Oncolytic adenovirus decreases the proportion of TIM-3(+) subset of tumor-infiltrating CD8(+) T cells with correlation to improved survival in patients with cancer

Background Oncolytic viruses are a potent form of active immunotherapy, capable of invoking antitumor T-cell responses. Meanwhile, less is known about their effects on immune checkpoints, the main targets for passive immunotherapy of cancer. T-cell immunoglobulin and mucin domain-3 (TIM-3) is a coinhibitory checkpoint driving T-cell exhaustion in cancer. Here we investigated the effects of oncolytic adenovirus on the TIM-3 checkpoint on tumor-infiltrating immune cells and clinical impact in patients with cancer receiving oncolytic immunotherapy. Methods Modulation of TIM-3 expression on tumor-infiltrating immune cells was studied preclinically in B16 melanoma following intratumoral treatment with Ad5/3 increment 24-granulocyte-macrophage colony-stimulating factor oncolytic adenovirus. We conducted a retrospective longitudinal analysis of 15 patients with advanced-stage cancer with tumor-site biopsies before and after oncolytic immunotherapy, treated in the Advanced Therapy Access Program (ISRCTN10141600, April 5, 2011). Following patient stratification with regard to TIM-3 (increase vs decrease in tumors), overall survival and imaging/marker responses were evaluated by log-rank and Fisher's test, while coinhibitory receptors/ligands, transcriptomic changes and tumor-reactive and tumor-infltrating immune cells in biopsies and blood samples were studied by microarray rank-based statistics and immunoassays. Results Preclinically, TIM-3(+) tumor-infiltrating lymphocytes (TILs) in B16 melanoma showed an exhausted phenotype, whereas oncolytic adenovirus treatment significantly reduced the proportion of TIM-3(+) TIL subset through recruitment of less-exhausted CD8(+) TIL. Decrease of TIM-3 was observed in 60% of patients, which was associated with improved overall survival over TIM-3 increase patients (p=0.004), together with evidence of clinical benefit by imaging and blood analyses. Coinhibitory T-cell receptors and ligands were consistently associated with TIM-3 changes in gene expression data, while core transcriptional exhaustion programs and T-cell dysfunction were enriched in patients with TIM-3 increase, thus identifying patients potentially benefiting from checkpoint blockade. In striking contrast, patients with TIM-3 decrease displayed an acute inflammatory signature, redistribution of tumor-reactive CD8(+) lymphocytes and higher influx of CD8(+) TIL into tumors, which were associated with the longest overall survival, suggesting benefit from active immunotherapy. Conclusions Our results indicate a key role for the TIM-3 immune checkpoint in oncolytic adenoviral immunotherapy. Moreover, our results identify TIM-3 as a potential biomarker for oncolytic adenoviruses and create rationale for combination with passive immunotherapy for a subset of patients.Peer reviewe

Local therapy with an engineered oncolytic adenovirus enables antitumor response in non- injected melanoma tumors in mice treated with aPD-1

Intratumoral immunotherapies are entering clinical use but concerns remain regarding their effects on non-injected tumors. Here, we studied the impact of local treatment with an adenovirus coding for TNFa and IL-2 on systemic antitumor response in animals receiving aPD-1 (anti-programmed cell death protein 1) therapy. Using bilateral murine melanoma models, we tested systemic tumor response to combined therapy with anti-PD-1 and an adenovirus coding for TNFa and IL-2 ("virus"). Virus was given intratumorally (to one of the two tumors only) and aPD-1 monoclonal antibody systemically. We evaluated both tumors' response to treatment, overall survival, metastasis development, and immunological mechanisms involved with response. Consistent tumor control was observed in both injected and non-injected tumors, including complete response in all treated animals receiving aPD-1+ virus therapy. Mechanistically, virus injections enabled potent effector lymphocyte response locally, with systemic effects in non-injected tumors facilitated by aPD-1 treatment. Moreover, adenovirus therapy demonstrated immunological memory formation. Virus therapy was effective in preventing metastasis development. Local treatment with TNFa and IL-2 coding adenovirus enhanced systemic response to aPD-1 therapy, by re-shaping the microenvironment of both injected and non-injected tumors. Therefore, our pre-clinical data support the rationale for a trial utilizing a combination of aPD-1 plus virus for the treatment of human cancer.Peer reviewe

