Interferons (IFNs) are the first group of cytokines that demonstrated efficacy in the treatment of malignancies. IFN signaling is mediated by binding of IFNs to their receptors and subsequent activation of Janus tyrosine kinase (JAK)-STAT signaling pathway (113). Interferon (IFN) has a strong antitumor effect and has been used in the treatment of pancreatic cancer. Some studies showed that IFN-α significantly prolongs survival rate (by 2 to 5 years) (114–116). However, there are limitations in using IFN-based therapies, including dose-limiting systemic toxicities and low intratumoral concentration of IFN because of its short half-life in the bloodstream (117, 118). In response to this, oncolytic adenoviruses have been engineered to express IFN, which showed positive outcomes in treatment of cancer.

Armstrong et al. (119, 120) has reported oncolytic adenoviruses expressing human IFN-α as a promising platform for selective, long-term expression of IFN in human pancreatic cancer tissues (119, 120). Armstrong et al. used the oncloytic adenovirus Ad5/Ad3-Cox2-ΔE3-ADP-IFN in their study, which was developed to selectively replicate within cancer cells expressing cyclooxygenase 2 (Cox2). In order to improve the infectivity and oncolysis of the oncolytic adenoviruses, they made genetic modification in the virus to include an Ad5/Ad3 chimeric fiber and overexpress the adenovirus death protein (ADP). The expression of adenovirus death protein (ADP) occurs during the late stage of infection a lytic infection. ADP promotes the release of progeny virus (virion) by accelerating the lysis and death of the host cell ( 121).

Similarly, other researchers have also reported as oncolytic adenoviruses expressing IFN-α have promising outcomes in treatment of cancer. The researchers reported that an oncolytic adenovirus (OAd-hamIFN) that was investigated in an immunocompetent Syrian hamster model of pancreatic ductal adenocarcinoma showed efficient viral replication in tumor, significant inhibition of tumor growth, and enhanced survival when used in combination with chemo therapies and radiation therapies (122, 123). Likewise, studies conducted by Tao etal. (124) have shown that significant tumor regression of bladder cancers occurred following administration of an adenovirus expressing human interferon α (Ad-IFNα) using a mouse superficial bladder cancer model in which human bladder tumors are growing (124).

Granulocyte macrophage colony-stimulating factor (GM-CSF) promotes activation of T-cells and maturation of dendritic cells (125, 126). Therefore, an immunotherapy using adenoviruses that express GM-CSF transgene can be an effective anti-tumor therapy. ONCOS-102 is an oncolytic adenovirus that contains GM-CSF transgene (127). A clinical study (phase-I) in patients with advanced solid tumors including colon, lung, and ovarian cancers demonstrated a strong immune cell infiltrate into tumors without dose-limiting toxicities (127).

The other oncolytic adenovirus that expresses immunostimulatoy cytokine is LOAd703. LOAd703 is armed with 4-1BBL and trimerized CD40L that was shown to replicate and kill pancreatic cancer cells via oncolysis in both in vitro and in vivo assays (128).

TILT-123 is an oncolytic adenovirus that incorporates transgenes for human tumor necrosis factor alpha (TNF-α) and interleukin-2 (IL-2). TNF-α and IL-2 were shown to be promising T cell stimulating factors when used in combination with adoptive cell therapy (129, 130).

Interleukin-12 (IL-12) is a proinflammatory cytokine that initiates antitumor immune responses by promoting the generation of tumor-specific cytotoxic T lymphocytes (CTLs) and activating natural killer (NK) cells and CD4+ T cells (131, 132). Several studies demonstrated promising antitumor effects of IL-12 in mice having solid tumor and hematologic malignancies (133–137). However, findings from clinical trials indicated severe side effects of systemic administration of IL-12 that markedly dampened hopes of the successful use of this cytokine in cancer patients (138). But, the use of Ads containing IL-12 transgene seems critical for maximizing the density of IL-12 that reaches the tumors and alleviating the toxicity.

Ads that were engineered to express IL-12 have been shown to enhance immune stimulation and antitumor effect in a clinical trial (139) and pre-clinical studies (140). Likewise, a study by Wang et al. showed that oncolytic adenovirus (Ad-TD-nsIL-12) expressing IL-12 induces strong antitumor immune response against pancreatic cancer in Syrian hamster models without toxicity (141). Similarly, a replication-deficient adenoviral vector encoding human IL-12 p70 transgene (Ad-RTS-hIL-12) was also shown to have no toxic effect in phase one clinical trial (142). A recent trial found that intratumoral injection of Ad-RTS-hIL-12 was safe in patients with recurrent glioblastoma (142).

