally, the Lyso-PS-mediated effect was shown to be antigen-specific as mice responded normally against a rechallenge with an unrelated antigen, ovalbumin. Furthermore, the hypo-responsiveness observed with Lyso-PS may involve interactions with a specific PS receptor, TIM-4, along with increasing regulatory T-cells. These data indicate that using Lyso-PS allows for a more homogenous formulation in order to induce tolerance towards therapeutic proteins.

Immunogenicity Challenges Associated with Subcutaneous Delivery of Therapeutic Proteins

“…SC co-administration of OPLS and rhGAA in a mixed formulation induced hyporesponsiveness to rhGAA in Pompe disease mice [ 211 ]. Furthermore, reverse vaccination by SC pre-administration of Lyso-phosphatidylserine (Lyso-PS)-containing nanoparticles loaded with FVIII significantly reduced anti-FVIII antibody response during re-exposure to FVIII intravenously, the mechanism of which involved a specific PS receptor, TIM-4 [ 212 ].

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De novo design of functional zwitterionic biomimetic material for immunomodulation

“…To examine whether the PS-mimetic polymers can emulate the modulating effect of apoptotic cells and elicit anti-inflammatory responses in activated macrophages, we investigated the potential effect of ZPS, NZPS, and MPC on RAW 264.7 macrophages in the presence of lipopolysaccharide (LPS), an immunostimulatory molecule. (10,25,50,100,200, and 1000 g/ml) for 18 hours followed by the stimulation of LPS (100 ng/ml) for 48 hours. The level of TNF- secretion in the supernatant was measured by an ELISA kit.…”

“…The zeta potential of anionic NZPS nanogel was negative (~−20 mv), while those of zwitterionic ZPS and MPC nanogels were close to neutral. RAW 264.7 macrophages (10 5 per well) were treated with nanogels composed of MPC, NZPS, or ZPS at various concentrations (10,25,50,100,200, and 1000 g/ml) for 18 hours followed by the stimulation of LPS (100 ng/ml). The status of macrophage was evaluated by measuring the production and release of proinflammatory cytokines [tumor necrosis factor- (TNF-), interleukin-6 (IL-6), and CXCL-10] in the supernatant.…”

“…Immunomodulatory effect test RAW 264.7 cells were seeded (10 5 per well) and were exposed to MPC, NZPS, or ZPS nanogel solution at various concentrations (10,25,50,100,200, and 1000 g/ml) for 18 hours. Then, these cells were stimulated by LPS solution (100 ng/ml) for another 48 hours, after which the cells were spun at 300g for 10 min.…”

Advances in knowledge of inhibitor formation in severe haemophilia A

“…How this method can be used to preferentially process the FVIII antigen has yet to be explored. Subcutaneous administration of a tolerogenic compound lyso‐phosphatidylserine complexed with FVIII later diminished systemic inhibitor responses after intravenous FVIII challenge (Glassman & Balu‐Iyer, ). Although primarily focussing on the tolerogenic properties of lyso‐phosphatidylserine, this suggests that sensitising PUPs with FVIII subcutaneously might be advantageous.…”