Oncolytic Adenovirus Type 3 Coding for CD40L Facilitates Dendritic Cell Therapy of Prostate Cancer in Humanized Mice and Patient Samples

Dendritic cell (DC)-based vaccines have shown some degree of success for the treatment of prostate cancer (PC). However, the highly immunosuppressive tumor microenvironment leads to DC dysfunction, which has limited the effectiveness of these vaccines. We hypothesized that use of a fully serotype 3 oncolytic adenovirus (Ad3-hTERT-CMV-hCD40L; TILT-234) could stimulate DCs in the prostate tumor microenvironment by expressing CD40L. Activated DCs would then activate cytotoxic T cells against the tumor, resulting in therapeutic immune responses. Oncolytic cell killing due to cancer cell-specific virus replication adds to antitumor effects but also enhances the immunological effect by releasing tumor epitopes for sampling by DC, in the presence of danger signals. In this study, we evaluated the companion effect of Ad3-hTERT-CMV-hCD40L and DC-therapy in a humanized mouse model and PC histocultures. Treatment with Ad3-hTERT-CMV-hCD40L and DC resulted in enhanced antitumor responses in vivo. Treatment of established histocultures with Ad3-hTERT-CMV-hCD40L induced DC maturation and notable increase in proinflammatory cytokines. In conclusion, Ad3-hTERT-CMV-hCD40L is able to modulate an immunosuppressive prostate tumor microenvironment and improve the effectiveness of DC vaccination in PC models and patient histocultures, setting the stage for clinical translation.Peer reviewe

Adenovirus Encoding Tumor Necrosis Factor Alpha and Interleukin 2 Induces a Tertiary Lymphoid Structure Signature in Immune Checkpoint Inhibitor Refractory Head and Neck Cancer

Immune checkpoint inhibitors (ICI) have provided significant improvement in clinical outcomes for some patients with solid tumors. However, for patients with head and neck cancer, the response rate to ICI monotherapy remains low, leading to the exploration of combinatorial treatment strategies. In this preclinical study, we use an oncolytic adenovirus (Ad5/3) encoding hTNF alpha and hIL-2 and non-replicate adenoviruses (Ad5) encoding mTNF alpha and mIL-2 with ICI to achieve superior tumor growth control and improved survival outcomes. The in vitro effect of Ad5/3-E2F-D24-hTNFa-IRES-hIL-2 was characterized through analyses of virus replication, transgene expression and lytic activity using head and neck cancer patient derived cell lines. Mouse models of ICI naive and refractory oral cavity squamous cell carcinoma were established to evaluate the local and systemic anti-tumor immune response upon ICI treatment with or without the non-replicative adenovirus encoding mTNF alpha and mIL-2. We delineated the mechanism of action by measuring the metabolic activity and effector function of CD3(+) tumor infiltrating lymphocytes (TIL) and transcriptomic profile of the CD45(+) tumor immune compartment. Ad5/3-E2F-D24-hTNFa-IRES-hIL-2 demonstrated robust replicative capability in vitro across all head and neck cell lines screened through potent lytic activity, E1a and transgene expression. In vivo, in both ICI naive and refractory models, we observed improvement to tumor growth control and long-term survival when combining anti-PD-1 or anti-PD-L1 with the non-replicative adenovirus encoding mTNF alpha and mIL-2 compared to monotherapies. This observation was verified by striking CD3(+) TIL derived mGranzyme b and interferon gamma production complemented by increased T cell bioenergetics. Notably, interrogation of the tumor immune transcriptome revealed the upregulation of a gene signature distinctive of tertiary lymphoid structure formation upon treatment of murine anti-PD-L1 refractory tumors with non-replicative adenovirus encoding mTNF alpha and mIL-2. In addition, we detected an increase in anti-tumor antibody production and expansion of the memory T cell compartment in the secondary lymphoid organs. In summary, a non-replicative adenovirus encoding mTNF alpha and mIL-2 potentiates ICI therapy, demonstrated by improved tumor growth control and survival in head and neck tumor-bearing mice. Moreover, the data reveals a potential approach for inducing tertiary lymphoid structure formation. Altogether our results support the clinical potential of combining this adenovirotherapy with anti-PD-1 or anti-PD-L1.Peer reviewe

