Primers used to clone CARG-2020, VLV-12 and VLV-GFP

Diagram showing major immune phenotypes in each phase of ovarian tumor progression.

A. Mean tumor burden over time of nude mice bearing OCSC1-F2 human ovarian tumors. mCherry fluorescent ROI was used to provide a measure of tumor burden. Data are presented as mean ± SEM. Treatments began on day 6 and continued through to day 12. B. Survival analysis using Kaplan-Meier.

Targeting multiple immune mechanisms may overcome therapy resistance and further improve cancer immunotherapy for humans. Here, we describe the application of virus-like vesicles (VLV) for delivery of three immunomodulators alone and in combination, as a promising approach for cancer immunotherapy. VLV vectors were designed to deliver single chain interleukin (IL)-12, short-hairpin RNA (shRNA) targeting programmed death ligand 1 (PD-L1), and a dominant-negative form of IL-17 receptor A (dn-IL17RA) as a single payload or as a combination payload. Intralesional delivery of the VLV vector expressing IL-12 alone, as well as the trivalent vector (designated CARG-2020) eradicated large established tumors. However, only CARG-2020 prevented tumor recurrence and provided long-term survival benefit to the tumor-bearing mice, indicating a benefit of the combined immunomodulation. The abscopal effects of CARG-2020 on the non-injected contralateral tumors, as well as protection from the tumor cell re-challenge, suggest immune-mediated mechanism of protection and establishment of immunological memory. Mechanistically, CARG-2020 potently activates Th1 immune mechanisms and inhibits expression of genes related to T cell exhaustion and cancer-promoting inflammation. The ability of CARG-2020 to prevent tumor recurrence and to provide survival benefit makes it a promising candidate for its development for human cancer immunotherapy.

Flow cytometry gating strategy CD8 T cells, Macrophages and MDSC.

A. Diagram of CARG-2020 vector and GFP control vector; B. TKO mouse ovarian cancer cells were treated in vitro with PBS Control or 1x105 PFU/cell of CARG-2020 for 24h. IL12 mRNA was quantified by qPCR and secreted IL-12p40 and p70 protein were quantified using xMAP technology; C. Expression of IL-12 and IL-17R ECD were detected by western blot analysis in total cell lysate. D. Secretion of IL-17 RA-ECD was detected by western blot analysis in the supernatant of cells treated with CARG-2020. Control cells were treated with VLV-GFP vector. Representative images of 3 independent experiments. E. Expression of PDL-1 following treatment with CARG-2020. Note the time dependent decrease on PDL-1 expression on cells treated with CARG-2020. Control=VLV-GFP. Representative imagesof 3 independent experiments.

Abstract Current immunotherapies have proven effective in strengthening antitumor immune responses, but constant opposing signals from tumor cells and the surrounding microenvironment eventually lead to immune escape. We hypothesized that in situ release of antigens and regulation of both the innate and adaptive arms of the immune system would provide a robust and long-term antitumor effect by creating immunologic memory against tumors. To achieve this, we developed CARG-2020, a genetically modified virus-like vesicle (VLV) that is a self-amplifying RNA with oncolytic capacity and encodes immune regulatory genes. CARG-2020 carries three immune modulators: (i) the pleiotropic antitumor cytokine IL12, in which the subunits (p35 and p40) are tethered together; (ii) the extracellular domain (ECD) of the protumor IL17RA, which serves as a dominant-negative antagonist; and (iii) a shRNA targeting PD-L1. Using a mouse model of ovarian cancer, we demonstrated the oncolytic effect and immune-modulatory capacities of CARG-2020. By enhancing IL12 and blocking IL17 and PD-L1, CARG-2020 successfully reactivated immune surveillance by promoting M1, instead of M2, macrophage differentiation, inhibiting MDSC expansion and establishing a potent CD8+ T cell–mediated antitumoral response. Furthermore, we demonstrated that this therapeutic approach provided tumor-specific and long-term protection against the establishment of new tumors. Our results provide a rationale for the further development of this platform as a therapeutic modality for ovarian cancer patients to enhance antitumor responses and prevent a recurrence.

A. Kaplan Meier curve comparing overall survival in mice treated with PBS Control, CARG-2020, VLV-IL-12, VLV-17R ECD, VLV-shRNA PD-L1, and VLV-GFP; p<0.0001. B. CARG-2020 but not VLV-IL-12 modulates the innate immune system. Peritoneal lavage from VLV-IL-12 and CARG-2020-treated mice (n=6) were analyzed by flow cytometry for macrophages using CD11b and CD206. VLV-IL-12 treatment is not able to increase the number of CD11blow/CD206- macrophages compared to CARG2020. Representative dot plots shown. Gating strategy is shown in Supp. Fig. 1. C. Decrease on Ly6C+/Ly6G+ MSC is observed only in the CARG2020 treated group but not in those treated with VLV-IL-12 group. Representative dot plots shown. Gating strategy is shown in Supp. Fig. 1.