In addition to IL-12, there are also other cytokines, such as IL-24 and IL-13 that have been used to arm adenoviruses and have shown promising immune-activating properties in multiple preclinical cancer models (143–145). RANTES is another cytokine engineered in Ad that has been shown to enhance anticancer effect. In murine models of mammary adenocarcinoma and lymphoma, Ad-RANTES-E1A eradicated established tumors and inhibited metastases by recruiting DCs, macrophages, NK cells, and CD8+ T cells into the immunologically cold tumors (146).

Bispecific T cell engager (BiTE) is a kind of artificial antibody that represents an innovative immunotherapy approach which enhances patients’ immune response to tumors. BiTE has dual antigen specificity, allowing them to bind to two unique antigens at the same time, i.e. the BiTE bind simultaneously to both tumor associated antigen and T cell (usually CD3), ultimately stimulating T-cell activation, tumor killing and cytokine production (147). BiTE has been shown to be promising immunotherapy for the treatment of cancer in preclinical and clinical studies (148, 149). The therapeutic efficacy of BiTE can be improved by using BiTE in conjunction with adenoviruses.

Pomés et al. tested weather an OAd expressing FAP-targeting BiTE improve antitumor efficacy (150). FAP-BiTE (FBiTE) comprises two single chain variable fragments (ScFvs) joined by a flexible Gly-Ser linker. One scFv arm binds mouse and human Fibroblast Activation Protein (m/hFAP), while the other binds human CD3epsilon on the T cell receptor (TCR). The study showed that the FBiTEs activate and re-direct T-cells to FAP+ cells, which leads to enhance cytotoxicity in vitro and improve antitumor efficacy in vivo.

Likewise, Alemany group engineered an oncolytic adenovirus expressing an EGFR-targeting BiTE that showed improved T cell-mediated killing of cancer cells both in vivo and in vitro (151). They also demonstrated that anti-EGFR BiTE-armed OAd in combination with adoptive CAR-T cell therapy results in improved antitumor efficacy and prolonged survival of mice as a result of intratumoral T cell activation by BiTE (152).

Similarly, another research group (Fisher group) that developed BiTE armed oncolytic adenovirus (EnAd-SA-EpCAM) reported promising results in use of BiTE expressing adenoviruses against cancer (153). The BiTE of EnAd-SA-EpCAM binds to epithelial cell adhesion molecule (EpCAM) in cancer cells. The Fisher group reported that the EnAd-SA-EpCAM effectively activate endogenous T cells within the immune-suppressive microenvironment and exhibited killing of endogenous tumor cells without the addition of exogenous T cells (153).

Recently, the Fisher group engineered another BiTE armed oncolytic adenovirus (EnAd-FAP-BiTE), which is targeted fibroblast activation protein (FAP) in cancer-associated fibroblasts (CAFs). CAFs are the main cellular component of solid tumor TME. The EnAd-FAP-BiTE induced the activation of tumor-infiltrating T cells that target and kill CAFs (154). Likewise, another BiTE armed adenovirus (ICO15K-FBiTE) that was developed by the Fisher research group was shown to enhance overall antitumor efficacy without increasing the toxicity in mouse model (154).

ADV/HSV-TK is an adenoviral vector expressing the herpes simplex virus (HSV) thymidine kinase (TK) gene. The HSV-TK protein has two principal functions, including 1. TK is a superantigen that stimulates a potent immune reaction and 2. A nucleotide analog product of prodrug phosphorylation lead to the death of dividing cancer cells (155). Herman et al. studied ADV/HSV-TK in combination with ganciclovir for the treatment of human prostate cancer (156). They reported that injection of ADV/HSV-TK into the prostate gland in the region with the greatest concentration of tumor cells resulted in significant reduction in tumor burden. Herman et al. also showed that ADV/HSV-TK was proven safe, with minimal toxicity (156). Likewise, other researchers also made similar observation on the efficacy and toxicology of ADV/HSV-TK against glioma, retinoblastoma, and mesothelioma (157–159).

