**2.3 PD-L1 inhibitors**

The Programmed Death receptor Ligand 1 (PD-L1) plays a vital role in the downregulation of T cell activation in the tumor microenvironment (TME). PD-L1 (B7-H1) and PD-L2 (B7-DC) are the two ligands known to bind to the PD-1 receptor described earlier [66, 83, 84]. Under normal physiological conditions, the PD-1/PD-L1 interaction moderates excessive immune cell activity, thereby preventing the development of autoimmunity and tissue destruction due to hyperactivation of the immune system. Cancer cells in the TME exploit this regulatory mechanism by overexpressing PD-L1 on their surface. The interaction between PD-L1 on tumor cells and PD-1 on cells (T cells) negatively regulates T-cell-mediated immune responses in the TME, resulting in T cell exhaustion and limitation of effector T cell responses [66, 84, 85]. Consequently, cancer development and progression are enhanced by maintaining tumor cell proliferation and survival. Therefore, the PD-L1 signaling represents an attractive target for novel anticancer therapy.

The development and clinical application of immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have significantly enhanced antitumor immunity, produced durable responses, and prolonged survival in cancer patients. Currently, there are three FDA-approved PD-L1 inhibitors, namely, Atezolizumab, Durvalumab, and Avelumab, for treating several solid cancers such as non-small cell lung cancer and metastatic melanoma [85] (**Table 1**). Atezolizumab was the first PD-L1 immune checkpoint inhibitor to be approved by the FDA in 2016 for the treatment of advanced or metastatic urothelial carcinoma (UC) [46]. Studies from clinical trial results revealed that treatment with Atezolizumab increased the ORR and was linked to the PD-L1 status of patients. Patients with less than 5% PD-L1 expression detected saw 9.5% ORR compared to 26% in patients with PD-L1 expression greater than 5% after the 14.4 month follow-up.

Atezolizumab (MPDL3280 or Tecentriq®, Genentech) is a fully humanized IgG1 monoclonal antibody. Its mechanism of action involves binding to PD-L1, thereby blocking PD-L1 interaction with the PD-1 receptor. The disruption of this interaction between immune (PD-1) and PD-L1-expressing tumor cells in the TME results in the reactivation of T-cell-mediated antitumor cytotoxicity. Clinical data have demonstrated that Atezolizumab is safe and efficacious in a wide range of solid tumors and hematologic malignancies [20, 46, 86]. Following its approval for the treatment of UC, the drug has been further approved for the treatment of non-small-cell and extensive stage lung cancer [87, 88]. The treatment of NSCLC and ES-SCLC with Atezolizumab improved the ORR by 17% compared to conventional chemotherapy.

Durvalumab (MEDI4736 or ImfinziTM, AstraZeneca) is another fully humanized IgG1 monoclonal antibody like Atezolizumab that binds with high affinity and specificity to PD-L1, blocking the interaction with PD-1. The US FDA first approved the immune checkpoint inhibitor in 2017 to treat locally advanced or metastatic urothelial carcinoma (UC) [89]. Following its approval, Durvalumab received further accelerated approval for treating unresectable stage III NSCLC following platinum-based chemotherapy and radiotherapy [90]. The introduction of Durvalumab in the treatment of UC and NSCLC improved the ORRs by 17% and 28.4%, respectively. In 2020, the drug was approved to treat extensive stage small cell lung cancer [54]. Currently, Durvalumab is being tested in combination with targeted therapies, chemotherapy, and immunotherapy to maximize its activity and improve patient survival rates.

