Development of large scale clinical-grade NK cell production and the diversity of allogeneic cell sources used for NK cell generation have supported advances in pre-clinical and clinical studies using NK cells for cancer immunotherapy [4, 5, 10, 11]. Nevertheless, enhancement of NK cell anti-tumor activity for adoptive transfer for treatment of solid tumors remains a challenge. Current therapies focus on potentiating NK cell activation or engagement with target cancer cells, like with use of stimulatory cytokines or bispecific and trispecific engager molecules, respectively [28,29,30,31].

From the immunotherapeutic perspective, these strategies do not provide alternatives for circumventing tumor microenvironment immunosuppression, which is a common hindrance of NK cell therapy success in solid tumors [32]. We have previously demonstrated that an adenovirus encoding a human variant IL-2 was capable of counteracting tumor immunosuppression in a hamster pancreatic model as a monotherapy [18]. Moreover, the vIL-2 virus has demonstrated to be safe as a monotherapy, and its backbone has been detected in high concentration in tumors upon systemic administration [18, 33]. In the present study, we propose the use of vIL-2 virus, Ad5/3-E2F-d24-vIL2, as a combination strategy to improve therapeutic response of allogeneic NK cells for the treatment of human OvCa tumors.

OvCa is the deadliest gynecological cancer, it presents high rates of tumor recurrence associated with poor 3-year overall survival, and over two thirds of patients already present an advanced stage of the disease at the time of cancer diagnosis [14, 34, 35]. Actually, in our study, most patients presented tumors at stage IVB, when the disease has spread beyond the organs in the abdomen [35]. Likewise, most of specimens collected, 9 out of 12, were derived from metastatic lesions. From the TME point of view, OvCa is a highly immunosuppressive tumor type characterized by the presence of T regulatory cells, MDSCs, and TAM that promote tumor growth and release of anti-inflammatory agents in the TME [13].

Nevertheless, baseline TME or initial histological state were not an impediment for efficient response in OvCa tumor digests. In fact, our results demonstrate that addition of Ad5/3-E2F-d24-vIL2 virus bolstered NK cell therapy killing effects in a set of OvCa human samples in ex vivo co-cultures regardless of tumor diagnosis or stage. Notably, we also observed better cancer control when the adenovirus was loaded with the vIL-2 cytokine transgene compared to its backbone counterpart in those co-cultures. Such results can be attributed to the selective mode of action of vIL-2 cytokine has on IL-2R of NK+, CD4+ T and CD8+ T cells triggering cell stimulation, compared to inactive effect in TReg cells [36]. Importantly, when said variant is expressed by an engineered oncolytic adenovirus, Ad5/3-E2F-d24-vIL2, additional downregulation of genes associated with MDSCs function is also observed [18], which makes this vectored viral approach particularly appealing for treatment of immunosuppressive tumors like OvCa. Of note, the impedance system used here to evaluate cell cytotoxicity has some limitations to differentiate adherence signals derived from cancer cells and immune cells, such as myeloid cells and NK cells. For this reason, the interpretation of some results like the ones obtained in ex vivo co-cultures of HUSOV5 and HUSOV15 can be difficult. On this regard, future studies investigating cancer cell molecular death like TUNEL staining might help to elucidate this matter.

Corroborating this notion, our analysis of immune cells in tumor co-cultures treated with vIL-2 virus and NK cell therapy showed increased levels of cytotoxic NK+ and CD8+ T cells, while no significant changes were observed in TReg cells. Interestingly, these findings differ from the analysis made in tumors from the OvCa PDX in vivo experiment, where significantly higher levels of TReg cells were found in vIL-2 virus plus NK cells treated animals compared to backbone control and mock groups. Perhaps explaining this finding, our vIL-2 virus therapeutic approach, similarly to other modified IL-2 cytokine candidates, does not prevent expression of wt IL-2 cytokine expression by the activated host immune cells, as well as it does not block the usage of wt IL-2 cytokine by immune cells, including TReg cells present in the TME [18, 37, 38]. Instead, our virus vector continuously produces high levels of vIL-2 cytokine in the TME, as previously demonstrated, that will be taken up by effector cells only, diminishing the overall TReg cell-derived immunosuppression [18]. Expression of wt IL-2 in healthy organs would be interesting to be evaluated in future studies.

Of note, our oncolytic adenovirus vector used to encode vIL-2 cytokine represents a therapeutic advantage for TME remodeling. Modifications made on the adenovirus structure have been optimized to promote increased virus infectivity and amplification in OvCa cells as well as to efficiently lyse cancer cells upon virus infection [22, 23]. In fact, adenovirus-mediated immunogenic cell death is an important mechanism for shedding of pro-inflammatory signals in the TME, such as cell danger signaling molecules, and subsequent engagement of the host immune system with anti-tumor response [16, 18, 39]. Overall, this goes in line with our findings in virus backbone treated groups, where partial control of OvCa progression ex vivo and in vivo can be linked to direct cancer cell debulking and immune response onset. Despite described benefits, our results demonstrate that only when loaded with vIL-2 transgene, the virus consistently reshapes the TME towards a pro-inflammatory state. These results corroborate with previous findings with vIL-2 virus treatment as a monotherapy, where high expression of CCL2, TNF-α, and IL-1β genes were detected in treated tumors [18]. Unfortunately, due to small tumors sizes, especially in vIL-2 virus combination group, the cytokine profiling study could not be performed.

