Tumor cells are recognized by T cells (44–46) through tumor-specific antigens (TSA) and tumor-associated antigens (TAA) (47, 48). TSAs are neoantigens, that are entirely absent in normal tissues and thus not tolerated by the immune system. Karasaki et al. (49) found in a group of NSCLC patients that a median of 46 potential neoantigens (pNeoAgs) are generated per one adenocarcinoma patient and 95.5 pNeoAgs per one squamous cell carcinoma (SCC) patient (50). Selection pressures from a diverse TME further affect neoantigen presentation, allowing immune escape and the clinical progression in NSCLC (50, 51). In contrast, TAAs refer to antigen molecules present on both tumor and some normal cells at various stages of development, maturation, or activation. In general, tumor cells express higher TAA levels in comparison to most normal tissues. The most frequently recognized TAAs in NSCLC patients are Aurora kinase A, p53, HER2/neu, and NY-ESO-1. DESTINY-Lung02 demonstrated deep and durable antitumor responses of Trastuzumab deruxtecan in patients with mutated HER2 NSCLC (52).

Previous studies have shown that T cell responses against one or more of these most common TAAs have significantly improved relapse-free survival in NSCLC patients (46), which could be attributed to tumor cell apoptosis induced by cytotoxic T lymphocyte (CTL), a main subset of tumor-infiltrating lymphocytes (TILs) (53). The TILs count was considerably linked with improved survival as well as tumor grade, size, vascular invasion, and stage of tumor differentiation in NSCLC (54). Additionally, the presence of TILs in the early of NSCLC was linked with an improvement in survival rate and reduced risk of systemic recurrence (55).

Recognition of tumor cells by the synergistic action of tumor antigen-specific and innate immunity could induce either complete or partial tumor elimination, with some tumor cells becoming dormant. The tumor dormancy is described as the equilibrium phase during which tumor cell outgrowth is inhibited by the activity of T cells, IL-12 and IFN-γ, indicating the dominant contribution of adaptive immunity. The tumor cells could remain in the equilibrium phase through the rest of the subject’s life. Nevertheless, constant immune pressure, particularly on genetically unstable tumor cells, could lead to the emergence of tumor cells, which are no longer controllable by the immune system, leading to the tumor escape phase with clinically manifested disease. The tumor cells escape due to either a) the loss of TAA or TSA expression or by deviations in antigen processing or presentation by tumor cells, b) the loss of sensitivity of tumor cells to immune-induced cell-elimination mechanisms, including overexpression of antiapoptotic proteins BCL2 and STAT3, or c) immunosuppressive action of TME (56). The suppression of the immune system could be mediated within the TME by a) inhibitory action of tumor and stromal cell-surface expressed receptors (immune checkpoints such as PD-1/PD-L1, suppressive major histocompatibility molecules HLA-E/G, ectoenzymes CD39/73, thrombospondin receptor (CD47), IL-2 receptor β subunit (CD122), or CD155) or by secreted factors, such as interleukin-10 (IL-10), and transforming growth factor-β (TGF-β), or b) by tumor-mediated attraction of immunosuppressive cell populations such as Treg cells, myeloid-derived suppressor cells (MDSC) of either monocytic or polymorphonuclear lineage, tumor-associated macrophages (TAMs), or tumor-associated neutrophils (TANs). Such immune cells could be attracted by a disturbance in the expression of many cytokines, chemokines and adhesive molecules within the TME (57–59). Treg cells, as one of the most discussed regulatory/immunosuppressive populations within TME, can inhibit the proliferation and activity of tumor-specific Th cells and CTL and NK cells by secretion of transforming growth factor-β (TGF-β), or immunosuppressive cytokines IL-10 and IL-35 and by surface expression of IL-2 receptor subunit α (CD25) (60). Treg cells could also modulate the activity of dendritic cells (61). The count of Treg cells within the tumor (62) can be used as an independent biomarker to predict the survival rate in NSCLC patients (63). Nevertheless, the Treg cells within a tumor consist of very heterogeneous populations of either phenotypically stable suppressive/regulatory Tregs (forkhead transcription factor 3 - FoxP3⁺⁺) and phenotypically unstable cells (FoxP3^+/-), which could, under specific TME environment, switch their phenotype to effector Th1, Th2, Th17, or Tfh cells contributing to tumor elimination. In general, TME Tregs are highly activated, and express high levels of inhibitory immune checkpoint ligands such as PD-1, cytotoxic T lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene-3 (LAG-3) and T cell immunoreceptor with Ig and ITIM domains (TIGIT), and also co-stimulatory molecules such as CD27, ICOS, OX40, 41BB, and GITR (64, 65). From a diagnostic point of view, the composition of Treg cell subsets in the blood, tissue, and tumor is distinct (66) ( Figure 1 ).

