**5. Turning gene therapy into immunotherapy: adenovirus-carrying ARF and interferon-beta**

One of the advantages of the suicide gene approach is the bystander effect that consists of a functional effect that may be seen even when only a small percent of cells has been transduced, and thus, tumor regression can occur. The most accepted hypotheses for this phenomenon of killing nontransduced tumor cells are passive diffusion of the drug, passage of the drug through gap junctions and release of soluble factors, forming a local bystander effect [89]. A different approach that relies on the bystander effect involves the use of mesenchymal stem cells (MSCs) to deliver drugs or vectors. The advantage in this case is that HSV-tk– modified MSCs could be effectively delivered to the area of interest and GCV could then be safely administrated systemically. HSV-tk–bearing MSCs home to and infiltrate the tumor region. Consequently, only tumor cells will be affected, while adjacent areas should remain

98 In Vivo and Ex Vivo Gene Therapy for Inherited and Non-Inherited Disorders

Alternatively, the bystander effect may be a consequence of an immune response initiated by suicide gene therapy *in vivo*, also known as a distant bystander effect. Several articles in the literature have demonstrated a relationship between HSV-tk and immune response. Also called gene-mediated cytotoxic immunotherapy, treatment with HSV-tk promotes innate immune stimulation and infiltration of T cells in tumors [89]. In a clinical trial treating prostate cancer, Ayala and collaborators used an adenoviral vector encoding HSV-tk. In addition to increased apoptosis and decreased microvessel density, they found circulating and activated CD8+ cells and increased IL-12, an important mediator of immune response to tumor cells and viral infection. They also found intratumor CD8+ cells, suggesting the occurrence of both local and systemic responses [91]. Combining suicide and immune gene therapy in an aggressive melanoma model, together HSV-tk and GM-CSF induced a meaningful systemic immune response that was stronger as compared to GM-CSF alone [92]. The induction of an immune response upon CD/5-FC may be less well known [93] but has also been reported [94, 95]. Adenoviral delivery of HSV-tk was tested in a phase III trial, showing increased time to death in patients with high-grade glioma, but it did not increase overall survival [96]; perhaps combining suicide gene therapy with an additional immunotherapy approach could improve response. For example, a current trial is testing the combination of HSV-tk with FMS-like tyrosine kinase 3 ligand (FLT3L) carried by adenoviral vectors in order to promote both tumor

Applied as a safety mechanism, HSV-tk is also used to control CAR-T cells. As described in more detail below, the successful clinical experience of engineered CAR-T cells is also associated with serious adverse events where the massive cell killing results in tumor lysis syndrome, an extreme elevation of plasma IL-6 concentrations that can lead to hypotension and respiratory distress in severe cases [98]. Accordingly, suicide gene therapy can be used to kill the CAR-T cells and thus stop the cytokine release syndrome [99]. In a myeloid leukemia model, Casucci and collaborators associated HSV-tk/GCV with CAR-T cells targeting the CD44v6 receptor and compared this approach with the use of the nonimmunogenic suicide gene iCas9 in an attempt to avoid an unwanted immune response, revealing that the second approach was more effective in containing the cytokine release syndrome [100]. At least three clinical trials utilizing iCas9 to control cell fate upon adoptive T cell transfer have been initi-

unharmed [90].

cell death and DC activity [97].

ated for the treatment of leukemia and lymphoma [79, 101].

Our own research has focused on the use of nonreplicating viral vectors for the transfer of tumor suppressor genes in combination with an immune-modulating gene (**Figure 1**). The goal is to induce both cell death and an immune response, thus overcoming the immunosuppressive tumor microenvironment and initiating the cancer immunity cycle. To this end, we have developed an improved vector system that promotes cooperation between gene function and vector performance.

We have constructed a series of viral vectors where transgene expression is controlled by the tumor suppressor p53, a powerful transcriptional regulator [54, 102, 103]. Moreover, placing the p53 cDNA under the control of the p53-responsive promoter (PGTxβ, or simply PG)

**Figure 1.** Schematic representation of our immunotherapy approach. (1) The adenoviral vectors encode either interferon-β (IFNβ) or p19ARF (alternate reading frame, p19ARF in mice and p14ARF in humans) where expression of the cDNA is controlled by a p53 responsive promoter, termed PG. (2) The combination of IFNβ + ARF induces tumor cell death by necroptosis and is associated with the release of immunogenic factors (such as HMGB1, ATP and calreticulin). (3) Immune cells are recruited and activated to attack the tumor.

establishes an autoregulatory, positive feedback mechanism that was shown to outperform vectors employing a constitutive promoter to express p53. That is, gene expression and cell killing *in vitro* and *in vivo* were superior when using our modified vectors to express p53 [104–106]. We have also looked to p19ARF (alternate reading frame, p19ARF in mice and p14ARF in humans), a functional partner of p53, to serve as the death-promoting factor in our approach and have observed that it is effectively expressed from our p53-responsive vectors in tumor cells that harbor wild type p53, resulting in activation of p53-mediated cell killing *in vitro* and *in vivo* [107]. Admittedly, cell killing mediated by the p53/ARF pathway alone has a limited, but recognized, role in promoting an antitumor immune response [108].

