**2. Mechanism of Action of Immune Checkpoint Inhibitors (ICIs)**

Although cancer cells are formed daily, almost all of them are properly eliminated through the host immune response. Immune responses to cancer cells are called cancer-immunity cycles and comprise seven phases: (1) release of cancer antigens by the death of cancer cells, (2) presentation of cancer antigens to T cells by antigen-presenting cells such as dendritic cells, (3) T cell activation (priming phase), (4) T cell migration, (5) T cell infiltration, (6) cancer cell recognition, and (7) attack and elimination of cancer cells (effector phase) [2]. However, cancer cells with low immunogenicity, which do not present cancer antigens, may evade this autoimmune response and survive for a longer duration (equilibrium phase) [2,3]. Further, immunosuppressive mechanisms activated upon the accumulation of mutations in cancer cells, the induction of regulatory T cells (Tregs) and immunosuppressive cells including myeloid-derived suppressor cells (MDSCs), and the expression of immune checkpoint molecules such as PD-L1 result in uncontrolled tumor growth (escape phase) [2,3]. Thus, certain cancers are detected only after the cancer cells approach the escape phase and undergo uncontrolled proliferation, having already established a system preventing them from being eliminated through the autoimmune response.

ICIs are drugs that block the immunosuppressive mechanisms of cancer cells (Figure 1). ICIs exert their antitumor effects by harnessing host autoimmune functions, as opposed to cytocidal anticancer drugs, which inhibit the cell cycle, and agents that directly attack cancer cells, such as molecularly targeted drugs that specifically bind to gene mutation sites and suppress proliferative signals. Currently, anti-PD-1/PD-L1 antibodies are clinically used to treat lung cancer and various other cancers. In lung cancer, PD-L1 expression is used as one of the biomarkers to distinguish the treatment indication cases. Microsatellite instability has also been used as a potential anti-PD-1/PD-L1 antibodies treatment biomarker in gastric cancer, mainly as a second-line treatment after standard treatment, in triple-negative breast cancer, and as a biomarker candidate in colorectal cancer. In 2011, monotherapy with ipilimumab, an anticytotoxic T-lymphocyte antigen 4 (CTLA-4) antibody, was approved by the food and drug administration (FDA) for advanced-stage malignant melanoma, and in 2015, the combination of nivolumab and ipilimumab was approved by the FDA for use in clinical practice. Studies comparing ipilimumab+nivolumab with sunitinib alone in renal cell carcinoma and the combination of ipilimumab+nivolumab in non-small cell lung cancer have shown favorable results [4,5]. Regarding the significance of ipilimumab in combination therapy, future results are awaited as to whether two-drug combinations of immune checkpoint inhibitors (ipilimumab and nivolumab combination therapy) can contribute to higher survival rates than either immune checkpoint inhibitors alone or immune checkpoint inhibitors in combination with chemotherapy. Since numerous aspects of the mechanism of action of ICIs in vivo are unclear, this review discusses the generally considered mechanisms.

**Figure 1.** Immune checkpoint inhibitors in cancer treatment. Notes: Inability to activate T cells in the tumor microenvironment through the suppressive effect of Tregs or through immune checkpoints allows cancer cells to escape immune attack, survive, and grow. B7 ligands expressed on antigen-presenting cells bind to TCR and induce T cell amplification and immune response. Alternatively, binding of B7 ligands to CTLA-4 expressed on T cells suppresses their activity. CTLA-4 also enhances the activity of Tregs leading to immunosuppressive activity. PD-1 is expressed on activated T cells. PD-1 binds to its PD-L1 leading to the anergy of T cells, further promoting inhibitory signals. Pharmacological inhibition of immune checkpoints with monoclonal antibodies restores T cell antitumor activity and relieves immunosuppression. Abbreviations: CTLA-4: cytotoxic T-lymphocyte antigen 4; MHC: major histocompatibility complex; PD-1: programmed cell death-1; PD-L1: programmed cell death-1 ligand; TCR: T cell receptor; Tregs: regulatory T cells; APC: antigen presenting cell.

Anti-PD-1/PD-L1 antibodies act in the effector phase of the cancer-immunity cycle. In the effector phase, effector T cells attack cancer cells. However, binding of PD-L1 expressed on the cancer cell surface to PD-1 expressed on the surface of effector T cells suppresses the attack by effector T cells on cancer cells. Anti-PD-1/PD-L1 antibodies pharmacologically prevent the PD-1/PD-L1 interaction, thus facilitating the attack by T cells. Furthermore, these antibodies are thought to inhibit the immune response in the priming phase of the cancer-immunity cycle [6].

