Immune checkpoint inhibitors in metastatic NSCLC: challenges and future directions

The therapeutic evolution of non-small cell lung cancer has been dramatic over the past decade with the discovery of oncogenic driver mutations and the advent of biomarker guided management. The journey to the discovery of immune checkpoints dates back to the 1980s, when scientists first discovered T-cell receptors (TCR) which eventually led to the understanding of the major signals required for T-cell activation.^1–3 By the mid 90's, CTLA-4 (cytotoxic T-lymphocyte–associated antigen 4), a homolog of CD28 was discovered which functioned as an inhibitory program on activated T-cells thereby serving as a inhibition to the T-cells.^4,5 In 1992, PD-1 (programmed cell death receptor 1), a transmembrane protein similar to CTLA-4, expressed on T-cells was discovered to negatively regulate the immune response of T-cells by interfering with receptor signaling.⁶ In 1999, B7-H1, also known as PD-L1 (programmed death ligand 1) was discovered and its blockade led to increased levels of IL-10 and IL-2, thereby stimulating T-cells.⁷ This led to the development of immune checkpoint inhibitors (ICIs). The CTLA-4 inhibitor ipilimumab was approved by the FDA in 2011 for the treatment of untreated metastatic melanoma in combination with chemotherapy after a landmark trial showed an OS benefit.⁸ The first ICI that was approved for the treatment of lung cancer was nivolumab. CHECKMATE-017 and CHECKMATE-057 were phase 3 trials that were designed to evaluate the efficacy of nivolumab in the management of squamous and non-squamous metastatic NSCLC and both trials showed an OS benefit leading to its approval in previously treated patients.^9,10 Similarly in 2016, pembrolizumab was approved after a trial showed an OS benefit in patients with previously treated metastatic NSCLC.¹¹ Currently, various approvals exist for the combination of ICIs with chemotherapy or ICI alone in the front-line management of metastatic NSCLC based on the PD-L1 status.¹² The approved immune checkpoint inhibitors include pembrolizumab, atezolizumab, cemiplimab, nivolumab/ipilimumab and durvalumab/tremelimumab. In table 1, we highlight the landmark trials that led to the approval of these agents in the management of metastatic NSCLC.

Despite these advances in the field of lung cancer and immunotherapy, several challenges continue to limit patient outcomes. In this review, we discuss the pressing challenges and controversies in the immunotherapy landscape in non-small cell lung cancer. We first discuss the issues with PD-L1 as an optimal biomarker and segue into the utility of using tumor mutational burden as a biomarker for immune checkpoint inhibitors. We also address other PD1/PD-L1 inhibitors in development and the lack of benefit associated with multiple agents of the same class with similar efficacy and safety profiles. Lastly, we also shine light on newer immunotherapy approaches and inform the reader on the future directions in this field.

There are several mechanisms which regulate the expression of PD-1/PD-L1 proteins in tumor cells (TCs).^34,35 PD-L1 is coded by CD274 gene located on chromosome 9p. Mutations in the JAK-STAT pathway result in the amplification and translocation of CD274 leading to its upregulation. Mutations in genes like TP53, KRAS, STK11 and NFE2L2, are known to predict the PD-L1 expression.³⁶ High levels of tumor PD-L1 expression has been shown to corroborate with superior response to ICIs. A positive PD-L1 expression is identified as a TPS ≥1% whereas high PD-L1 expression is identified at a cut off ≥50%. Studies describe a wide range of PD-L1 positivity in NSCLC from 24-60% with prevalence in the <1%, 1-49% and ≥50% groups being 33-60%, 38-42% and 13-28% respectively.^37–39

One of the mechanisms of PD-L1 expression is guided by the exposure to IFN-γ released by the T effector cells which increases the expression of PD-L1 on other cellular compartments including tumor infiltrating immune cells (ICs). Therefore, quantification of PD-L1 expression not only includes the tumor proportions score (TPS) but also the degree of PD-L1 expressed on ICs. The significance of immune cells expression of PD-L1 is an evolving subject but it has been hypothesized that the PD-L1 protein of ICs binds with the PD-1 of T cells to facilitate the immune evasion.⁴⁰

Various FDA approved assays for PD-L1 measurement were compared in the blueprint studies to promote consistency in testing. The assays include: 22C3 pharmDx Dako (pembrolizumab and cemiplimab), SP263 (atezolizumab) and 28-8 pharmDx Dako (nivolumab/ipilimumab). SP142 Ventana assay (atezolizumab) is the only assay approved for testing of PD-L1 expression on ICs.^41,42 The Blueprint 1 study demonstrated that the three assays 22C3, 28–8, and SP26 were comparable in their assessment of PD-L1 expression on TCs, whereas the SP-142 PD-L1 assay stained fewer TCs.⁴³ Blueprint 2 confirmed these results and demonstrated that a 5th assay, the 73-10 assay showed greater staining of the TCs. However, when studied in ICs, low level of staining and concordance was noted between all 5 assays.⁴⁴ In the real world, more cost-effective and easily available laboratory developed tests (LDTs) are also used for assessing PD-L1 expression. The concordance of such tests with the studied assays have not been fully validated and remains a challenge.^45,46

Despite the volume of studies correlating response to immunotherapy with PD-L1 expression, this remains a controversial biomarker. Intra-tumoral temporal heterogeneity, spatial heterogeneity and variable concordance between FDA approved assays and LDTs raises questions regarding the predictive and prognostic value of this biomarker.^47–50 In KEYNOTE-024, the overall response rate (ORR) of patients with a PD-L1 TPS ≥ 50% to pembrolizumab was only 45% meaning that even in patients with a pre-defined high PD-L1 expression, a majority of the population did not respond to the check-point inhibitor as a monotherapy.¹⁴ Similarly, in CHECKMATE 227, the objective response rate in the PD-L1 <1% was around 27% and a significant improvement in overall survival was noted with dual check-point inhibition when compared to chemotherapy.¹⁸ These varying responses with no clear correlation with the use of dual ICI versus a single ICI adds to the skepticism regarding PD-L1 as a predictive biomarker.

The inter-tumoral discordance in PD-L1 expression of primary tumor when compared metastatic sites particularly the brain has also been highlighted.^51–54 Studies have shown that various factors affect tumor heterogeneity such as histology, surgical status, targeted therapy use, prior chemotherapy, immunotherapy use and antibiotic use.⁵⁵ Herbst et al. demonstrated the dynamic variability of PD-L1 expression in patients treated with atezolizumab, showing an increase in PD-L1 expression with decrease in tumor volume.^55,56

The utility of PD-L1 as a predictive biomarker in patients with other driver mutations also has limitations. A meta-analysis showed that the use of ICIs had no OS benefit in the second line setting in NSCLC patients with an EGFR mutation.⁵⁷ Another phase-2 trial studying pembrolizumab in PD-L1 positive and EGFR mutant NSCLC was ceased due to futility.⁵⁸ The reason behind the lack of response to ICIs in EGFR mutant NSCLC has been thought to be due to low PD-L1 expression in tumors harboring an EGFR mutation.⁵⁹ However, there are studies showing conflicting results and others showing a lack of correlation between PD-L1 expression in tumors with driver mutations.^60–63

Despite the breadth of data depicting PD-L1 to be an imperfect biomarker, obtaining the PD-L1 expression status is the standard of care in the management of patients with metastatic NSCLC.