With the advances in molecular and cellular biology as well as animal models for cancer research, the perspective of taking oncolytic virotherapy (OV) from bench to the bedside became feasible. For example, a report in 1991 described the construction of a modified herpes simplex virus (HSV-1), which was thymidine kinase-negative and attenuated for neurotoxic-

Gene-based Interventions for Cancer Immunotherapy http://dx.doi.org/10.5772/intechopen.80386

By definition, OV encompasses native or genetically engineered viruses whose replication is restricted to tumor cells. As per the immunotherapy trend, OV is increasingly gaining attention due to its performance in clinical trials where it is used to treat several types of cancers. With the 2015 approval of Imlygic (talimogene laherparepvec, OncoVex, T-VEC, an HSV-based oncolytic virus) by the FDA and the EMA (European Medicines Agency) for the treatment of unresectable melanoma, the principle of taking advantage of viral replication in

Even in the absence of tools to genetically modify viruses in order to make them safer, in the 1950s, Alice Moore observed that it was possible to generate virus strains with higher oncolytic capacity and more tumor specificity through adaptation. In particular, the oncolytic features of Russian encephalitis virus were enhanced after 20–30 passages in the Sarcoma 180 cell line as compared to the original strain, leading to the idea that the tumor cells could exert an evolutionary pressure upon the virus, favoring those particles adapted to replicate in the

After the development of techniques for the manipulation of DNA, these tools were used to break down the barriers for the development of virotherapy. Thus, undesirable virulence could be mitigated by eliminating key genes from the viral genome, generating attenuated viruses. The viral genome often codes important proteins that regulate its replication in postmitotic cells. For example, the thymidine kinase (TK) gene is associated with DNA synthesis and cell cycle progression [123]. Taking advantage of this information, Martuza and collaborators showed that HSV lacking the gene coding for TK could replicate in dividing cells, but replication was hampered in quiescent cells, in line with the need for selective replication in tumor cells. In an animal model of glioma, locally administrated mutant HSV led to inhibition of tumor growth and showed decreased neurotoxicity [121]. Alternatively, the viral life cycle may be guided by cellular or virus-encoded microRNAs that alter the level of expression of

In addition to the aforementioned approaches, tumor selectivity may be achieved by directing the interactions between the virus particle and the target cell. The retargeting of the viral particles can be achieved in different ways, such as the genetic modification of viral proteins so that they gain specificity for a particular cell surface protein. Alternatively, the use of bispecific adapters mediates the interaction of native capsid proteins with a specific cellular receptor. The virus may also be detargeted, that is, modified so that it no longer interacts with

Besides the transductional targeting, the tropism can be also altered at the transcriptional level by using a tissue-specific promoter to regulate the expression of genes critical for viral

ity [121]; thus, a critical step was taken to advance the technology of OV.

order to treat cancer is now an established therapeutic approach.

**6.1. Targeting and mechanism of OV**

tumor [122].

cell-specific proteins [124].

nontumor cells [125].

In order to activate the immune response against the tumor, we have added interferon-β (IFNβ) to our therapeutic approach since it is a central player in innate and adaptive immunity [109]. Indeed, the combination of p19Arf and IFNβ is better able to induce melanoma cell death both *in vitro* and *in vivo* [110, 111]. Strikingly, the mechanism of cell death involves necroptosis with liberation of the classic markers of immunogenic cell death [111]. In a mouse model of melanoma, we have confirmed the induction of an antitumor immune response in vaccine and immunotherapy settings, with critical involvement of NK cells, CD4+ and CD8+ T cells [112]. In a mouse model of lung carcinoma, we have shown that *in situ* gene therapy can bring about an antitumor immune response with critical involvement of neutrophils [113]. Together these studies show that our gene transfer approach is an effective immunotherapy [114, 115]. The results to date are promising and research will continue to evolve, with critical development using clinically relevant models, such as testing with patient-derived tumor samples as well as alternative animal models, including canines [116].

