**3. Cancer vaccines**

Some of these strategies involve the application of soluble antibody molecules that specifically recognize and bind TAAs, resulting in blocked receptor signaling and/or passive immunotherapy. In particular, targeting tumor cells by engaging surface antigens differentially expressed in cancers has been widely used. For example, rituximab targets CD20 in non-Hodgkin B cell lymphoma. At least nine monoclonal antibodies (mAbs) targeting six TAAs (HER2/Neu, EGFR, VEGF, CD20, CD52 and CD33) are approved for the treatment of solid

Approved by the Food and Drug Administration (FDA) in 2011, ipilimumab is a mAb against cytotoxic T lymphocyte–associated protein 4 (CTLA-4), a negative checkpoint of T cell function. Thus, checkpoint blockade with ipilimumab releases the brakes of the immune system, promoting T cells to combat cancer cells, and has already benefited thousands of patients with advanced melanoma, a disease that typically kills in less than a year [20]. Additional targets of immune checkpoint therapy include programmed cell death protein 1 (PD1) and its ligand PD-L1, which are even more effective and have fewer side effects as compared to anti-CTLA4 [21]. Moreover, checkpoint inhibitors may be used in combination with each other or with other therapies resulting in the induction of sustained antitumor responses in a wide variety of tumors [22–25]. Checkpoint blockade has undoubtedly been one of the most impressive advancements in cancer therapeutics in recent years, prolonging and saving the lives of many cancer patients. Even so, this approach does not directly induce a *de novo* immune response

Vaccines are strategies to activate effector immune cells upon stimulation with tumor antigens, promoting the patient's own immune system to mount an immune response against neoplastic cells. Numerous vaccine approaches have been attempted and share the goal of providing effective target antigens while reverting, perhaps, the immunosuppressive tumor microenvironment and activating the ability of DCs to present these antigens. One example is GVAX (Cell Genesys, Inc., South San Francisco, CA), a polyvalent vaccine derived from a cultured cancer cell line expressing a plurality of shared tumor antigens. In addition, the cells have been genetically modified to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF), an immune-modulatory cytokine that can activate antigen-presenting cells (APCs) locally at the vaccine site. Indeed, autologous and/or allogeneic GM-CSF-secreting tumor cell vaccines have demonstrated evidence of immunologic responses in patients with various types of cancers, for example, chronic myeloid leukemia [26], melanoma [27], pancre-

Oncolytic virotherapy (OV) is a novel form of cancer therapy that employs native or engineered viruses that selectively replicate in and kill cancer cells. OVs act as immunotherapies, promoting antitumor responses due to the viral infection of tumor cells and their acute lysis. An example of this therapy is an intralesional injection with talimogene laherparepvec (Imlygic, T-VEC, Amgen, Thousand Oaks, CA), a genetically engineered oncolytic HSV (herpes simplex virus), with mutations in infectious cell proteins (ICPs) 34.5 and 47, and express-

Alternatively, the patient's own T cells or NK cells may be used as a therapeutic agent. Such adoptive cell therapy (ACT) involves the recovery and *ex vivo* expansion of the patient's cells, providing the opportunity for selection and activation of tumor-specific populations, before

and hematological malignancies [19].

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

but releases experienced T cells from inhibitory signaling.

atic adenocarcinoma [28] and prostate cancer [29].

ing US11 and GM-CSF [30].

Genetic instability intrinsic to cancer generates innumerable missense mutations in tumor cells and thus generates specific targets for T cell immunity [37]. Since these neoantigens are not expressed in normal somatic cells, they are inviting targets for the development of cancer vaccines and rational combinations of immunotherapies [38].

Although the term vaccine initially referred to the use of prophylactic immunizations for bacterial or viral infections, there are vaccines for therapeutic purposes, especially when we refer to cancer. This strategy has been gaining prominence lately as it offers the opportunity for a lasting effector response and with far fewer side effects than established traditional treatments, such as chemotherapy. In general terms, cancer vaccines seek to restore the ability of the immune system to recognize and eliminate neoplastic cells. In addition, the possibility of generating memory T cells favors long-lasting protective effects, including the prevention of metastasis after primary remission, which would greatly increase the survival and quality of life of these patients.

