Glycoprotein D (gD)
Mechanism of Action and Efficacy
Glycoprotein D (gD) is a viral fusion protein that herpes simplex virus (HSV) utilizes to infect human cells upon binding to Herpes Virus Entry Mediator (HVEM). HVEM is a ubiquitous protein found on different types of human cells including antigen presenting cells (APC). HVEM on APCs interacts with naïve T cells through a number of ligands that control CD8+ T cell activation (Figure 1A) - B and T-Lymphocyte Attenuator (BTLA) binding results in inhibition, LIGHT binding, which occurs at a different domain on HVEM than that of BTLA, produces stimulation and when both ligands are bound to HVEM in a tri-molar complex, BTLA’s inhibitory signaling predominates. HSV gD and BTLA bind to the same domain on HVEM, however gD binds with higher affinity and thereby blocks the HVEM-BTLA interaction (Figure 1B). Virion has strategically utilized gD as a novel checkpoint inhibitor of the immunosuppressive BTLA-HVEM pathway. A specific genetic construct of gD, fused to a disease-specific antigen of choice, is inserted into a vaccine vehicle and upon vaccination the gD protein is expressed on the APC’s surface where it can bind to HVEM and thereby block its interactions with BTLA without reducing LIGHT binding (Figure 1C). Part of the gD-antigen fusion protein is degraded and then enters the antigen presentation pathways so that the same cells that express gD-HVEM-LIGHT complexes also express antigenic peptides displayed on major histocompatibility antigens. Concomitant display of gD and the antigen blocks the BTLA-HVEM interactions, thereby removing BTLA’s inhibitory signal and allowing LIGHT’s co-stimulatory signaling to predominate – CD8+ T cell responses to the vaccine antigen at the time of T cell stimulation are more potent and sustained and they are broadened to subdominant epitopes, which renders them more resistant to immune exhaustion within an immunosuppressive microenvironment. This novel mechanism of early checkpoint inhibition that acts locally at the onset of the immune response has shown promising therapeutic efficacy in several settings, including: animal models of fast and slow growing tumors, with viral or human cancer antigens, in young and older animals, and when inserted into DNA or adenovirus vaccine delivery platforms (both human and chimpanzee). In addition, cancer vaccines based on DNA or adenovirus vectors that only had marginal effects on tumor progression were rendered highly efficacious upon insertion of gD.
Figure 1: Glycoprotein D (gD) - Mechanism of Action for Checkpoint Inhibition
The best example of gD as a checkpoint inhibitor is shown using a transgenic mouse model of HPV-16 E7-induced thyroid adenocarcinoma (Lasaro et al. Mol. Ther. 2011). In contrast to most mouse models of transplanted cancer cells where tumors rapidly appear and mice have to be euthanized after relatively short periods of time, these thyroid tumors, which are caused by the HPV-16 E7 oncoprotein, appear at roughly 6 months of age. They grow slowly and a tumor microenvironment that results in T cell exhaustion, a condition commonly found in human cancers. As can be seen in Figure 2, the thyroids of non-genetically altered mice are small and have consistent texture throughout the tissues (far right column). In contrast, genetically altered mice have by one year of age large thyroids that largely consist of tumor cells (far left column). Upon vaccination with an HPV-16 E7 vaccine (directly targeting the protein causing the thyroid tumors) via an adenoviral vector (which induces potent CD8+ T-cells) the thyroid tumors show little to no decrease in size or improvements upon histologic evaluation (second from left column). When gD is added to this same vaccine, the tumors shrink and when boosted with a second gD HPV-16 E7 adenovirus vaccine, the tumors shrink even more (third and fourth columns from the left).
Figure 2: Efficacy of gD-antigen Vaccination in Slow Growing Tumors When Added to Ineffective Adenovirus Vaccine
In both cases, thyroids show large areas of necrosis and fibrosis indicating immune-mediated destruction of tumor cells. The thyroids of non-gD treated animals have low numbers of interferon gamma producing HPV-16 E7 specific CD8+ T cells and high numbers of regulatory T cells, findings consistent with an immunosuppressive tumor microenvironment (Figure 3). In contrast, tumors of mice vaccinated with the gD-E7 expressing vaccine have high frequencies of interferon gamma producing HPV-16 E7 specific CD8+ T cells and a delayed infiltration of regulatory T cells, results consistent with checkpoint inhibition. The impact of the gD-E7 vaccine on tumor size, histology and within the thyroid tumor microenvironment persist out to 6-months, even in the single gD vaccinated animals.