Supplementary Figure S2 with legend

Abstract IFN regulatory factor 1 (IRF1) can promote antitumor immunity. However, we have shown previously that in the tumor cell, IRF1 can promote tumor growth, and IRF1-deficient tumor cells exhibit severely restricted tumor growth in several syngeneic mouse tumor models. Here, we investigate the potential of functionally modulating IRF1 to reduce tumor progression and prolong survival. Using inducible IRF1 expression, we established that it is possible to regulate IRF1 expression to modulate tumor progression in established B16-F10 tumors. Expression of IRF2, which is a functional antagonist of IRF1, downregulated IFNγ-induced expression of inhibitory ligands, upregulated MHC-related molecules, and slowed tumor growth and extended survival. We characterized the functional domain(s) of IRF2 needed for this antitumor activity, showing that a full-length IRF2 was required for its antitumor functions. Finally, using an oncolytic vaccinia virus as a delivery platform, we showed that IRF2-expressing vaccinia virus suppressed tumor progression and prolonged survival in multiple tumor models. These results suggest the potency of targeting IRF1 and using IRF2 to modulate immunotherapy.

Supplementary Video 2 MPG file - 1158K, 3-dimensional tumor model of 10 cm HCC tumor depicted in Fig. 4b (baseline; 5 days post JX-594 treatment). Beige = perfused; green = hypoperfused; red = necrotic

Supplementary Fig. from CNOB/ChrR6, a new prodrug enzyme cancer chemotherapy

IFNγ resistance in E.G7-OVA and P815 tumors.

Characterization of myeloid cells in Luc-E.G7 tumor bearing eyes.

Supplementary Fig. from CNOB/ChrR6, a new prodrug enzyme cancer chemotherapy

Background: TGFB1 mediated immune resistance is one of the major mechanisms of immune suppression utilized across multiple tumor types. Immune resistance imparted by TGFB1 is mediated through its pleiotropic effects on vasculature, fibrogenesis and regulatory/effector immune cells within the tumor microenvironment. Blockade of TGFB1 (TGFBi) will likely improve response to immunotherapy. IL-12 is a cytokine that through IFNg induction, promotes type 1 inflammatory response, M1 macrophages and effector CD8 T cell response. Combining TGFB1 blockade with IL-12 may maximize therapeutic benefits through simultaneously reducing immunosuppression and enhancing anti-tumor immune response. Current studies have developed a vaccinia-based immunotherapy, combining enhanced systemic virus delivery to CXCR3 ligand rich tumors and locally expressed IL-12 and TGFBi within the tumor microenvironment, for efficient control of multiple tumor models. Methods: An oncolytic vaccinia virus expressing CXCR3, IL-12 and a TGFB1 antagonizing mini-monomer was constructed (VET3-TGI) and the expression and function of the transgenes were confirmed. Using in vivo mouse RENCA, EMT-6 and MC38 tumor models, the functionality and therapeutic efficacy of VET-TGI were tested with comparison to control virus. Post-mortem analysis was used to analyze the impact of VET3-TGI on immune/stromal/endothelial milieu of the tumors and to determine toxicity profile. Results: VET3-TGI infected cells expressed CXCR3 and showed enhanced migration to CXCR3 ligands in vitro and improved systemic delivery to tumors expressing CXCR3 ligands in vivo, even in the face of pre-existing anti-viral immunity. IL-12 expression and TGFBi blockade of TGFB1 mediated suppression of CD8 T cell proliferation were confirmed in vitro. In vivo mouse studies using EMT6, RENCA and MC38 tumor models demonstrated potent therapeutic activity, including 100% CRs, even at doses several logs below equivalent clinical doses and in multiple models. Mechanism of activity studies suggested that the therapeutic efficacy of VET3-TGI is associated with considerable modification of the tumor microenvironment. In addition, preliminary toxicity studies demonstrated the safety of VET3-TGI in mouse models. Conclusions: VET3-TGI demonstrated an ability to reduce immunosuppression and dramatically enhance antitumor immune response leading to safe and potent therapeutic activity in multiple mouse tumor models. This data led to the selection of VET3-TGI as our lead clinical candidate. A human version of the virus is currently undergoing clinical manufacture and toxicology testing. Citation Format: Ravikumar Muthuswamy, Steve Thorne. The oncolytic virus VET3-TGI both blocks TGF-beta signaling and activates type 2 IFN responses, resulting in potent therapeutic responses in multiple mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6789.