Acquired somatic mutations in tumor DNA can potentially be translated into neo-proteins on the cellular surface which act as neoantigens. These neoantigens are recognized by the T cells using the MHC pathways, subsequently activating the immune cascade. Tumor cells utilize different pathways to evade this immune surveillance, like the PD-1/PD-L1 and CTLA-4 axis. Therefore, in addition to PD-L1, the neoantigen load or the bulk of mutations can also have a predictive role as a biomarker.

Tumor mutational burden (TMB) is defined as the total number of acquired nonsynonymous somatic mutations per megabase of tumor DNA.⁶⁴ The burden of mutations was initially measured using the whole exon sequencing on both tumor DNA as well as matched normal DNA. Though only non-synonymous mutations lead to changes in protein structures thereby contributing to tumor immunogenicity, synonymous mutations have also been used in the calculations of TMB by different platforms.⁶⁵ The rationale to include all mutations is to improve the resolution of TMB (using the synonymous mutations as a surrogate for total mutations burden), especially in samples with lowed DNA load.⁶⁵ Recently specific gene directed next generation sequencing has been validated for TMB measurement and two panels (for tissue TMB) have been approved by the FDA, F1CDx (pembrolizumab in solid tumors with high TMB) by Foundation Medicine Inc. and MSK-IMPACT by the Memorial Sloan Kettering Cancer Center.

The method of calculation of TMB is variable. Different assays have used different methods resulting in poor reproducibility of the results.⁶⁶ Several assays use proprietary germline variant datasets for filtering germline mutations and some assays use paired tumor and normal tissue to subtract germline alterations and calculate the TMB. Tumor-only sequencing methods have been shown to overestimate TMB compared to the germline-sequenced and subtracted TMB methods. This may particularly impact minority races that have a low representation in most germline variant libraries.^67–69 Other issues with TMB calculation are the inclusion of different mutations types (most assays use single nucleotide variants but some also include insertions and deletions, or synonymous mutations), corrections for formalin induced DNA damage (different assays use different methods for compensations for formalin induced deamination), size of the captured coding regions, and the pre-analytical processing which is different for different assays.^65,70–73

Blood based assays (like NCC-GP150) to measure TMB using circulating tumor DNA has been tested as surrogate of tissue samples. Though no assay has been approved by the FDA, significant correlation (both technical and clinical) has been demonstrated between the blood TMB (bTMB) and tumor TMB (tTMB) though some studies have shown a low concordance owing to tumor heterogeneity.^74,75 bTMB has been demonstrated to be a predictor of clinical response to ICI in NSCLC cases.^76,77 Tumor heterogeneity has been graded using several methods and has been demonstrated to influence both tTMb and bTMB.⁷⁸ Theoretically bTMB represents a more holistic picture of the cellular and genomic diversity within the tumor tissue, but there are limited studies testing this concept. In a study with 32 operated cases of NSCLC muti-region tTMB was found to correlate with both single region tTMB as well as bTMB and high intra-tumoral heterogeneity cases were found to have higher chances of high-TMB when evaluated using muti-regions of tumor tissue.⁷⁹

The prognostic role of TMB in NSCLC has also been analyzed in systematic review including eight cohorts. Using different cut offs, amongst the high TMB groups (≥10 mut/Mb or ≥ 243 somatic mutations or ≥20 mut/Mb), ICIs were superior to chemotherapy in terms of ORR, PFS and OS. This finding was not seen in the low TMB subgroup.^17,18,29,80 In addition, though PFS has been shown to be higher in subjects with high-TMB in tumor tissue, the impact on OS has not seen similar improvements, thereby questioning the role of tTMB in patient selection.^18,81

No concordance between PD-L1 levels (assessed using 22C3 platforms) and TMB (assessed using whole exome sequencing) in NSCLC cases have been found.^29,82 Even though there have been no head-to-head comparisons, PFS and ORR amongst patients with high PD-L1 expression has been found to be higher than in patients with high TMB.^76,83

While the large number of FDA approved agents available for targeting the PD-1/PD-L1/CTLA-4 pathway has opened the doors in-terms of providing various options for the care of patients, they have also been a source of confusion for many oncologists practicing in the United States with regard to teasing out the differences in these regimens, their approved indications, and maneuvering the nuances in choosing the right ICIs for their patients.⁸⁴ While there are no head-to-head trials comparing various ICIs, a recent meta-analysis has compared various approved ICI regimens and suggested that there may be differences in outcomes based on the PD-L1 status and metastatic sites of patients.⁸⁵

There is a palpable need to move the needle forward in the treatment of metastatic NSCLC by unlocking the potential of other immune checkpoints. There have been several ongoing trials targeting other pathways with emerging results. One such pathway that is being studied is the T cell immunoglobulin and ITIM domain (TIGIT). The TIGIT protein expression is mainly in T-lymphocytes and NK cells. It competes with CD226 to deliver an inhibitory signal, thereby causing an immunosuppressive effect by decreasing T-cell activation, function and inhibiton of NK cell activity.^86–88 Interim results from the phase-3 trial SKYSCRAPER (NCT04294810) assessing the combination of atezolizumab combined with tiragolumab (anti-TIGIT) in metastatic NSCLC did not meet the co-primary endpoint of PFS. Due to immature OS data, the study is being continued. The recently presented interim analysis results of the phase-2 ARC-7 study showed a superior PFS and OS of the doublet domvanalimab (anti-PD-1) plus zimberelimab (anti-TIGIT) and the triplet domvanalimab plus zimberelimab plus etrumadenant (A2a/b adenosine receptor antagonist) when compared to domvanalimab alone in patients with PD-L1 high metastatic NSCLC.⁸⁹ In the phase-2 CITYSCAPE trial, an improvement in PFS was seen with the incorporation of anti-TIGIT tiragolumab plus atezolizumab compared to ICI alone.⁹⁰

The challenges associated with ICIs in low and middle-income countries (LMIC) are different. The easy availability of ICIs that exist in upper- middle income and high-income countries have not percolated into LMICs due to multiple barriers such as cost, availability, physician preference and lack of ethnic-diverse representation in clinical trials. Ravikrishna et al., recently showed that in a 5-year time period including more than 15,000 patients in India whom were eligible for ICIs, only about 2.8% received them.⁹¹ Nazha et al., have described the involvement of Asian representation in trials to be around 6%.⁹² In China, there are five PD/PD-L1 agents (camrelizumab, sintilimab, tislelizumab, toripalimab, sugemalimab) have been granted approval. However, these drugs have not made their way to many other Asian countries. The Chinese anti-PD-1 drug sintilimab, was recently denied approval by the FDA approval based on the ORIENT-11 trial,⁹³ partially due to a majority of the patients enrolled being non-US based.⁸⁴ The manufacturer of this drug had proposed a reduction of the prices of other similar drugs in the market by 40-90%, speaking to the much-discussed speculation regarding improvement in competitive pricing with the development of "me too" drugs. However, whether this theory holds true or not is still debatable with existing evidence suggesting that "brand-brand" competition is historically not known to lower the prices of drugs of the same class.⁹⁴ The emergence of these "me too" drugs while, unlikely to be beneficial in markets such as the United States and European Union, could solve a pressing issue in other countries where ICIs are not easily accessible. Manufacturing companies can seek to include ethnically diverse populations from countries like India, for example, where patient recruitment is unlikely to be a major issue. This will solve the issues of finding a niche for these "me too" drugs while also improving access to equitable care across other countries.

