Abstract
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.
Introduction
Therapeutic proteins are one of the fastest growing class of drugs, as they offer treatment options for several diseases with minimal off-target effects. However, the unwanted immune responses against these life-saving therapies not only impacts their safety profile but also efficacy1,2,3. For example, about 89–100% of Pompe disease patients who receive recombinant acid alpha glucosidase (GAA) as enzyme replacement therapy develop anti-GAA antibodies4,5. Once a sustained immune response is established, the efficacy of this life-saving therapy is compromised and tolerance inducing regimens using bortezomib in combination with rituximab, methotrexate, and intravenous immunoglobulin are attempted to rescue these high titer patients6. However, the use of such immunosuppressive agents poses a risk for secondary infections. In the case of Hemophilia A (HA), a bleeding disorder, about one third of the severe HA patients receiving replacement therapy using recombinant Factor VIII (FVIII) develop neutralizing anti-FVIII antibodies, referred to as inhibitors, that abrogate the biological activity and hemostatic efficacy of the administered FVIII7,8,9. Clinical options after the development of antibodies are very costly (over $700,000/year/patient) and, in some patients, ineffective10. Therefore, strategies to prevent unwanted immune responses against therapeutic proteins are desirable for clinical management, patient care, and cutting health care costs.
Our previous studies have shown that phosphatidylserine (PS) can convert an immunogen to a tolerogen, thereby leading to the reduction of unwanted immune responses. The subcutaneous administration of FVIII and GAA in the presence of nanoparticles containing double-chain PS species were able to induce immunological hypo-responsiveness in relevant mouse models11,12,13,14. Furthermore, in vitro studies demonstrated that PS promotes phenotypic changes in dendritic cells (DCs) and converts them to the tolerogenic type15. Surprisingly when given via oral route, nanoparticles containing double-chain PS did not induce tolerance to the same proteins as seen with subcutaneous administration. This is possibly due to the uptake and mechanistic differences of oral tolerance compared to other routes of administration. PS externalization to the outer leaflet of bilayer membrane and PS surface density are critical determinants promoting differential recognition by immune-regulatory PS receptors including T-cell immunoglobulin and mucin domain containing 4 (TIM-4)16,17. Additionally, the uptake of PS-containing particles across the intestinal wall is postulated to be facilitated by microfold (M) cells, which predominantly express the scavenger receptor class B type 1 (SR-B1), clusterin, and annexin V18. These proteins have been demonstrated to bind to and/or interact with PS exposed on the outer leaflet of apoptotic cells or PS-containing liposomes, which aids the phagocytosis and internalization of cells or vesicles expressing PS19,20,21,22. Therefore, we rationally designed and tested a nanoparticle platform with the optimal structural (single-acyl chain lysophosphatidylserine [Lyso-PS] derivative) and biophysical characteristics, including size and PS surface exposure. This platform serves as a tolerogenic form of protein for a novel lipid-based immunotherapy to deliver proteins in the presence of PS to prevent their immunogenicity. The rationale for this approach is that pre-exposure of a protein in the presence of PS induces tolerance and blocks the patients’ immune response towards that protein prior to the initiation of the therapy. The utilization of oral administration would also allow for the exposure of formulations to the mucosal immune system, which has evolved as an effective, default tolerogenic site23,24. Furthermore, oral administration can offer clinical translatability due to the simplicity, convenience, and high patient compliance.
Results
Design of lipid nanoparticles for immunotherapy
PS is an anionic phospholipid that is generally present in the inner leaflet of a healthy cell, but flips to the outer leaflet when cells undergo apoptosis25,26. This externalization of PS sends an “eat me” signal to phagocytes for a clearance of cell debris in an immunologically silent manner, therefore “maintaining” tolerance towards self-proteins by immunological ignorance termed as a “tolerate/ignore me” signal27. However, our efforts to reduce immunogenicity of therapeutic proteins demonstrated that the externalization of PS promotes an active learning process through induction of antigen-specific tolerance14. It has been identified that TIM-4 is one of the PS receptors, which is exclusively expressed on antigen presenting cells (APCs)28,29,30. Studies from our lab have shown that PS-mediated tolerance induction involves the interaction between PS nanoparticles and TIM-414. Interestingly, TIM-4 is sensitive to PS density exposed on the surface of bilayer membrane16,17 and mediates differential recognition of apoptotic events over other forms of PS externalization to promotes tolerance28,31. Additionally, particle size and PS exposure on the outer leaflet of the particles are critical biophysical parameters for cellular uptake and receptor binding17,25,32,33, that in turn influence the biological functions of PS-containing vesicles. Therefore, we designed a nanoparticle containing PS with optimal biophysical characteristics and PS externalization for a more effective tolerance induction. While the structure of PS is generally known to consist of a serine headgroup and two fatty acid acyl chains connected together by a glycerol backbone, several derivatives of PS also exist in which they differ in the lengths of fatty acid acyl chain, degree of unsaturation, and number of acyl chains. The PS derivative with only one fatty acid chain is termed Lyso-PS. Since phosphatidylcholine is the major lipid component providing structural framework of biological membranes, we use dimyristoylphosphatidylcholine (DMPC) as the base phospholipid to form bilayer of the nanoparticles. Therefore, pure DMPC nanoparticles are used as the control. Unlike the structure of PS and Lyso-PS, DMPC contains a net neutral charge with zwitterionic choline headgroup. After testing various formulations comprised of different PS species, we found that DMPC nanoparticles containing single-chain 18:1 Lyso-PS had a significantly smaller mean particle size (112 ± 9.6 nm) compared to pure DMPC (189 ± 24.9 nm) and DMPC containing double-chain brain PS or double-chain 18:1 PS (169 ± 9.3 nm and 169 ± 0.4 nm, respectively) despite the identical preparation procedure (thin film method followed by extrusion through 200 nm polycarbonate membranes) (Table 1). The decrease in particle size of Lyso-PS nanoparticles is likely due to the increase in surface curvature of the vesicles, caused by the partitioning of the cone-shaped Lyso-PS into DMPC bilayers. In contrast, the partitioning of cylindrical-shaped double-chain PS into DMPC bilayers increased the fluidity of the nanoparticles, but did not alter the particle curvature or size.