Avelumab (MSB0010718C or Banvecio®, Merck and Pfizer) is another fully humanized IgG1 monoclonal antibody that binds to PD-L1. Banvecio® binds and blocks PD-L1 expressed in tumor cells resulting in T-cell-mediated antitumor immune response, particularly T cell reactivation and cytokine production [91]. The FDA accelerated the approval of Avelumab for treating 12-year-old and older patients with *Current Advances in Immune Checkpoint Therapy DOI: http://dx.doi.org/10.5772/intechopen.107315*

metastatic Merkel cell carcinoma (MCC) in March 2017 [49]. The approval was based on the observed improved ORRs by 31.8% compared to chemotherapy. Avelumab was further approved in May 2017 for the treatment of locally advanced or metastatic UC with disease progression during or following platinum-based chemotherapy [50]. The treatment improved ORR by 18.2%. Avelumab's most recent approval is for the treatment of renal cell carcinoma [51]. Avelumab is currently being tested in combination with traditional cancer therapies in emerging new small molecules (that have synergistic or complementary functions) in clinical trials. Several other PD-L1 immune checkpoint inhibitors are currently in preclinical and early-phase clinical trials [83].

#### **2.4 LAG-3 inhibitors**

The lymphocyte activation gene-3 (LAG-3) (CD223) is a membrane receptor protein that is predominately expressed by activated CD4+ and CD8+ T cells, regulatory T cells (Tregs), and natural killer (NK) cells. LAG-3 can also be expressed to a lower extent by B cells and plasmacytoid dendritic cells (DCs) [92]. It interacts with its primary ligand, the major histocompatibility complex class II (MHC-II) (**Figure 1A**), as well as other ligands, including galectin-3, liver sinusoidal endothelial cell lectin (LSECtin), α-synuclein, and fibrinogen-like protein 1 (FGL1). These interactions result in immune cell exhaustion and decreased cytokine secretion [92–95]. Blocking LAG-3 alone cannot reverse T cell exhaustion; however, combining it with a PD-1 inhibitor has been shown to decrease tumor size [96]. Therefore, in March 2022, the combination of Relatlimab (anti-LAG-3) and Nivolumab (anti-PD-1) was approved by the FDA for the treatment of advanced or metastatic melanoma (**Figure 1E**) [56]. The most common adverse reactions (≥20%) were musculoskeletal pain, fatigue, rash, pruritus, and diarrhea. The most common laboratory abnormalities (≥20%) were decreased hemoglobin, decreased lymphocytes, increased aspartate aminotransferase (AST), increased alanine aminotransferase (ALT), and decreased sodium. Currently, there are 17 small molecule drugs targeting LAG-3 in clinical trials comprising of mono and combination treatments (**Table 2**). Furthermore, Tebotelimab (MGD013) is a bispecific DART molecule designed to independently or coordinately block PD-1 and LAG-3 and is being investigated in patients with HER2-positive gastric cancer or gastroesophageal junction cancer (GEJ) (NCT04082364).

#### **3. Immune checkpoint inhibitors in phase III clinical trials**

Clinical trials are underway on novel immune checkpoint inhibitors and new combinations of already FDA-approved ICIs. Novel emerging immune checkpoint inhibitors include drugs that target lymphocyte activation gene-3 (LAG-3), T cell immunoglobulin and ITIM domain (TIGIT), T cell immunoglobulin and mucin domain containing-3 (TIM-3), V-domain immunoglobulin suppressor of T cell activation (VISTA), B7 homolog 3 protein (B7-H3), inducible T cell costimulatory (ICOS), and B and T lymphocyte attenuator (BTLA). Currently, at least nine novel ICIs have reached Phase III clinical trials (**Table 2**). We note that in addition to the drugs listed in **Table 2**, there are more than 50 other agents (antibodies and small molecules) targeting immune checkpoint proteins that are in Phase I and II [106].


#### **Table 2.**

*Immune checkpoint inhibitors in phase III clinical trials.*

#### **4. Resistance to immune checkpoint inhibitors**

One of the most significant challenges in immune checkpoint therapy is the development of resistance, whether it is primary (the patient never responds to treatment) or acquired (the patient initially responds to treatment but stops responding after the commencement of therapy). Resistance can also be intrinsic or extrinsic to tumor cells [107]. Intrinsic resistance occurs when cancer cells alter processes related to immune recognition, cell signaling, gene expression, and DNA damage response. Resistance to immune checkpoint inhibitors is associated with loss of immunogenic neoantigens, an increase of immunosuppressive cells, and the upregulation of alternate immune checkpoint receptors [27, 108]. Response to ICIs can also vary by tumor type, with the highest response rates found in tumors with a high mutational burden, such as melanoma, lung, and bladder cancers.