TAM and tumor stromal cells have been suggested to release IL-10 and TGF-β. Furthermore, tumors and other cells present in the TME (dendritic cells, macrophages or endothelial cells) increase expression of the indoleamine-2,3-dioxygenase (IDO) – an immunosuppressive enzyme that cause a decrease in tryptophan amino acid concentration in extracellular environment (essentially required for the T cell function), induce the tumor-specific T cells exhaustion, and promote activation of Treg populations (67).

Spatial tumor heterogeneity occurs in most tumor types, including NSCLC, at both genetic and immunological levels, leading to a heterogeneous immune response toward individual cancer cells. This may be one of the main reasons for resistance development in cancer against immunotherapy, as it is very difficult to evaluate the actual status of immune response from available tumor tissue samples. For example, assessing the immune status in the center and periphery of the tumor as well as in distinct parts of the tumor can give completely different results. Furthermore, at different time points during various phases of therapy, temporal tumor heterogeneity plays an important role. Considering the findings mentioned above, small biopsies can give false results compared to surgically resected specimens.

NSCLC tumors can contain a high number of immune cells, including B cells, macrophages, Th cells, CTL, dendritic cells, NK cells, neutrophils, basophils, eosinophils, mast cells, myeloid-derived suppressor cells (MDSC), tumor-associated neutrophils (TANs), tumor-associated macrophages (TAMs) and other. 47% of all leukocytes, characterized by surface expression of CD45⁺, are represented by T cells, of which 26% are CD4⁺ T cells, followed by 22% of CD8⁺ T cells (68, 69). CD8⁺ T lymphocyte population present in NSCLC tumors has heterogeneous PD-1 expression levels. The most important CD8⁺ cells are those with high PD-1 expression since they show a high tumor recognition capacity which recruits the immune cells to tertiary lymphoid structures (TLS). The presence of PD-1 high CD8⁺ T cells was reported to be associated with favorable response and survival of NSCLC patients treated with PD-1 inhibitors (70). The percentage of B cells varies from 4.4 to 15.9% between studies (69, 71). This significant variance in B cell proportions has been suggested due to different tumor tissue sampling, while B cells reside primarily at the periphery of the tumor tissue, where they cluster with DCs and T cells (69). DCs represent 2.1% of all immune cells in NSCLC tumors of which plasmocytoid DC (pDC; HLA-DR⁺CD123⁺) represent 1.2% of all leukocytes, myeloid DC (mDC2; HLA-DR⁺CD11c⁺CD1c⁺) represent 0.8% and CD141⁺mDC (mDC1; HLA-DR⁺CD11c⁺CD141⁺) represent 0.1% of all leukocytes (72). mDCs present antigen to naïve T cells and contribute to CTL generation, pDCs produce type I IFNs, thus enhancing the antitumor immunity (73). Macrophages represent 4.7% and NK cells represent 4.5% of the immune cells in NSCL. Granulocytes are represented mainly by neutrophils (8.6%), followed by mast cells (1.4%), basophils (0.4%) and eosinophils (0.3%) (69).

