Plasma membrane vesicles decorated with glycolipid-anchored antigens and adjuvants via protein transfer as an antigen delivery platform for inhibition of tumor growth

Abstract

Antigen delivered within particulate materials leads to enhanced antigen-specific immunity compared to soluble administration of antigen. However, current delivery approaches for antigen encapsulated in synthetic particulate materials are limited by the complexity of particle production that affects stability and immunogenicity of the antigen. Herein, we describe a protein delivery system that utilizes plasma membrane vesicles (PMVs) derived from biological materials such as cultured cells or isolated tissues and a simple protein transfer technology. We show that these particulate PMVs can be easily modified within 4 h by a protein transfer process to stably incorporate a glycosylphosphatidylinositol (GPI)-anchored form of the breast cancer antigen HER-2 onto the PMV surface. Immunization of mice with GPI-HER-2-modified-PMVs induced strong HER-2-specific antibody responses and protection from tumor challenge in two different breast cancer models. Further incorporation of the immunostimulatory molecules IL-12 and B7-1 onto the PMVs by protein transfer enhanced tumor protection and induced beneficial Th1 and Th2-type HER-2-specific immune responses. Since protein antigens can be easily converted to GPI-anchored forms, these results demonstrate that isolated plasma membrane vesicles can be modified with desired antigens along with immunostimulatory molecules by protein transfer and used as a vaccine delivery vehicle to elicit potent antigen-specific immunity.

Cell-derived plasma membrane vesicles are permeable to hydrophilic macromolecules

“…These findings are of particular relevance to studies using GPMVs for drug encapsulation (27,28), therapeutic targeting (26,27), and studies of membrane permeation (26,(29)(30)(31)(32). Our suggestion for possible approaches to avoid leakage in GPMVs may be of value therein.…”

Exosome as a Vehicle for Delivery of Membrane Protein Therapeutics, PH20, for Enhanced Tumor Penetration and Antitumor Efficacy

“…However, studies have shown that GPI‐anchored proteins can be enriched on the lipid rafts of exosomes without any alteration of their intrinsic functionality . Indeed, plasma membrane‐derived vesicles were recently developed for the delivery of antigens conjugated to GPI …”

Tumor Membrane Vesicle Vaccine Augments the Efficacy of Anti-PD1 Antibody in Immune Checkpoint Inhibitor-Resistant Squamous Cell Carcinoma Models of Head and Neck Cancer

“…Tumor tissue was homogenized, and membranes were isolated by centrifugation over a 41% sucrose gradient. These TMVs were incorporated with immunoaffinity-purified murine GPI-B7-1 and GPI-IL-12 molecules (GPI-ISMs) by protein transfer [26]. GPI-ISM incorporation was confirmed by flow cytometry analysis using fluorochrome-conjugated antibodies.…”

“…Briefly, cells isolated from in vivo studies were pre-incubated with Fc receptor blocking antibody in FACS buffer (PBS with 2% BCS, 5 mM EDTA, 0.05% sodium azide) at 4 • C for 10 min to block nonspecific binding of monoclonal antibodies to immune cells. Fluorochrome-conjugated primary antibodies were added and incubated for 30 min with shaking at 4 • C. Cells were washed three times with FACS buffer and analyzed the cells using a FACSCalibur, BD LSRII (BD Biosciences, San Jose, CA, USA) or a CYTEK Aurora (CYTEK, Fremont, CA, USA) flow cytometer [26,34]. Protein expression profiles of TMVs were also analyzed by staining with fluorochrome-conjugated antibodies after blocking the Fc receptors with Fc blocking antibody [26].…”