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Multiple species of oncolytic viruses (OVs) have been shown to modulate the tumor microenvironment (TME) by increasing immune cell infiltration and activating stimulatory immune responses, leading to the induction of a tumor-specific immune response. Many engineered OVs achieve tumor specificity by either gene deletion or mutations and are then armed with immunomodulatory transgenes to promote anti-tumor immune responses. In an alternative approach, OVs derived from Codagenix’s codon/codon pair modification platform aim to leverage the natural immunostimulatory capacity of selected viral species for efficacy and take advantage of defects in innate immune sensing and apoptosis mechanisms in cancer cells as well as receptor overexpression for tumor selectivity. CodaLytic is a novel virotherapeutic derived from influenza virus strain A/California/07/2009, that is synthetically engineered to contain over 600 silent mutations in hemagglutinin and neuraminidase genes and is being developed as a novel immunotherapeutic for breast cancer. In the orthotopic EMT6 triple-negative breast cancer model, known for its moderate sensitivity to immunotherapies, intratumoral injection of 108 PFU three times a week for up to 4 weeks as a monotherapy led to significant tumor growth inhibition by 76% (p < 0.001 vs vehicle control), translating into a significant survival benefit with a 66% cure rate. Intravenous rechallenge of EMT6 long-term survivors led to a 27-fold reduction in lung nodule formation and a tumor-specific interferon-γ memory response was observed ex vivo in their splenocytes. Anti-tumor efficacy after CodaLytic treatment was accompanied by a change in the composition of the tumor immune infiltrate with significant increases in T, B and NK cells and increased gene expression of pathways and genes related to T cell effector function, dendritic cell activation, antigen presentation and chemoattraction. In immunotherapy-resistant orthotopic 4T1 tumors, combination of CodaLytic with a CTLA-4 inhibitory antibody, but not anti-PD-1 blockade – a combination that had demonstrated combination benefit in several other preclinical models – led to reduction in tumor growth by 75% (p < 0.0001 vs vehicle control and PD-1 combination group). Median overall survival improved from 22 to 30 days with addition of CTLA-4 blockade. Triple combination including PD-1 inhibition led to improved long-term survival beyond 50 days with 30% complete regressions. In summary, these preclinical data demonstrate CodaLytic’s ability to induce broad innate and adaptive changes in the breast cancer TME, resulting in anti-tumor efficacy and prolonged survival. Characterization of the tumor immune infiltrate after CTLA-4 combination and pharmacodynamic changes achieved after CodaLytic/checkpoint combination treatment in human breast cancer tumoroids will further support identification of correlates of efficacy with translational implications. Together with preclinical toxicology data and demonstrated clinical safety of this attenuated influenza virus after intranasal administration in healthy individuals, CodaLytic emerges as a promising novel viroimmunotherapeutic agent and is planned to enter a phase 1 clinical trial in early 2023. Citation Format: Marcin Stawowczyk, Yiwen Zhao, Katie Pfeffer, Juliana Tafrova, James Rodriguez, Chen Yang, Nusrat Jahan, Sybil A. Tasker, Steffen Mueller, J. Robert Coleman, Johanna K. Kaufmann. Preclinical development of CodaLytic™, a codon-modified influenza virus, as a novel virotherapeutic agent for breast cancer immunotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-04.

Multiple oncolytic viruses have been shown to induce various beneficial changes in the tumor microenvironment (TME), which ultimately support induction of anti-tumor immune responses. We have previously demonstrated that CodaLytic, a codon-modified influenza virus, can kickstart the cancer immunity cycle at various steps in several mouse models with differing baseline immune contextures. Here, we are confirming the mechanisms of action of the virus in combination with immune checkpoint inhibition in murine and human preclinical models. Efficacy after intratumoral injection of 108 PFU CodaLytic in combination with αPD-1 and/or αCTLA-4 checkpoint blockade was determined in αPD-1-resistant B16-F10 melanoma and 4T1 triple-negative breast cancer mouse models. Changes in the tumor immune infiltrate after treatment were characterized using flow cytometry. Primary human tumoroids were incubated with CodaLytic +/- 10 μg/ml pembrolizumab using the 3D-Explore platform by Nilogen Oncosystems. Tumor cell killing (TCK) was detected by high-content imaging at 72h and effects on the TME were characterized by cytokine release and RNA sequencing at 24h and/or 48h. In B16-F10, addition of CodaLytic to αPD-1 blockade rescued non-significant tumor growth inhibition (TGI) with αPD-1 alone, achieving 86% TGI (p < 0.01), 40% complete regressions and 60% survival beyond 45 days (69% TGI, 20% regressions and 30% long-term survival after CodaLytic monotherapy). Mechanistically, CodaLytic-containing regimens increased tumor immune infiltration, in particular with CD8+ T cells and cross-presenting dendritic cells (DCs). 4T1 tumors did not significantly respond to αPD-1, αCTLA-4 or CodaLytic monotherapies, but achieved 86% TGI in combination with 50% long-term survival past 55 days. CTLA-4 blockade emerged as the initial driver of slowed tumor growth and αPD-1 drove long-term survival, which was further augmented by CodaLytic addition. Infiltration with CD45+ leukocytes and cross-presenting DCs correlated with tumor volumes. The benefit of CodaLytic combination treatment was also demonstrated ex vivo in human tumoroid cultures. Meaningful TCK was observed in 50% of specimens vs 17% with pembrolizumab and 33% with CodaLytic alone. In this system without immune cell recruitment from a systemic reservoir, sustained release of CXCL9 and CXCL10 and temporal decreases of MIP-1β and CCL2 associated with response. Additional transcriptional analyses are ongoing. Taken together, this and additional preclinical data confirms the emergence of CodaLytic as a potent immunostimulatory agent capable of restoring immune cell infiltration and anti-tumor efficacy when used in combination with checkpoint inhibition in PD-1 resistant models. This data supports future development of CodaLytic for immuno-virotherapy of tumor types in which checkpoint therapy is less established. Citation Format: Yiwen Zhao, Nusrat Jahan, Marcin Stawowczyk, Katie Pfeffer, Juliana Tafrova, James Rodriguez, Chen Yang, Sybil A. Tasker, Steffen Mueller, J. Robert Coleman, Johanna K. Kaufmann. The codon-modified influenza virus CodaLytic™rescues immunostimulatory activity and anti-tumor efficacy in PD-1-refractory tumor 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 689.

