Latest Immunotherapy Methods in Non - small Cell Lung Cancer Treatment

Pharmacotherapy for advanced lung cancer, especially for non-small cell lung cancer (NSCLC) has progressed dramatically in past 10 years. The prognosis of patients with advanced NSCLC has improved immensely due to treatment with molecular target drugs and immune checkpoint inhibitors. Numerous clinical trials have shown that immunotherapy using immune checkpoint inhibitors is highly effective in the treatment of patients with advanced NSCLC. The 5-year survival rate is dismal in patients with advanced NSCLC treated with conventional cytotoxic chemotherapy. Alternatively, advanced NSCLC patients treated with nivolumab, an immune checkpoint inhibitor, have exhibited a 20% chance of survival for 5 years. However, immune checkpoint inhibitors are ineffective in many patients. Therefore, efforts have been taken to develop methods predicting the effects of immune checkpoint inhibitors and to explore new combination therapies using immune checkpoint inhibitors. Moreover, since immune checkpoint inhibitors may cause serious immune-related adverse events ( e.g. , pneumonitis, enterocolitis, encephalitis), it is important to develop methods that control these adverse events. More patients with lung cancer are expected to benefit from immunotherapies.


Introduction
Lung cancer remains as the primary leading cause of cancer-related mortality worldwide due to poor prognosis. Non-small-cell lung cancer (NSCLC), the most common histologic subtype of lung cancer, often presents as a locally advanced or metastatic disease 1) . In the absence of driver mutations, first-line platinum-based chemotherapy has been the standard care for patients with NSCLC. Despite the improvements in systemic therapy, the survival rate for patients with stage IV disease is poor, with fewer than 5% living 5 years after diagnosis 2) .
Recent breakthroughs in immunotherapy have brought changes in the treatment of lung cancer during clinical practice. Since 2015, four immune checkpoint inhibitors (ICIs) namely, anti pro-grammed death 1 (anti-PD-1) antibodies nivolumab and pembrolizumab and anti programmed death ligand 1 (anti-PD-L1) antibodies atezolizumab and durvalumab, were approved for treatment of NSCLC in Japan. More recently, pembrolizumab or atezolizumab in combination with platinum doublet chemotherapy has been approved as firstline therapy for advanced NSCLC.
Currently available immune checkpoint inhibitors for treatment of NSCLC are reviewed below, followed by a discussion of biomarkers associated with treatment responses.

Nivolumab
Nivolumab is a human antibody (IgG4) specific for human PD-1. It binds PD-1 with a high affinity and prevents the interaction of PD-1 with its 444 Health  ligands PD-L1 or PD-L2, thereby enhancing tumor antigen-specific T cell proliferation 3) . In phase I and II trials, nivolumab showed durable antitumor activity and a cumulative response rate of about 18% in all NSCLC subtypes 4)-6) . In two phase III studies comparing the efficacies of nivolumab versus docetaxel in advanced squamous (Check-Mate 017) 7) and nonsquamous (CheckMate 057) 8) NSCLC that were resistant to platinum-based chemotherapy, nivolumab was found to be significantly better than docetaxel in response rate, overall survival (OS), and progression-free survival (PFS), regardless of intratumoral PD-L1 expression levels (Table-1). The overall response rate was 19-20% in the nivolumab treated group versus 9-12% in docetaxel treated group. Based on these results, nivolumab was approved in Japan in 2015 as a second-line monotherapy for pretreated advanced NSCLC. CheckMate 026 trial is the only completed phase III clinical trial that has evaluated nivolumab as first-line therapy. This trial compared nivolumab regimen with a traditional platinumbased chemotherapy regimen in patients with advanced NSCLC who showed PD-L1 tumor cell expression of 5% or greater. However, no significant difference in PFS and OS between the two study groups were observed 9) . Based on the negative results of the study, nivolumab has not been approved for first-line use in advanced NSCLC.

Pembrolizumab
Pembrolizumab is a humanized IgG4 monoclonal antibody specific for human PD-1. In a randomized phase II and III trial (KEYNOTE-010) with the patients with NSCLC, who were previously treated with platinum-based chemotherapy and were PD-L1-positive in tumor cells based on immunohistochemical analysis using 22C3 (≥1%) 10) , patients were randomly assigned to three arms: pembrolizumab at 2 mg/kg, pembrolizumab at 10 mg/kg, and docetaxel at 75 mg/m 2 . The results showed that, among patients with at least 50% of tumor cells expressing PD-L1, OS and PFS were significantly longer in the groups treated with pembrolizumab at 2 mg/kg and 10 mg/kg than the group treated with docetaxel (Table-1). In another phase III trial (KEYNOTE-024) with the patients with advanced NSCLC who had no previous chemotherapy and no driver mutation for target therapies in their tumors but had at least 50% PD-L1 tumor cells, patients were randomly assigned to the treatment with either pembrolizumab (200 mg every 3 weeks) or platinum-based chemotherapy 11) . The results revealed that both PFS and estimated OS at 6 months were significantly higher in the pembrolizumab group than in the chemotherapy group (Table-1). These results led to approval of pembrolizumab as second-line therapy for advanced NSCLC with PD-L1 expression of ≥ 1% and firstline therapy for NSCLC with expression of PD-L1 of ≥ 50%.

