Fibroblast activation protein (FAP) is overexpressed in cancer-associated fibroblasts and is an interesting target for cancer immune therapy, with prior studies indicating a potential to affect the tumor stroma. Our aim was to extend this earlier work through the development of a novel FAP immunogen with improved capacity to break tolerance for use in combination with tumor antigen vaccines. We used a synthetic consensus (SynCon) sequence approach to provide MHC class II help to support breaking of tolerance. We evaluated immune responses and antitumor activity of this novel FAP vaccine in preclinical studies, and correlated these findings to patient data. This SynCon FAP DNA vaccine was capable of breaking tolerance and inducing both CD8 and CD4 immune responses. In genetically diverse, outbred mice, the SynCon FAP DNA vaccine was superior at breaking tolerance compared with a native mouse FAP immunogen. In several tumor models, the SynCon FAP DNA vaccine synergized with other tumor antigen-specific DNA vaccines to enhance antitumor immunity. Evaluation of the tumor microenvironment showed increased CD8 T-cell infiltration and a decreased macrophage infiltration driven by FAP immunization. We extended this to patient data from The Cancer Genome Atlas, where we find high FAP expression correlates with high macrophage and low CD8 T-cell infiltration. These results suggest that immune therapy targeting tumor antigens in combination with a microconsensus FAP vaccine provides two-fisted punch-inducing responses that target both the tumor microenvironment and tumor cells directly. .

Antibody-based immune therapies targeting the T cell checkpoint molecules CTLA-4 and PD-1 have impacted cancer therapy. However, this immune therapy requires complex manufacturing and frequent dosing, limiting the global use of this treatment. Here we focused on the development of a DNA-encoded monoclonal antibody (DMAb) approach for delivery of anti-CTLA-4 monoclonal antibodies in vivo. With this technology, engineered and formulated DMAb plasmids encoding IgG inserts were directly injected into muscle and delivered intracellularly by electroporation, leading to in vivo expression and secretion of the encoded IgG. DMAb expression from a single dose can continue for several months without the need for repeated administration. Delivery of an optimized DMAb encoding anti-mouse CTLA-4 IgG resulted in high serum levels of the antibody as well as tumor regression in Sa1N and CT26 tumor models. DNA-delivery of the anti-human CTLA-4 antibodies ipilimumab and tremelimumab in mice achieved potent peak levels of approximately 85μg/mL and 58μg/mL, respectively. These DMAb exhibited prolonged expression, with maintenance of serum levels at or above 15μg/mL for over a year. Anti-human CTLA-4 DMAbs produced in vivo bound to human CTLA-4 protein expressed on stimulated human PBMC and induced T cell activation in a functional assay ex vivo. In summary, direct in vivo expression of DMAb encoding checkpoint inhibitors serve as a novel tool for immunotherapy that could significantly improve availability and provide broader access to such therapies.

Pharmaceuticals and, as such, receive salary and benefits, including ownership of stock and stock options. KM receives grants and consulting fees from Inovio Pharmaceuticals related to DNA vaccine development. DBW has received grant funding, participates in industry collaborations, has received speaking honoraria, and has received fees for consulting, including serving on scientific review committees and board series. Remuneration received by DBW includes direct payments, stock, or stock options, and, in the interest of disclosure, he notes potential conflicts associated with his work with Inovio Pharmaceuticals and possibly others. MCW and DBW have a pending US patent, 62750213.

Immune checkpoint blockade antibodies are setting a new standard of care for cancer patients. It is therefore important to assess any new immune-based therapies in the context of immune checkpoint blockade. Here, we evaluate the impact of combining a synthetic consensus TERT DNA vaccine that has improved capacity to break tolerance with immune checkpoint inhibitors. We observed that blockade of CTLA-4 or, to a lesser extent, PD-1 synergized with TERT vaccine, generating more robust anti-tumor activity compared to checkpoint alone or vaccine alone. Despite this anti-tumor synergy, none of these immune checkpoint therapies showed improvement in TERT antigen-specific immune responses in tumor-bearing mice. αCTLA-4 therapy enhanced the frequency of T-bet/CD44 effector CD8 T cells within the tumor and decreased the frequency of regulatory T cells within the tumor, but not in peripheral blood. CTLA-4 blockade synergized more than Treg depletion with TERT DNA vaccine, suggesting that the effect of CTLA-4 blockade is more likely due to the expansion of effector T cells in the tumor rather than a reduction in the frequency of Tregs. These results suggest that immune checkpoint inhibitors function to alter the immune regulatory environment to synergize with DNA vaccines, rather than boosting antigen-specific responses at the site of vaccination.

Purpose: Cancer/testis antigens have emerged as attractive targets for cancer immunotherapy. Clinical studies have targeted MAGE-A3, a prototype antigen that is a member of the MAGE-A family of antigens, in melanoma and lung carcinoma. However, these studies have not yet had a significant impact due to poor CD8+ T cell immunogenicity, platform toxicity, or perhaps limited target antigen availability. In this study we develop an improved MAGE-A immunogen with cross-reactivity to multiple family members. Experimental Design: In this study we analyzed MAGE-A expression in The Cancer Genome Atlas and observed that many patients express multiple MAGE-A isoforms, not limited to MAGE-A3, simultaneously in diverse tumors. Based on this, we designed an optimized consensus MAGE-A DNA vaccine capable of cross-reacting with many MAGE-A isoforms, and tested immunogenicity and anti-tumor activity of this vaccine in a relevant autochthonous melanoma model. Results: Immunization of this MAGE-A vaccine by electroporation in C57Bl/6 mice generated robust IFN-γ and TNF-α CD8+ T cell responses as well as cytotoxic CD107a/IFN-γ/T-bet triple-positive responses against multiple isoforms. Furthermore, this MAGE-A DNA immunogen generated a cross-reactive immune response in 14 out of 15 genetically diverse, outbred mice. We tested the anti-tumor activity of this MAGE-A DNA vaccine in Tyr::CreER;BRAFCa/+;Ptenlox/lox transgenic mice that develop melanoma upon tamoxifen induction. The MAGE-A DNA therapeutic vaccine significantly slowed tumor growth and doubled median mouse survival. Conclusions: These results support the clinical use of consensus MAGE-A immunogens with the capacity to target multiple MAGE-A family members to prevent tumor immune escape.