BackgroundOncolytic viruses (OVs) of multiple species have been demonstrated to induce beneficial changes in the tumor microenvironment (TME), increasing immune cell infiltration and activating stimulatory immune responses, which ultimately support induction of an anti-tumor immune response. The majority of OVs are attenuated by either gene deletion or mutations and then armed with immunomodulatory transgenes to promote anti-tumor immune responses. Here, we describe a next-generation OV that is rationally attenuated via codon-pair deoptimization and capable of activating anti-tumor immune responses in a mouse model of triple-negative breast cancer without the need of transgenes.MethodsHemagglutinin and neuraminidase genes of influenza virus strain A/California/07/2009 were codon-pair deoptimized using an algorithm to design synthetic viral genomes, yielding CodaLytic, a genetically stable OV with over 600 silent mutations across the two genes. For in vivo studies, EMT6 cells were implanted into inguinal mammary fad pads and treated intratumorally with CodaLytic three times a week for up to 4 weeks for efficacy studies or for up to 5 doses for pharmacodynamic readouts. Tumor infiltrating immune cells were characterized by flow cytometry or RNA was isolated for transcriptomic analysis. Anti-tumor memory was assessed by intravenous EMT6 rechallenge and interferon-γ ELISpot in splenocytes of long-term survivors.ResultsCodaLytic treatment of orthotopic EMT6 tumors led to dose-dependent tumor growth retardation and increased survival with significant tumor growth inhibition of 60% and 40–60% complete regressions at 108 pfu/dose across repeat experiments. Intravenous rechallenge of long-term survivors led to a 27-fold reduction in lung nodule formation (colony mean 0.75 vs 19.92 in naïve control animals, p = 0.005). Anti-tumor efficacy after CodaLytic treatment was accompanied by a change in the composition of the tumor immune infiltrate with significant increases in CD4+ T, B and NK cells and increased gene expression of interferon-γ, MHC-II and CCL-5. Further evidence of induction of anti-tumor immunity was an EMT6-specific interferon-γ recall response in splenocytes from long-term survivors.ConclusionsThese data demonstrate the induction of innate and adaptive changes in the TME and anti-tumor efficacy after intratumoral treatment of EMT6 tumors with CodaLytic. Additional holistic gene expression analysis is ongoing to further characterize the mechanisms of immune activation. Taken together with preclinical safety data and demonstrated clinical safety and immunogenicity of this attenuated influenza virus after intranasal administration in healthy individuals, CodaLytic is a promising immunotherapeutic to be further developed as monotherapy and in combination with immune checkpoint inhibitors or other modalities.Ethics ApprovalAll animal studies were conducted in compliance with protocol 2019-01-17-COD-1, approved by the Mispro Biotech Services Institutional Animal Care and Use Committee.