Atezolizumab
Atezolizumab is an anti-PD-L1 antibody. This drug was recently approved as a second-line therapy for patients with advanced NSCLC based on two randomized trials (OAK and POPLAR) with a total of 1,137 patients with advanced NSCLC, which demonstrated consistent results in efficacy and safety of atezolizumab in the treatment of NSCLC (Table-1). In comparison with docetaxel, treatment with atezolizumab resulted in improvement of OS in these two trials 12) 13) . This improvement in OS was associated with an increased expression of PD-L1 in tumor cells and tumorinfiltrating immune cells.

Durvalumab
Durvalumab is a PD-L1 specific human IgG1 monoclonal antibody. This drug was recently approved for the treatment of patients with unresectable stage III NSCLC based on the PACIFIC trial. In this phase III trial of the patients with stage III NSCLC who did not show disease progression after two or more cycles of platinumbased chemotherapy, durvalumab was found to have significantly better PFS, response rate, and OS when compared with placebo (Table-1) 14) . The PFS and OS benefits of durvalumab were observed irrespective of PD-L1 expression before chemoradiotherapy, based on the stratification of PD-L1 as ≥ 25% or < 25%.

Immunotherapy in combination with chemotherapy
In patients with advanced NSCLC, three studies have shown an OS benefit from adding anti-PD-1/ PD-L1 antibody to standard chemotherapy: KEY-NOTE-189, IMpower 150, and KEYNOTE-407 (Table-2) 15)-17) . Based on these results, these combination therapies were approved in Japan as first-line therapy for patients with advanced NSCLC.
The KEYNOTE-189 phase III trial compared a platinum plus pemetrexed with either pembrolizumab 200 mg or placebo in previously untreated patients with advanced non-squamous NSCLC. The study was positive for both the coprimary endpoints, that is, PFS and OS. Median PFS was 8.8 months versus 4.9 months [HR 0.52; 95% confidence interval (CI), 0.43-0.64; p < 0.001] in the pembrolizumab and placebo arms, respectively. The median OS was not reached in the pembrolizumab arm compared with 11.3 months in the placebo arm (HR 0.49; 95% CI, 0.38-0.64; p < 0.001). Importantly, the improvement in OS with pembrolizumab was seen across all levels of PD-L1 expression, including PD-L1 negative 15) .
The three-arm IMpower 150 study compared atezolizumab plus carboplatin, paclitaxel, and bevacizumab (ABCP); and atezolizumab plus carboplatin and paclitaxel (ACP) to carboplatin, paclitaxel, and bevacizumab (BCP; reference arm). The PFS and OS were significantly longer with the ABCP arm than with the BCP control arm. However, the ACP arm did not show a survival benefit compared with the BCP control arm. This result suggests a benefit to use bevacizumab with atezolizumab. Although, there are no data to directly compare ABCP and ACP. Therefore, an effect of bevacizumab in this combination therapy is unclear. The rate of grade ≥3 treatment-related AEs was slightly higher for ABCP than with BCP (58.5% vs. 50.0%, respectively) in the IMpower150 study, whereas the rate of treatment-related deaths was similar for the two regimens (2.8% vs. 2.3%, respectively). The rate of AEs leading to discontinuation of study treatment was higher with ABCP (32.6% vs. 24.9%) 16) .
For the patients with metastatic squamous NSCLC histology, the KEYNOTE-407 study has shown that pembrolizumab added to a standard  Although the incidence of grade ≥3 AEs was similar for the two treatment arms, the rate of discontinuation of treatment due to AEs was higher for the pembrolizumab regimen (23.4% vs. 11.8%, respectively) 17) .