## **6. Oncolytic virotherapy**

In 1892, viruses were first noted by humans and it took only a few years for researchers to raise the possibility that some viral infections may interfere in the clinical outcomes of some patients with different types of cancers. In 1904, a transitory spontaneous remission of acute leukemia in a patient after infection with influenza was reported, prompting the observation of additional occurrences of this type and paving the way for the concept of virotherapy [117]. One of the first reports of viruses being deliberately applied as a therapeutic approach for cancer dates back to 1949, when Herman A. Hoster and colleagues evaluated the clinical outcome of 21 Hodgkin's disease patients after intentional exposure to Hepatitis B virus [118]. Some years after that, Newman and Southam evaluated the use of several different viruses (vaccinia, mumps, West Nile, dengue, among others) for the treatment of advanced cancer in 57 patients, though no remarkable clinical outcome was observed [119].

Concomitant with the expansion of knowledge in the field of virology, additional protocols describing novel attempts to establish cancer virotherapy were reported, including the use of an array of different virus species, such as adenovirus, Coxsackie, and Epstein-Barr. Despite the new investigations in the 1970s, the threshold of "transitory response" could not be surpassed due to adverse events, such as neurotoxicity, possibly associated with technological limits related to the handling of viruses, for example, the lack of genetic engineering tools needed for the development and testing of more effective and safer versions [120].

With the advances in molecular and cellular biology as well as animal models for cancer research, the perspective of taking oncolytic virotherapy (OV) from bench to the bedside became feasible. For example, a report in 1991 described the construction of a modified herpes simplex virus (HSV-1), which was thymidine kinase-negative and attenuated for neurotoxicity [121]; thus, a critical step was taken to advance the technology of OV.

By definition, OV encompasses native or genetically engineered viruses whose replication is restricted to tumor cells. As per the immunotherapy trend, OV is increasingly gaining attention due to its performance in clinical trials where it is used to treat several types of cancers. With the 2015 approval of Imlygic (talimogene laherparepvec, OncoVex, T-VEC, an HSV-based oncolytic virus) by the FDA and the EMA (European Medicines Agency) for the treatment of unresectable melanoma, the principle of taking advantage of viral replication in order to treat cancer is now an established therapeutic approach.

#### **6.1. Targeting and mechanism of OV**

establishes an autoregulatory, positive feedback mechanism that was shown to outperform vectors employing a constitutive promoter to express p53. That is, gene expression and cell killing *in vitro* and *in vivo* were superior when using our modified vectors to express p53 [104–106]. We have also looked to p19ARF (alternate reading frame, p19ARF in mice and p14ARF in humans), a functional partner of p53, to serve as the death-promoting factor in our approach and have observed that it is effectively expressed from our p53-responsive vectors in tumor cells that harbor wild type p53, resulting in activation of p53-mediated cell killing *in vitro* and *in vivo* [107]. Admittedly, cell killing mediated by the p53/ARF pathway alone has a

In order to activate the immune response against the tumor, we have added interferon-β (IFNβ) to our therapeutic approach since it is a central player in innate and adaptive immunity [109]. Indeed, the combination of p19Arf and IFNβ is better able to induce melanoma cell death both *in vitro* and *in vivo* [110, 111]. Strikingly, the mechanism of cell death involves necroptosis with liberation of the classic markers of immunogenic cell death [111]. In a mouse model of melanoma, we have confirmed the induction of an antitumor immune response in vaccine and immunotherapy settings, with critical involvement of NK cells, CD4+ and CD8+ T cells [112]. In a mouse model of lung carcinoma, we have shown that *in situ* gene therapy can bring about an antitumor immune response with critical involvement of neutrophils [113]. Together these studies show that our gene transfer approach is an effective immunotherapy [114, 115]. The results to date are promising and research will continue to evolve, with critical development using clinically relevant models, such as testing with patient-derived tumor

In 1892, viruses were first noted by humans and it took only a few years for researchers to raise the possibility that some viral infections may interfere in the clinical outcomes of some patients with different types of cancers. In 1904, a transitory spontaneous remission of acute leukemia in a patient after infection with influenza was reported, prompting the observation of additional occurrences of this type and paving the way for the concept of virotherapy [117]. One of the first reports of viruses being deliberately applied as a therapeutic approach for cancer dates back to 1949, when Herman A. Hoster and colleagues evaluated the clinical outcome of 21 Hodgkin's disease patients after intentional exposure to Hepatitis B virus [118]. Some years after that, Newman and Southam evaluated the use of several different viruses (vaccinia, mumps, West Nile, dengue, among others) for the treatment of advanced cancer in

Concomitant with the expansion of knowledge in the field of virology, additional protocols describing novel attempts to establish cancer virotherapy were reported, including the use of an array of different virus species, such as adenovirus, Coxsackie, and Epstein-Barr. Despite the new investigations in the 1970s, the threshold of "transitory response" could not be surpassed due to adverse events, such as neurotoxicity, possibly associated with technological limits related to the handling of viruses, for example, the lack of genetic engineering tools

needed for the development and testing of more effective and safer versions [120].

limited, but recognized, role in promoting an antitumor immune response [108].