Supplementary Methods PDF file - 44K, GLP toxicology study, In vivo imaging and mouse immunohistochemical analyses

Background The clinical success of oncolytic viral immunotherapies will likely require development of technologies to achieve targeted intravenous delivery to the tumor bed, and the expression of novel therapeutic transgene combinations to simultaneously destroy the tumor through multiple mechanisms. Methods VET3-TGI has been designed using the VETTM viral backbone to achieve systemic delivery, even in the face of pre-existing anti-viral immunity. This was achieved through insertion of the chemokine receptor CXCR3 into the viral backbone. CXCR3 is then expressed in hematopoietic cells that become infected soon after intravenous delivery of the viral therapy, resulting in directed trafficking of these cells towards tumors expressing matching chemokines. This was demonstrated in vitro and in vivo in both naïve and immunized or pre-treated mouse tumor models. Further, VET3-TGI contains a novel therapeutic transgene combination, combining a TGFb1 inhibitor (TGFb mini-monomer, TGFbMM) with IL12 expression. Counteracting the immunosuppressive activity of TGFb1 and enhancing Type-1 immune responses in the tumor microenvironment led to greatly increased anti-tumor immunity and durable complete responses. Results The therapeutic efficacy of VET3-TGI over control viruses was tested in multiple pre-clinical in vivo mouse tumor models (including B16, MC38, RENCA, EMT6), and potent therapeutic activity was demonstrated, including 100% CRs in multiple models, even at doses several logs below expected equivalent clinical doses. Post-mortem analysis showed that VET3-TGI reduced systemic toxicity and improved systemic delivery. Analysis of the tumor microenvironment revealed profound changes with VET3-TGI treatment, including greatly enhanced infiltration of CD3+CD8+ T cell, polarization to a type-1 immune response and concomitant decreases in TGFb1associated genes. Conclusions Altogether, VET3-TGI demonstrated good ability to counter TGFb1 mediated immunosuppression and dramatically enhanced anti-tumor immune responses leading to safe and potent therapeutic activity in multiple mouse tumor models. Based on the above, VET3-TGI was selected as a lead clinical candidate and clinical manufacture and toxicology testing is undergoing with a human version of this virus.

Inflammatory gene expression within intraocular Luc-E.G7 tumors.

Supplementary Figure legends

While checkpoint blockade immunotherapies have widespread success, they rely on a responsive immune infiltrate; as such, treatments enhancing immune infiltration and preventing immunosuppression are of critical need. We previously generated αPD-1 resistant variants of the murine HNSCC model MEER. While entirely αPD-1 resistant, these tumors regress after single dose of oncolytic vaccinia virus (VV). We then generated a VV-resistant MEER line to dissect the immunologic features of sensitive and resistant tumors. While treatment of both tumor types induced immune infiltration and IFNγ, we found a defining feature of resistance was elevation of immunosuppressive cytokines like TGFβ, which blunted IFNγ signaling, especially in regulatory T cells. We engineered VV to express a genetically encoded TGFβRII inhibitor. Inhibitor-expressing VV produced regressions in resistant tumor models and showed impressive synergy with checkpoint blockade. Importantly, tumor-specific, viral delivery of TGFβ inhibition had no toxicities associated with systemic TGFβ/TGFβR inhibition. Our data suggest that aside from stimulating immune infiltration, oncolytic viruses are attractive means to deliver agents to limit immunosuppression in cancer.

Supplementary Figure 1 PDF file - 74K, VEGF- and FGF-2 stimulate vaccinia replication

P815 tumor cell line authentication.