**2. Identification of the GK-1 peptide**

**1. Introduction**

66 Cancer Survivorship

**1.1. Immunomodulation**

Immunosurveillance comprises interactions between the immune system and cancer cells that take place even before the tumor formation [1, 2]. This process includes the recognition and control of transformed cells through antitumor immune responses, with three related outcomes: elimination, equilibrium, and escape [1–5]. In this regard, stimulating the innate immune system by immunogenic cells plays a role in the removal of incipient tumors, activating cells from the adaptive response like T and B cells, as well as promoting acute inflammation due to the concomitant production of immunostimulatory cytokines. Nevertheless, some transformed cells may not be eliminated. This escape phase of immunosurveillance is characterized by tumor growth promotion through a phenomenon called tumor-induced tolerance, which involves an increased expression of immunosuppressive components such as myeloidderived suppressor cells (MDSC), regulatory T cells (Tregs), as well as T cell exhaustion and the production of immunosuppressive soluble factors [6, 7]. Indeed, some of these cells could be used as prognosis factors, since increased numbers of Treg and MDSC cells are related to a poorer outcome in cancer patients [8–13]; by contrast, a Th1 response is associated with a good prognosis in melanoma, breast, head, neck, colorectal, prostatic, and renal cancer [14–16].

The immune response can be modulated by compounds capable of enhancing (immunopotentiation) or diminishing (immunosuppression) the immune response, either in an antigenspecific or in a nonspecific manner; the latter implies that the immune system requires to be stimulated to restore the patient's immunocompetence. Immunomodulators are biological or nonbiological substances that can modify one or more components of the immunoregulatory network to achieve a specific antitumor immunity, such as inducing effector tumor-specific cytotoxic T lymphocytes (CTLs), activating macrophages and natural killer (NK) cells, and/or

Immunomodulators include adjuvants, vaccines, and immunoglobulins used to prevent or treat infectious diseases. They are characterized by their ability to activate cells of the innate immune system, mainly dendritic cells (DCs) and macrophages. Some examples of this type of agents are pathogen-associated molecular patterns (PAMPs) and molecules like squalene,

Anticancer strategies based on peptides have several advantages over other chemotherapeutic approaches, like being nongenotoxic or possessing adjuvant properties; they also have a strong specificity, high affinity, good tissue penetration, and low toxicity with respect to small-molecule drugs and monoclonal antibodies [23–26]. Examples of anticancer peptides are (1) necrotic peptides (some of them are expressed in a wide diversity of species, including insects, fish, amphibians, and mammals, e.g., cecropins A and B found in mammals and various insects) [27]; (2) apoptotic peptides, cationic peptides known as host defense peptides (HDP) such as the bovine lactoferricin, magainin 2, hCAP109-135 (comprising the C-terminal

aluminum salts, and peptides, which are often used as adjuvants in vaccines [21, 22].

promoting the production of inflammation mediators [17–21].

**1.2. Peptide-based therapies**

Based on the nonspecific reactivity and immunopotentiator properties of GK-1, our group has been studying it as a promising adjuvant for cancer immunotherapy. This 18-amino acid peptide was first derived from the KETc7 protein, isolated from a *Taenia crassiceps* cysticercus cDNA library [48]; KETc7 is part of a broad family of proteins associated with membrane processes [49]. When searching for T cell epitopes *in silico*, GK-1 exhibited a strong association with MHC-I and, to a lesser extent, with MHC-II [49]. The immunomodulatory properties of GK-1 are associated with an efficient activation of cells involved in antigen presentation (such as DCs) by promoting the expression of the costimulatory molecules CD86 and MHC-II, as well as the secretion of soluble pro-inflammatory factors like IFN-γ, TNF-α, and CCL2 [50]. GK-1-treated DCs enhanced the proliferative response of antigen-specific CD4+ T cells both *in vivo* and *in vitro* [50]. GK-1 also induced the proliferation of CD8+ T cells and higher IFN-γ levels [51] even in the absence of adjuvant [52]. Considering that this peptide can promote APC function and enhance Th1 cell effector pathways, its capacity as an adjuvant of the influenza vaccine was evaluated. GK-1 increased the levels of specific IgG antibodies *in vivo,* before and after infection, in a murine model of influenza in elderly mice [53], favoring virus clearance after infection in both young and aged mice, which could be associated with an early infiltrate of mononuclear cells (lymphocytes and macrophages) to the lung parenchyma following the GK-1 peptide coadministration. Furthermore, lung histological examination showed better preserved alveolar spaces and less congested alveolar walls with respect to the vaccine-only animals [53].
