**7. CAR-T cells**

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,

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

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 establish-

**6.2. Oncolytic virotherapy makes its mark: oncolytics with regulatory approval for** 

with hepatobiliary tumors not eligible for surgical resection [138].

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

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

leading to an adenovirus that is only able to replicate in prostate cells [126].

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

infection, plays an important role.

ment of a memory immune response [130].

**the treatment of cancer**

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

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 strategy [142].

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 domain, thus ensuring full activation of the T cell. The third generation included other transduction signaling domains, preferentially originating from transmembrane proteins derived from the TNF superfamily, such as CD27, 4-1BB and OX40. All of them can transduce signals resulting in survival, proliferation and maintenance of T cells. The fourth generation uses a vector to deliver, in addition to CAR, cytokine genes, such as IL-2 or IL-12, whose expression changes the tumor microenvironment in favor of T cell activity [144].

research conducted by Smith and colleagues [148], they have developed an approach that may show a way around this problem. In a mouse model, they have modified the circulating T cells within the animal's own body. The strategy is based on the transfection of the CAR gene using β-amino-ester–based nanoparticles. For this, nanocarriers were coated with CD3, a lymphocyte surface antigen. The recognition of this antigen induces the endocytosis of the nanocarriers by the lymphocytes. Furthermore, peptides containing microtubule-associated sequences (MTAS) and nuclear localization signals (NLS) were added to the polymer, facilitating the rapid import of its genetic load through microtubule transport machinery. Alternative approaches include the use of viral vectors and the use of transposon/transposase systems, such as sleeping beauty, that promote integration of the CAR sequence in the host DNA [148]. Instead of a complicated scenario of transporting of patients' cells to and from specialized facilities, methodology enabling *in situ* modification of T cells implies that nanoparticles, virus and other vectors containing the CAR sequence can be produced in a central location, packaged and shipped to any hospital. All that is needed is a syringe to inject the vector into the bloodstream of the patient. As the nanoparticles are stable, this enables long-term storage, reducing the cost of this medical technology and permitting the sale of CAR cell therapies at

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

Clearly, cancer immunotherapy can be achieved by a variety of interventions that share the common goal of boosting the antitumor immune response. These modalities may target distinct points along the cancer immunity cycle, from inducing immunogenic cell death, promoting antigen presentation and culminating in activation of innate and adaptive responses, including cytolytic T cell activity, which can then further promote antitumor immunity since tumor cell killing would reinitiate and propagate the cycle [149]. Moreover, distinct points in the cancer immunity cycle may be targeted simultaneously, enhancing even more the antitu-

As shown here, gene transfer plays a critical role in several key cancer immunotherapies. Vaccines, suicide gene therapy, simultaneous induction of cell death and immune response, OV and CAR-T cells all benefit from gene transfer. While the gene transfer technology will continue to evolve, the therapeutic benefit of genetically modifying cells in order to alter their function will certainly continue to be a central theme in cancer immunotherapy. The approval of Imlygic (FDA and EMA), Yescarta and Kymriah (FDA and EMA), as well as the commercialization of Oncorine (China) show that immunotherapies involving some component of

In addition, we expect that future approaches will rely on multiple immunotherapies that work in harmony. For example, checkpoint blockade along with the gene transfer interventions should bring about strategic combinations of inducing cell death, tumor-specific immune response and maintenance of cytolytic T cell activity. Challenges remain to be addressed, such as avoiding adverse effects, proper monitoring criteria, identification of adequate biomarkers

more affordable prices.

**8. Conclusions**

mor response.

gene transfer are now well established.

The first insight into the development of a chimeric transmembrane receptor that could activate cytotoxic T cells came in 1989 by Gross and colleagues. And in 2017, the FDA approved the first two CAR-T cell therapies in rapid succession. These CARs target CD19, a molecule expressed only in B-lymphocytes, an approach shown to be a powerful second-line treatment against B cell acute lymphoblastic leukemia (B-ALL) (Kymriah—tisagenlecleucel, August 2017 [145]) and certain B cell lymphomas (Yescarta—axicabtagene ciloleucel, September 2017 [146]). While both present a scFv against CD19, Kymriah uses the 4-1BB whereas Yescarta uses CD28 as costimulatory domains. The success in clinical trials ranged from 70 to 94%, making these treatments a breakthrough in gene and immunotherapy [144]. However, there are cytotoxic effects that in some cases can be intense, caused by the killing of large numbers of cancer cells that release cytokines and waste products, leading to harmful consequences in the patients. Thus, much more is needed to understand and manage the side effects of these new and promising therapies, such as the inclusion of a suicide gene to eliminate overactive CAR-T cells [147].

Despite the incredible potential of this therapeutic strategy, CAR-T cells have some limitations that prevent their effective use in the fight against a wide range of tumors. Among them, the most troubling is the lack of a perfect antigen present only in tumor cells but not in other tissues. Tandem CAR and inhibitory chimeric antigen receptors (iCAR) are some of the strategies with the greatest potential to overcome this barrier. Tandem CAR consists of two chimeric receptors designed to provide costimulatory signals in response to the recognition of two different antigens [144]. Only after the recognition of both signals are the tandem CAR cells activated. On the other hand, iCAR aims to inhibit T cell activity as soon as the second specific antigen is recognized [144].This second antigen does not exist in tumor cells, so when the iCAR-T cells find it, they are inhibited and leave nontumor cells unscathed.

In some studies, the inhibitory molecules used in the construction of iCAR-T cells are derived from the intracellular domains of proteins often expressed by tumors and whose function is to evade the immune system. Well-known examples are the receptors CTLA-4 and PD-1 that reduce the potency of TCR signaling. The fusion of their intracellular domain to a CAR also inhibits signaling, resulting in decreased cytokine production, limited lymphocyte motility and reduced target cell lysis [144].

Another hindrance to the application of CAR cell therapies is their large-scale production. The usual steps to produce CAR cells are based on extraction of cells from the patient, genetic engineering of NK or T cells, expansion and infusion in the patient. Due to the laborious process, few health care institutions are prepared to produce CAR cells. And off-site preparation of the CAR cells presents extensive logistical challenges. Thus, the production of the CAR cells is one of the principle factors that promote the high cost of this therapy. In a remarkable research conducted by Smith and colleagues [148], they have developed an approach that may show a way around this problem. In a mouse model, they have modified the circulating T cells within the animal's own body. The strategy is based on the transfection of the CAR gene using β-amino-ester–based nanoparticles. For this, nanocarriers were coated with CD3, a lymphocyte surface antigen. The recognition of this antigen induces the endocytosis of the nanocarriers by the lymphocytes. Furthermore, peptides containing microtubule-associated sequences (MTAS) and nuclear localization signals (NLS) were added to the polymer, facilitating the rapid import of its genetic load through microtubule transport machinery. Alternative approaches include the use of viral vectors and the use of transposon/transposase systems, such as sleeping beauty, that promote integration of the CAR sequence in the host DNA [148].

Instead of a complicated scenario of transporting of patients' cells to and from specialized facilities, methodology enabling *in situ* modification of T cells implies that nanoparticles, virus and other vectors containing the CAR sequence can be produced in a central location, packaged and shipped to any hospital. All that is needed is a syringe to inject the vector into the bloodstream of the patient. As the nanoparticles are stable, this enables long-term storage, reducing the cost of this medical technology and permitting the sale of CAR cell therapies at more affordable prices.