There continues to be progress in the field of checkpoint inhibition as numerous other immune checkpoints have been identified as potential targets for inhibition and drug development. Table 2 highlights many of the emerging checkpoints.

The TACTI-002 (NCT03625323) study studied the combination of the soluble anti-LAG-3 protein, eftilagimod alpha with pembrolizumab in the front-line management of metastatic non-small cell lung cancer showed an ORR in the intention to treat (ITT) population of 37% with a disease control rate was around 73% with more mature data pending.¹¹⁵ Modest results have also been seen in trials studying the combination of TIM-3 with PD-L1 inhibition due to potential synergistic effects of the two drugs. One phase II trial (NCT02608268) studied MBG453 (anti-TIM-3) plus spartalizumab (anti-PD-1) in patients with progressive metastatic NSCLC and demonstrated a durable clinical benefit in about 42% of the NSCLC.¹¹⁶ There are several studies evaluating the anti-tumor effects of 4-1BB agonists alone and in combination with ICIs.¹² Two drugs, urelumab (BMS-663513) and utomilumab (PF-05082566) with initial results showed only a modest response in solid tumors.^117,118 Monalizumab, an anti-NKG2A humanized monoclonal antibody is being studied in the non-metastatic setting in the management of NSCLC. The interim analysis from the phase-2 COAST study demonstrated promising results with a superior ORR of monalizumab plus durvalumab when compared to durvalumab alone.¹¹⁹ Figure 1 shows newer checkpoints and their ligands.

The road thus far, has been challenging with many of the mentioned therapies showing suboptimal activity when used alone. The synergistic activity of these drugs when used with existing checkpoint inhibitors remains to be further explored with multiple concerns regarding tolerability and more severe immune related adverse events. There is a need for further research in identifying biomarkers and for the development of strategies that can help turn "cold" tumor microenvironments (TME) to "hot" TME, thereby increasing the response to ICIs.¹²¹

Immunotherapy in lung cancer still has a long way to go. The immune checkpoint inhibitors, especially PD-1/PDL1 targeted agents, have been shown to significantly improve clinically relevant endpoints. But despite the overwhelming success, patient selection remains an imperfect facet. Research efforts need to be focused on rational combination strategies developed on the basis of improved tumor biology understanding. Different biomarkers with different assays have been tested and approved but the search of an ideal biomarker remains ongoing. Deeper insight into the immune evasion mechanisms of tumor cells might add to the understanding and development of molecules leading to superior outcomes.

In addition to the immune checkpoint inhibitors, various other forms of immunotherapies have also been investigated in different phases of clinical trials like tumor directed monoclonal antibodies, tumor vaccines, T cell therapies, nanomaterials and bi-specific T-cell engagers (BiTEs) and drugs targeting other checkpoints.

Chimeric antigen receptor (CAR)-T cell therapy, part of adoptive cell transfer therapy, utilizes the genetically modified T cells for their actions against tumor cells. Gene coding the modified chimeric proteins are inserted into the autologous T cell using viral or non-viral vectors. These proteins are able to bind to various cell surface antigens or receptors present on tumor cells.¹²² CAR-T cells have been demonstrated to be successful in hematological malignancies but their response rates in solid tumors have been low, probably due to low levels of tumor infiltrations, lack of tumor specific antigens and risk of cytokine release syndrome.¹²³ Clinical trials for CAR-T cells in lung cancer have tested their role in malignant pleural mesothelioma due to the availability of specific MSLN antigen but role of CAR-T cells in NSCLC has yet not been tested in clinical trials.¹²² Additionally, TCR (T cell receptor) gene engineered T cells, which have TCR specifically directed towards cancer antigens have also been tested in preclinical models. One of the antigens used for the development of TCRs has been Kita-Kyushu Lung Cancer Antgen-1 which is one of its family proteins, not expressed on healthy tissues.¹²⁴ PD-1 gene disrupted T cells (using CRISPR-Cas9 technology) have also been tested in phase I trial and demonstrated to be safe and feasible.¹²⁵ Another form of cellular immunotherapy is TILs (tumor infiltrating lymphocytes). These lymphocytes are usually very few hence, for using this method TILs are expanded in vitro and later reinjected in large amounts. Autologous TIL therapy requires prior lymphocyte depletion. The acceptable safety profile of TIL therapy has been demonstrated in phase 1 study where TILs were administered with nivolumab.¹²⁶

Bispecific T cell engager (BiTE) platforms is another of targeted immunotherapy where two different antigens are linked together. One end of the protein binds with the tumor antigens whereas the other end binds with the T cells and leads to immune activation.¹²⁷ AMG 757 (Tarlatamab) is a bispecific T-cell engager targeting delta like ligand 3 (DLCC3) in SCLC. Phase I results of AMG 757 have demonstrated acceptable safety profile and trial is still ongoing.¹²⁸ Bispecific antibody, Y111, which targets PD-L1 and CD3 has also been tested in preclinical setting where it has been demonstrated to be effective in inducing tumor cell cytotoxicity.¹²⁹

In addition to the BiTEs, the bispecific monoclonal antibodies which have two different targets have also been discovered and tested. Amivantamab, targeting EGFR and MET has been given an accelerated approval by the FDA for exon 20 insertion mutations, which are inherently resistant to the conventional EGFR TKIs,¹³⁰ following the results of CHRYSALIS trial which demonstrated an ORR of 40% and DOR of 11.1 months.¹³¹ Another example is Zenocutuzumab (MCLA-128) which is a NRG1 fusion targeting bispecific antibody being tested in NRG1 positive solid tumors.¹³²

Apart from the conventional biomarkers, neoantigen load, ctDNA and MSI-H/MMR (mismatch repair) are also being studied as potential biomarkers for ICIs therapy.¹³³ Newer methods of resistance are also being studied and targeted like STK11/LKB1¹³⁴ and JAK2/STAT.¹³⁵

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No artificial intelligence tools were used in the writing, editing, or creation of this manuscript.

Concept and design: AS, KP, PKS

Data acquisition: AS, KP, PKS

Data analysis and interpretation: AS, KP, PKS

Drafting of the manuscript: AS, KP, PKS

Critical revision of the manuscript: AS, KP, PKS

All authors (AS, KP, PKS) approved the final version of the manuscript and agree to be accountable for all aspects of the work, in accordance with the International Committee of Medical Journal Editors criteria.

