Immunotherapy with tumor-infiltrating lymphocytes (TIL) or oncolytic adenoviruses, have shown promising results in cancer treatment, when used as separate therapies. When used in combination, the antitumor effect is synergistically potentiated due oncolytic adenovirus infection and its immune stimulating effects on T cells. Indeed, studies in hamsters have shown a 100% complete response rate when animals were treated with oncolytic adenovirus coding for TNFa and IL-2 (Ad5/3-E2F-D24-hTNFa-IRES-hIL2; TILT-123) and TIL therapy. In humans, one caveat with oncolytic virus therapy is that intratumoral injection has been traditionally preferred over systemic administration, for achieving sufficient virus concentrations in tumors, especially when neutralizing antibodies emerge. We have previously shown that 5/3 chimeric oncolytic adenovirus can bind to human lymphocytes for avoidance of neutralization. In this study, we hypothesized that incubation of oncolytic adenovirus (TILT-123) with TILs prior to systemic injection would allow delivery of virus to tumors. This approach would deliver both components in one self-amplifying product. TILs would help deliver TILT-123, whose replication will recruit more TILs and increase their cytotoxicity. In vitro, TILT-123 was seen binding efficiently to lymphocytes, supporting the idea of dual administration. We show in vivo in different models that virus could be delivered to tumors with TILs as carriers.

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.

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 hTNFα and hIL-2 and non-replicate adenoviruses (Ad5) encoding mTNFα 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 naïve 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 mTNFα 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 naïve 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 mTNFα 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 mTNFα 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 mTNFα 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.

IntroductionImmune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer, but preclinical testing of hypotheses such as combination therapies has been complicated, in part due to species incompatibility issues. For example, one of few known permissive animal models for oncolytic adenoviruses is the Syrian hamster, for which an ICI, mainly an anti-PD-L1 monoclonal antibody (mAb) was not previously available. In this study, we developed an anti-Syrian hamster PD-L1 mAb to enable the evaluation of safety and efficacy, when combining anti-PD-L1 with an oncolytic adenovirus encoding tumour necrosis factor alpha (TNFα) and interleukin-2 (IL-2) (Ad5/3-E2F-D24-hTNFα-IRES-hIL-2 or TILT-123).MethodsRecombinant Syrian hamster PD-L1 was expressed and mice immunized for mAb formation using hybridoma technology. Clonal selection through binding and functional studies in vitro, in silico and in vivo identified anti-PD-L1 clone 11B12-1 as the primary mAb candidate for immunotherapy modelling. The oncolytic virus (OV) and ICI combination approach was then evaluated using 11B12-1 and TILT-123 in a Syrian hamster model of pancreatic ductal adenocarcinoma (PDAC).ResultsSupernatants from hybridoma parent subclone 11B12B4 provided the highest positive PD-L1 signal, on Syrian hamster PBMCs and three cancer cell lines (HT100, HapT1 and HCPC1). In vitro co-cultures revealed superior immune modulated profiles of cell line matched HT100 tumour infiltrating lymphocytes when using subclones of 7G2, 11B12 and 12F1. Epitope binning and epitope prediction using AlphaFold2 and ColabFold revealed two distinct functional epitopes for clone 11B12-1 and 12F1-1. Treatment of Syrian hamsters bearing HapT1 tumours, with 11B12-1 induced significantly better (p<0.05) tumour growth control than isotype control by day 12. 12F1-1 did not induce significant tumour growth control. The combination of 11B12-1 with oncolytic adenovirus TILT-123 improved tumour growth control further, when compared to monotherapy (p<0.05) by day 26.ConclusionsNovel Syrian hamster anti-PD-L1 clone 11B12-1 induces tumour growth control in a hamster model of PDAC. Combining 11B12-1 with oncolytic adenovirus TILT-123 improves tumour growth control further and demonstrates good safety and toxicity profiles.