Abstract 5673: Large established B16 tumors in mice are eradicated by ZVex® (dendritic cell-targeting lentiviral vector) and G100 (TLR4 agonist) combination immunotherapy through increasing tumor-infiltrating effector T cells and inducing antigen spreading
Authors: Albershardt Tina C. , Parsons Andrea J. , Leleux Jardin , Berglund Peter , Meulen Jan ter 
Published: July 2017

Abstract
INTRODUCTION: Effective immunotherapy requires the presence of effector T cells penetrating the tumor. ZVex is a hybrid lentiviral vector platform that targets dendritic cells in vivo to express genes of tumor-associated antigen (TAA)-of-interest and activate TAA-specific CD8 T cells. G100 is the intratumoral (IT) injection of formulated glucopyranosyl lipid A (a synthetic TLR4 agonist) and has been shown to induce T cell homing chemokines, CXCL9 and CXCL10. We report here that G100 promoted an inflamed tumor microenvironment (TME) and improved infiltration of ZVex-induced TAA-specific CD8 T cells to the TME, thereby eradicating large established B16 tumors. This was previously achieved only with a complex vaccine/anti-tumor antibody/checkpoint inhibitor/IL-2 regimen (Moynihan, Nature, 2016).
RESULTS: B16-OVA tumor-bearing mice were randomized into 4 treatment cohorts: 1) untreated; 2) ZVex expressing ovalbumin (ZVex/OVA), subcutaneously (SC); 3) G100 (IT); 4) ZVex/OVA (SC) and G100 (IT) combination. While mice in Cohorts 2 and 3 exhibited delayed tumor growth, nearly all mice (16/18) from Cohort 4 had completely regressed tumors and survived tumor-free until end of study (109 days). CD8 T cell depletion abrogated this anti-tumor response. Cohort 4 mice with regressed tumors were then randomized and re-challenged with either a) B16-OVA or b) parental B16 (lacking expression of the ZVex-targeted antigen, OVA): a) B16-OVA re-challenge was rejected by 100% of recipient mice - consistent with induction of T cell memory; and b) B16 re-challenge was rejected by 30%-50% of recipient mice - demonstrating functional antigen spreading. T cell receptor deep sequencing showed that ZVex/OVA alone increased tumor-infiltrating T cell clones specific for OVA, validated by pMHC-multimer staining. ZVex/OVA and G100 combination expanded additional T cell clones, further evident of antigen spreading. Lastly, the TME of Cohort 4 mice showed the most profound pro-inflammatory changes, as assessed by RNA transcriptional profiling.
CONCLUSIONS: These data collectively demonstrate that anti-tumor efficacy observed in Cohort 4 mice was mediated largely by ZVex/OVA-induced effector T cells and that TME modulation with G100 drastically shifted the TME to a more inflamed milieu, promoting T cell proliferation, antigen spreading, and generation of immunological memory. To our knowledge, this is the first time that large established B16 tumors have been completely eradicated using a combination of systemic and in situ immunizations, a potentially effective strategy to convert non-inflamed tumors to inflamed tumors. Both ZVex and G100 are in multiple clinical trials, and their combination is currently being investigated in a phase I trial in soft tissue sarcoma patients with injectable tumors.
Citation Format: Tina C. Albershardt, Andrea J. Parsons, Jardin Leleux, Peter Berglund, Jan ter Meulen. Large established B16 tumors in mice are eradicated by ZVex® (dendritic cell-targeting lentiviral vector) and G100 (TLR4 agonist) combination immunotherapy through increasing tumor-infiltrating effector T cells and inducing antigen spreading [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5673. doi:10.1158/1538-7445.AM2017-5673