1. Sharpe AH. Mechanisms of costimulation. Immunol Rev. 2009;229(1):5-11. doi:10.1111/j.1600-065X.2009.00784.x

2. Jenkins MK, Schwartz RH. Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo. J Exp Med. 1987;165(2):302-319. doi:10.1084/jem.165.2.302

3. Mueller DL, Jenkins MK, Schwartz RH. An accessory cell-derived costimulatory signal acts independently of protein kinase C activation to allow T cell proliferation and prevent the induction of unresponsiveness. J Immunol. 1989;142(8):2617-2628. doi:10.4049/jimmunol.142.8.2617

4. Walunas TL, Lenschow DJ, Bakker CY, et al. CTLA-4 can function as a negative regulator of T cell activation. Immunity. 1994;1(5):405-413. doi:10.1016/1074-7613(94)90071-X

5. Krummel MF, Allison JP. CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. J Exp Med. 1995;182(2):459-465. doi:10.1084/jem.182.2.459

6. Sharma P, Allison JP. Immune checkpoint targeting in cancer therapy: toward combination strategies with curative potential. Cell. 2015;161(2):205-214. doi:10.1016/j.cell.2015.03.030

7. Dong H, Zhu G, Tamada K, Chen L. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med. 1999;5(12):1365-1369. doi:10.1038/70932

8. Robert C, Thomas L, Bondarenko I, et al. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med. 2011;364(26):2517-2526. doi:10.1056/NEJMoa1104621

9. Brahmer J, Reckamp KL, Baas P, et al. Nivolumab versus docetaxel in advanced squamous-cell non–small-cell lung cancer. N Engl J Med. 2015;373(2):123-135. doi:10.1056/NEJMoa1504627

10. Borghaei H, Paz-Ares L, Horn L, et al. Nivolumab versus docetaxel in advanced nonsquamous non–small-cell lung cancer. N Engl J Med. 2015;373(17):1627-1639. doi:10.1056/NEJMoa1507643

11. Herbst RS, Baas P, Kim D, et al. Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial. Lancet. 2016;387(10027):1540-1550. doi:10.1016/S0140-6736(15)01281-7

12. National Comprehensive Cancer Network. NCCN clinical practice guidelines in oncology: non-small cell lung cancer. Accessed July 27, 2026. https://www.nccn.org/professionals/physician_gls/default.aspx

13. Reck M, Rodríguez-Abreu D, Robinson AG, et al. Pembrolizumab versus chemotherapy for PD-L1–positive non–small-cell lung cancer. N Engl J Med. 2016;375(19):1823-1833. doi:10.1056/NEJMoa1606774

14. Reck M, Rodríguez-Abreu D, Robinson AG, et al. Five-year outcomes with pembrolizumab versus chemotherapy for metastatic non–small-cell lung cancer with PD-L1 tumor proportion score ≥ 50%. J Clin Oncol. 2021;39(21):2339-2349. doi:10.1200/JCO.21.00174

15. Mok TSK, Wu Y, Kudaba I, et al. Pembrolizumab versus chemotherapy for previously untreated, PD-L1-expressing, locally advanced or metastatic non-small-cell lung cancer (KEYNOTE-042): a randomised, open-label, controlled, phase 3 trial. Lancet. 2019;393(10183):1819-1830. doi:10.1016/S0140-6736(18)32409-7

16. Gandhi L, Rodríguez-Abreu D, Gadgeel S, et al. Pembrolizumab plus chemotherapy in metastatic non–small-cell lung cancer. N Engl J Med. 2018;378(22):2078-2092. doi:10.1056/NEJMoa1801005

17. Carbone DP, Reck M, Paz-Ares L, et al. First-line nivolumab in stage IV or recurrent non–small-cell lung cancer. N Engl J Med. 2017;376(25):2415-2426. doi:10.1056/NEJMoa1613493

18. Hellmann MD, Paz-Ares L, Bernabe Caro R, et al. Nivolumab plus ipilimumab in advanced non–small-cell lung cancer. N Engl J Med. 2019;381(21):2020-2031. doi:10.1056/NEJMoa1910231

19. Paz-Ares L, Ciuleanu T, Cobo M, et al. First-line nivolumab plus ipilimumab combined with two cycles of chemotherapy in patients with non-small-cell lung cancer (CheckMate 9LA): an international, randomised, open-label, phase 3 trial. Lancet Oncol. 2021;22(2):198-211. doi:10.1016/S1470-2045(20)30641-0

20. Socinski MA, Jotte RM, Cappuzzo F, et al. Atezolizumab for first-line treatment of metastatic nonsquamous NSCLC. N Engl J Med. 2018;378(24):2288-2301. doi:10.1056/NEJMoa1716948

21. Jotte R, Cappuzzo F, Vynnychenko I, et al. Atezolizumab in combination with carboplatin and nab-paclitaxel in advanced squamous NSCLC (IMpower131): results from a randomized phase III trial. J Thorac Oncol. 2020;15(8):1351-1360. doi:10.1016/j.jtho.2020.03.028

22. Jassem J, de Marinis F, Giaccone G, et al. Updated overall survival analysis from IMpower110: atezolizumab versus platinum-based chemotherapy in treatment-naive programmed death-ligand 1–selected NSCLC. J Thorac Oncol. 2021;16(11):1872-1882. doi:10.1016/j.jtho.2021.06.019

23. Herbst RS, Giaccone G, de Marinis F, et al. Atezolizumab for first-line treatment of PD-L1–selected patients with NSCLC. N Engl J Med. 2020;383(14):1328-1339. doi:10.1056/NEJMoa1917346

24. West H, McCleod M, Hussein M, et al. Atezolizumab in combination with carboplatin plus nab-paclitaxel chemotherapy compared with chemotherapy alone as first-line treatment for metastatic non-squamous non-small-cell lung cancer (IMpower130): a multicentre, randomised, open-label, phase 3 trial. Lancet Oncol. 2019;20(7):924-937. doi:10.1016/S1470-2045(19)30167-6

25. Nishio M, Barlesi F, West H, et al. Atezolizumab plus chemotherapy for first-line treatment of nonsquamous NSCLC: results from the randomized phase 3 IMpower132 trial. J Thorac Oncol. 2021;16(4):653-664. doi:10.1016/j.jtho.2020.11.025

26. Kim ES, Velcheti V, Mekhail T, et al. Blood-based tumor mutational burden as a biomarker for atezolizumab in non-small cell lung cancer: the phase 2 B-F1RST trial. Nat Med. 2022;28(5):939-945. doi:10.1038/s41591-022-01754-x

27. Peters S, Dziadziuszko R, Morabito A, et al. Atezolizumab versus chemotherapy in advanced or metastatic NSCLC with high blood-based tumor mutational burden: primary analysis of BFAST cohort C randomized phase 3 trial. Nat Med. 2022;28(9):1831-1839. doi:10.1038/s41591-022-01933-w

28. Johnson ML, Cho BC, Luft A, et al. Durvalumab with or without tremelimumab in combination with chemotherapy as first-line therapy for metastatic non-small-cell lung cancer: the phase III POSEIDON study. J Clin Oncol. 2023;41(6):1213-1227. doi:10.1200/JCO.22.00975

29. Rizvi NA, Cho BC, Reinmuth N, et al. Durvalumab with or without tremelimumab vs standard chemotherapy in first-line treatment of metastatic non-small cell lung cancer: the MYSTIC phase 3 randomized clinical trial. JAMA Oncol. 2020;6(5):661-674. doi:10.1001/jamaoncol.2020.0237