Intratumor immune cells are able to form tertiary lymphoid structures (TLSs) placed at the periphery of tumor tissue (74). TLSs comprise a B cell area, neighboring a T cell area covering clusters of mature DCs and T cells (71, 75). Previous studies have established the TLSs as predictive biomarkers for the efficacy of immunotherapy in sarcoma, melanoma, and renal cell carcinoma (76, 77). Their critical role as a biomarker in NSCLC is under investigation (78, 79).

PD-1/PD-L1 signaling has an important function in immune tolerance due to its ability to suppress the immune response. Its impairment leads to the induction of autoimmunity, progression of chronic infections and tumor growth ( Figure 2 ).

PD-1 signaling affects both the adaptive and innate immune responses. Its expression was originally linked to the regulation of activity of T cells, B cells, DCs, macrophages, monocytes, and NK cells (80, 84) ( Figure 3 ), and is regulated by 10 transcription factors: eight activators (c-fos/AP-1, STAT3, STAT4, ISGF3, FoxO1, Notch, NFATc1, and NF-κB) and two inhibitors (Blimp-1 and T-bet) (85). Populations of developing thymocytes and resting naïve T cells show low basal-level expression of PD-1 (86), which has been linked to immune tolerance (87). During an acute infection or the initial stage of tumor development, the generated pathogen/tumor-derived antigens stimulate the maturation of T lymphocytes towards effector and memory subsets. Effector T cells target the infected/tumor cells and eradicate them. In contrast to acute T cell activation, permanent TCR stimulation promotes T cell exhaustion associated with upregulating surface co-inhibitory receptors such as PD-1, TIM-3 and LAG-3, which prevents the generation of autoreactive T cells.

PD-1 interacts with PD-1 ligands (PD-L1 and PD-L2). PD-L1 is expressed in T cells, B cells, macrophages, DCs, bone marrow-derived mast cells, and tumor cells (88) ( Figure 3 ). In contrast, PD-L2 expression is greatly limited, as it is mainly detected on activated macrophages, DCs, and some tumor cells (89). DCs have the potential to express PD‐L1 on their surface (90), and studies on other cancers, including colorectal cancer, have discovered that PD‐L1 positive DCs are better predictors of response to anti-PD‐L1 immunotherapy compared to PD‐L1⁺ tumor cells (91).

Cancer cells utilize the expression of PD-L1 as an “adaptive immune mechanism” to escape from the immune anti-tumor responses (80). 55% of NSCLC patients are positive for PD-L1 (92). The expression of PD-L1 is typically linked to the characteristics of the immune microenvironment enriched with CD8⁺ T cells, generation of Th1 cytokines, chemical factors, interferons and specific gene expression (93). PD-L1 expression, therefore, can be categorized as constitutive and inducible based on the extrinsic or intrinsic stimuli. The tumor cells exhibit constitutive PD-L1 expression mediated by dysregulation of tumor suppressor genes or oncogenic signaling pathways. For example, activation of the PI3K/AKT signaling pathway can enhance the expression of PD-L1 via mediating an increase in extrinsic signaling or a decrease in negative regulators, like PTEN. The downregulation of PTEN has been suggested to activate the PI3K/AKT which further accelerates the PD-L1 expression (94). The other signaling pathway involved in the upregulation of PD-L1 is the MAPK signaling pathway (95), and recently the JAK/STAT pathway has also been stated to produce PD-L1 expression in cancers. Additionally, fibroblast growth factor receptor 2 (FGFR2) signaling was also documented to substantially activate the JAK/STAT3 signaling pathway, which in turn caused enhanced expression of PD-L1. Altogether, PI3K/AKT, MAPK, and JAK/STAT3 pathways lead to overexpression of Myc, an oncogenic transcription factor, which binds to the PD-L1 promoter and enhances its expression. Increased Myc is observed in ∼70% of tumors (96, 97). Moreover, genetic, or pharmacological deactivation of Myc has been reported to attenuate the mRNA expression of PD-L1 levels and regenerate the anti-tumor responses in the TME (98). PD-L1 expression, on the other hand, can also be upregulated by DNA double-strand breaks which trigger the STAT signaling pathway via ataxia-telangiectasia mutated (ATM) and Rad3-related checkpoint kinase 1 (Chk1) (99). Other genetic drivers contributing to the high expression of PD-L1 are the mutations in ALK, the HIF1/2α and nuclear factor κB (NF-κB) (96). The tumor-immune cells microenvironment augments PD-L1 expression with pro-inflammatory cytokines, such as IFN-γ, tumor necrosis factor α (TNF-α), IL-10, interleukin-1 (IL-1) and interleukin-6 (IL-6) (100). IFN-γ regulates PD-L1 expression preferentially on immune cells, whereas PD-L1 expression on tumor cells is epigenetically regulated and associated with hypomethylation of PD-L1 promoter (101). In some NSCLC cases, PD-L1 amplification of the genomic region on chromosome 9p24.1 appeared and led to enhanced PD-L1 expression (102). The role of PD-L1 in cellular membrane of tumor cells is well known. Currently, the role of PD-L1 in nucleus of tumor cells were described. Hypoxia present in rapidly growing tumor tissue induce histone deacetylase 2 (HDAC2)-dependent deacetylation of membrane PD-L1, and promotes its nuclear translocation. Nuclear PD-L1 can evade conventional anti-PD1/PD-L1 therapies and can effectively promote tumorigenesis by regulation of angiogenesis by EGR1 regulation (103) ( Figure 2 ).