One of the earliest reports of cancer immunotherapy was conducted by William B. Coley. After observing that established tumors associated with fever or infection generally had higher rates of spontaneous regression, Streptococcus (Coley's toxin) was injected into an inoperable bone tumor. Despite generating data with difficult interpretation, it sparked a debate and numerous other fronts of investigation [39]. Corroborating this hypothesis, Lamm et al. demonstrated that Bacillus Calmette-Guerin (BCG) could be used to activate the immune system and thus enable the treatment of bladder cancer. This therapy, approved by the FDA, is still in clinical use [40].

#### **3.1. Improving vaccine efficacy**

In both of the pioneering works described above, bacterial components having immunostimulatory properties were used. It is now clear that the formulation of vaccines should include adjuvants, important components for immunomodulatory actions or acting as delivery systems for vaccine antigens [41–43]. The adjuvants' property of modulating the immune system is in part due to their interaction with the receptors of pathogen-associated molecular patterns (PAMPs). Toll-like receptor (TLR) and the Nod-like (nucleotide oligomerization domain) receptor families, for example, mediate the cellular response to PAMPs [44, 45]. Different classes of TLRs each recognize a specific molecular pattern. Briefly, TLRs 1, 2, 4, 5 and 6 recognize molecular patterns associated with bacteria. On the other hand, TLRs 3 and 7 are specialized in the recognition of molecular patterns associated with viral dsRNA and ssRNA, respectively. While TLRs 8, 9 and 13 recognize patterns of viruses and bacteria concomitantly, associated with ssRNA, DNA CpG patterns and ribosomal RNA sequences, respectively [46, 47]. The possibility of synergy when different innate receptors are stimulated may further enhance the adaptive immune response [48].

Several vaccine strategies may be employed for delivery of tumor antigens, adjuvants and modulators of the immune response (**Table 2**). Each strategy has its strengths and weaknesses. Even when meticulously planned, the actual response seen in clinical trials is often unpredictable. The vaccine regimen, number of doses, dosage, route of administration and adjuvant employed are variables that directly influence the type and intensity of the immune response generated. Another important point to be weighed is the mechanism of action, including (i) passive therapies based on the transfer of molecules (such as antibody or cytokine therapies) or mature immune effector cells for example transfer of adoptive T cells, or even CAR-T cell–based therapy; or, (ii) active therapies including classical therapeutic vaccines and those based on DCs to establish effector immune responses against tumors.

Protein-based immunotherapy combines peptides and/or proteins, aiming to activate antitumor immune responses. This strategy has been particularly effective in preventing oncogenic virus infection, as has been seen with Gardasil and Cervarix, which block HPV-associated cervical cancer [49]. The immune responses to structural proteins or viral oncoproteins are likely to be more effective since these antigens are foreign in the body. However, cancer-associated proteins or epitopes, being self-antigens, are naturally less immunogenic and typically associated with immune tolerance; consequently, they are less effective in eliciting immune responses in preclinical cancer models. In this way, delivery systems involving peptides, proteins and DNA/RNA vaccines, although classically used, may be poorly immunogenic and require appropriate pairing with adjuvants [50–52].

On the other hand, delivery systems based on viral vectors can be used for this purpose and may offer greater immunogenicity. Considering that many viral vectors come from pathogenic viruses such as lentivirus, retroviruses and adenoviruses, there is already a line of defense against these "intruders" that can be raised during immunotherapy. This strategy has inherent advantages, such as the possibility of activating innate immune responses due to a variety of viral molecular patterns that are agonists of TLRs, attracting and helping to mature cells of the adaptive immune response. As for the safety of these vectors, genetic engineering techniques allow the removal of specific genes related to pathogenicity, making them innocuous and safe for human use [53, 54]. In the last few years, several virus-driven therapies have been approved for human use, showing substantial progress in the field of gene therapy. Such approaches include Glybera for lipoprotein lipase deficiency and the oncolytic

virotherapy Imlygic [55, 56], CAR-T cell immunotherapies Kymriah and Yescarta [57], as well as Strimvelis for the treatment of ADA-SCID (severe combined immunodeficiency due to adenosine deaminase deficiency) [58] and Luxturna for the treatment of Leber's congenital

**Type Generic mechanism Clinical** 

Cytokine therapy Modulate positively antitumor immunity. 721 Proleukin (IL2r)

overexpressed proteins in tumors.

cytokines, among other transgenes.

antitumor immune responses.

loaded dendritic cells for the correct antigen presentation, and consequent generation of effector T cells against the

To provide T lymphocytes with lithic capacity directed at tumor cells.

of costimulatory molecules.