30. Sezer A, Kilickap S, Gümüş M, et al. Cemiplimab monotherapy for first-line treatment of advanced non-small-cell lung cancer with PD-L1 of at least 50%: a multicentre, open-label, global, phase 3, randomised, controlled trial. Lancet. 2021;397(10274):592-604. doi:10.1016/S0140-6736(21)00228-2

31. Gogishvili M, Melkadze T, Makharadze T, et al. Cemiplimab plus chemotherapy versus chemotherapy alone in non-small cell lung cancer: a randomized, controlled, double-blind phase 3 trial. Nat Med. 2022;28(11):2374-2380. doi:10.1038/s41591-022-01977-y

32. Zhou C, Wu L, Fan Y, et al. Sintilimab plus platinum and gemcitabine as first-line treatment for advanced or metastatic squamous NSCLC: results from a randomized, double-blind, phase 3 trial (ORIENT-12). J Thorac Oncol. 2021;16(9):1501-1511. doi:10.1016/j.jtho.2021.04.011

33. Yang Y, Wang Z, Fang J, et al. Efficacy and safety of sintilimab plus pemetrexed and platinum as first-line treatment for locally advanced or metastatic nonsquamous NSCLC: a randomized, double-blind, phase 3 study (Oncology pRogram by InnovENT anti-PD-1-11). J Thorac Oncol. 2020;15(10):1636-1646. doi:10.1016/j.jtho.2020.07.014

34. Cha J, Chan L, Li C, Hsu JL, Hung M. Mechanisms controlling PD-L1 expression in cancer. Mol Cell. 2019;76(3):359-370. doi:10.1016/j.molcel.2019.09.030

35. Ju X, Zhang H, Zhou Z. Regulation of PD-L1 expression in cancer and clinical implications in immunotherapy. Am J Cancer Res. 2020;10(1):1-11.

36. Scheel AH, Ansén S, Schultheis AM, et al. PD-L1 expression in non-small cell lung cancer: correlations with genetic alterations. Oncoimmunology. 2016;5(5):e1131379. doi:10.1080/2162402X.2015.1131379

37. Yu H, Boyle TA, Zhou C, Rimm DL, Hirsch FR. PD-L1 expression in lung cancer. J Thorac Oncol. 2016;11(7):964-975. doi:10.1016/j.jtho.2016.04.014

38. Ullah A, Pulliam S, Karki NR, et al. PD-L1 over-expression varies in different subtypes of lung cancer: will this affect future therapies? Clin Pract. 2022;12(5):653-671. doi:10.3390/clinpract12050068

39. Aggarwal C, Abreu DR, Felip E, et al. Prevalence of PD-L1 expression in patients with non-small cell lung cancer screened for enrollment in KEYNOTE-001, -010, and -024. Ann Oncol. 2016;27:vi363. doi:10.1093/annonc/mdw378.14

40. Kowanetz M, Zou W, Gettinger SN, et al. Differential regulation of PD-L1 expression by immune and tumor cells in NSCLC and the response to treatment with atezolizumab (anti–PD-L1). Proc Natl Acad Sci USA. 2018;115(43):E10119-E10126. doi:10.1073/pnas.1802166115

41. US Food and Drug Administration. List of cleared or approved companion diagnostic devices (in vitro and imaging tools). In Vitro Diagnostics. Updated 2022. Accessed July 27, 2026. https://www.fda.gov/medical-devices/in-vitro-diagnostics/list-cleared-or-approved-companion-diagnostic-devices-in-vitro-and-imaging-tools#CDx_Table

42. Twomey JD, Zhang B. Cancer immunotherapy update: FDA-approved checkpoint inhibitors and companion diagnostics. AAPS J. 2021;23(2):39. doi:10.1208/s12248-021-00574-0

43. Hirsch FR, McElhinny A, Stanforth D, et al. PD-L1 immunohistochemistry assays for lung cancer: results from phase 1 of the Blueprint PD-L1 IHC Assay Comparison Project. J Thorac Oncol. 2017;12(2):208-222. doi:10.1016/j.jtho.2016.11.2228

44. Tsao MS, Kerr KM, Kockx M, et al. PD-L1 immunohistochemistry comparability study in real-life clinical samples: results of Blueprint Phase 2 Project. J Thorac Oncol. 2018;13(9):1302-1311. doi:10.1016/j.jtho.2018.05.013

45. Adam J, Le Stang N, Rouquette I, et al. Multicenter harmonization study for PD-L1 IHC testing in non-small-cell lung cancer. Ann Oncol. 2018;29(4):953-958. doi:10.1093/annonc/mdy014

46. Scheel AH, Baenfer G, Baretton G, et al. Interlaboratory concordance of PD-L1 immunohistochemistry for non-small-cell lung cancer. Histopathology. 2018;72(3):449-459. doi:10.1111/his.13375

47. McLaughlin J, Han G, Schalper KA, et al. Quantitative assessment of the heterogeneity of PD-L1 expression in non–small-cell lung cancer. JAMA Oncol. 2016;2(1):46-54. doi:10.1001/jamaoncol.2015.3638

48. Nakamura S, Hayashi K, Imaoka Y, et al. Intratumoral heterogeneity of programmed cell death ligand-1 expression is common in lung cancer. PLoS One. 2017;12(10):e0186192. doi:10.1371/journal.pone.0186192

49. Ben Dori S, Aizic A, Sabo E, Hershkovitz D. Spatial heterogeneity of PD-L1 expression and the risk for misclassification of PD-L1 immunohistochemistry in non-small cell lung cancer. Lung Cancer. 2020;147:91-98. doi:10.1016/j.lungcan.2020.07.012

50. Bodor JN, Boumber Y, Borghaei H. Biomarkers for immune checkpoint inhibition in non–small cell lung cancer (NSCLC). Cancer. 2020;126(2):260-270. doi:10.1002/cncr.32468

51. Mansfield A, Aubry M, Moser J, et al. Temporal and spatial discordance of programmed cell death-ligand 1 expression and lymphocyte tumor infiltration between paired primary lesions and brain metastases in lung cancer. Ann Oncol. 2016;27(10):1953-1958. doi:10.1093/annonc/mdw289

52. Suda K, Mitsudomi T. Inter-tumor heterogeneity of PD-L1 status: is it important in clinical decision making? J Thorac Dis. 2020;12(5):1770-1775. doi:10.21037/jtd-20-1661

53. Haragan A, Field JK, Davies MP, Escriu C, Gruver A, Gosney JR. Heterogeneity of PD-L1 expression in non-small cell lung cancer: implications for specimen sampling in predicting treatment response. Lung Cancer. 2019;134:79-84. doi:10.1016/j.lungcan.2019.06.005

54. Saito Y, Horiuchi S, Morooka H, et al. Inter-tumor heterogeneity of PD-L1 expression in non-small cell lung cancer. J Thorac Dis. 2019;11(12):4982-4991. doi:10.21037/jtd.2019.12.24

55. Zhao X, Bao Y, Meng B, et al. From rough to precise: PD-L1 evaluation for predicting the efficacy of PD-1/PD-L1 blockades. Front Immunol. 2022;13:920021. doi:10.3389/fimmu.2022.920021

56. Herbst RS, Soria J, Kowanetz M, et al. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature. 2014;515(7528):563-567. doi:10.1038/nature14011

