Mechanism of anionic phospholipids mediated mitigation of immunogenicity of recombinant human factor VIII.

摘要

The primary objective of this research work was to take a rational and stepwise approach to investigate the mechanism of phosphatidylserine/phosphatidylinositol (PS/PI) effect on reducing Factor VIII immunogenicity.;The initial step involved in the generation of therapeutic protein immunogenicity is antigen uptake and presentation of antigenic epitopes by professional antigen presenting cells (APC). Since, dendritic cells (DC) are a type of professional APC, we established a protocol in our lab for the culturing and characterization of murine bone-marrow derived DC in chapter 2. Further, as cell surface expression level of phenotypic markers is indicative of the level of DC maturation and activation, we used flow-cytometry analyses to examine the expression level of these markers. We also investigated whether DC can discriminate between self- and non-self antigens based on their prior immunological knowledge of the antigen. Hence in chapter 3, we tested the ability of DC obtained from both; wild-type and HA mice, on up-regulation of cell-surface co-stimulatory markers upon free FVIII exposure. .;In chapter 4, we conducted cell-based studies to deduce PS effect on FVIII uptake and presentation by DC, T-cell proliferation and cytokines secretion. The FVIII-PS exposed DCs were co-cultured with FVIII primed murine splenic CD4+ T-cells. The data showed that PS was internalized by DC and hence, may not interfere with FVIII uptake, however, PS exposure led to down-regulation of DC co-stimulatory signals that implied inefficient T-cell activation ability of these DC. This was further evident from significant suppression of T-cell proliferation upon DC -- T-cell co-culture. Additionally, significant increase in the secretion level of immuno-suppressive TGF-beta and IL-10 cytokines and corresponding decrease in pro-inflammatory IL-6 and IL-17 cytokines was observed in the supernatants of the FVIII-PS exposed group.;In chapter 5, we investigated PI effect on influencing FVIII uptake, DC maturation, T-cell proliferation and cytokines secretion profiles. We observed that unlike PS, there was limited uptake of PI particles by DC. Thus, uptake of FVIII by DC can be reduced by associating FVIII with PI, thereby preventing DC maturation. Even though PS and PI differ in their effects at the initial stage of antigen-uptake, both can reduce FVIII immune response by regulating DC function and subsequent reduction in T-cell proliferation.;In chapter 6, we developed and tested a 'Reverse Vaccination (TM)' strategy to utilize PS and PI to induce FVIII immune tolerance in previously untreated, naive HA mice. Mice pre-immunized with FVIII-PS were hypo-responsive to future challenges of free FVIII and demonstrated significantly lower anti-FVIII Nab titers. FVIII-PI pre-immunized mice also elicited reduction in FVIII immunity; however, the reduction in titer levels was not statistically significant. Thus, it was inferred that PS and perhaps PI, induce hypo-responsiveness in the naive HA mice towards FVIII and have the potential to induce immunological tolerance towards FVIII in naive HA mice.;The C2 domain of FVIII binds to O-Phospho-L-Serine (OPLS), the head-group of PS, and previous results examining the utility of OPLS as an excipient showed that OPLS enhanced FVIII stability upon subjecting to thermal stress, as well as, subcutaneous administration of FVIII-OPLS in HA mice resulted in lower FVIII antibodies development. Since, FVIII is currently administered in the clinic via the intravenous (i.v.) route; we studied the influence of FVIII-OPLS complexation on FVIII immune response, T-cell proliferation and FVIII pharmacokinetics (PK) following i.v. administration in naive HA mice in chapter 7. OPLS was found to significantly reduce FVIII immunogenicity and T-cell proliferation, and it had no detrimental effects on the PK of FVIII. Thus, the results suggest that OPLS could be highly beneficial as an excipient in future FVIII preparations. (Abstract shortened by UMI.).