Adenovirus Encoding Tumor Necrosis Factor Alpha and Interleukin 2 Induces a Tertiary Lymphoid Structure Signature in Immune Checkpoint Inhibitor Refractory Head and Neck Cancer

Immune checkpoint inhibitors (ICI) have provided significant improvement in clinical outcomes for some patients with solid tumors. However, for patients with head and neck cancer, the response rate to ICI monotherapy remains low, leading to the exploration of combinatorial treatment strategies. In this preclinical study, we use an oncolytic adenovirus (Ad5/3) encoding hTNF alpha and hIL-2 and non-replicate adenoviruses (Ad5) encoding mTNF alpha and mIL-2 with ICI to achieve superior tumor growth control and improved survival outcomes. The in vitro effect of Ad5/3-E2F-D24-hTNFa-IRES-hIL-2 was characterized through analyses of virus replication, transgene expression and lytic activity using head and neck cancer patient derived cell lines. Mouse models of ICI naive and refractory oral cavity squamous cell carcinoma were established to evaluate the local and systemic anti-tumor immune response upon ICI treatment with or without the non-replicative adenovirus encoding mTNF alpha and mIL-2. We delineated the mechanism of action by measuring the metabolic activity and effector function of CD3(+) tumor infiltrating lymphocytes (TIL) and transcriptomic profile of the CD45(+) tumor immune compartment. Ad5/3-E2F-D24-hTNFa-IRES-hIL-2 demonstrated robust replicative capability in vitro across all head and neck cell lines screened through potent lytic activity, E1a and transgene expression. In vivo, in both ICI naive and refractory models, we observed improvement to tumor growth control and long-term survival when combining anti-PD-1 or anti-PD-L1 with the non-replicative adenovirus encoding mTNF alpha and mIL-2 compared to monotherapies. This observation was verified by striking CD3(+) TIL derived mGranzyme b and interferon gamma production complemented by increased T cell bioenergetics. Notably, interrogation of the tumor immune transcriptome revealed the upregulation of a gene signature distinctive of tertiary lymphoid structure formation upon treatment of murine anti-PD-L1 refractory tumors with non-replicative adenovirus encoding mTNF alpha and mIL-2. In addition, we detected an increase in anti-tumor antibody production and expansion of the memory T cell compartment in the secondary lymphoid organs. In summary, a non-replicative adenovirus encoding mTNF alpha and mIL-2 potentiates ICI therapy, demonstrated by improved tumor growth control and survival in head and neck tumor-bearing mice. Moreover, the data reveals a potential approach for inducing tertiary lymphoid structure formation. Altogether our results support the clinical potential of combining this adenovirotherapy with anti-PD-1 or anti-PD-L1.Peer reviewe

Local delivery of interleukin 7 with an oncolytic adenovirus activates tumor-infiltrating lymphocytes and causes tumor regression

Cytokines have proven to be effective for cancer therapy, however whilst low-dose monotherapy with cytokines provides limited therapeutic benefit, high-dose treatment can lead to a number of adverse events. Interleukin 7 has shown promising results in clinical trials, but anti-cancer effect was limited, in part due to a low concentration of the cytokine within the tumor. We hypothesized that arming an oncolytic adenovirus with Interleukin 7, enabling high expression localized to the tumor microenvironment, would overcome systemic delivery issues and improve therapeutic efficacy. We evaluated the effects of Ad5/3-E2F-d24-hIL7 (TILT-517) on tumor growth, immune cell activation and cytokine profiles in the tumor microenvironment using three clinically relevant animal models and ex vivo tumor cultures. Our data showed that local treatment of tumor bearing animals with Ad5/3- E2F-d24-hIL7 significantly decreased cancer growth and increased frequency of tumor-infiltrating cells. Ad5/3-E2F-d24-hIL7 promoted notable upregulation of pro-inflammatory cytokines, and concomitant activation and migration of CD4+ and CD8 + T cells. Interleukin 7 expression within the tumor was positively correlated with increased number of cytotoxic CD4+ cells and IFNg-producing CD4+ and CD8+ cells. These findings offer an approach to overcome the current limitations of conventional IL7 therapy and could therefore be translated to the clinic.Peer reviewe