100 In Vivo and Ex Vivo Gene Therapy for Inherited and Non-Inherited Disorders

samples as well as alternative animal models, including canines [116].

57 patients, though no remarkable clinical outcome was observed [119].

**6. Oncolytic virotherapy**

Even in the absence of tools to genetically modify viruses in order to make them safer, in the 1950s, Alice Moore observed that it was possible to generate virus strains with higher oncolytic capacity and more tumor specificity through adaptation. In particular, the oncolytic features of Russian encephalitis virus were enhanced after 20–30 passages in the Sarcoma 180 cell line as compared to the original strain, leading to the idea that the tumor cells could exert an evolutionary pressure upon the virus, favoring those particles adapted to replicate in the tumor [122].

After the development of techniques for the manipulation of DNA, these tools were used to break down the barriers for the development of virotherapy. Thus, undesirable virulence could be mitigated by eliminating key genes from the viral genome, generating attenuated viruses. The viral genome often codes important proteins that regulate its replication in postmitotic cells. For example, the thymidine kinase (TK) gene is associated with DNA synthesis and cell cycle progression [123]. Taking advantage of this information, Martuza and collaborators showed that HSV lacking the gene coding for TK could replicate in dividing cells, but replication was hampered in quiescent cells, in line with the need for selective replication in tumor cells. In an animal model of glioma, locally administrated mutant HSV led to inhibition of tumor growth and showed decreased neurotoxicity [121]. Alternatively, the viral life cycle may be guided by cellular or virus-encoded microRNAs that alter the level of expression of cell-specific proteins [124].

In addition to the aforementioned approaches, tumor selectivity may be achieved by directing the interactions between the virus particle and the target cell. The retargeting of the viral particles can be achieved in different ways, such as the genetic modification of viral proteins so that they gain specificity for a particular cell surface protein. Alternatively, the use of bispecific adapters mediates the interaction of native capsid proteins with a specific cellular receptor. The virus may also be detargeted, that is, modified so that it no longer interacts with nontumor cells [125].

Besides the transductional targeting, the tropism can be also altered at the transcriptional level by using a tissue-specific promoter to regulate the expression of genes critical for viral replication. As an example, in order to produce adenovirus whose replication is restricted to prostate cancer cells, expression of the *E1A* adenoviral gene (essential for regulating adenoviral replication) was placed under the control of the prostate-specific antigen (PSA) promoter, leading to an adenovirus that is only able to replicate in prostate cells [126].

and stimulation of an antitumor immune response [30]. After showing safety and antitumor activity in experimental models [30], Imlygic was then administered in a phase I clinical trial, in patients with cutaneous or subcutaneous metastases from refractory head and neck carcinoma, melanoma, breast and gastrointestinal adenocarcinoma, being well tolerated and provoking only mild adverse events (local erythema and fever) [139]. Encouraged by these results, efficacy was assessed in a phase II clinical trial carried out with 50 stages III and IV melanoma patients. In this study, mild adverse events were observed and there was a 26% Response Evaluation Criteria in Solid Tumors (RECIST) response rate, including 8 complete and 5 partial responses [140]. Based on these positive results, an open-label phase III study was carried out where therapy with Imlygic was compared to treatment with GM-CSF, revealing high tolerance to the treatment, and a higher durable response rate (DRR) and also overall survival compared to the GM-CSF treatment, results that culminated in the first FDA

Gene-based Interventions for Cancer Immunotherapy http://dx.doi.org/10.5772/intechopen.80386

An emerging and exciting subject in cellular therapies relies on the engineering of cytotoxic T cells and natural killer cells so they can recognize specific antigens on the cell membrane and induce cell death without reliance on MHC or costimulator expression. Even though infiltrating T cells may recognize tumor antigens, they may be unable to induce a cytotoxic response due to a strong inhibitory microenvironment [142]. The modification of patients' T cells to express a chimeric antigen receptor (CAR) creates the opportunity to induce a strong

