RHDV VLP Mediated Delivery of α-Galactosylceramide for Immunotherapy

Abstract

The immune system has the potential to detect and destroy tumour cells, however naturally occurring anti-tumour immunity is not always capable of controlling tumour growth. Immunotherapy can harness the anti-tumour function of the immune system to its full potential and is a promising strategy for the treatment of cancer. Virus-like particles (VLP) are a genome-free subclass of viral nanoparticle derived from the capsid of the parental virus. Incorporation of antigenic peptide sequences into the VLP capsid proteins increases the immunogenicity of the peptide by adopting the ‘viral fingerprint’ of the VLP. VLP derived from the Rabbit Haemorrhagic Disease Virus (RHDV) are been shown to be effective vehicles for the delivery of heterologous antigenic peptide sequences to generate T-cell mediated anti-tumour activity. Inclusion of adjuvants in protein-based vaccines can promote the generation of effective and long-lived anti-tumour responses. The glycolipid α-galactosylceramide activates natural killer T (NKT) cells to express pro-inflammatory cytokines and co-stimulatory molecules that licence antigen-presenting cells and enhance the priming of effective cytotoxic T lymphocytes (CTLs). Therefore, α-GalCer is an effective adjuvant to include in immunotherapies to generate anti-tumour immune responses.

Co-delivery of antigenic peptide sequences in intimate association with adjuvants has been shown to be an effective way of generating anti-tumour immunity by closely mimicking the natural pathway of CTL generation against viral threats. RHDV binds carbohydrate moieties on the surface of epithelial cells to initiate infection. RHDV VLP maintained this affinity for carbohydrate moieties and was able to bind α-GalCer. The complexed particle of RHDV VLP and α-GalCer (VLP/α-GalCer) was able to generate CTL activity against the VLP-incorporated antigen gp33 and protect against a subcutaneous challenge of the B16 tumour expressing the gp33 antigen. It was estimated that a standard 100 μg of VLP/α-GalCer contained the equivalent of 20 ng unassociated α-GalCer. Comparison of vaccination with VLP/α-GalCer and VLP + 20 ng α-GalCer demonstrated a 10-fold benefit to VLP-mediated delivery at the level of gp33-specific CTL activity. This reinforced the concept of co-delivery being beneficial to the generation of CTL responses and justified use of the VLP/α-GalCer as a composite particle.

To understand the mechanism behind the generation of gp33-specific CTL activity, the cellular requirements were determined. RHDV VLP was found predominately within the marginal zone of the spleen after intravenous administration. A subset of langerin+ dendritic cells (DC) located within the marginal zone was shown to be essential for generation of CTLs in response to vaccination with VLP/α-GalCer. The essential role of these DC could not be pinpointed to a single phenotype. Instead, it appeared to be collection of qualities including location, expression of co-stimulatory molecules in response to α-GalCer and potential to express chemokines that contributed to the essential role of the langerin+ DC.

To investigate the potential of the RHDV VLP/α-GalCer vaccine for use in the clinic the immune response to a prime-boost protocol was explored. Multiple doses of α-GalCer are known to ‘stun’ NKT cells into a state of long-term unresponsiveness called ‘anergy’. Nanoparticle mediated delivery of α-GalCer has been shown to permit multiple doses of α-GalCer without causing anergy. However, multiple doses of VLP/α-GalCer rendered the NKT cells anergic and this translated to compromised CTL activity, demonstrating that VLP/α-GalCer could only be used as a single dose vaccine. Antibody responses generated against the capsid protein of RHDV VLP measured as total VLP-specific IgG and was not increased by the inclusion of α-GalCer. A primary vaccination with RHDV VLP followed by a booster vaccination with VLP.gp33/α-GalCer significantly improved the generation of gp33-specific CTL response, suggesting that anti-vector immunity is beneficial for generating CTL responses against RHDV VLP. Therefore, a prime-boost protocol was feasible for RHDV/α-GalCer when α-GalCer was only included in the boost.

These data clearly demonstrate that RHDV VLP complexed with the adjuvant α-GalCer is able to generate effective anti-tumour immunity to a greater degree than either component or both components simply co-administered. Therefore, co-delivery antigen and adjuvant on a VLP scaffold in the form of VLP/able to is a promising, novel immunotherapeutic for the treatment of cancer.