57. Lee CK, Man J, Lord S, et al. Checkpoint inhibitors in metastatic EGFR-mutated non–small cell lung cancer—a meta-analysis. J Thorac Oncol. 2017;12(2):403-407. doi:10.1016/j.jtho.2016.10.007

58. Lisberg A, Cummings A, Goldman J, et al. A phase II study of pembrolizumab in EGFR-mutant, PD-L1+, tyrosine kinase inhibitor naïve patients with advanced NSCLC. J Thorac Oncol. 2018;13(8):1138-1145. doi:10.1016/j.jtho.2018.03.035

59. Dong Z, Zhang J, Liu S, et al. EGFR mutation correlates with uninflamed phenotype and weak immunogenicity, causing impaired response to PD-1 blockade in non-small cell lung cancer. Oncoimmunology. 2017;6(11):e1356145. doi:10.1080/2162402X.2017.1356145

60. Azuma K, Ota K, Kawahara A, et al. Association of PD-L1 overexpression with activating EGFR mutations in surgically resected nonsmall-cell lung cancer. Ann Oncol. 2014;25(10):1935-1940. doi:10.1093/annonc/mdu242

61. D'Incecco A, Andreozzi M, Ludovini V, et al. PD-1 and PD-L1 expression in molecularly selected non-small-cell lung cancer patients. Br J Cancer. 2015;112(1):95-102. doi:10.1038/bjc.2014.555

62. To KKW, Fong W, Cho WCS. Immunotherapy in treating EGFR-mutant lung cancer: current challenges and new strategies. Front Oncol. 2021;11:635007. doi:10.3389/fonc.2021.635007

63. Koh J, Go H, Keam B, et al. Clinicopathologic analysis of programmed cell death-1 and programmed cell death-ligand 1 and 2 expressions in pulmonary adenocarcinoma: comparison with histology and driver oncogenic alteration status. Mod Pathol. 2015;28(9):1154-1166. doi:10.1038/modpathol.2015.63

64. Goodman AM, Kato S, Bazhenova L, et al. Tumor mutational burden as an independent predictor of response to immunotherapy in diverse cancers. Mol Cancer Ther. 2017;16(11):2598-2608. doi:10.1158/1535-7163.MCT-17-0386

65. Sholl LM, Hirsch FR, Hwang D, et al. The promises and challenges of tumor mutation burden as an immunotherapy biomarker: a perspective from the International Association for the Study of Lung Cancer Pathology Committee. J Thorac Oncol. 2020;15(9):1409-1424. doi:10.1016/j.jtho.2020.05.019

66. Ning B, Liu Y, Wang M, Li Y, Xu T, Wei Y. The predictive value of tumor mutation burden on clinical efficacy of immune checkpoint inhibitors in melanoma: a systematic review and meta-analysis. Front Pharmacol. 2022;13:748674. doi:10.3389/fphar.2022.748674

67. Asmann YW, Parikh K, Bergsagel PL, et al. Inflation of tumor mutation burden by tumor-only sequencing in under-represented groups. npj Precis Oncol. 2021;5(1):22. doi:10.1038/s41698-021-00164-5

68. Parikh K, Huether R, White K, et al. Tumor mutational burden from tumor-only sequencing compared with germline subtraction from paired tumor and normal specimens. JAMA Netw Open. 2020;3(2):e200202. doi:10.1001/jamanetworkopen.2020.0202

69. Nassar AH, Adib E, Abou Alaiwi S, et al. Ancestry-driven recalibration of tumor mutational burden and disparate clinical outcomes in response to immune checkpoint inhibitors. Cancer Cell. 2022;40(10):1161-1172.e5. doi:10.1016/j.ccell.2022.08.022

70. Kazdal D, Endris V, Allgäuer M, et al. Spatial and temporal heterogeneity of panel-based tumor mutational burden in pulmonary adenocarcinoma: separating biology from technical artifacts. J Thorac Oncol. 2019;14(11):1935-1947. doi:10.1016/j.jtho.2019.07.006

71. Moens LN, Falk-Sörqvist E, Ljungström V, et al. HaloPlex targeted resequencing for mutation detection in clinical formalin-fixed, paraffin-embedded tumor samples. J Mol Diagn. 2015;17(6):729-739. doi:10.1016/j.jmoldx.2015.06.009

72. Buchhalter I, Rempel E, Endris V, et al. Size matters: dissecting key parameters for panel-based tumor mutational burden analysis. Int J Cancer. 2019;144(4):848-858. doi:10.1002/ijc.31878

73. Chan T, Yarchoan M, Jaffee E, et al. Development of tumor mutation burden as an immunotherapy biomarker: utility for the oncology clinic. Ann Oncol. 2019;30(1):44-56. doi:10.1093/annonc/mdy495

74. Wang Z, Duan J, Cai S, et al. Assessment of blood tumor mutational burden as a potential biomarker for immunotherapy in patients with non–small cell lung cancer with use of a next-generation sequencing cancer gene panel. JAMA Oncol. 2019;5(5):696-702. doi:10.1001/jamaoncol.2018.7098

75. Davis AA, Chae YK, Agte S, et al. Association of circulating tumor DNA (ctDNA) tumor mutational burden (TMB) with DNA repair mutations and response to anti-PD-1/PD-L1 therapy in non-small cell lung cancer (NSCLC). J Clin Oncol. 2017;35(15 suppl):11537. doi:10.1200/JCO.2017.35.15_suppl.11537

76. Gandara DR, Paul SM, Kowanetz M, et al. Blood-based tumor mutational burden as a predictor of clinical benefit in non-small-cell lung cancer patients treated with atezolizumab. Nat Med. 2018;24(9):1441-1448. doi:10.1038/s41591-018-0134-3

77. Zhang N, Zhang J, Wang G, et al. Predictive efficacy of blood-based tumor mutation burden assay for immune checkpoint inhibitors therapy in non-small cell lung cancer: a systematic review and meta-analysis. Front Oncol. 2022;12:795933. doi:10.3389/fonc.2022.795933

78. Fridland S, Choi J, Nam M, et al. Assessing tumor heterogeneity: integrating tissue and circulating tumor DNA (ctDNA) analysis in the era of immuno-oncology - blood TMB is not the same as tissue TMB. J Immunother Cancer. 2021;9(8):e002551. doi:10.1136/jitc-2021-002551

79. Zhang Y, Chang L, Yang Y, et al. The correlations of tumor mutational burden among single-region tissue, multi-region tissues and blood in non-small cell lung cancer. J Immunother Cancer. 2019;7(1):98. doi:10.1186/s40425-019-0581-5

80. Galvano A, Gristina V, Malapelle U, et al. The prognostic impact of tumor mutational burden (TMB) in the first-line management of advanced non-oncogene addicted non-small-cell lung cancer (NSCLC): a systematic review and meta-analysis of randomized controlled trials. ESMO Open. 2021;6(3):100124. doi:10.1016/j.esmoop.2021.100124

81. Ready N, Hellmann MD, Awad MM, et al. First-line nivolumab plus ipilimumab in advanced non–small-cell lung cancer (CheckMate 568): outcomes by programmed death ligand 1 and tumor mutational burden as biomarkers. J Clin Oncol. 2019;37(12):992-1000. doi:10.1200/JCO.18.01042