Transmembrane CAR receptors have two main functions: the first is to recognize a specific antigen present only in the membrane of tumor cells. The second is to induce signal transduction independently of other costimulatory signals, culminating in the release of cytotoxic signals and T cell proliferation [144]. Physiologically, the activation of a cytotoxic T cell is mediated by a T cell receptor (TCR) in an MHC-dependent context. Though this antigenreceptor interaction is insufficient to bring about cell killing, it is imperative that other transmembrane receptors interact, authorizing T cells to exert their cytotoxic function. Moreover, the tumor has several mechanisms to evade T cell responses, from losing the MHC complex to expressing inhibitory molecules that induce T cell exhaustion and anergy. Therefore, modifying the TCR so they do not depend upon other authorizing signals has proven an exciting

Structurally, a CAR has an extracellular component responsible for recognizing the antigen of interest, comprised of a single-chain variable fragment (scFv), followed by a spacer region whose length may vary, a transmembrane region (TM), and an intracellular domain composed of one or more signaling components associated with T cell activation. The first generation included, on the intracellular domain, the ζ-chain, a portion of the T cell receptor responsible for its activity. Improved understanding of the complementary signals needed for activation lead to the development of second-generation CARs, which include a CD28 costimulatory

cytotoxic response against the tumor even in the face of negative signals [143].

and EMA approval of an OV [141].

**7. CAR-T cells**

strategy [142].

Viruses themselves are entities capable of subverting the cell replication machinery and making a favorable environment for their own replication, which occasionally leads to cell death by lysis when the new viral particles are released and the infection cycle continues, increasing the initial quantity of viral particles that is then only limited by the decreased number of target cell as well as by the direct action of the immune system through an antiviral response. In addition to lysis due to viral replication, some viruses can produce proteins that trigger molecular pathways that lead to cell death, as is the case for adenovirus, whose E3-11.6 K transcript is found to be important for the lysis of infected cells [127, 128]. However, more recently, it was found that the immune system, concomitant to the intrinsic effect of oncolytic infection, plays an important role.

After infection, more precisely after cell lysis, the release of intracellular content participates in the activation of both innate and adaptive immune responses against tumor- and virus-associated antigens, potentially reverting the intrinsic immune tolerance of the tumor microenvironment [129]. After rupture of the cellular membrane by the virions, the following release of PAMPs and DAMPs induces the activation of type I interferon, Toll-like receptor–mediated molecular pathways and the production of cytokines, which culminate in the recruitment and activation of antigen-presenting cells (APCs) and the subsequent establishment of a memory immune response [130].

#### **6.2. Oncolytic virotherapy makes its mark: oncolytics with regulatory approval for the treatment of cancer**

In 2005, Oncorine (H101, Onyx-015), an adenovirus-based oncolytic developed by Shanghai Sunway Biotech, was approved by State Food and Drug Administration, China (SFDA), for the treatment of head and neck squamous cell carcinoma [131]. Oncorine is a modified adenovirus whose E1B and E3 genes are deleted, leading to a virus that, it was originally thought, should only replicate in cells that lack p53 activity, mainly tumor cells [132, 133], though other mechanisms have been proposed for Oncorine's tumor selectivity [134]. Its precursor, Onyx-15, showed good performance in clinical trials, especially when combined with additional therapeutic approaches, and was well tolerated and safe [135], with no therapy-associated severe adverse events when administered intratumorally in gliomas [136]. In addition to its safety profile, Onyx-15 administration may be associated with some clinical improvement for patients with metastatic colorectal cancer who failed the first-line therapy [137] and those with hepatobiliary tumors not eligible for surgical resection [138].

In 2015, the FDA and the EMA approved an OV based on a modified herpes simplex virus (HSV-1) for the treatment of melanoma. Imlygic (OncoVex, T-VEC, talimogene laherparepvec) expresses granulocyte-macrophage colony-stimulating factor (GM-CSF), while viral genes ICP34.5 and ICP47 were deleted, modifications that conferred better replication in tumor cells and stimulation of an antitumor immune response [30]. After showing safety and antitumor activity in experimental models [30], Imlygic was then administered in a phase I clinical trial, in patients with cutaneous or subcutaneous metastases from refractory head and neck carcinoma, melanoma, breast and gastrointestinal adenocarcinoma, being well tolerated and provoking only mild adverse events (local erythema and fever) [139]. Encouraged by these results, efficacy was assessed in a phase II clinical trial carried out with 50 stages III and IV melanoma patients. In this study, mild adverse events were observed and there was a 26% Response Evaluation Criteria in Solid Tumors (RECIST) response rate, including 8 complete and 5 partial responses [140]. Based on these positive results, an open-label phase III study was carried out where therapy with Imlygic was compared to treatment with GM-CSF, revealing high tolerance to the treatment, and a higher durable response rate (DRR) and also overall survival compared to the GM-CSF treatment, results that culminated in the first FDA and EMA approval of an OV [141].