Immune checkpoints are regulators of the immune system. Immune checkpoints pathways prevent the immune system from attacking its own cells. Immune checkpoints works by means of immune check point proteins, including programmed cell death 1 protein (PD-1) and programmed cell death protein-ligand 1 (PD-L1), and cytotoxic T-lympotcyte antigen-4 (CTLA-4). One of the mechanisms by which tumor cells evade immunesurveillance is by activation of immune checkpoint pathways that suppress antitumor immune responses. PD-1 is the transmembrane programmed cell death 1 protein, which interacts with PD-L1 (PD-1 ligand 1, or CD274). The binding of PD-L1 on cancerous cell with PD-1 on T-cell surface results in inhibition of T-cell activity. Therefore, antibodies that bind with PD-L1 and or PD-1 can block the interaction of PD-L1 and PD-1, allowing T-cells to attack the tumor.

Immune checkpoint inhibitors therapy is a kind of immunotherapy that work by blocking the binding of checkpoint proteins (PD-1, PD-L and CTLA-4) with partner proteins so that T-cell became free and active to attack cancer cells. Ipilimumab is an anti-CTLA-4 antibody (that blocks CTLA-4 ligand and prevents inhibition T-cells) that was approved by the United States food and drug administrations (U.S. FDA) in 2010 for the treatment of advanced melanoma (171). However, the systemic administrations of immune checkpoint inhibitors have been shown to cause severe immune-related adverse events (172–174). One of the strategies that can be employed to overcome these obstacles of using immune checkpoint inhibitors (such as anti-PD-L1, anti-PD-1 and anti-CTLA-4) in cancer therapy is to utilize immune checkpoint inhibitors in combination with oncolytic adenoviruses.

ETBX-011(also called Ad5-CEA) is an adenoviral vector-based cancer vaccine that is engineered to express a modified carcinoembryonic antigen (CEA) which contains the highly immunogenic epitope CAP1-6D. CEA is found in different kinds of cancer cells. ETBX-011 induces potent CEA-specific cell-mediated immune responses with antitumor activity (183). ETBX-011 is well-tolerated in metastatic colorectal cancer patients and has potential survival benefit (184, 185). This study showed evidence of a potential survival benefit: 25 patients treated at least twice with ETBX-011 exhibited a 12-month overall survival probability of 48% and a mean overall survival of 11 months (184, 185).

In order to overcome the challenge posed by tumor heterogeneity, such as the diversity of tumor associated antigens (TAA), a Tri-Ad vaccine (a combination of ETBX-011 with three different human TAA-expressing Ad vector vaccines (ETBX-011, ETBX-061, and ETBX-051) have been developed and tested in phase I clinical trial. The first, ETBX-061, is an Ad5-based adenovirus vector vaccine with the same backbone as the ETBX-011, but expressing a modified human mucin 1 (MUC1) gene. The modified MUC1 gene contains agonist epitopes designed to increase CTL antitumor immune responses. The other, ETBX-051 or Ad5-brachyury, encodes the entire brachyury gene with a deletion of 25 amino acids involved in DNA binding, and modified to express an enhancer T cell epitope (186). The Tri-Ad vaccine regimen induces antitumor cytotoxic T cell (CTL) responses and was proven safe and well tolerated in treatment of advanced cancer (187).

Ad5-PSA is replication-deficient adenoviral vector that is engineered to express human prostate specific antigen (PSA). Ad5-PSA stimulates potent anti-PSA T cell responses and causes the destruction of PSA-secreting tumor cells both in preclinical (188) and clinical (189–191). Furthermore, Ad5-PSA prolongs survival and was demonstrated safe in patients with recurrent and hormone refractory prostate cancer (189–191).

Ad-E6E7 is another replication-deficient adenovirus-based anti-cancer vaccine, which expresses human papillomavirus (HPV) genes E6 and E7 (192, 193). MG1-E6E7 is an oncolytic maraba virus strain which also expresses the HPV genes E6 and E7. The combination of Ad-E6E7 and MG1-E6E7 induced potent tumor-specific responses in various mouse cancer models (194). In addition, the combination of Ad-E6E7 with MG1-E6E7 was proven to significantly prolong survival of mice with HPV-associated cancer (194).

Cappuccini et al. investigated the immunogenicity and efficacy of ChAdOx1–MVA against prostate cancer in mouse model (195). They reported that the ChAdOx1–MVA induced tumor specific cell mediated immune response. Furthermore, the ChAdOx1–MVA was proven to prolong the survival of the mice when used in combination with anti-PD-1 antibody (195).

The other approach to use adenoviral vectors for anticancer vaccine is the use Dendritic cells (DC)-based adenoviral vaccines. In this approach, DCs are transduced ex vivo with Ads encoding cancer specific antigens. The advantageous of transducing DC ex vivo instead of injecting Ads in vivo include: 1. Ex vivo transduction of DC overcomes pre-existing anti-viral immunity and induce effective anti-tumor responses (196, 197) and 2. Ex vivo transduced DC induces lower anti-viral antibody responses than that of Ads injected in vivo (196, 198).

