*4.2.3 Immune checkpoint inhibitors*

Several inhibitory receptors and ligands expressed on T cells, antigen-presenting cells, and tumor cells have recently been important elements of immunosuppression in the tumor microenvironment. Because of their biological role as regulators of T cell activation, these receptor/ligand pairs have been termed "immune checkpoints". Immune checkpoints are cell membrane proteins involved in the regulation of the immune response. Multiple controls or "checkpoints" are present or activated to ensure that the immune-inflammatory response is not continuously activated after tumor antigens have generated a response. Immune checkpoints are signals that can halt an existing immune response. The over -expression of these signals by tumor cells affects tumor cell-specific T-cell immunity in the cancer microenvironment. The aim of treatments involving inhibition of the immune checkpoint is to use and strengthen the immune system by disrupting the negative immune system. In 2011, the drug called Ipilimumab was used in clinical use for melanoma patients by using immune control point drugs. As of March 2019, 7 immunotherapy drugs based on checkpoints are used in clinical practice. Monoclonal antibodies that bind to immune checkpoints bind with cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), programmed cell death protein 1 (PD-1), and programmed cell death ligand (PDL-1) [33].


### *4.2.4 Cytokines*

They are the main regulators of innate and adaptive immune systems that allow cells of the immune system to communicate in paracrine and autocrine systems over short distances. Unlike other therapeutic agents, these molecules directly stimulate immune cells, for example Interleukin-21 (IL-21) can act as agents involved in active immunotherapy [36]. The use of cytokines in cancer immunotherapy showed tumor regression, prevention of metastasis formation, improvement of immunological memory and decrease in risk of disease recurrence with increased survival. The use of cytokine (IL-2, GM-CSF, IFN-α) -based biological therapy in combination with conventional therapies is under clinical development [37]. In 1986, IFN-α became the first FDA approved cytokine for the treatment of leukemia.

Subsequently, IL-2 was approved by the FDA in 1992 for metastatic kidney cancer and 1998 for advanced melanoma treatment [36].

#### **4.3 Combinational immunotherapy**

Combinational immunotherapy refers the use of a different anticancer agent for treatments of cancer. Conjugation of IL-2 and HER-2 monoclonal antibody proved to be a very forceful combination in immunotherapy. Lately, PD-1 and CTLA-4 conjugation has been examined. The results revealed that the combined system was safe and had no significant toxic effect [38].

### **5. Nanoparticles in cancer immunotherapy**

Nanoparticle-based biomaterials have a critical role in cancer immunotherapy compared with conventional drugs [39]. Immunotherapy often targets tumor cells, immune and stromal cells in the tumor microenvironment [40]. Additionally, side reactions occurring due to the interactions between nanoparticles (NPs) and cells can be adjusted by modifications of nanoparticles [41]. Nanoparticle-based drug delivery systems can improve the solubility, *in vivo* stability, and pharmacokinetic profile. Also, they protect drugs from premature release and degradation in the living system. These systems can be designed according to the microenvironment of the target such as pH, redox potential or enzymes, and external dynamics such as light, electrical and magnetic fields. Targeted delivery with NPs can also reduce toxicity and immune-related side effects [2]. The size and the shape of the NP are very effective in therapeutic efficacy by changing its pharmacokinetics, transportation, and cellular uptake [42]. Recent advances in nanoparticle formulations have generated a wide range of other shapes like rods, prisms, cubes, stars, and discs out of spherical. It is considered as non-spherical particles have higher blood circulation periods, prolonged margination effects, and higher penetration capacities within solid tissues and tumors [43]. The charge of NP has great priority in the transition of it into cells. Besides, NP-ligand coupling conditions and the elasticity of NP upgrade transportation and accumulation of NP in the living system [44, 45]. Generally, it is well known that cationic NPs create a higher immune response than neutral or anionic NPs [43]. The size, shape, elasticity, optical, magnetic, and electrical properties of nanoparticles can be modified to increase the usage of NPs in cancer therapy as a carrier [2, 41, 46]. High specificity, efficacy, diagnosing, imaging, and therapeutic properties make NPs candidates in immunotherapy for effective cancer treatment. Liposomes, micelles, polymeric, metallic, and inorganic NPs have a wide range of usage in cancer immunotherapy [44].

#### **5.1 Classification of nanoparticles**

The nanoparticles are generally categorized into tree class as organic, inorganic, and carbon-based. Dendrimers, micelles, and liposomes are the most widely known organic nanoparticles. These biodegradable, non-toxic, and capsule-shaped nanoparticles appear to be an ideal choice for drug delivery due to their sensitivity to thermal and electromagnetic radiation. Inorganic nanoparticles, metal, and metal oxide-based NPs do not contain carbon in their structure. Aluminum, cadmium, cobalt, copper, gold, iron, lead, silver, and zinc can be used to fabricate metallic NPs in 10 to 100 nm size range. Carbon-based nanoparticles, fullerenes, graphene, carbon nanotubes (CNT), and carbon nanofibers, are build up from carbon in nanosize [47].
