**4. Translational E**ff**orts and Clinical Development of Oncolytic Adenoviruses**

Numerous clinical trials with oAds have been reported and several studies are currently ongoing. Without the claim of being exhaustive, Table 1 gives an overview of currently running clinical trials listed on https://clinicaltrials.gov. In the following section, some examples of oAds that have already entered clinical trials are described in more detail.

In preclinical studies, the telomerase-dependent OBP-301/telomelysin showed growth suppression in a panel of tumor cells and in xenograft models of lung cancer [38]. It was also demonstrated that oAds spread to the lymph nodes yielding an antimetastatic effect [50]. Safety of OBP-301 has been confirmed in phase I studies in various advanced solid tumors [51] and is currently being tested in phase II studies in metastatic melanoma, and in esophagogastric cancers in combination with pembrolizumab. It has been observed in bilateral, syngeneic models of colorectal and pancreatic cancer that a variant of OBP-301 in combination with an antibody targeting programmed cell death protein-1 (PD-1) yielded an abscopal effect on non-treated tumors confirming that oAds are promising agents to immunize tumors for checkpoint inhibitors [52].

DNX2401 (tasadenoturev), a delta24-RGD adenovirus, contains delta24-E1A to facilitate selective replication in Rb-dysfunctional cells. The ability to infect tumor cells is enhanced through the integration of an RGD-motif in the fiber. DNX-2401 treatment was effective in preclinical glioma models and showed immunoactivating properties in syngeneic pancreas tumors in mice [53,54]. Regarding therapeutic efficacy, Lang et al. reported that after a single intratumoral injection of DNX-2401 in glioma, 20% of patients survived more than three years, and also almost complete responses could be observed, resulting in a progression-free survival of more than three years [25]. Investigations on tumor specimens from patients receiving a neoadjuvant treatment suggest that DNX-2401 replicates and spreads within the tumor. Signs of effective immune activation, such as infiltration of CD8 T cells and T-bet+ cells, have been reported. DNX-2401 is now under clinical investigation with pembrolizumab in brain cancers. A further variant expressing OX40L (DNX-2440) has already been generated and subjected to clinical testing.

VCN-01 (Ad-E2F-∆24RGD-PH20) is also an oAd for use in Rb-dysfunctional tumors. In this virus, ∆24-E1A is transcriptionally controlled by a promoter harboring E2F-1 responsive elements to boost replication in tumor cells through a positive feedback loop. The virus additionally expresses a hyaluronidase for improved virus spreading. VCN-01 has shown tumor selectivity in vitro, antitumoral effects in murine xenograft models, and increased spreading of virus infection [55,56]. VCN-01 effectively killed patient-derived retinoblastoma in vitro. Intravitreous administration in retinoblastoma xenografts led to tumor necrosis, improved ocular survival, and prevented dissemination. Data from a phase I trial showed the feasibility of vitreous administration and antitumor activity in vitreous seeds. Local inflammation of the retina has been observed, but no systemic complications occurred [57]. VCN-01 is currently involved in clinical studies in pancreatic cancer in combination with gemcitabine and nab-paclitaxel and also in head and neck cancer in combination with pembrolizumab.



**Table 1.** *Cont.*



**Table 1.** *Cont.*


cell lung cancer; TNBC, triple negative breast cancer, PDAC, pancreatic adenocarcinoma; DIPG, diffuse intrinsic pontine glioma; GM-CSF, granulocyte-macrophage stimulating factor; 5-FU, 5-fluorouracil; 5-FC, 5-fluorocytosine; HAIC, hepatic artery infusion chemotherapy; SBRT, stereotactic body radiation therapy; hMSC, human mesenchymal stem cells; DCVAC/PCa, autologous dendritic cells pulsed with killed LNCaP prostate cancer cells; TIL, tumor-infiltrating lymphocytes; TNFα, tumor necrosis factor alpha; IL2, interleukin 2; IL-12, interleukin 12; HSV-tk, herpes simplex thymidine kinase; CXCL, chemokine ligand; FAP, fibroblast activation protein; IFNα, interferon alpha; CAR, chimeric antigen receptor.

CG0070 and ONCOS-102 are both selective for Rb-dysfunctional tumor cells and express granulocyte-macrophage colony-stimulating factor (GM-CSF). CG0070, an oAd5 that uses an E2F-responsive promoter for control of E1A, has been developed for application in non-muscle invasive bladder cancer (NMIBC) [58]. A phase II trial has shown an overall complete response rate of 47% at 6 months in patients with Bacillus Calmette–Guerin (BCG)-unresponsive NMIBC with acceptable toxicity [59]. ONCOS-102 is an Ad5/3 fiber chimeric oAd with favorable toxicity data in a phase I study [47]. Clinical studies of ONCOS-102 combined with chemotherapy in mesothelioma, with pembrolizumab in advanced melanoma, and together with a dendritic cell (DC)-vaccine for treatment of prostate cancer are ongoing. LoAd703 is an oAd5 containing chimeric fibers with an Ad35 knob and is additionally armed with the costimulatory factors CD40L and 4-1BBL. Intratumoral injection of LoAd703 inhibited tumor growth in a syngeneic pancreatic tumor model in mice, which could be further enhanced with gemcitabine [48]. LoAd infection promoted lymphocyte migration and stimulated DCs resulting in the activation of natural killer (NK) cells and the triggering of tumor-directed T cell responses. Currently, the safety and viroimmunotherapeutic activity of LoAd703 in combination with atezolizumab are being clinically investigated in several cancer entities including pancreatic cancer.

Enadenotucirev (or EnAd, formerly Colo-Ad1) has been generated by in vitro chimerization using adenovirus serotypes 3 and 11, and subsequent coevolution by serial passaging in colon cancer cells [60]. Safety of intravenous injections has been demonstrated in a phase I study [61]. An additional phase I study is being carried out in colorectal cancer patients prior to surgical removal and in combination with chemoradiotherapy. A further advancement is the EnAd-variant NG-641, expressing a fibroblast activation protein (FAP)-CD3 bispecific T-cell engager (BiTE), the chemokines chemokine ligand 9 (CXCL9) and CXCL10, and interferon alpha (IFNα), in an approach to attract and stimulate T cells to attack both tumor cells and cancer-associated fibroblasts (CAFs). This variant and also the variant NG-350A, which expresses a costimulatory, agonistic CD40 antibody, are in clinical testing.