82. Peters S, Cho BC, Reinmuth N, et al. Tumor mutational burden (TMB) as a biomarker of survival in metastatic non-small cell lung cancer (mNSCLC): blood and tissue TMB analysis from MYSTIC, a phase III study of first-line durvalumab ± tremelimumab vs chemotherapy. Cancer Res. 2019;79(13 suppl):CT074. doi:10.1158/1538-7445.AM2019-CT074

83. Yoh K, Matsumoto S, Furuya N, et al. Comprehensive assessment of PD-L1 expression, tumor mutational burden and oncogenic driver alterations in non-small cell lung cancer patients treated with immune checkpoint inhibitors. Lung Cancer. 2021;159:128-134. doi:10.1016/j.lungcan.2021.07.015

84. Ferrara R, Ricciuti B, Ambrogio C, Trapani D. The anti–programmed cell death protein-1/programmed death-ligand 1 me-too drugs tsunami: hard to be millennials among baby boomers. J Thorac Oncol. 2023;18(1):17-20. doi:10.1016/j.jtho.2022.11.001

85. Siciliano M, Caridà G, Ciliberto D, et al. Efficacy and safety of first-line checkpoint inhibitors-based treatments for non-oncogene-addicted non-small-cell lung cancer: a systematic review and meta-analysis. ESMO Open. 2022;7(3):100465. doi:10.1016/j.esmoop.2022.100465

86. Le Mercier I, Lines JL, Noelle RJ. Beyond CTLA-4 and PD-1, the generation Z of negative checkpoint regulators. Front Immunol. 2015;6:418. doi:10.3389/fimmu.2015.00418

87. Lozano E, Dominguez-Villar M, Kuchroo V, Hafler DA. The TIGIT/CD226 axis regulates human T cell function. J Immunol. 2012;188(8):3869-3875. doi:10.4049/jimmunol.1103627

88. Qin S, Xu L, Yi M, Yu S, Wu K, Luo S. Novel immune checkpoint targets: moving beyond PD-1 and CTLA-4. Mol Cancer. 2019;18(1):155. doi:10.1186/s12943-019-1091-2

89. Johnson ML, Fox W, Lee Y, et al. ARC-7: randomized phase 2 study of domvanalimab + zimberelimab ± etrumadenant versus zimberelimab in first-line, metastatic, PD-L1-high non-small cell lung cancer (NSCLC). J Clin Oncol. 2022;40(36 suppl):397600. doi:10.1200/JCO.2022.40.36_suppl.397600

90. Cho BC, Abreu DR, Hussein M, et al. Tiragolumab plus atezolizumab versus placebo plus atezolizumab as a first-line treatment for PD-L1-selected non-small-cell lung cancer (CITYSCAPE): primary and follow-up analyses of a randomised, double-blind, phase 2 study. Lancet Oncol. 2022;23(6):781-792. doi:10.1016/S1470-2045(22)00226-1

91. Ravikrishna M, Abraham G, Patil VM, et al. Checkpoint inhibitor accessibility in 15,000+ Indian patients. J Clin Oncol. 2022;40(16 suppl):9012. doi:10.1200/JCO.2022.40.16_suppl.9012

92. Nazha B, Mishra M, Pentz R, Owonikoko TK. Enrollment of racial minorities in clinical trials: old problem assumes new urgency in the age of immunotherapy. Am Soc Clin Oncol Educ Book. 2019;39:3-10. doi:10.1200/EDBK_100021

93. [Duplicate of reference 33 — Yang Y et al., J Thorac Oncol 2020. Please remove and renumber.]

94. Sarpatwari A, DiBello J, Zakarian M, Najafzadeh M, Kesselheim AS. Competition and price among brand-name drugs in the same class: a systematic review of the evidence. PLoS Med. 2019;16(7):e1002872. doi:10.1371/journal.pmed.1002872

95. Sakuishi K, Apetoh L, Sullivan JM, Blazar BR, Kuchroo VK, Anderson AC. Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity. J Exp Med. 2010;207(10):2187-2194. doi:10.1084/jem.20100643

96. Jia K, He Y, Dziadziuszko R, et al. T cell immunoglobulin and mucin-domain containing-3 in non-small cell lung cancer. Transl Lung Cancer Res. 2019;8(6):895-906. doi:10.21037/tlcr.2019.11.17

97. Huang C, Workman CJ, Flies D, et al. Role of LAG-3 in regulatory T cells. Immunity. 2004;21(4):503-513. doi:10.1016/j.immuni.2004.08.010

98. Andreae S, Piras F, Burdin N, Triebel F. Maturation and activation of dendritic cells induced by lymphocyte activation gene-3 (CD223). J Immunol. 2002;168(8):3874-3880. doi:10.4049/jimmunol.168.8.3874

99. Maçon-Lemaître L, Triebel F. The negative regulatory function of the lymphocyte-activation gene-3 co-receptor (CD223) on human T cells. Immunology. 2005;115(2):170-178. doi:10.1111/j.1365-2567.2005.02145.x

100. Kisielow M, Kisielow J, Capoferri-Sollami G, Karjalainen K. Expression of lymphocyte activation gene 3 (LAG-3) on B cells is induced by T cells. Eur J Immunol. 2005;35(7):2081-2088. doi:10.1002/eji.200526090

101. Kouo T, Huang L, Pucsek AB, et al. Galectin-3 shapes antitumor immune responses by suppressing CD8+ T cells via LAG-3 and inhibiting expansion of plasmacytoid dendritic cells. Cancer Immunol Res. 2015;3(4):412-423. doi:10.1158/2326-6066.CIR-14-0150

102. Melero I, Shuford WW, Newby SA, et al. Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors. Nat Med. 1997;3(6):682-685. doi:10.1038/nm0697-682

103. Kim AMJ, Nemeth MR, Lim S. 4-1BB: a promising target for cancer immunotherapy. Front Oncol. 2022;12:968360. doi:10.3389/fonc.2022.968360

104. André P, Denis C, Soulas C, et al. Anti-NKG2A mAb is a checkpoint inhibitor that promotes anti-tumor immunity by unleashing both T and NK cells. Cell. 2018;175(7):1731-1743.e13. doi:10.1016/j.cell.2018.10.014

105. Fu Y, Lin Q, Zhang Z, Zhang L. Therapeutic strategies for the costimulatory molecule OX40 in T-cell-mediated immunity. Acta Pharm Sin B. 2020;10(3):414-433. doi:10.1016/j.apsb.2019.08.010

106. De Giglio A, Di Federico A, Nuvola G, Deiana C, Gelsomino F. The landscape of immunotherapy in advanced NSCLC: driving beyond PD-1/PD-L1 inhibitors (CTLA-4, LAG3, IDO, OX40, TIGIT, vaccines). Curr Oncol Rep. 2021;23(11):126. doi:10.1007/s11912-021-01124-9

107. Vigano S, Alatzoglou D, Irving M, et al. Targeting adenosine in cancer immunotherapy to enhance T-cell function. Front Immunol. 2019;10:925. doi:10.3389/fimmu.2019.00925