**Table 2.** Type of gene transfer used in vaccines and immunotherapy against cancer.

recognizes proteins/tumor epitopes, being endowed with lytic capacity independent

Peptides/proteins Provide epitopes for specific antitumor immune responses.

Antibody therapy Selectively target dysfunctional or

DNA/RNA vaccines Provide epitopes for select antitumor immune responses.

Adenovirus Gene transfer, including TK, CD and

Oncolytic virus Selective infection in tumors promoting cell death.

Tumor cells Provide wide range of epitopes for select

Dendritic cells Provide mature, activated and antigen-

tumors.

CAR-T T lymphocytes engineered *in vitro* that

clinicaltrials.gov, search performed April, 2018.

**trialsa**

446/278 Gardasil

142/80 –

193 –

78 –

77 –

342 Kymriah

74 Imlygic

574 Sipuleucel-T

**FDA approved**

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

4187 Rituximab, bevacizumab,

ipilimumab, pembrolizumab

The efficiency of immunotherapies may be increased by applying combinations of different strategies. The combination of antibody therapy, cytokine therapy and checkpoint blockade with other immunotherapeutic strategies has been shown to increase antitumor activity [60–62]. Antibody therapy often targets tumor antigens and/or tumor-promoting proteins. Some antibodies act as blockers of the function of their targets, while others may act as agonists. Additionally, the binding of these antibodies to their targets may direct opsonization or

amaurosis [59].

Transfer of adoptive T

**Protein based**

**Gene based**

**Cell based**

cells

a

**Recombinant virus based**


**Table 2.** Type of gene transfer used in vaccines and immunotherapy against cancer.

adjuvants, important components for immunomodulatory actions or acting as delivery systems for vaccine antigens [41–43]. The adjuvants' property of modulating the immune system is in part due to their interaction with the receptors of pathogen-associated molecular patterns (PAMPs). Toll-like receptor (TLR) and the Nod-like (nucleotide oligomerization domain) receptor families, for example, mediate the cellular response to PAMPs [44, 45]. Different classes of TLRs each recognize a specific molecular pattern. Briefly, TLRs 1, 2, 4, 5 and 6 recognize molecular patterns associated with bacteria. On the other hand, TLRs 3 and 7 are specialized in the recognition of molecular patterns associated with viral dsRNA and ssRNA, respectively. While TLRs 8, 9 and 13 recognize patterns of viruses and bacteria concomitantly, associated with ssRNA, DNA CpG patterns and ribosomal RNA sequences, respectively [46, 47]. The possibility of synergy when different innate receptors are stimulated may further

Several vaccine strategies may be employed for delivery of tumor antigens, adjuvants and modulators of the immune response (**Table 2**). Each strategy has its strengths and weaknesses. Even when meticulously planned, the actual response seen in clinical trials is often unpredictable. The vaccine regimen, number of doses, dosage, route of administration and adjuvant employed are variables that directly influence the type and intensity of the immune response generated. Another important point to be weighed is the mechanism of action, including (i) passive therapies based on the transfer of molecules (such as antibody or cytokine therapies) or mature immune effector cells for example transfer of adoptive T cells, or even CAR-T cell–based therapy; or, (ii) active therapies including classical therapeutic vaccines and those

Protein-based immunotherapy combines peptides and/or proteins, aiming to activate antitumor immune responses. This strategy has been particularly effective in preventing oncogenic virus infection, as has been seen with Gardasil and Cervarix, which block HPV-associated cervical cancer [49]. The immune responses to structural proteins or viral oncoproteins are likely to be more effective since these antigens are foreign in the body. However, cancer-associated proteins or epitopes, being self-antigens, are naturally less immunogenic and typically associated with immune tolerance; consequently, they are less effective in eliciting immune responses in preclinical cancer models. In this way, delivery systems involving peptides, proteins and DNA/RNA vaccines, although classically used, may be poorly immunogenic and

On the other hand, delivery systems based on viral vectors can be used for this purpose and may offer greater immunogenicity. Considering that many viral vectors come from pathogenic viruses such as lentivirus, retroviruses and adenoviruses, there is already a line of defense against these "intruders" that can be raised during immunotherapy. This strategy has inherent advantages, such as the possibility of activating innate immune responses due to a variety of viral molecular patterns that are agonists of TLRs, attracting and helping to mature cells of the adaptive immune response. As for the safety of these vectors, genetic engineering techniques allow the removal of specific genes related to pathogenicity, making them innocuous and safe for human use [53, 54]. In the last few years, several virus-driven therapies have been approved for human use, showing substantial progress in the field of gene therapy. Such approaches include Glybera for lipoprotein lipase deficiency and the oncolytic

based on DCs to establish effector immune responses against tumors.

require appropriate pairing with adjuvants [50–52].

enhance the adaptive immune response [48].