An oncolytic adenovirus coding for a variant interleukin 2 cytokine improves response to chemotherapy through enhancement of effector lymphocyte cytotoxicity, fibroblast compartment modulation and mitotic slippage

Pancreatic ductal adenocarcinoma (PDAC) is a highly treatment-resistant cancer. Currently, the only curative treatment for PDAC is surgery, but most patients are diagnosed with metastatic disease and thus outside the scope of surgery. The majority of metastatic patients receive chemotherapy, but responses are limited. New therapeutics are thus urgently needed for PDAC. One major limitation in treating PDAC has been the highly immunosuppressive tumor microenvironment (TME) which inhibits anti-cancer immune responses. We have constructed an oncolytic adenovirus coding for a variant the interleukin 2 molecule, Ad5/3-E2F-d24-vIL2 (also known as TILT-452, and “vIL-2 virus”), with preferential binding to IL-2 receptors on the surface of effector lymphocytes over T regulatory cells (T regs). In the present study this virus was evaluated in combination with nab-paclitaxel and gemcitabine chemotherapy in Panc02 mouse model. Ad5/3-E2F-d24-vIL2 showed marked PDAC cell killing in vitro, alongside induction of mitotic slippage and immunogenic cell death in PDAC cell lines, when combined with chemotherapy. Increased survival was seen in vivo with 80% of animals surviving long term, when compared to chemotherapy alone. Moreover, combination therapy mediated enhanced tumor growth control, without observable toxicities in internal organs or external features. Survival and tumor control benefits were associated with activation of tumor infiltrating immune cells, downregulation of inhibitory signals, change in fibroblast populations in the tumors and changes in intratumoral cytokines, with increased chemokine amounts (CCL2, CCL3, CCL4) and anti-tumor cytokines (IFN-γ and TNFα). Furthermore, vIL-2 virus in combination with chemotherapy efficiently induced tumor protection upon rechallenge, that was extended to a previously non-encountered cancer cell line. In conclusion, Ad5/3-E2F-d24-vIL2 is a promising immunotherapy candidate when combined with nab-paclitaxel and gemcitabine

Oncolytic Adenovirus Coding for a Variant Interleukin 2 (vIL-2) Cytokine Re-Programs the Tumor Microenvironment and Confers Enhanced Tumor Control

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Viral Vectors Expressing Interleukin 2 for Cancer Immunotherapy

Interleukin 2 (IL-2) plays a crucial role in T cell growth and survival, enhancing the cytotoxic activity of natural killer and cytotoxic T cells and thus functioning as a versatile master proinflammatory anticancer cytokine. The FDA has approved IL-2 cytokine therapy for the treatment of metastatic melanoma and metastatic renal cell carcinoma. However, IL-2 therapy has significant constraints, including a short serum half-life, low tumor accumulation, and life-threatening toxicities associated with high doses. Oncolytic viruses (OVs) offer a promising option for cancer immunotherapy, selectively targeting and destroying cancer cells while sparing healthy cells. Numerous studies have demonstrated the successful delivery of IL-2 to the tumor microenvironment without compromising safety using OVs such as vaccinia, Sendai, parvo, Newcastle disease, tanapox, and adenoviruses. Additionally, by engineering OVs to coexpress IL-2 with other anticancer transgenes, the immune properties of IL-2 can be further enhanced. Preclinical and clinical studies have shown promising antitumor effects of IL-2-expressing viral vectors, either alone or in combination with other anticancer therapies. This review summarizes the therapeutic potential of IL-2-expressing viral vectors and their antitumor mechanisms of action.Peer reviewe