e13109 Background: Triple negative breast cancer (TNBC) is resistant to classical hormone-based therapy, prone to relapse and aggressive metastasis. Oncolytic viruses (OVs) when injected into the tumor, can promote local lysis of tumor cells and drive presentation of tumor antigens, thereby making tumors visible to the immune system. CodaLytic is a re-coded influenza A virus that has a high frequency of disfavored codons and codon pairs which attenuate the virus yet preserve all antigens and immune stimulation properties of the wild type influenza virus. CodaLytic is produced from the same master virus seed as CodaVax, a universal influenza vaccine candidate currently in Phase I clinical trials under a US IND. Methods: BALB/c mice were implanted with EMT6 mouse TNBC cells into a mammary fat pad. Six days post-implantation, when tumors became palpable, treatment was initiated by intratumoral administration of PBS (mock) or CodaLytic (108 PFU in 50 µL). Mice were treated three times a week for a total of three weeks. Tumor growth was monitored daily for 33 days and animals were euthanized if the tumor volume exceeded 500 mm3. Survivors were challenged via flank or intravenous (IV) injection of EMT6 cells. Two weeks after IV challenge, lungs were removed and compared to the lungs of control mice. Results: At the end of the initial experiment (day 33), 80% of CodaLytic-treated mice were alive with 70% of survivors being tumor-free as compared to mock, of which none survived. Survivors on day 33 had a median decrease in tumor size of 75%. CodaLytic stimulated lasting anti-tumor immunity as 100% of CodaLytic-treated survivors failed to establish tumors following challenge with EMT6 cells via flank injection. To model metastasis, surviving treated mice were also challenged with EMT6 cells delivered 2x104 cells IV. IV challenged mice had over 10-fold fewer nodules in their lungs as compared to naïve controls and 40% of the animals remained tumor free, whereas 100% of the control animals developed tumors. Additionally, CodaLytic-treated survivors showed no weight loss after IV re-challenge compared to controls which lost an average of 12% body mass. Conclusions: CodaLytic OV treatment of TNBC increased survival, led to complete tumor clearance in the EMT6 mouse model, and induced systemic anti-tumor immunity that has potential for preventing and treating metastatic disease.

Inflammation is inextricably associated with primary tumor progression. However, the contribution of inflammation to tumor outgrowth in metastatic organs has remained underexplored. Here, we show that extrinsic inflammation in the lungs leads to the recruitment of bone marrow-derived neutrophils, which degranulate azurophilic granules to release the Ser proteases, elastase and cathepsin G, resulting in the proteolytic destruction of the antitumorigenic factor thrombospondin-1 (Tsp-1). Genetic ablation of these neutrophil proteases protected Tsp-1 from degradation and suppressed lung metastasis. These results provide mechanistic insights into the contribution of inflammatory neutrophils to metastasis and highlight the unique neutrophil protease-Tsp-1 axis as a potential antimetastatic therapeutic target.

The ability of interferons (IFNs) to inhibit viral replication and cellular proliferation is well established, but the specific contribution of each IFN-stimulated gene (ISG) to these biological responses remains to be completely understood. In this report we demonstrate that ISG54, also known as IFN-induced protein with tetratricopeptide repeats 2 (IFIT2), is a mediator of apoptosis. Expression of ISG54, independent of IFN stimulation, elicits apoptotic cell death. Cell death and apoptosis were quantified by propidium iodide uptake and annexin-V staining, respectively. The activation of caspase-3, a key mediator of the execution phase of apoptosis, was clearly apparent in cells expressing ISG54. The anti-apoptotic B cell lymphoma-xl (Bcl-xl) protein inhibited the apoptotic effects of ISG54 as did the anti-apoptotic adenoviral E1B-19K protein. In addition, ISG54 was not able to promote cell death in the absence of pro-apoptotic Bcl family members, Bax and Bak. Analyses of binding partners of ISG54 revealed association with two homologous proteins, ISG56/IFIT1 and ISG60/IFIT3. In addition, ISG60 binding negatively regulates the apoptotic effects of ISG54. The results reveal a previously unidentified role of ISG54 in the induction of apoptosis via a mitochondrial pathway and shed new light on the mechanism by which IFN elicits anti-viral and anti-cancer effects.

Inflammation is known to be inextricably associated with tumor progression. However, the role of inflammation in metastatic initiation and outgrowth remains largely unexplored. In our study, we show that CXCR2-CXCL2-dependent recruitment of neutrophils supported a pro-metastatic phenotype in acutely inflamed lungs. The increased influx of Ly6G+ neutrophils into inflamed lungs was accompanied by a loss of thrombospondin-1 (Tsp-1) protein. In vitro, the degranulation of neutrophils was marked by an increase in the activity of the neutrophil proteases elastase and cathepsin-G, leading to degradation of Tsp-1, which was protected by using the protease inhibitor Sivelestat. Whereas genetic ablation of either cathepsin-G or elastase in vivo partially impaired metastasis, pharmacological inhibition of both neutrophil proteases, using the small molecule drug Sivelestat, dramatically suppressed metastasis. We have uncovered a novel pathway in which the serine proteases, cathepsin-G and elastase produced by degranulating neutrophils, promote a metastasis-conducive microenvironment through the cleavage of Tsp-1. Our results provide mechanistic insights into how inflammatory components of the premetastatic niche can support metastatic outgrowth, and they suggest that the neutrophil protease-Tsp-1 axis presents a therapeutic target against metastatic cancers. Citation Format: Tina El Rayes, Raul Catena, Sharrell Lee, Dingcheng Gao, Marcin Stawowczyk, Nasser Altorki, Vivek Mittal. Neutrophil serine proteases, cathepsin-G and elastase, promote metastasis through cleavage of thrombospondin-1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2073. doi:10.1158/1538-7445.AM2014-2073