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

virotherapy Imlygic [55, 56], CAR-T cell immunotherapies Kymriah and Yescarta [57], as well as Strimvelis for the treatment of ADA-SCID (severe combined immunodeficiency due to adenosine deaminase deficiency) [58] and Luxturna for the treatment of Leber's congenital amaurosis [59].

The efficiency of immunotherapies may be increased by applying combinations of different strategies. The combination of antibody therapy, cytokine therapy and checkpoint blockade with other immunotherapeutic strategies has been shown to increase antitumor activity [60–62]. Antibody therapy often targets tumor antigens and/or tumor-promoting proteins. Some antibodies act as blockers of the function of their targets, while others may act as agonists. Additionally, the binding of these antibodies to their targets may direct opsonization or 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].

Sipuleucel-T (Provenge), a dendritic cell-based vaccine for the treatment of metastatic castration-resistant prostate cancer, is the only example approved for use in humans. Its manufacture is done in a personalized manner, which involves the extraction of the patient's peripheral blood mononuclear cells (PBMCs) by leukapheresis, transport of the cells to Dendreon's facility (New Jersey, USA) for in vitro culture, maturation of DCs and loading with PA2024 (hybrid protein of GM-CSF and prostate-specific prostatic acid phosphatase, PAP) before returning

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

Three phase 3 clinical trials supported the approval of sipuleucel-T by the FDA [81–83]. These studies have demonstrated that sipuleucel-T extended the survival of treated patients by 4.1 months when compared to the control group that received cells processed in a manner similar to sipuleucel-T, however, without activation due to the absence of the recombinant protein. Although this gain in survival seems promising, none of these studies showed significant increase in time to disease progression [84]. However, no side effects were observed in most cases, and T-lymphocyte proliferation was also detected, factors contributing to FDA approval [84]. In practice, the logistics of sending temperature- and time-sensitive material from widely distributed health care institutions to and from a single processing center made this immunotherapeutic strategy cumbersome and relatively expensive, since the total cost of treatment with sipuleucel-T has been reported to be \$93,000 to \$140,000.00 [80, 85]. Despite the prolonged survival and increased quality of life, this therapeutic option was not sustained

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].

the cells to the hospital where they will be administered to the patient [80].

and was discontinued.

**4. Suicide gene therapy**

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

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, in turn, is one of the major antitumor defenses.

Autologous dendritic cell vaccines can be prepared from the patient's peripheral blood, with isolation of CD14<sup>+</sup> cells and *in vitro* treatment with GM-CSF and IL-4 for differentiation and 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), tissue rejection by antigenic determinants (HLA) may function as an adjuvant.

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 or serious side effects.

Sipuleucel-T (Provenge), a dendritic cell-based vaccine for the treatment of metastatic castration-resistant prostate cancer, is the only example approved for use in humans. Its manufacture is done in a personalized manner, which involves the extraction of the patient's peripheral blood mononuclear cells (PBMCs) by leukapheresis, transport of the cells to Dendreon's facility (New Jersey, USA) for in vitro culture, maturation of DCs and loading with PA2024 (hybrid protein of GM-CSF and prostate-specific prostatic acid phosphatase, PAP) before returning the cells to the hospital where they will be administered to the patient [80].

Three phase 3 clinical trials supported the approval of sipuleucel-T by the FDA [81–83]. These studies have demonstrated that sipuleucel-T extended the survival of treated patients by 4.1 months when compared to the control group that received cells processed in a manner similar to sipuleucel-T, however, without activation due to the absence of the recombinant protein. Although this gain in survival seems promising, none of these studies showed significant increase in time to disease progression [84]. However, no side effects were observed in most cases, and T-lymphocyte proliferation was also detected, factors contributing to FDA approval [84]. In practice, the logistics of sending temperature- and time-sensitive material from widely distributed health care institutions to and from a single processing center made this immunotherapeutic strategy cumbersome and relatively expensive, since the total cost of treatment with sipuleucel-T has been reported to be \$93,000 to \$140,000.00 [80, 85]. Despite the prolonged survival and increased quality of life, this therapeutic option was not sustained and was discontinued.