In contrast, tumors with lower tumor mutational burden (TMB), such as prostate and pancreas, show a lower response [109]. However, ICI response can vary among tumors with a similar TMB, thus suggesting that response to ICI is influenced by several other factors [110]. These factors may include PD-L1 expression or induction, deficiencies in DNA mismatch repair (MMR), levels of tissue-specific neoantigens and tumor-

#### *Current Advances in Immune Checkpoint Therapy DOI: http://dx.doi.org/10.5772/intechopen.107315*

infiltrating lymphocytes (TILs), endogenous retroviruses (RVs) epigenetic alterations, and oncogenic alterations [27, 108, 111, 112]. Extrinsic resistance occurs external to tumor cells throughout the T cell activation process. Tumors can have different immunophenotypes, such as variation in type, density, and location of immune infiltrates, and these differences can affect the response to ICI therapy. In general, inflamed tumors generally respond better to ICI therapy [113, 114]. In addition, the tumor microenvironment (TME) also plays a big role in treatment response, contributing to both primary and acquired resistance. The TME is complex and comprises various immune and stromal cells, the extracellular matrix, surrounding vasculature, and cytokines [114, 115]. This scenario further complicates the development of drug resistance.

Resistance can also be attributed to contextual factors, which include the gut microbiome, expression of human endogenous retroviruses, and gender. The response to ICI therapy influenced by gut microbiomes is thought to involve the activation of dendritic cells, upregulation of MHC-II, and the increased levels of effector T cells [107, 116–118]. High expression of human endogenous retroviruses (RVs) in tumors resulted in a phenotype consistent with immune checkpoint activation in various cancer types. Furthermore, the abnormal expression of ERVs appears to activate epigenetic changes such as histone methylation [111, 119]. Overall, the abnormal expression of ERVs indicates a positive response to ICI treatment.

With overall response rates for most cancers to FDA-approved drugs generally being between 10 and 50%, this indicates that in at least 50% of patients, either primary or acquired resistance is occurring. Two of the most promising strategies by which we can overcome resistance are combinational therapy and identifying predictive biomarkers of ICI therapy.

#### **5. Combinational therapy as a strategy to overcome resistance**

In the past decades, patients diagnosed with various cancers that did not respond well to traditional methods such as chemotherapy and radiotherapy received very poor prognoses. Moreover, these conventional cancer therapy methods are also known to cause damage to healthy normal cells. Since then, various cancer therapies targeting disordered proteins, immune cells, and components of the tumor microenvironment (TME) have been developed to improve prognosis. Small molecules and immunotherapy have drastically improved the prognosis for some patients. Despite that, a limited number of patients obtain benefits from the treatment. This is attributed mainly to low response and acquired resistance during the treatment, and severe side effects also lead to unfavorable outcomes. To overcome this, researchers are investigating the potential of combining ICIs with various other treatments, including chemo/radiotherapy and targeted therapies. Immunotherapy based on single targets often results in serious side effects, unresponsiveness, or overreaction. In contrast, combinational immunotherapies show synergistic outcomes with higher efficacy and safety. Strategies combining immunotherapy and conventional therapies like radiotherapy and/or chemotherapy have demonstrated promising clinical and basic research results. However, the underlying mechanisms are still unclear.

#### **5.1 Combination of two or more immune checkpoint inhibitors**

Checkpoint inhibitors that target CTLA-4 and the PD-1/PD-L1 axis are promising candidates for combination immunotherapy. The rationale behind the dual