The level of PD-L1 is tightly connected with its stability. The fully glycosylated form of PD-L1 was estimated to have a half-life of 12 h, whereas non-glycosylated PD-L1 showed rapid proteolysis with a half-life of 4 h. Furthermore, decorated glycans were predicted to protect PD-L1 against proteasome degradation, and thus improving its molecular contacts with PD-1 on CD8⁺ T cells (104). In contrast, glycosylation of PD-1 has been a topic of discussion as some studies claim the highly glycosylated PD-1 protein is essentially required in PD-1/PD-L1 interaction (105) while others deciphered the distant location of glycosylation sites from the PD-L1 binding pocket on PD-1 and suggested no direct role of glycosylation in PD-1/PD-L1 interaction (106). Studies describing glycosylation level of PD-L1 in NSCLC tumor cells still need to be completed. We hypothesize that NSCLC tumor cells not only constitutively express PD-L1, but also exhibit a high level of glycosylation in comparison with other tumors.

Higher levels of both PD-L1 and IDO proteins in tumor tissue are independent negative prognostic factors for overall survival in resected NSCLC patients. Immunosuppressive microenvironment - exhausted T-cells together with a decrease in tryptophan, promotes tumor growth, increases the risk of progression and unfavorable prognosis (107).

CTLA-4 (cluster of differentiation 152, CD152) has been identified as the surface receptor of activated T cells and is constitutively expressed on Treg cells, where constitutive expression of CTLA-4 is controlled by FoxP3 (108, 109), and essentially contributes to the regulation of T cell homeostasis and self-tolerance. The current study of Hiroshi Saijo shows that anti-PD-1 antibody monotherapy might be less effective against large NSCLC due to the infiltration of Treg cells. Therefore, it might be appropriate for large NSCLC to select a treatment including an anti-CTLA-4 antibody, which can target Treg cells (110). In naïve T cells, CTLA-4 is primarily located in the cytoplasm since it is exposed to constitutive endocytosis from the plasma membrane, which results in the internalization of 90% CTLA-4 (111). The functional activity of the CTLA-4 receptor primarily includes competition with CD28 receptors for binding to B7 ligands (B7-1/CD80 and B7-2/CD86) on APC. CTLA-4 and CD28 facilitate opposing functions in T cell activation. In naïve T cells, stimulatory signals resulting from both TCR and CD28:B7 binding induce upregulation of CTLA-4 and its transfer to plasmatic membrane. CTLA-4 is known to interact with B7 ligands with higher affinity and avidity than CD28 (111, 112). The functional role of CTLA-4 is to inhibit the priming, activation and migration of T cells (111). In the case of Treg-expressed CTLA-4 interaction with APC-expressed CD80/CD86 induces the upregulation and secretion of indoleamine 2,3-dioxygenase by APC, contributing to the creation of an extrinsic immunosuppressive environment. CTLA-4/B7 function in different phases in comparison to the PD-1/PD-L1 axis, and thus synergistic inhibition of both signaling pathways offers a potential solution to reduce tumor resistance during ICI monotherapy (113, 114). Although CTLA4 expression was found in multiple tumor cells including NSCLC, its function in these cells is largely unknown and is correlated with poor prognosis (115). In NSCLC, CTLA-4 positivity was identified on tumor epithelial cells as well as on tumor stromal compartments with relatively homogenous distribution. The prognostic impact of CTLA-4 on tumor epithelial cells is favorable, since 5-year OS is 29% in CTLA-4 positive vs 19% in CTLA-4 negative primary tumors (115).