Current immunotherapies have proven effective in strengthening antitumor immune responses. However, constant opposing signals from tumor cells and the surrounding microenvironment eventually lead to immune escape. We hypothesize that regulating both the innate and adaptive immune system will provide a robust and long-term antitumoral effect by creating an immunological memory against the tumor. To achieve this, we developed CARG-2020, a virus-like vesicle (VLV), delivering three immune modulators (IL-12, IL-17 antagonist, and shRNA-PD-L1). IL-12 is expected to promote T cell expansion. IL-17 antagonist is expected to prevent a tumor-promoting microenvironment. Finally, shRNA-PD-L1 is expected to preclude pro-tumor macrophages and reverse T cell exhaustion.

Background: Immune therapy has been proven successful in multiple types of cancer, but not ovarian. Indeed, ovarian cancer is classified as a “cold tumor” with most tumors exhibiting low levels of T cell infiltrates and poor responses to immune checkpoint inhibitors. It is therefore important to develop better immune modulatory modalities that can improve clinical responses. In this study we evaluated the efficacy of CARG-2020, a virus-like vesicle (VLV) delivering three immune modulators (single chain IL-12, IL-17 antagonist and shRNA for PD-L1) in eliciting an effective and sustained anti-tumoral response to prevent recurrent disease in a syngeneic mouse model of ovarian cancer. Materials and Methods: Triple knockout (TKO; p53LSL-R172H/Dicerflox/flox/Ptenflox/flox) mouse ovarian cancer cells stably expressing the mCherry fluorescent protein were injected i.p. in C57BL/6 mice. Tumor growth was monitored by live imaging using mCherry fluorescence as surrogate for i.p. tumor burden. Treatment commenced when mCherry region of interest (ROI) area reached 20,000 photons/sec. Treatment groups were as follows (n=6/group): 1) PBS Control; 2) 1x10^6 PFU CARG-2020; 3) 1x10^6 PFU VLV-GFP (vector only control). All treatments were given i.p. for a total of 3 doses given 72h apart. Percentage of effector memory, central memory, and naïve CD8 T cells were measured by flow cytometry by staining for CD8, CD44, and CD62L. Results: Treatment with CARG-2020 induced a significant decrease in i.p. tumor burden compared to PBS Control (p=0.0044) and VLV-GFP (p=0.0011). Complete disease regression was observed in all CARG-2020-treated mice with a significant impact on overall survival conferring 100% protection (p=0.0008). Rechallenge of these animals with the same cancer cells failed to form tumors and analysis of splenocytes showed high levels of CD8+/CD44+/CD62- cytolytic effector memory T cells compared to tumor-bearing control and tumor-bearing VLV-GFP-treated mice (p=0.0011). Transfer of splenocytes from long term survivor mice into a new set of tumor-bearing, treatment-naïve mice, induced complete disease regression (p<0.0001, compared to PBS Control) and improved overall survival (p=0.0019, compared to PBS Control). Conclusion: We report the successful anti-tumoral effect of CARG-2020 by modulating the immune response in a syngeneic mouse model of recurrent ovarian cancer. CARG-2020, by enhancing the expression of IL-12 and blocking IL-17 and PD-L1, provided a long-term protection associated with the expansion of cytolytic effector memory CD8+T cells, which in addition to its antitumoral effect, provides protection against recurrent disease. Our results provide rationale for the further development of this platform as a therapeutic modality for ovarian cancer patients. Citation Format: Ayesha B. Alvero, Alexandra Fox, Bhaskara Madina, Marie Krady, Timur O. Yarovinski, Valerian Nakaar, Bijan Almassian, Gil Mor. CARG-2020 a novel immunemodulatory approach to prevent recurrent ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5370.

