1116 VAX014 as a novel oncolytic agent for STING and RIG-I-positive solid tumors

<h3>Background</h3> Oncolytic virus (OV)-based therapies have demonstrated considerable promise for treating various solid tumor types. However, mounting evidence indicates OVs are negatively impacted by Type I interferon(s) (IFN) produced in solid tumors with functional cytosolic nucleic acid sensing mechanisms, including the cGAS/STING and/or RIG-I pathways.^{1 2} VAX014 is a novel, clinical stage oncolytic agent based on recombinant bacterial minicells (rBMCs) and is designed to target and deliver a pre-formed oncolytic protein toxin, perfringolysin O (PFO), directly to integrin-expressing tumor cells.^{3 4} Here we characterize the interaction and impact of tumor intrinsic STING and RIG-I on the antitumor activity of VAX014 <i>in vitro</i> and <i>in vivo</i>. <h3>Methods</h3> Targeted deletion of STING or RIG-I in wild type MB49 murine urothelial carcinoma cells was performed via CRISPR-Cas9. Following extensive orthogonal <i>in vitro</i> functional characterization to confirm genotype/phenotype, MB49^{STING KO} and MB49^{RIG-I KO} tumor cell lines were utilized in <i>in vivo</i> preclinical pharmacology studies in wild type C57BL/6 mice to assess any influence of tumor intrinsic STING and/or RIG-I on the efficacy of VAX014 following intratumoral (i.t.) administration to intradermal (i.d.) tumors. Individual tumor growth rates and survival curves were plotted against respective saline and wild type controls. <h3>Results</h3> <i>In vitro</i> analysis demonstrated wild type MB49 cells express STING and RIG-I (but not cGAS) and upregulate PD-L1 and MHC-I in a Type I IFN and Tank-binding kinase-1 (TBK-1) dependent manner following treatment with VAX014. Targeted genetic ablation of either STING or RIG-I (MB49^{STING KO} and MB49^{RIG-I KO}) reduced this response. In MB49^{STING KO} cells, pharmacologic inhibition of TBK-1 led to complete elimination of Type I IFN production and subsequent PD-L1/MHC-I upregulation, as did pharmacologic inhibition of STING in MB49^{RIG-I KO} cells. <i>In vivo,</i> both the MB49^{STING KO} and MB49^{RIG-I KO} tumor cell lines had similar i.d. tumor growth rates to wild type MB49. Consistent with previous work, weekly i.t. administration of VAX014 to wild type MB49 tumors led to a 100% durable complete response (CR) rate. In contrast, the loss of tumor intrinsic STING or RIG-I reduced CR rates and lengthened the time to respond in MB49^{STING KO} or MB49^{RIG-I KO} tumors. <h3>Conclusions</h3> VAX014 activates both the murine STING and RIG-I pathways and the presence of tumor-intrinsic STING and/or RIG-I leads to optimal antitumor activity of VAX014 following i.t. administration. This unique mechanism pairs STING and RIG-I agonism and subsequent Type I IFN production with oncolysis-mediated availability of tumor antigens, which together, may lead to better antitumor T cell priming. <h3>Acknowledgements</h3> We would like to acknowledge Steven N. Fiering, Ph.D and Trevor Hallam, Ph.D for useful discussions, review, and interpretation of study data. <h3>Ethics Approval</h3> This study was approved by San Diego State University’s Institutional Animal Care and Use Committee under approved Animal Protocol number IACUC-22–064. <h3>References</h3> Kaufman HL, Shalhout SZ, Iodice G. Talimogene Laherparepvec: Moving From First-In-Class to Best-In-Class. <i>Front. Mol. Biosci</i>. 2022;<b>9</b>:834841 Li Q, Tan F, Wang Y, Liu X, Kong X, Meng J, Yang L, Cen S. The gamble between oncolytic virus therapy and IFN. <i>Front. Immunol</i>. 2022;<b>13</b>:971674 Tsuji S, Reil KA, Nelson K, Proclivo VH, McGuire KL, Giacalone MJ. Intravesical VAX014 Synergizes with PD-L1 Blockade to Enhance Local and Systemic Control of Bladder Cancer. <i>Cancer Imunol. Res</i>. 2022;<b>10</b>:978–95. Reil KA, Tsuji S, Molina E, Nelson KL, McGuire KL, Giacalone MJ. Intralesional administration of VAX014 facilitates in situ immunization and potentiates immune checkpoint blockade in immunologically cold tumors. <i>J. Immunother Cancer</i>. <b>11</b>:e006749