Another interesting aspect noticed in OvCa co-cultures was the high intensity of CD158b in NK+ cells in the combination therapy. CD158b is part of the family of killer cell immunoglobulin-like receptors (KIR), a group of transmembrane proteins that modulate NK cell cytotoxicity particularly through inhibitory signaling interaction with HLA-ABC receptors [1, 40]. In the cancer context, CD158b upregulation has been negatively associated with NK cell activation and production of CD107, IFN-γ, and perforin even when tumors are exposed to exogenous IL-2 and IL-15 cytokines [40, 41]. In our co-cultures, we hypothesize that augmented CD158b intensity in NK+ cells could indicate a progressive transition of NK+ cells from a cytotoxic to a baseline state, in view of the time point selected for immune cells analysis and the efficient cell killing by the combination approach already observed at earlier hours. However, future studies should investigate the potential effect of the virus backbone and vIL-2 virus monotherapies might have on the modulation of CD158 molecule in NK cells present in OvCa tumors.

Enabling the full potential of adoptive NK cell therapy in vivo represents one of the main current challenges for NK cell therapy success. Here, we demonstrated that vIL-2 virus efficiently increased allogeneic NK cell anti-tumor control in an OvCa PDX mouse model. Animals receiving the combination of vIL-2 virus plus NK cells had the best tumor control compared to the other experimental groups. Such improvement was associated with the ability of the vIL-2 transgene to enhance the cytotoxic potential of NK+ cells, CD4+T, and CD8 + T infiltrating the immunosuppressive OvCa TME. Altogether, these results confirm our ex vivo findings and endorse the therapeutic advantage of using our vIL-2 virus candidate to potentiate allogeneic adoptive NK cell therapy for the treatment of OvCa tumors. Of note, differences on proportion of detected CD4 + T and CD8+ T cells in the ex vivo and in vivo studies can be partially explained by the PBMCs expansion protocol utilised in the latter case prior mice injection. Addition of exogenous wt IL-2 cytokine to cell cultures can condition T cells response more promptly to further cytokine exposure [25, 42]. While in the OvCa ex vivo co-cultures, no cytokine stimulation was done prior the treatment with the combination therapy.

From the NK cell immunotherapeutic perspective, continuous production of vIL-2 cytokine by the adenovirus vector is determinant for sustained NK cell anti-tumor response, although no increase on the NK cell proportions was observed at the time point studied. Possibly due to the regular NK cell life-spam programming after response to target cancer cells [43]. In our in vivo animal experiment, a single dose of allogeneic NK cells was sufficient to promote continued tumor response when cell therapy was used in conjunction with vIL-2 virus. In contrast, in an iPSC-derived NK cell therapy, best anti-tumor response was obtained when a total of 3 doses of NK cells were administrated together with 5 doses of IL-2 cytokine injections into pre-irradiated mice bearing OvCa tumors [8].

Similarly, multiple doses of CD34+ hematopoietic progenitor cell (HPC)-derived NK cells and IL-15 were given to animals bearing OvCa tumors treated with gemcitabine to improve tumor response [11]. Taken together, the data presented here highlights the prospective potential of our vIL-2 virus candidate to unleash the therapeutic potential of NK cells for OvCa treatment, by allowing optimized use of NK cells, with reduced rounds of cell transfer and absence of need for exogenous cytokine therapy. In the clinical context, the latter is particularly relevant in view of eventual constraints with NK cell availability for adoptive transfer and frequent toxicity associated with systemic administration of human stimulatory cytokines [5, 19].

Considering the key role NK cells have in the clearance of viral infections, proposing an adenovirus candidate as a combination strategy for NK cell adoptive therapy could seem like a counter-intuitive approach. In our results, however, the administration of said therapies together resulted in improved cancer cell killing and control of treated OvCa tumors. Of note, adenoviruses possess their own mechanisms for immune evasion, in particular, the E3 region hosts genes closely associated with expression of immunoregulatory proteins such as the E3/glycoprotein19K that binds to MHC I in the endoplasmic reticulum preventing the antigen presentation of viral peptides on the cell surface and activation of CD8+ T cells [6, 44].

To escape NK cell response, adenovirus 5 acts by downregulating co-stimulatory proteins MICA/MICB, CD112, and CD155 and upregulates HLA-E: a non-classical HLA with negative effects on NK cell activation [6, 44, 45]. Importantly, the partially deleted E3 region in the vIL-2 virus construct is replaced by the vIL-2 cytokine transgene, which in turn should facilitate the recognition of virus-infected cells by the host´s lymphocytes. Challenging these expectations, the vIL-2 virus downregulated MHC I (HLA-ABC) intensity in most of the samples studied. CD155 and MICA/MICB values oscillated up and down, while HLA-E intensity remained unchanged in infected OvCa tumor digests. This data suggests that absence of immunoregulatory protein E3/glycoprotein19K is not sufficient to evade NK cells recognition and activation upon vIL-2 virus infected OvCa cells.

In conclusion, our results demonstrate that Ad5/3-E2F-d24-vIL2 is a powerful approach for enabling allogeneic NK cell therapy. Ad5/3-E2F-d24-vIL2 efficiently counteracted the immunosuppressive human OvCa TME by enhancing NK cell and T lymphocyte cytotoxicity, while maintaining tumor-infiltrating TReg levels comparable to NK cell monotherapy. Of note, this preclinical study paves the way for clinical trials with Ad5/3-E2F-d24-vIL2 in combination with NK cells, as well as NK cells derived products such as CAR-NKs, iPSC-NKs and other forms of engineered NK cell therapy.