**4. Suicide gene therapy**

complement-mediated lysis and thereby contribute to the elimination of tumor cells. Another aspect of passive immune therapies is the use of recombinant cytokines, such as IL-2, IL-12 and interferon-α, β and γ [63]. Although both strategies can modulate the immune system to bring improvements, their action is temporary and can only be palliative, requiring successive doses and may provoke serious adverse effects [64, 65]. Checkpoint blockade has been gaining prominence recently and also encompasses the use of monoclonal antibody inhibitors of negative modulators of immune function, such as anti-PD-1, PDL1 and CTLA4 [66–68].

The presentation of antigens is a crucial event in the genesis of adaptive immune responses. Antigen-presenting cells (APCs) capture proteins in peripheral tissues, process them by proteolytic digestion and, after migrating to secondary lymphoid organs, present them to T lymphocytes in the context of class I or II MHC molecules [69]. In addition to the MHC molecules (HLA in humans), a number of costimulators (such as CD80, CD86, CD40, CD83 and CD14) are also required, important for the complementation of the biochemical signals necessary for the activation of T lymphocytes upon recognition of the presented antigens [70–72]. The maturation of cytotoxic T lymphocytes is central to the generation of adaptive immunity and,

Autologous dendritic cell vaccines can be prepared from the patient's peripheral blood, with

maturation of monocyte-derived DCs (Mo-DCs). Next, different techniques can be used to "load" the tumor antigens into the DCs, such as peptides, proteins, DNA or RNA transfection, exosomes or exposure to tumor cell lysates [73, 74]. In addition to the changes that occur in the tumor microenvironment, the tumor is also capable of inducing systemic changes in the host's immune system, so that the monocytes from cancer patients may result in DCs with altered phenotype and cytokine production, negatively impacting immunotherapy [15]. Thus, immunotherapy with allogeneic DCs represents an interesting alternative. In addition to offering greater availability of DCs (since healthy donors have higher monocyte counts),

Barbuto et al. used an interesting strategy for the construction of DC-based therapeutic vaccines for cancer. Healthy donor monocytes are differentiated and matured *ex vivo* and are subsequently fused to tumor cells by electrical shock, resulting in a hybrid cell. These hybrids are gamma irradiated, to prevent replication, and then administered back to the patient, seeking the generation of immune responses against neoplasms. Although the hybrids were shown to offer limited improvement of mortality rates, longer survival of the treated patients was achieved [75, 76]. Another phase I study in melanoma patients employed immunotherapy using plasmacytoid and myeloid DCs (pDC and mDC, respectively). The results were promising and indicated a survival time of more than 2 years in most of their patients [77, 78].

Currently, more than 500 clinical trials using dendritic cells are being conducted for the treatment of various forms of cancer in different countries. Most of these (324) are in the US, followed by the European Union (120) and China (72) [79]. Although results are very heterogeneous, there is a consensus that the use of these therapies in humans does not present risks

tissue rejection by antigenic determinants (HLA) may function as an adjuvant.

cells and *in vitro* treatment with GM-CSF and IL-4 for differentiation and

**3.2. Modified dendritic cells as therapeutic vaccines**

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

in turn, is one of the major antitumor defenses.

isolation of CD14<sup>+</sup>

or serious side effects.

In cancer gene therapy, different approaches can be used to kill tumor cells. Suicide gene therapy (also called gene-directed enzyme prodrug therapy) is one example where a viral or bacterial gene is introduced in the cancer cell such that it can convert a nontoxic prodrug into its lethal form. The most famous system used in this strategy is herpes simplex virus thymidine kinase gene (HSV-tk) and ganciclovir (GCV) as the prodrug. Expression of the HSV-tk gene leads to production of the enzyme that turns GCV into GCV monophosphate. After this first conversion, cellular kinases metabolize GCV monophosphate into GCV triphosphate, which is an analogue of deoxyguanosine triphosphate. GCV triphosphate causes tumor cell death upon its incorporation into DNA and consequent inhibition of DNA replication [86]. Another example of a suicide gene is the cytosine deaminase gene (CD) of *Escherichia coli* that catalyzes the hydrolytic deamination of cytosine into uracil, converting the nontoxic antifungal agent 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU). This process causes cell death by three main pathways: thymidylate synthase inhibition, formation of (5-FU) RNA and of (5-FU) DNA complexes [86]. More recent systems were developed, including an engineered version of human thymidylate kinase (TMPK) and the prodrug azidothymidine (AZT), which was first tested in leukemia model *in vitro* and *in vivo*. Native TMPK catalyzes AZT into AZT monophosphate, the toxic compound, only very slowly, so the engineering of TMPK allows it to act more robustly [87, 88]. In another example, the iCas9 system consists of inducible expression of the caspase-9 gene and administration of the small molecule chemical inducer of dimerization (CID) that leads to caspase-9 dimerization, thus promoting apoptosis [86].

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 unharmed [90].

In summary, suicide gene therapy is an approach that involves death mechanisms and immunotherapy. The strategy is still evolving from the initial trials and may be an interesting option

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

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

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.

**5. Turning gene therapy into immunotherapy: adenovirus-carrying** 

against cancer and for the improved safety of CAR-T cell therapy.

**ARF and interferon-beta**

and vector performance.

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 cell death and DC activity [97].

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

In summary, suicide gene therapy is an approach that involves death mechanisms and immunotherapy. The strategy is still evolving from the initial trials and may be an interesting option against cancer and for the improved safety of CAR-T cell therapy.