e14560 Background: Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in humans. Treatment of late-stage CRC remains ineffective, even with the use of latest immunotherapies. Oncolytic viruses have shown limited use for the treatment of cancers, and further improvement of these agents with immune-modulating activities may prove crucial for patients with CRC and other malignancies. To this end, we developed CARG-2020 as an artificial virus for infectious diseases and immuno-oncology (AVIDIO) that employs virus-like vesicles (VLV). VLVs, which are membrane-encapsulated RNA replicons, are oncolytic and can deliver multiple genes resulting in the modulation of several independent immune pathways. Methods: The AVIDIO platform is comprised of in vitro evolved RNA-dependent RNA polymerase from an alphavirus, Semliki Forest virus, and envelope glycoproteins from vesicular stomatitis virus, which together form VLVs. Unarmed or empty vector (VLV), VLV armed with IL-12 (VLV-IL-12) and a VLV that simultaneously expresses IL-12, a dominant-negative form of IL-17 receptor A (dn-1L17RA) and shRNAs targeting PD-L1 (CARG-2020), were given intratumorally to test their therapeutic potential against established (500-600mm 3 ) MC38 tumors in mice. We used tumor growth measurements and analyses of tumor-infiltrating cells after consecutive treatments with these agents to monitor their antitumor and immunomodulatory activities, respectively. Results: Both VLV-IL-12 and CARG-2020 regressed the tumors to undetectable levels in most mice harboring syngeneic MC38 tumors when given intratumorally. CARG-2020, carrying IL-12, dn-IL17RA and PD-L1 shRNA, exerts broader spectrum of immuno-responses and higher number of complete response rates compared with VLV-IL-12. Treatment of primary tumors with VLV-IL-12 or CARG-2020 also significantly repressed the growth of secondary tumors on the other flank of mouse’s body, suggesting an effective systemic immunity elicited by these two agents against the same type of tumors. In addition to the marked local and systemic activation of Th1 cells and CD8 + T cells by both vectors, CARG-2020 also downregulated PD-L1 expression in tumors, and suppressed expression of IL-17A-activated chemokines CXCL1 and CXCL2 that are known to promote cancer development and therapy resistance. Conclusions: As an oncolytic RNA replicon, AVIDIO platform-derived CARG-2020 encodes IL-12, dn-IL-17RA and PD-L1 shRNAs which are expressed concurrently within the same vector. CARG-2020 modulates IL-12, IL-17RA and PD-L1 signaling, and exerts broad immune modulation in tumor microenvironment. Treatment with CARG-2020 eliminates the majority of grafted large tumors in mice, and is effective against both primary and distal tumors. Based on this impressive efficacy results, further development of CARG-2020 in colorectal patients is warranted.

Background: Ovarian cancer is the most lethal of all gynecologic malignancies with a grim 5-year survival rate of ~47%. Mortality occurs in the setting of recurrent disease wherein the co-presentation of chemoresistance and carcinomatosis limits the value of standard of care. It is therefore imperative to develop approaches that can prevent recurrent ovarian cancer. Oncolytic viruses are genetically modified replication competent viruses that can selectively infect cancer cells to induce cell lysis and death. This approach has proven to be clinically safe making it a promising approach to complement standard of care. The objective of this study is to evaluate the efficacy of CARG-2020, a virus-like vesicles (VLV) delivering three immune-modulators including IL-12, IL-17 antagonist and shRNA-PD-L1 in preventing recurrent disease in a syngeneic mouse model of recurrent ovarian cancer. Materials and Methods: In vitro: Triple knockout (TKO; p53LSL-R172H/Dicerflox/flox/Ptenflox/flox) mouse ovarian cancer cells were seeded in 96-well plates and treated with different doses of CARG-2020. Cell death was determined using the Incucyte imaging system. In vivo: TKO ovarian cancer cells stably expressing the mCherry fluorescent protein were injected i.p. in C57bl/6 mice (day 0) to mimic the establishment of recurrent disease. Treatment commenced on day 3 with the treatment group receiving three doses of 1x10^8 PFU of CARG-2020 given every other day (n=5). Control group received PBS (n=5). Both treatments were given i.p. Tumor growth was monitored by live imaging using mCherry fluorescence ROI area. Progression-free survival (PFS) was defined as the day ROI area reached 2,000. Results: CARG-2020 (1x10^5 PFU) demonstrated cytolytic effect in vitro and induced 100% cell death within 24h. In vivo, CARG-2020 induced significant decrease in i.p. tumor growth (p = 0.0128) compared to PBS control. Treatment with CARG-2020 completely delayed the establishment of recurrent disease (PFS, p = 0.0003). Whereas PBS control group demonstrated median PFS of 20 days, mice in the CARG-2020 group remained disease-free until day 60. Conclusion: CARG-2020 is able to prevent the establishment of recurrent disease in a syngeneic mouse model of recurrent ovarian cancer. Our results provide rationale for the further development of this platform as a therapeutic modality for ovarian cancer patients. Citation Format: Ayesha B. Alvero, Alexandra Fox, Bhaskara Madina, Valerian Nakaar, Timur Yarovinsky, Marie Krady, Bijan Almassian, Gil Mor. CARG-2020 artificial oncolytic virus delivering three immune-modulators prevents tumor recurrence in a syngeneic model of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1150.