Biomarker
A lot of clinical trials have shown that anti-PD-1/ PD-L1 therapy is a promising treatment strategy with unprecedented survival benefits in selected NSCLC patients. Nevertheless, a substantial proportion of patients who received PD-1/PD-L1 inhibitors showed little to no benefit. Therefore, it is necessary to identify biomarkers for the establishment of valid predictors of treatment responses. In this regard, the expression of immunosuppressive molecules including PD-L1, PD-1, and indoleamine 2, 3-dioxygenase (IDO), mutational landscape and mutational load 18) , and mismatch repair deficiency (MMRD) 19) have been examined as potential predictors of response to anti-PD-1/PD-L1 treatments.
Among these potential predictors, PD-L1 expression has been employed as a biomarker in clinical trials of anti-PD-1/PD-L1 therapy for NSCLC treatment 10) 11) . Several clinical trials have reported remarkable therapeutic outcomes caused by immune checkpoint blockades in NSCLC patients guided by PD-L1 IHC assays. Currently, four commercial IHC-based bioassays (22C3, 28-8, SP263, and SP142) are approved as diagnostic assays for detecting PD-L1 expression in tumors, thereby guiding the use of PD-1/PD-L1 inhibitors alone or in combination with other therapeutic agents with different cutoff values in distinct types of lung cancer. PD-L1 is not expressed in most normal tissues. However, its expression can be induced by cancer drivers 20) 21) or by IFNγ 22) 23) . IFNγ-induced PD-L1 expression has a unique histopathological pattern, which is usually focal rather than diffuse and is expressed in cells adjacent to tumor-infiltrating lymphocytes (TILs), as observed in most human cancers 24)-26) . Therefore, both increased TIL levels and increased expression of PD-L1 in the tumor tissues can serve as surrogate biomarkers of immunogenicity of cancer cells and interactions between cancer cells and immune cells or of the presence of adaptive immune resistance 25) 27) 28) . It has been proposed that, based on the presence or absence of PD-L1 expression and TILs, the tumor microenvironment can be classified into four groups 29) 30) : 1) TIL and PD-L1 double-positive, suggesting the presence of adaptive immune resistance; tumor will likely benefit from PD-L1/ PD-1 blockade therapy; 2) TIL and PD-L1 doublenegative, indicating immune ignorance or lack of detectable immune reaction; tumor will likely not benefit from ICI therapy; 3) TIL-negative but PD-L1-positive; indicating that PD-L1 expression in cancers is independent of IFNγ but is intrinsic through oncogenic signaling; tumor may not respond to immune checkpoint inhibitors; and 4) TIL-positive but PD-L1-negative, indicating that other immunosuppressive mechanisms may mediate immune tolerance; targeting alternative immunosuppressive pathways will be required to restore anticancer immunity.
The presence of tumor-specific antigens and the interaction of immune cells with tumor antigens are the two basic principles of cancer immunology. Tumor antigens can derive from mutant proteins, overexpressed or dysregulated embryonic proteins, and oncogenic viral proteins. Increased nonsynonymous mutation burden in tumor tissues was expected to increase neoantigens in tumor, leading to a stronger immune response against cancer cells. Indeed, clinical trials in lung cancer and melanoma have shown that high tumor mutation burden (TMB) was significantly associated with better objective response, durable clinical benefit, and prolonged PFS for patients with ICIs 18) 31) . Analysis of the data of ICI therapy available in the literature for 27 cancer types revealed a significant correlation between the TMB and the objective response rate of anti-PD-1/PD-L1 therapies 32) . In a randomized phase III trial with advanced NSCLC, nivolumab as first-line therapy was found to be not superior to chemotherapy in PFS or in response rate in patients whose tumor had PD-L1 expression of ≥5%. However, nivolumab was found to have higher response rate and longer PFS than chemotherapy among patients with high TMB 33) .
However, each biomarker is insufficient to predict the efficacy of immune checkpoint inhibitors. Development of more accurate biomarkers is expected.

Future directions and conclusion
Conventional cancer treatments, including surgical resection, chemotherapy, and/or radiotherapy, have shown modest progress in NSCLC survival over the past two decades. The introduction of targeting agents, such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) or anaplastic lymphoma kinase (ALK) inhibitor, further offered significant improvements in NSCLC survival carrying an EGFR mutation or ALK rearrangement. To date, there are four anti-PD-1/ PD-L1 agents namely pembrolizumab, nivolumab, atezolizumab, and durvalumab approved for functioning as first-or second-line NSCLC therapy. Therefore, we expect these agents to improve clinical efficacy. However, there are a lot of problems in the treatments using immune checkpoint inhibitors as described previously.
There are grave challenges in anti-PD-1/PD-L1 therapies for NSCLC patients. These include identification of patients who may benefit from the therapy and minimizing the development of acquired resistance to therapy. Therefore, in order to improve the therapeutic efficacy and reduce adverse effects of anti-PD-1/PD-L1 blockades, continual efforts are required to identify novel predictive biomarkers for patient selection, by utilizing the rapid development in computational models and high-throughput sequencing techniques for effective and personalized immunotherapy. In addition, based on our deeper understanding of immune escape mechanism and its role in the biological behavior of NSCLC, a combinatory approach on the basis of anti-PD-1/PD-L1 therapy, such as combination with chemotherapy, targeted therapy, radiotherapy, and other immunotherapies, is predicted to establish landmarks for treatment of NSCLC. For instance, in NSCLC patients with high tumor burden or rapid disease progression, a combination of anti-PD-1/PD-L1 therapy and targeted therapy may be an option. To this end, it is important to comprehend the mechanisms of resistance to anti-PD-1/PD-L1 agents and to identify patients who may potentially benefit from this therapeutic schedule. In this respect, the discovery of novel biomarkers and/or development of precise companion diagnostic assays become critical for patient selection. Currently, however, the expression of PD-L1 is only the tip of the iceberg in the predictive index of anti-PD-1/PD-L1 therapy; it is necessary to combine the multiple indexes to make the best predictive ability, indicating an importance of personalized biomarkers in guiding anti-PD-1/PD-L1 immune checkpoint blockade therapy for NSCLC.