checkpoint inhibitor treatment is the synergy of inhibiting both CTLA-4 and PD-1 with Nivolumab plus Ipilimumab which was the first combination immunotherapy to be licensed in the US and Europe and has been used in the treatment of melanoma for several years [120]. Clinical studies have shown that the combination of Ipilimumab with Nivolumab significantly improved overall survival rates to 57% compared to Nivolumab (43%) and Ipilimumab (25%) alone in melanoma patients after a 6.5-year follow-up to assess efficacy and safety [120]. Following its first approval for the treatment of advanced melanoma in 2017, the combination is now used for the treatment of advanced RCC, HCC microsatellite instability-high (MSI-H), or mismatch repair deficient (dMMR) metastatic colorectal carcinoma (CRC), NSCLC, and malignant pleural mesothelioma (MPM) as shown in **Table 1** [61, 63, 121, 122]. Combination therapy has significantly improved the clinical outcomes for most patients. Longterm follow-up (42 months) in RCC patients revealed an improved overall response rate of 42% (Nivolumab + Ipilimumab) versus 26% in patients treated with Sunitinib, a small molecule monotherapy [123]. Furthermore, durable long-term efficacy was observed, especially among patients with more than 1% PD-L1 expression [62].

More recently, the combination of Relatlimab and Nivolumab, known as Opdualag, was approved by the FDA for treating advanced or metastatic melanoma in patients aged 12 years and older. The approval was based on results from the RELA-TIVITY-047 clinical trials [98]. The combination treatment with Relatlimab +Nivolumab was at 47.7% compared to 36% in the Nivolumab monotherapy group after 12 months of follow-up. As described in the introduction, Relatlimab inhibits LAG-3 while Nivolumab inhibits PD-1, which are both often expressed by immune cells in the TME (**Figure 1E**). The expression of PD-1 and LAG-3 negatively regulates T cell tumor infiltration and proliferation, respectively. Combination immunotherapy has become an attractive avenue for the treatment of resistant cancers following the Ipilimumab + Nivolumab treatment of various cancers. Currently, several Phase III/IV clinical trials are ongoing to test the safety and efficacy of dual checkpoint inhibitor therapy combining two or more ICIs as listed in **Table 3**.

## **5.2 Combination of immune checkpoint inhibitors with conventional therapies (chemotherapy/radiotherapy and small molecules)**

In some instances, chemotherapeutic agents have appeared to impact the immune system positively. The positive effects of standard chemotherapy on tumor immunity are mainly reflected in inducing immunogenic cell death and disrupting tumor escape strategies. Experimental data have shown that some anticancer chemotherapeutic agents can stimulate naïve immune cells to induce immunogenic cancer cell death [133]. For this reason, chemotherapy in combination with immune checkpoint inhibitors is an attractive strategy for synergistic combination treatment in cancer. Several studies using murine models have shown that chemotherapeutic agents such as cyclophosphamide, fluorouracil (5-FU), and Gemcitabine can reduce Tregs, improve circulating NK cells, and augment tumor-infiltrating T cells, respectively [134, 135]. Indeed, a combination of PD-L1 inhibitor (Nivolumab) plus Gemcitabine and Cisplatin significantly improved the ORR over monotherapy in a Japanese Phase I clinical trial [136]. Since then, several ICI and chemotherapy combination treatments have been investigated to improve patient response rate and survival.

To date, there are several ICI and chemotherapy/radiation combination therapies that have been approved by the FDA. Others are currently in Phase III/IV clinical trials as listed in **Table 3**. Pembrolizumab combined with standard chemotherapy has


#### **Table 3.**

*Current combination therapies.*

become the first such combination therapy to be licensed for first-line use in patients with metastatic non-squamous NSCLC in the US and Europe after a trial showed that the combination enhanced overall survival at 12 months by 69.2% compared to 49.4% in the monotherapy group [137]. Since then, Pembrolizumab in combination with Axitinib, a vascular endothelial growth factor (VEGF) inhibitor has been further approved for the treatment of RCC. The ORR favored the Pembrolizumab/Axitinib group (59.3%) over the sunitinib group (35.7%). Atezolizumab and Durvalumab, both targeting the PD-L1, have been FDA-approved in combination with chemotherapy as a first-line treatment for advanced SCLC [138]. The approval was based on the IMpower133 and CASPIAN clinical trials which both evaluated Atezolizumab and Durvalumab, respectively, in combination with etoposide and carboplatin-based chemotherapy. Both studies revealed improved overall survival (OS) by Atezolizumab + chemotherapy (12.3 months); Durvalumab + chemotherapy (13 months) compared to chemotherapy alone (10 months) [54, 139].
