*5.6. Arming with Transgenes Addressing T Cell Costimulation or Immune Checkpoints*

Pharmacological blockade of inhibitory immune checkpoints or activation of costimulatory receptors are potent strategies to activate antitumor T cells. Dias et al. used a full length anti-CTLA4 monoclonal antibody expressed by Ad5/3-∆24-CTLA to combine oncolysis and checkpoint inhibition [111]. Intratumoral expression allowed high local levels of the checkpoint inhibitor. In patient-derived peripheral blood mononuclear cells (PBMCs), the authors observed T cell activation and αCTLA4-mediated apoptosis. The PD-1/PD-L1 is an inhibitory checkpoint regulating the activity of peripheral T cells. In prostate cancer models, Tanoue et al. showed that an oAd combined with a helper-Ad expressing a PD-L1-blocking mini-antibody supported the intratumoral activity of adoptively transferred CAR T cells [112]. The specific benefit of viral delivery was confirmed by the demonstration that local expression of the PD-L1-blocking minibody was superior compared with systemic infusion of αPD-L1 IgG. An equivalent approach using helper-dependent adenoviruses for expression of the PD-L1 blocking mini-antibody and IL-12-p70 for immune stimulation augmented the activity and persistence of CAR T cells in murine models of head and neck squamous cell carcinoma (HNSCC) [113].

CD40 is a costimulatory receptor expressed on antigen-presenting cells (APCs), mostly B cells, macrophages and DCs. Interaction of CD40 with its ligand CD40L induces cytokine production, increases MHC class II-dependent antigen presentation and thus supports the priming and expansion of T cells. Tumor infection with the CD40L-armed AdEHCD40L reduced the growth of xenografted human myeloma [114]. The oncolytic Ad5/3-hTERT-E1A-hCD40L (CGTG-401) induced multiple antitumor effects including reduced tumor growth via apoptosis, increased number of cytotoxic CD8 T

cells in the tumor, and upregulation of TH1 associated cytokines [115]. Administration of CGTG-401 in nine patients with advanced solid tumors demonstrated that the treatment was well tolerated, and immunological responses could be confirmed [116]. The Hemminki group also recently showed that a CD40L-expressing oAd enabled effective antitumoral DC-therapies in humanized mice [117].

APCs express the co-stimulatory molecule 4-1BB ligand (4-1BBL), and 4-1BB antibodies are known to stimulate potent antitumor immune responses. The oncolytic adenovirus LoAd703, armed with 4-1BBL together with a trimerized CD40L, is currently being tested in clinical trials as described above [48]. A further approach studying the combined expression of 4-1BBL and IL-12 (Ad-∆B7-IL12/4-1BBL) demonstrated a synergistic enhancement of IFNγ levels compared to single cytokine viruses and supported the administration of DCs through an enhanced TH1-mediated antitumor immune response [118].

The costimulatory OX40 ligand (OX40L) binds to OX40 on T cells and promotes T cell activation. Application of the OX40L-expressing oAd Delta-24-RGDOX showed intratumoral activation of lymphocytes and the development of a tumor-specific CD8 T-cell immune memory in syngeneic mouse models of glioma [119]. Delta24-RGDOX is currently being tested in clinical trials (see above).

Another co-stimulatory receptor is glucocorticoid-induced TNFR family-related gene (GITR). Stimulation with GITR-ligand (GITRL) leads to activation and proliferation of antigen-primed CD4 and CD8 T cells. Glioma treatment with the GITRL-armed oAd Delta24-GREAT resulted in expansion and activation of T cells with a high frequency of central memory CD8 T cells [120].
