Phosphatidylserine-mediated oral tolerance.

Phosphatidylserine (PS) is an anionic phospholipid exposed on the surface of apoptotic cells. The exposure of PS typically recruits and signals phagocytes to engulf and silently clear these dying cells to maintain tolerance via immunological ignorance. However, recent and emerging evidence has demonstrated that PS converts an "immunogen" into a "tolerogen", and PS exposure on the surface of cells or vesicles actively promotes a tolerogenic environment. This tolerogenic property depends on the biophysical characteristics of PS-containing vesicles, including PS density on the particle surface to effectively engage tolerogenic receptors, such as TIM-4, which is exclusively expressed on the surface of antigen-presenting cells. We harnessed the cellular and molecular mechanistic insight of PS-mediated immune regulation to design an effective oral tolerance approach. This immunotherapy has been shown to prevent/reduce immune response against life-saving protein-based therapies, food allergens, autoantigens, and the antigenic viral capsid peptide commonly used in gene therapy, suggesting a broad spectrum of potential clinical applications. Given the good safety profile of PS together with the ease of administration, oral tolerance achieved with PS-based nanoparticles has a very promising therapeutic impact.

Immunogenicity of Therapeutic Biological Modalities - Lessons from Hemophilia A Therapies.

The introduction and development of biologics such as therapeutic proteins, gene-, and cell-based therapy have revolutionized the scope of treatment for many diseases. However, a significant portion of the patients develop unwanted immune reactions against these novel biological modalities, referred to as immunogenicity, and no longer benefit from the treatments. In the current review, using Hemophilia A (HA) therapy as an example, we will discuss the immunogenicity issue of multiple biological modalities. Currently, the number of therapeutic modalities that are approved or recently explored to treat HA, a hereditary bleeding disorder, is increasing rapidly. These include, but are not limited to, recombinant factor VIII proteins, PEGylated FVIII, FVIII Fc fusion protein, bispecific monoclonal antibodies, gene replacement therapy, gene editing therapy, and cell-based therapy. They offer the patients a broader range of more advanced and effective treatment options, yet immunogenicity remains the most critical complication in the management of this disorder. Recent advances in strategies to manage and mitigate immunogenicity will also be reviewed.

Biophysical Characterization of Tolerogenic Lipid-Based Nanoparticles Containing Phosphatidylcholine and Lysophosphatidylserine.

Autoimmune conditions, allergies, and immunogenicity against therapeutic proteins are initiated by the unwanted immune response against self and non-self proteins. The development of tolerance induction approaches can offer an effective treatment modality for these clinical conditions. We recently showed that oral administration of lipidic nanoparticles containing phosphatidylcholine (PC) and lysophosphatidylserine (Lyso-PS) converted an immunogen to a tolerogen and induced immunological tolerance towards several antigens. While the biophysical properties such as lamellar characteristics of this binary lipid system are critical for stability, therapeutic delivery, and mechanism of tolerance induction, such information has not been thoroughly investigated. In the current study, we evaluated the lamellar phase properties of PC/Lyso-PS system using orthogonal biophysical methods such as fluorescence (steady-state, anisotropy, PSvue, and Laurdan), dynamic light scattering, and differential scanning calorimetry. The results showed that Lyso-PS partitioned into the PC bilayers and led to changes in the particles' lamellar phase properties, lipid-packing, and lipid-water dynamics. Additionally, the biophysical characteristics of PC/Lyso-PS system are different from the well-studied PC/double-chain phosphatidylserine (PS) system. Notably, the incorporation of Lyso-PS significantly reduced the hydrodynamic diameter of PC particles. Results from the in vivo uptake study and intestinal loop assay utilizing flow cytometry analysis also indicated that the uptake of Lyso-PS-containing nanoparticles by immune cells in the gut and Peyer's patches is significantly higher than that of double-chain PS due to the differential transport through microfold cells. It was also found that the acyl chain mismatch between PC and Lyso-PS is critical for the miscibility and particle stability. Collectively, the results suggest that these biophysical characteristics likely influence the in vivo behaviors and contribute to the oral tolerance property of PC/Lyso-PS system.

Tolerogenic form of Factor VIII to prevent inhibitor development in the treatment of Hemophilia A.

BACKGROUND: The development of antidrug antibodies, also termed inhibitors, against administered factor VIII (FVIII) is one of the major complications in the clinical management of hemophilia A. Once formed, these inhibitory antibodies abrogate the activity of FVIII, resulting in loss of hemostatic efficacy and patients are subjected to increased risk of bleeding tendencies. Current treatment options after inhibitor development are expensive and ineffective in some cases. Therefore, treatment strategies that can prevent inhibitor formation is an effective approach in the management of hemophilia A. OBJECTIVES: We aimed to evaluate and discuss the use of a tolerogenic form of FVIII as an immunotherapy strategy to prevent inhibitor risk. METHODS: FVIII was associated with nanoparticles containing lysophosphatidylserine (Lyso-PS) and administered to hemophilia A mice via intravenous route. These animals then received weekly rechallenge injections with free FVIII, and plasma was collected at the end of the study to evaluate for inhibitor development. To investigate whether Lyso-PS nanoparticles influence the plasma survival of FVIII, a pharmacokinetic study following a single intravenous administration of FVIII in the presence and absence of Lyso-PS nanoparticles was performed. For dosing convenience, the tolerogenic effect of Lyso-PS nanoparticles following oral administration was also examined. RESULTS AND CONCLUSIONS: The results demonstrated that FVIII associated with Lyso-PS nanoparticles significantly reduced inhibitor development while improving plasma survival of FVIII following intravenous administration, suggesting a multifunctional FVIII form to improve clinical utility. Additionally, reduction in inhibitor formation can also be achieved using Lyso-PS nanoparticles through the user-friendly oral route of administration.

Rational design of a nanoparticle platform for oral prophylactic immunotherapy to prevent immunogenicity of therapeutic proteins.

The safety and efficacy of several life-saving therapeutic proteins are compromised due to their immunogenicity. Once a sustained immune response against a protein-based therapy is established, clinical options that are safe and cost-effective become limited. Prevention of immunogenicity of therapeutic proteins prior to their initial use is critical as it is often difficult to reverse an established immune response. Here, we discuss a rational design and testing of a phosphatidylserine-containing nanoparticle platform for novel oral prophylactic reverse vaccination approach, i.e., pre-treatment of a therapeutic protein in the presence of nanoparticles to prevent immunogenicity of protein therapies.