T cell immunoglobulin and mucin domain 3 (TIM-3) is a member of the TIM family of genes known for their expression on T cells in allergy and autoimmune diseases. It was initially identified as a specific cell surface marker of interferon (IFN-γ)-producing Th1 and CTL cells (116, 117). TIM-3 has been studied as a negative regulator which essentially contributes to immune tolerance. For instance, upregulated TIM-3 expression is noticed in exhausted CD8⁺ T cells in chronic infections and tumors, while its expression on CD4⁺ T cells is correlated with poor prognosis of NSCLC (118). Tumor cells could inhibit T cells by surface expression or by releasing several TIM-3 ligands such as PtdSer, HMGB1, or Galectin-9 (119). In their study, Zhuang et al., 2012 show TIM-3 positively stained on 92.9% of TILs and most infiltrated tumor-associated macrophages (TAMs) in lung cancer tissues, and 87% of NSCLC primary tumors were positive for TIM-3 (120). Another, more comprehensive study shows TIM-3 located on 25.3% of TILs in NSCLC tumors (121). There are currently a number of clinical trials investigating the use of TIM-3 blockers in NSCLC. Most of them aim for combination of two targets - TIM-3 and PD-1 (e.g. NCT06162572, NCT03708328, NCT04931654, source clinicaltrials.gov).

LAG-3 is an immune inhibitory receptor that is expressed on the surface of activated T cells, pDC, NK cells and B cells. Important roles of LAG-3 are inhibiting Th1 cell proliferation and reducing IL-2, IFN-γ, and TNF secretions (113). The presence of LAG-3 on TILs in tumor tissues has been linked to the poor prognosis of NSCLC (122, 123). LAG-3 is established to regulate the T cell-induced immune response in three ways: (i) directly by stopping the T cells activation and proliferation through negative regulation of T cells, (ii) by enhancing the inhibitory activities of Tregs, and (iii) by preventing the activation of T cells via regulation of APCs (124). LAG-3 ligands such as MHC II, Galectin-3, LSECtin, and FGL1 are expressed on DCs and some other cells. Importantly, the expression of FGL1 was confirmed on lung adenocarcinoma cells (125) which indicates another mechanism of tumor-mediated immune cell suppression. LAG3 is strongly associated with proliferation in NSCLC and significant LAG3 co-expression with other ICI targets suggests its plausible use in clinical trial selection and patient stratification for combination immunotherapy strategies (126). LAG-3 is often connected with poor prognosis (123).

In newly diagnosed NSCLC patients, PD-1, LAG-3 and TIM-3 were detected in TILs from 55%, 41.5%, and 25.3%, respectively. Primarily, PD-1 and LAG-3 were localized on T cell subsets while higher expression of TIM-3 was noticed in macrophages and NK cells. Co-expression of PD-1, LAG-3, and TIM-3 has been related to the activation of T cells, proliferation, and higher expression of proapoptotic markers (FAS/BIM) (121). Thus, combination therapy is a promising target, which is already present in many clinical studies (ClinicalTrials.gov).

