**2. Role of immunotherapy in TNBC**

The immune system is known to kill tumor cells by a process called immunosurveillance in which the immune cells target and kill the tumor cells by two ways; either directly or indirectly by releasing soluble chemicals. The cells involved are cytotoxic T lymphocytes (CTL), dendritic cells (DC), macrophages, Natural killer cells (NK) etc. As described earlier, the cancer cells are known to evade the host's immune responses in that the host's immune system identify the tumor cells as self due to which the tumor cell is favored to escape, grow, proliferate and metastasize to distant organs. Furthermore, as the tumor develops, they modify the immune cells for their own benefit like they modify TAMs and recruit them to the tumor

microenvironment to release chemicals that suppress the immune system further enhancing the suitable environment for the tumor cells to survive and proliferate [21]. Therefore, targeting this strategy of immune evasion by cancer cells i.e. modulating the immune system is imperative for the development of therapeutics against tumors. In addition, the currently available treatment options like chemotherapy, radiotherapy are known to be ineffective because of the induction of relapse and recurrence, development of resistance, lack of specificity in addition to side effects and toxicity that leads to tumor development and metastasis in secondary sites. In view of this, immunotherapy is considered to be the most reliable therapeutic approach in terms of target specificity by targeting different immune cells, their functional attributes to block the development and spread of aggressive tumors and as a non-toxic anti-cancer therapeutic strategy. Moreover, immunotherapy has emerged as the fourth most important treatment for cancer after surgery, chemotherapy and radiotherapy and has shown effective treatment responses among patients (**Figure 2**) [22].

Recently immunotherapy was developed as an effective treatment strategy against cancers with a goal to design therapeutics that can effectively enhance the immune system in terms of its specificity and strength its response towards the evading tumors [23]. In the year 2018, James P. Allison and Tasuku Honjo won the Nobel Prize in Physiology and Medicine for discovering a treatment for cancer by downregulating the negative immunomodulation. In their study, they demonstrated that the immune checkpoints like PD-1 (programmed cell death protein1) and CTLA-4 (cytotoxic T lymphocytes associated protein 4) act as "brake" in immune system as they may reactivate T cells by immune checkpoint inhibition, hence eliciting an improved immune response against malignant tumors [24]. The significance of immune checkpoint inhibitors as potential therapeutics has proven in various studies. Many studies have revealed that PD-1 inhibition promotes effective immune responses against cancers [25]. Accumulating studies on PD-1 signaling suppression has revealed that the patient's clinical response to immunotherapy depends upon the effectiveness of T-cells to penetrate the tumor [26]. In the past decade many immune system components have been explored as adoptive immunotherapies like cytotoxic T cells, TILs, anti-CD3 monoclonal antibody-induced killer cells and activated killer cells but they showed less efficiency as therapeutics because of their low anti-tumor functions [27]. However, an *in-vitro* study has suggested the cytokine-induced killer (CIK) cells to a promising target for utilization as immunotherapeutic target because of its higher proliferation rate, hence more effectiveness towards eradicating cancer [21]. CIKs contribute to sturdy cytolytic activities towards tumors as these are non-major Histocompatibility complex- restricted cells that can express both natural killer cell and T cell markers such as CD56 and CD3 [28]. Furthermore, CIKs are known to improve the immune response in patients by regulating and therefore, increase the efficacy of immune function [29]. However, study of CIK cell therapy in breast cancer, particularly in TNBC has been limited. Despite that evidences have reported that the association of CIKs with chemotherapy may result in synergistic effects, supported by an *in-vitro* and *in-vivo* study against cancer stem cells that were resistant to chemotherapy. Therefore, strongly suggests that combined therapy might improve therapeutic efficacy in patients having TNBC, as chemotherapy has shown to regulate the patient's immune status [30].

### **3. Immune checkpoints in immunotherapy**

Immune checkpoints comprise of a collection of different regulatory proteins in the adaptive system that regulate the immune system functions i.e. anti-tumor *Integrating Immunotherapy with Chemotherapy: A New Approach to Drug Repurposing DOI: http://dx.doi.org/10.5772/intechopen.100183*

activity and self-tolerance. They are known to function by coordinating the frequency, magnitude and type of immune response either via positive or negative regulation. There are mainly two immune checkpoint s studied namely PD-1/PD-L1 and CTLA-4, as their presence in the TME prevents to elicit an anti-tumor response via negative regulators of immune activation [31].