Virus-like vesicles (VLV) are hybrid vectors based on an evolved Semliki Forest virus (SFV) RNA replicon and the envelope glycoprotein (G) from vesicular stomatitis virus (VSV). Previously, we showed that VLV can be used to express protein antigens and generate protective antigen-specific CD8(+)T cells. This report describes VLV vectors designed for enhanced protein expression and immunogenicity. Expressing hepatitis B virus (HBV) middle S antigen (MHBs) from VLV using a dual subgenomic promoter significantly increased MHBs-specific CD8(+)T cell and antibody production in mice. Furthermore, envelope glycoprotein switch from VSV Indiana to the glycoprotein of Chandipura virus enabled prime-boost immunization and further increased responses to MHBs. Therapeutic efficacy was evaluated in a mouse model of chronic HBV infection initiated by HBV delivery with adeno-associated virus. Mice with lower or intermediate HBV antigen levels demonstrated a significant and sustained reduction of HBV replication following VLV prime-boost immunization. However, mice with higher HBV antigen levels showed no changes in HBV replication, emphasizing the importance of HBV antigenemia for implementing immunotherapies. This report highlights the potential of VLV dual promoter vectors to induce effective antigen-specific immune responses and informs the further development and evaluation of hybrid viral vaccine platforms for preventative and therapeutic purposes.

e15210 Background: Colorectal cancer is the third deadliest of all cancers causing more than 50,000 deaths per year in the U.S. Oncolytic viruses have seen limited use for the treatment of cancers, and further improvement of these methods with immune-modulating activities may prove crucial for the effectiveness of these agents in the treatment of human malignancies. To this end, we developed an artificial virus for infectious diseases and immuno-oncology (AVIDIO) platform that employs virus-like vesicles (VLV) for both the delivery of immunomodulatory agents to tumors and oncolytic activity. Methods: The AVIDIO platform is comprised of in vitro evolved RNA-dependent RNA polymerase from an alphavirus, Semliki forest virus, and envelope glycoproteins from vesicular stomatitis virus, which together form VLVs. Both unarmed VLVs and VLVs armed with the p35 subunit of IL-12 (VLV-IL12p35), an immunomodulatory cytokine that can induce Th1-mediated immunity, were tested for oncolytic activity against various cancer cell lines, including MC38 colorectal cancer cells, in vitro. Using the MC38 syngeneic murine tumor model, we evaluated the antitumor activity of VLV-IL-12p35 in vivo. We used tumor growth measurements and analyses of tumor-infiltrating cells after consecutive treatments with VLV-IL-12p35 to monitor its antitumor and immunomodulatory activities, respectively. Results: VLV-IL-12p35 showed robust oncolytic activity against MC38 cells in vitro, killing over 80% of cells within 24 h. Treatment of intradermal MC38 tumors by intra-tumoral delivery of VLV-IL-12p35 resulted in more than 65% suppression of tumor growth within 2 weeks ( p< 0.05). VLV-IL-12p35-treated tumors also harbored significantly more CD8+ T cells, IFN-gamma-producing CD4+ T cells, and reduced numbers of Foxp3+ regulatory T cells. Conclusions: Our results show that VLV-IL-12p35 derived from the AVIDIO platform has oncolytic activity in vitro and antitumor and immunomodulatory activities in vivo. Therefore, AVIDIO is a promising platform for the delivery of immunomodulatory agents to tumors. Further optimization of the platform, including the addition of other immunomodulatory agents, is in progress to advance the AVIDIO platform to clinical applications for colorectal cancer.

Infections with hepatitis B virus (HBV) can initiate chronic hepatitis and liver injury, causing more than 600,000 deaths each year worldwide. Current treatments for chronic hepatitis B are inadequate and leave an unmet need for immunotherapeutic approaches. We designed virus-like vesicles (VLV) as self-amplifying RNA replicons expressing three HBV antigens (polymerase, core, and middle surface) from a single vector (HBV-VLV) to break immune exhaustion despite persistent HBV replication. The HBV-VLV induces HBV-specific T cells in naive mice and renders them resistant to acute challenge with HBV. Using a chronic model of HBV infection, we demonstrate efficacy of HBV-VLV priming in combination with DNA booster immunization, as 40% of treated mice showed a decline of serum HBV surface antigen below the detection limit and marked reduction in liver HBV RNA accompanied by induction of HBsAg-specific CD8 T cells. These results warrant further evaluation of HBV-VLV for immunotherapy of chronic hepatitis B.