Ad-transduced DCs have shown positive outcomes against both solid tumor and hematologic cancers (4, 66, 199). Butterfield et al. (199) tested an autologous DCs transduced ex vivo with Ads encoding the full-length melanoma antigen MART-1/Melan-A. They reported that the autologous DC-based adenoviral vaccine significantly induced cell mediated immune response (CD+8-T cells response) in metastatic melanoma patients (199). Similarly, in patients with advanced NSCLC, injections of autologous DCs resulted in induction of systemic tumor antigen-specific immune responses with enhanced CD8+T-cell infiltration (4).

Pellom et al. evaluated PRGN-2009, a therapeutic gorilla adenovirus HPV vaccine containing multiple cytotoxic T cell epitopes of the viral oncoproteins HPV 16/18 E6 and E7, including T cell enhancer agonist epitopes. The study revealed that PRGN-2009 treatment reduced tumor volume and increased CD8+ and CD4+ T cells in the tumor microenvironment of humanized mice bearing the human cervical tumor SiHa. The PRGN-2009 monotherapy in the syngeneic TC-1 model also reduced tumor volumes and weights, generated high levels of HPV16 E6–specific T cells, and increased multifunctional CD8+ and CD4+ T cells in the tumor microenvironment (40).

Non-human adenoviruses have been used to overcome the challenge of preexisting human adenoviral immunity. The commonly used nonhuman adenovirus vectors include gorilla adenovirus and chimpanzee-derived adenovirus vector (ChAd) (40). Several chimpanzee adenoviral vector-based vaccines, such as ChAd7 for Ebola virus, ChAd6 for rabies, and ChAd6, ChAd7, and ChAd9 for malaria, have shown high efficacy in animal models (216). Furthermore, ChAd63-based malaria and ChAd3-based hepatitis C virus vaccines have shown to be safe and highly immunogenic in phase I clinical trial (217, 218). Despite low seroprevalence of ChAd vectors in humans, pre-existing cross-reactive T cells against many conserved viral antigens are still a major concern. HAd-induced ChAd cross-reactive T cells have been reported against ChAd6, ChAd7, ChAd24, ChAd32, and ChAd68 (35, 38, 219, 220).

In addition to chimpanzee adenoviruses, several other adenoviruses derived from animals such as bovine, porcine, ovine, canine, and fowl are have been explored for vector development (221–224). The human population lack nAbs against these adenoviruses, and therefore, the vectors derived from these adenoviruses could be more efficacious in comparison to HAd and ChAd. The mouse models with experimentally induced pre-existing immunity by the administration of HAd vector demonstrated a lack of nAbs and CD4+ T cells against PAd3 and BAd3 (225). Furthermore, BAd3- or PAd3-based influenza virus vaccine demonstrated high efficacy even in the presence of pre-existing HAd5 immunity. There was also no effect of pre-existing HAd5 immunity on transgene expression, immunogenicity, and efficacy in animal models.

Heterologous prime-boost strategy is another strategy to avoid pre-existing adenoviral vector immunity. Unlike homologous prime-boost immunizations, in this strategy, the priming and boosting are done by using different antigen delivery vehicle and/or vectors derived from either different serotypes of the same species or vectors from completely different host species. For instance priming with ChAd68 and boosting with ChAd1 or priming with DNA and boosting with HAd5 (226). Studies have shown that the heterologous prime-boost induces more robust immune responses compared to single vaccination or homologous prime-boost immunizations. The cellular immune responses induced by DNA prime and HAd boost were not affected by pre-existing HAd5 immunity. A preclinical study involving Plasmodium or SARS antigens encoded by Modified Vaccinia Ankara (MVA)/adenoviral vector as prime/boost showed induction of robust T cell and Ab responses of higher magnitude compared to Ad/DNA regimens (218, 227–229).

Studies showed that different route of vaccinations can overcome the detrimental effect of pre-existing immunity. This result is partly due to evasion of tissue-resident Ad-specific T cells when using different routes of immunization. Tissue resident CD8 memory T cells are not systemic and do not prevent Ad vector infection in distant tissues (230–232). In preclinical study, HAd5-induced protective immune responses by intranasal/intratracheal immunization were not affected by pre-existing HAd5 immunity that had been induced by intramuscular administration of an unrelated HAd5 vector (233).