**8. Conclusions**

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

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

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

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

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

the iCAR-T cells find it, they are inhibited and leave nontumor cells unscathed.

changes the tumor microenvironment in favor of T cell activity [144].

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

CAR-T cells [147].

and reduced target cell lysis [144].

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 antitumor response.

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 gene transfer are now well established.

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 and definition of a reasonable price tag for cutting edge, personalized interventions. Thus, immunotherapies require further study. As such future developments unfold, gene transfer technologies are expected to remain as crucial components of cancer immunotherapy.

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