In contrast, anti-CTLA-4 antibodies act during antigen presentation in the priming phase, wherein dendritic cells present antigens to and activate T cells. T-cell activation requires both T-cell receptors (TCRs) and the MHCI-cancer antigen complex on the dendritic cells (principal stimulation), accompanied by the interaction between B7 (CD80/86) and CD28 on dendritic and T cells, respectively (costimulation) [7]. CTLA-4, like CD28, is expressed on the T cell surface and binds B7 with a stronger affinity than that of CD28. Thus, when CTLA-4 is upregulated, it remains bound to B7 and the costimulatory signal is not transmitted, resulting in the suppression of T cell activation [8]. Anti-CTLA-4 antibodies inhibit the binding of CTLA-4 and B7, resulting in enhanced binding of CD28 and B7, which stimulates T-cell activation and exerts antitumor effects (Figure 1) [9]. Furthermore, CTLA-4 is present on Treg surfaces, induced by cancer cells, and inhibits T-cell activation by binding to B7 on dendritic cells [10]. Thus, anti CTLA-4 antibodies are also thought to exert antitumor effects by facilitating the binding of Tregs to CTLA-4 and directly eliminating Tregs.

#### **3. Changes in Treatment of Lung Cancer without Driver-Oncogene Mutations**

Second-line therapy following platinum-based chemotherapy has long been cytotoxic therapies such a docetaxel (DTX).

In 2014, nivolumab, the world's first ICI targeting PD-1, emerged as a novel therapeutic agent for malignant melanoma. In 2015, a phase-III comparative study of DTX and nivolumab as secondary treatments for squamous and non-squamous lung cancers was conducted in the CheckMate017 (NCT01642004) and CheckMate057 (NCT01673867) studies, respectively; both studies reported that nivolumab significantly prolonged overall survival (OS) compared to DTX (CheckMate017: 6.0 mo vs. 9.2 mo, Hazard Ratio (HR) 0.59; CheckMate057: 9.4 mo vs. 12.2 mo, HR 0.73) [11,12]. Considering these findings, the indication of nivolumab was also expanded to the second-line treatment of NSCLC, and ICIs were approved for the first time for lung cancer treatment.

In 2016, another anti-PD-1 antibody, pembrolizumab, has been reported, and a phase-III comparative study of DTX and pembrolizumab as second-line therapy in NSCLC with PD-L1 ≥ 1% reported that pembrolizumab significantly prolonged patient survival compared to DTX (8.5 mo vs. 10.4 mo (pembro 2 mg/kg) /12.7 mo (pembro 10 mg/kg) [13]. Furthermore, the OAK trial (NCT02008227) compared the second-line NSCLC anti PD-L1 antibodies atezolizumab and DTX, and showed that atezolizumab prolonged survival significantly (9.6 mo vs. 13.8 mo, HR 0.73) [14]. Based on these results, pembrolizumab and atezolizumab, in addition to nivolumab, were introduced as the second-line treatment for NSCLC.

Subsequent to being established as a standard-of-care treatment for second-line therapy, in the KEYNOTE-024 study (NCT02142738) (2016), pembrolizumab significantly prolonged the overall survival (OS) of patients (10.3 mo vs. 6.0 mo, HR 0.60) [15] upon platinum-based chemotherapy as first-line therapy in a PD-L1 ≥ 50% NSCLC without driver mutations, and was approved for the first time as first-line treatment for NSCLC. In 2018, KEYNOTE-189 trial (NCT02578680) and KEYNOTE-407 trials (NCT02775435) assessed the efficacy of the combination of platinum-based chemotherapy and ICIs and approved this combination therapy as first-line treatment of lung cancer, as it significantly prolonged the OS compared to platinum-based chemotherapy alone (KEYNOTE-189 not reached (NR) vs. 11.3 mo, HR 0.49; KEYNOTE-407 15.9 mo vs. 11.3 mo, HR 0.64) [16,17] by combining pembrolizumab with platinum-based chemotherapy for non-squamous-cell lung cancer and squamous cell lung cancer, respectively. In the same year, maintenance therapy with chemoradiotherapy (CRT) followed by durvalumab drastically improved the progression-free survival (PFS) in comparison with CRT alone in unresectable stage III NSCLC in the PACIFIC study among patients with locally advanced lung cancer (16.8 mo vs. 5.6 mo, HR 0.52) [18], and thus, ICIs contributed to advancements in the standard-of-care treatment for locally advanced NSCLC for the first time in 20 y.

In 2019, the IMpower133 trial (NCT02763579) reported that the combination of atezolizumab with platinum-based chemotherapy, as first-line treatment of small-cell lung cancer (SCLC), prolonged both the PFS and OS (PFS 4.3 mo vs. 5.2 mo, HR 0.77; OS 10.3 mo vs. 12.3 mo, HR 0.70) [19]. ICIs are expected to be used to treat SCLC.

Thus, since 2016, ICIs have been widely used therapeutics in different settings from first-line to second-line and onwards, for locally advanced to advanced-stage NSCLC and SCLC and lung cancer.

#### **4. Immune Combination Therapy**
