*5.4. Arming with Immunostimulatory Cytokines and Chemokines*

Corresponding to the diversity of immune mechanisms that can be dysfunctional in tumors, various immunostimulatory transgenes have been integrated into oAds to stimulate effective antitumor immune responses. Since systemic administration of potent immunostimulatory factors, such as type I Interferons, tumor necrosis factor alpha (TNFα), or interleukin 12 (IL-12), may have considerable side effects, delivery by oAds provides an attractive option to focus cytokine activity on the target tumor.

In tumor cells, IFNs exert pleiotropic effects including the activation of the immune proteasome, the upregulation of major histocompatibility complex (MHC) class I and II, and potent activation of NK cells and cytotoxic T lymphocytes (CTLs). Shashkova et al. integrated IFNα into an oAd (KD3-IFN), which should render replication more sensitive to the IFNα response in normal cells [81]. The authors were able to confirm a decreased off-target toxicity in HCC xenografts in nude mice and in an immunocompetent model of kidney cancer in Syrian hamsters. A cyclooxygenase (Cox) 2-dependent oAd expressing IFNα was capable of inhibiting tumor growth in a Syrian hamster model of pancreatic cancer [82]. In an immunocompetent mouse model of Lewis lung carcinoma, co-application of an oAd in combination with a non-replicating Ad-IFNβ has been investigated [83]. This binary strategy prolonged interferon expression and improved antitumoral immune responses. Efficient delivery of a non-replicating transgenic adenovirus by coinfection with an oAd has been initially shown in an approach of cancer gene therapy [84]. Regarding armed virotherapeutic vectors, this binary approach is particularly promising for immunostimulatory transgenes. Assuming that enough events with single virus transduction will occur, the binary method holds promise to maintain cytokine expression beyond clearance of the oAd.

The potent antitumor functions of TNFα have been well known for decades. Loco-regional delivery of TNFα by oAds promises potent antitumor activity with limited side effects. Hirvinen et al. showed that the TNFα-armed Ad5/3-E2F-delta24 vector led to increased tumor destruction due to TNFα-mediated apoptosis, immunogenic cell death, and induction of antitumor immune responses, including tumor-antigen-specific T cells [85]. A corresponding virus with additional expression of interleukin 2 (IL-2) (Ad5/3-E2F-D24-hTNFα-IRES-hIL2 or TILT-123) is currently under clinical investigations. IL-2 is a central cytokine for survival and proliferation of T cells qualifying TILT-123 to augment the transfer of tumor infiltrating lymphocytes (TIL). In an immunocompetent Syrian hamster tumor model, concomitant transfer of TILs and virus application resulted in a 100% cure of treated animals [86]. The virus has also been used to support tumor infiltration with chimeric antigen receptor (CAR) transgenic T cells [87]. By using an ex vivo ovarian cancer (OVCA) model derived from patient samples, enhanced levels of proinflammatory signals (IFNγ, CXCL10, TNFα and IL-2) associated with a concomitant activation of CD4 and CD8 TILs could be observed when tumor cells were infected with TILT-123 [88]. In response to autologous, T cell-depleted OVCA cultures, which had been infected with TILT-123, TILs secreted high levels of IFNγ. These observations confirmed the use of TILT-123 in adoptive cell transfer.

Several oAds have been armed with IL-12, an essential cytokine involved in inflammation and proliferation of effector T cells and NK cells. Using the hypoxia-dependent Ad-DhscIL12 in a Syrian hamster model of pancreatic cancer, Bortolanza et al. showed active viral replication and enhanced transgene expression in vivo resulting in potent antitumor effects and less toxicity due to shorter systemic exposure [89]. Lee et al. investigated the oAd YKL-IL12/B7 expressing IL-12 and B/7-1 (CD80), a ligand of the costimulatory CD28 receptor, on T cells. In a syngeneic murine B16-F10 melanoma, the virus showed effective tumor growth inhibition including complete tumor regressions and improved survival [90]. Using the oncolytic Ad-TD-nsIL12, which expresses a non-secreted version of IL-12, Wang et al. were able to reduce off-target toxicity of IL-12 [91].

IL-24 is an immunomodulatory cytokine with profound antitumor effects through immune activation, induction of tumor cell apoptosis and inhibition of angiogenesis. IL-24-expressing oAds have shown antitumor efficacy in vitro and in xenografts in mice [92,93]. IL-4 has been used to promote intratumoral leukocyte infiltration [94]. The cytokine IL-18 induces IFNγ production through T cells and NK cells. Using the IL-18-armed ZD55 in xenograft models, Zheng et al. could observe stronger antitumor responses and inhibition of tumor angiogenesis [95]. Choi et al. generated oAd expressing IL-12 in combination with IL-18, or IL-23, respectively, and demonstrated enhanced antitumor efficacy in B16-F10 melanoma associated with an improved Th1/Th2 cytokine ratio and infiltration of NK and T cells [96,97]. Cytokines have also been combined with the chemokine CCL21, which binds to CCR7 on naïve T cells and DCs and promotes their attraction to the tumor [98,99].

Alternative options for immune arming are factors that directly target immunosuppression in the tumor microenvironment. Seth et al. have targeted transforming growth factor β (TGFβ) with a soluble TGFβ-receptor II protein fused to a human immunoglobulin (IgG) Fc fragment [100]. By using an oAd expressing sTGFβRII-Fc (rAd.sT), the authors showed in a xenograft mouse model tumor regression in 85% of treated animals. rAd.sT enhanced the efficacy of concomitant anti-PD-1 and anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) treatment in an immunocompetent 4T1 breast cancer model [101].