## **3.1 PD-1**

PD-1 also known as CD279 was first discovered in the year 1992 [32]. It is a 55 kDa transmembrane protein comprising of 288 amino acids with an extracellular N-terminal domain, a cytoplasmic tail at each N and C end, a transmembrane domain respectively with two tyrosine bases [33]. PD-1 are expressed on a number of immune cells like macrophages, B lymphocytes, activated T cells, Dendritic cells, natural killer cells, activated T cells and monocytes. However, they are highly expressed on specific T-cells. PD-1 is known to act as an inhibitor of both innate and adaptive immune responses [34]. It is supposed its transcription is triggered by many transcription factors such as NOTCH, nuclear factor of activated T cells (NFAT), Interferon (IFN), Forkhead box protein (FOXO1) and interferon regulatory factor 9 (IRF9) [35]. PD-1 expression is highly increased during acute infection and also when there happens to be leakage from cancer cells. PD-1 function in both beneficial and harmful manner to the immune system as it plays a significant role in maintaining immune tolerance by regulation of the harmful and inefficient immune responses while also interfering with the classical protective role of immune system by negative regulation [36–38]. A higher PD-1 expression has been seen in TNBC patients in comparison to non-TNBCs and has been associated with larger tumors, higher histological grades, increased TILs etc. [39].

### **3.2 PD-L1**

PD-L1 is a ligand to PD-1. It belongs to the B7 series and is also known as B7-H1 and CD279. It is a transmembrane glycoprotein as is PD-1, containing

#### **Figure 3.**

*Represents PD-1 mediated T cell inhibition. The binding of PD-L1 expressed on tumor cells binds to its receptor PD-1 on T cells delivering an inhibitory signal to T cells that lead to T cell exhaustion and ineffective T cells.*


#### **Table 1.**

*Comparison of immune checkpoints CTLA-4 and PD-1.*

290 amino acids with IgC domains in its extracellular portion. The cells that express PD-L1 include: activated B and T cells, epithelial cells, macrophages and dendritic cells, particularly at the time of inflammatory responses. The PD-L1 expression is connected with the production of Th1 cytokines, presence of CD8 T cells, interferon, other chemical factors as well as expression of specific genes i.e. all these are responsible for the over expression of PD-L1 and further malignant disease progression, which we will be discussing later in the chapter. Therefore, inhibiting the particular pathways for instance, on activation the NK and T cells secrete interferon-gamma that induces PD-L1 expression on the cells including tumor cells etc. has been shown to promote strong antitumor responses among patients.

The PD-L1 is utilized by the opportunistic tumor cells to evade immune response by mimicking the "Adaptive immune process". Furthermore, PD-L1 is known to act as a pro-tumorigenic factor activating survival and proliferating signaling pathways by receptor binding, hence implicating its greater role in tumor proliferation and metastasis (**Figure 3**). In addition, PD-L1 also acts in a non- immune pattern by inducing epithelial to mesenchymal transition exerting in the tumor cells stem cell like characteristics promoting metastasis and disease progression **Table 1** [41].

#### **3.3 CTLA-4**

CTLA-4 is a member of the CD28 family and is considered to be the "leader" of the immune checkpoint inhibitors as it potentially stops autoreactive T cells in the lymph nodes at the initial stages of development [42, 43]. It is the first immune checkpoint discovered among other immune checkpoints. It is a transmembrane receptor of T cells and it is a leukocyte differentiation antigen that regulates the immune process by negative regulation by competing and binding to the B7 receptor, as it is a CD28 homolog [40]. CTLA-4 plays a significant role to prevent self-reactive immune responses particularly by increasing immunosuppressive Treg. Activity and downregulation of the T effector cell function [14].

*Integrating Immunotherapy with Chemotherapy: A New Approach to Drug Repurposing DOI: http://dx.doi.org/10.5772/intechopen.100183*
