3.3.1. Cytokines

Cytokines are polypeptides or glycoproteins that cause growth, differentiation and inflammatory or anti-inflammatory signals to different types of cells, which are released at a particular time in response to a specific stimulus and have a limited half-life time in the circulation [77]. Target cells of cytokines express high affinity membrane receptors, which activate intracellular signalling when they bind to cytokines, producing modifications in gene transcription that will determine the cellular response. The receptors receive information about the concentration and time of exposure to different cytokines, which implies a high degree of complexity. Due to all these features, cytokines play important roles as modulating agents that are involved in immune homeostasis by regulating inflammatory response, specific immune response, tolerance mechanisms, and promoting effective pathogen control. Hence,

the administration of cytokines allows for the manipulation of the immune system in auto-immune disorders, infectious diseases, increasing the efficiency of the vaccines (due to inherent adjuvants disorders) and in the therapy of cancer [78].

**Figure 6.** Schematic classification of immunotherapies designed for cancer.

The ability of cytokines to enhance the immune response against cancer and the development of recombinant DNA technology has allowed for preclinical and clinical investigation of the anti-tumoral activity of several recombinant human cytokines since the 1980s [77]. Several cytokines, among others including IL-2, IL-12, IL-15, IL-21, GM-CSF and INF-α, have demonstrated efficacy in preclinical models of murine cancer [79]; however, cytokines have shown limitations, such as their short half-life and narrow therapeutic framework, with low anti-tumour efficacy in their use as monotherapy agent. So far, only a few cytokines showed clinical benefit, which were IL-2 and IFN-α, being approved by the Food and Drug Administration (FDA) as anti-tumoral therapies. In the case of IL-2, it was approved for the treatment of advanced renal cell carcinoma and metastatic melanoma; regarding IFN-α, it was approved for the treatment of hairy cell leukemia, follicular non-Hodgkin's lymphoma, melanoma, and AIDS-related Kaposi's sarcoma [77].

In the case of IL-2, which has been approved by FDA for the treatment of advanced renal cell carcinoma and metastatic melanoma. The identification of IL-2 as a therapeutic agent began in the 1960s, when a factor capable of stimulating lymphocyte division in antigen-activated leukocyte culture supernatants was discovered. In 1969, it was demonstrated that human lymphocyte media contained this factor and could be used to maintain T-cell cultures for more than nine months without the need for repetitive antigenic stimulation. This technique was used to cultivate tumour-reactive cytotoxic T cells. This allowed a more in-depth study of this lymphocyte growth factor, thus giving it the name IL-2 [80], which was approved for the treatment of metastatic renal cell cancer in 1992 and advanced melanoma in 1998. IL-2 has opposite functions, acting as a T-cell growth factor during the initiation of the immune response, but is also essential for terminating the T-cell response, maintaining self-tolerance. This cytokine acts as a growth factor for T CD4+ cells and NK cells and promotes the clonal expansion of antigen activated CD8 T cells. In addition, it facilitates the production of antibodies by B cells that have been previously stimulated by factors such as CD40L. With respect to its immune response attenuation function, IL-2 plays an essential role in the maintenance of peripheral Tregs cells, as well as in the Activation-Induced Cell Death (AICD) of Fas-mediated T CD4+ cells. In IACD, receptor-mediated stimulation of T CD4+ cells with high antigen concentrations induces the expression

of IL-2 and their receptors, which interacts and activate the T cell cycle. This antigen activation in turn increases transcription and expression of Fas Ligand (FasL), resulting in T cell death [79].

Regarding IFN-α, it was approved for the treatment of hairy cell leukemia, follicular non-Hodgkin's lymphoma, melanoma, and AIDS-related Kaposi's sarcoma. IFN-α belongs to IFN type I, a family of cytokines synthesized by different cells in response to viral infections and immune stimulation [79]. IFNs of this type induce the expression of MHC class I molecules in tumour cells, involved in the maturation of DCs, activate B and T cells and increase the number of cytotoxic cells. Specifically, IFN-α has pro-apoptotic and anti-proliferative activity, but also presents anti-angiogenic activity on the tumour vasculature. The use of IFN-α was approved in 1986 for the treatment of hairy cell leukemia [77], as it produced a sustained improvement in granulocyte, platelet count and hemoglobin levels in 77% of LCH patients treated [81] and has since been used in the treatment of hematologic malignancies and solid tumours [77], such as chronic myeloid leukemia, AIDS-related Kaposi's sarcoma, renal cell cancer, and in the case of stage II and III melanoma has been used as adjuvant therapy [79].

In contrast, administration of IL-2 and IFN-α has a low response rate and high toxicity associated with high doses, making targeted therapy and checkpoint inhibitors a better option currently for these tumours [77].

A drawback of treatments with cytokines is that, for some of them, positive actions are accompanied by the induction of immune checkpoint cytokines, such as the inhibitory factors IL-10 or TGFβ [79]. IL-10 is released by innate and adaptive immune cells to regulate the activity of pro-inflammatory cytokines; but also as an immunosuppressive cytokine, because it decreases the antigen-presenting activity of dendritic cells (DCs) and inhibits cytotoxic function and cytokine release from T and NK cells (depending on the microenvironment). In chronic infections and cancer, CD8+ T cells exhibit autocrine activity mediated by IL-10, inhibiting their antigen-induced apoptosis, thus prolonging the efficacious activity of cytotoxic lymphocytes. TGFβ has a dual role in the tumour process, since at the beginning of tumorigenesis, TGFβ is an inhibitor of tumour development by blocking the cell cycle; nevertheless, in later stages, the cells develop mechanisms of resistance against the TGFβ´s effects. This resistance mechanism begins to promote tumour progression and mediates the epithelium-mesenchyme transition. In addition, TGFβ promotes the release of angiogenic factors (such as vascular endothelial growth factor (VEGF)), and the recruitment of Treg cells, neutrophils, macrophages (with pro-tumour polarization), myeloid-derived suppressor cells (MDSC) and tolerogenic DCs, in turn decreasing the functions of NK cells and CD8 T lymphocytes [77].

In summary, cytokines have demonstrated anti-tumour therapeutic activity in murine models and in the clinical treatment of certain specific human cancers. Moreover, IL-2 and IFN-α have been approved for the treatment of selected malignancies. In contrast, cytokines in monotherapy have not met all the expectations efficiency as has been observed in preclinical experiments. This is because they are often associated with severe dose-limiting toxicities, and are known to induce immunosuppressive humoral factors, suppressive cells and immune checkpoints. Normally, soluble cytokines act over short distances, in a paracrine or autocrine manner; therefore, to achieve effective intra-tumoral concentrations they must be administered parenterally at high doses, which increases the potential for systemic toxicities, such as hypotension, acute renal failure, respiratory failure and neuropsychiatric symptoms in severe situations. They also do not induce a tumour-specific immune response. To avoid these drawbacks, new mutant engineered cytokines (supercins), chimeric antibody-cytokine fusion proteins (immunocins) or even the combination of cytokines with other therapies such as checkpoint inhibitors, among other novel strategies, are being investigated in an attempt to increase their anticancer efficacy [82]. However, due to these limitations, it has been necessary to develop more tumour-specific immunotherapeutic agents with greater effectiveness and less associated toxicity that are currently being used with better results, and the employment of cytokines in immunotherapy has taken a back seat.