108. Leone RD, Lo Y, Powell JD. A2aR antagonists: next generation checkpoint blockade for cancer immunotherapy. Comput Struct Biotechnol J. 2015;13:265-272. doi:10.1016/j.csbj.2015.03.008

109. Wang J, Wu G, Manick B, et al. VSIG-3 as a ligand of VISTA inhibits human T-cell function. Immunology. 2019;156(1):74-85. doi:10.1111/imm.13001

110. Nowak EC, Lines JL, Varn FS, et al. Immunoregulatory functions of VISTA. Immunol Rev. 2017;276(1):66-79. doi:10.1111/imr.12525

111. Le Mercier I, Chen W, Lines JL, et al. VISTA regulates the development of protective antitumor immunity. Cancer Res. 2014;74(7):1933-1944. doi:10.1158/0008-5472.CAN-13-1506

112. Janakiram M, Shah UA, Liu W, Zhao A, Schoenberg MP, Zang X. The third group of the B7-CD28 immune checkpoint family: HHLA2, TMIGD2, B7x, and B7-H3. Immunol Rev. 2017;276(1):26-39. doi:10.1111/imr.12521

113. Suh W, Gajewska BU, Okada H, et al. The B7 family member B7-H3 preferentially down-regulates T helper type 1–mediated immune responses. Nat Immunol. 2003;4(9):899-906. doi:10.1038/ni967

114. Prasad DVR, Nguyen T, Li Z, et al. Murine B7-H3 is a negative regulator of T cells. J Immunol. 2004;173(4):2500-2506. doi:10.4049/jimmunol.173.4.2500

115. Felip E, Majem M, Doger B, et al. A phase II study (TACTI-002) in first-line metastatic non–small cell lung carcinoma investigating eftilagimod alpha (soluble LAG-3 protein) and pembrolizumab: updated results from a PD-L1 unselected population. J Clin Oncol. 2022;40(16 suppl):9003. doi:10.1200/JCO.2022.40.16_suppl.9003

116. Mach N, Curigliano G, Santoro A, et al. Phase II study of MBG453 + spartalizumab in patients with non-small cell lung cancer (NSCLC) and melanoma pretreated with anti–PD-1/L1 therapy. Ann Oncol. 2019;30:v491-v492. doi:10.1093/annonc/mdz253.028

117. Segal NH, Logan TF, Hodi FS, et al. Results from an integrated safety analysis of urelumab, an agonist anti-CD137 monoclonal antibody. Clin Cancer Res. 2017;23(8):1929-1936. doi:10.1158/1078-0432.CCR-16-1272

118. Segal NH, He AR, Doi T, et al. Phase I study of single-agent utomilumab (PF-05082566), a 4-1BB/CD137 agonist, in patients with advanced cancer. Clin Cancer Res. 2018;24(8):1816-1823. doi:10.1158/1078-0432.CCR-17-1922

119. Herbst RS, Majem M, Barlesi F, et al. COAST: an open-label, phase II, multidrug platform study of durvalumab alone or in combination with oleclumab or monalizumab in patients with unresectable, stage III non–small-cell lung cancer. J Clin Oncol. 2022;40(29):3383-3393. doi:10.1200/JCO.22.00227

120. BioRender. Created with BioRender.com. Accessed July 27, 2026. https://www.biorender.com

121. Liu Y, Sun Z. Turning cold tumors into hot tumors by improving T-cell infiltration. Theranostics. 2021;11(11):5365-5386. doi:10.7150/thno.58390

122. Drougkas K, Karampinos K, Karavolias I, et al. Comprehensive clinical evaluation of CAR-T cell immunotherapy for solid tumors: a path moving forward or a dead end? J Cancer Res Clin Oncol. 2023;149(6):2709-2726. doi:10.1007/s00432-022-04547-4

123. Qu J, Mei Q, Chen L, Zhou J. Chimeric antigen receptor (CAR)-T-cell therapy in non-small-cell lung cancer (NSCLC): current status and future perspectives. Cancer Immunol Immunother. 2021;70(3):619-631. doi:10.1007/s00262-020-02735-0

124. Marcinkowski B, Stevanović S, Helman SR, et al. Cancer targeting by TCR gene-engineered T cells directed against Kita-Kyushu lung cancer antigen-1. J Immunother Cancer. 2019;7(1):229. doi:10.1186/s40425-019-0678-x

125. Lu Y, Xue J, Deng T, et al. Publisher correction: safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer. Nat Med. 2020;26(7):1149. doi:10.1038/s41591-020-0973-6

126. Creelan BC, Wang C, Teer JK, et al. Tumor-infiltrating lymphocyte treatment for anti-PD-1-resistant metastatic lung cancer: a phase 1 trial. Nat Med. 2021;27(8):1410-1418. doi:10.1038/s41591-021-01462-y

127. Einsele H, Borghaei H, Orlowski RZ, et al. The BiTE (bispecific T-cell engager) platform: development and future potential of a targeted immuno-oncology therapy across tumor types. Cancer. 2020;126(14):3192-3201. doi:10.1002/cncr.32909

128. Owonikoko TK, Champiat S, Johnson ML, et al. Updated results from a phase 1 study of AMG 757, a half-life extended bispecific T-cell engager (BiTE) immuno-oncology therapy against delta-like ligand 3 (DLL3), in small cell lung cancer (SCLC). J Clin Oncol. 2021;39(15 suppl):8510. doi:10.1200/JCO.2021.39.15_suppl.8510

129. Yang R, Shen S, Gong C, et al. Bispecific antibody PD-L1 x CD3 boosts the anti-tumor potency of the expanded Vγ2Vδ2 T cells. Front Immunol. 2021;12:654080. doi:10.3389/fimmu.2021.654080

130. Vyse S, Huang PH. Targeting EGFR exon 20 insertion mutations in non-small cell lung cancer. Signal Transduct Target Ther. 2019;4(1):5. doi:10.1038/s41392-019-0038-9

131. Park K, Haura EB, Leighl NB, et al. Amivantamab in EGFR exon 20 insertion–mutated non–small-cell lung cancer progressing on platinum chemotherapy: initial results from the CHRYSALIS phase I study. J Clin Oncol. 2021;39(30):3391-3402. doi:10.1200/JCO.21.00662

132. Schram AM, O'Reilly EM, O'Kane GM, et al. Efficacy and safety of zenocutuzumab in advanced pancreas cancer and other solid tumors harboring NRG1 fusions. J Clin Oncol. 2021;39(15 suppl):3003. doi:10.1200/JCO.2021.39.15_suppl.3003

133. Wang D, Wu X, Sun Y. Therapeutic targets and biomarkers of tumor immunotherapy: response versus non-response. Signal Transduct Target Ther. 2022;7(1):331. doi:10.1038/s41392-022-01136-2

134. Mamdani H, Matosevic S, Khalid AB, Durm G, Jalal SI. Immunotherapy in lung cancer: current landscape and future directions. Front Immunol. 2022;13:823618. doi:10.3389/fimmu.2022.823618

135. Qian F, Han B. Mechanisms of resistance to immune checkpoint inhibitors and strategies to reverse drug resistance in lung cancer. Chin Med J (Engl). 2020;133(20):2444-2455. doi:10.1097/CM9.0000000000001124