OX40, a member of the TNFR family, is involved in the activation of anti-tumor immune response. Its expression is induced on the T cell surface after antigen recognition and coincides with the expression of OX40 ligand (OX40L) mainly on the activated B cells, APCs, NK cells and macrophage cells (113, 127). Activation of OX40 can enhance Th1/Th2-mediated antitumor immune response and, conversely, suppress the suppressive function of Treg cells (127). Enhanced OX40 signaling induced by OX40 agonist antibodies has been observed to augment the T cell-mediated anti-tumor immunity and support enhanced T cell infiltration into tumors, which further results in tumor regression and prolonged survival. Biological activity and the role of the soluble form of OX40 and OX40L in patients with a solid tumor are not well investigated, however, the elevated levels of OX40 and OX40L in serum can be linked with poor prognosis and may display the immune-exhausted status against lung adenocarcinoma (128). In NSCLC, high OX-40 expression in the tumor immune infiltrate is associated with a favorable prognosis. Its prognostic utility is independent of PD-L1 and other common markers of immune activation. High OX-40 expression potentially identifies a unique subgroup of NSCLC that may benefit from co-stimulation with OX-40 agonist antibodies and potentially enhance the efficacy of existing immune checkpoint therapies (129).

TIGIT is an inhibitory immune receptor from the poliovirus receptor (PVR) family of immunoglobulin proteins (113). The biological role of TIGIT involves interactions with the CD155 expressed on APCs or tumor cells to down-regulate the functions of NK cells and T cells (130). Like in other types of cancers, enhanced expression of TIGIT was observed in NSCLC and can be associated with increased levels of other immune inhibitory receptors, including PD-1, LAG-3, TIM-2. We can expect that dual inhibition of immune receptors can offer a favorable immunotherapy. As a mice experiment has already shown, the positive effect of dual blockade TIGIT and PD-1/PD-L1 resulted in complete tumor rejection and overall prolonged survival (131). In NSCLC, the prognostic value of TIGIT is still questionable, however, high TIGIT density correlates with advanced TNM score (132). Blocking TIGIT in NSCLC requires further investigation, but TIGIT will likely belong to promissing ICI targets in combination therapy.

Pembrolizumab (Keytruda, Merck Sharp & Dohme) is a humanized monoclonal IgG4 antibody against the immune checkpoint protein PD-1. Pembrolizumab was first approved in the United States (US) by the Food and Drug Administration (the FDA) in September, 2014 and later in Europe by the European Medicines Agency (EMA) in July, 2015. In, 2019, the FDA and EMA approved Keytruda as first-line treatment in metastatic NSCLC where tumor proportion score (TPS) is greater than 50% (82) and second-line treatment for patients with PD-L1 expression TPS exceeding 1%. For evaluation of the PD-L1 TPS in NSCLC tissue samples, the FDA approved a validation assay from Dako/Agilent for qualitative assessment of PD-L1 protein expression in formalin-fixed paraffin-embedded (FFPE) tissue samples of NSCLC patients. This assay utilizes monoclonal mouse anti-PD-L1 antibody clone 22C3, designed for immunohistochemical (IHC) analysis. In, 2018, the FDA approved Keytruda in combination with pemetrexed and platinum chemotherapy as the first-line treatment of patients with metastatic nonsquamous NSCLC, with no EGFR or ALK genomic tumor aberrations, and in the same year, in combination with carboplatin and either paclitaxel or paclitaxel protein-bound, as first-line treatment of patients with metastatic squamous NSCLC ( Table 2 ).

Nivolumab (Opdivo, Bristol-Myers Squibb Company), IgG4 anti-PD-1 monoclonal antibody was approved by the FDA and EMA (2015) for treatment of both squamous and non-squamous NSCLC, and as second-line therapy in patients with metastatic NSCLC (153). In, 2020, nivolumab was approved in combination with Ipilimumab as first-line treatment for patients with metastatic NSCLC whose tumor cells express PD-L1≥1%. On March 4, 2022, the FDA and EMA approved nivolumab with platinum-doublet chemotherapy in adult patients with resectable NSCLC in the neoadjuvant setting ( Table 2 ).

Cemiplimab (Libtayo, Regeneron Pharmaceuticals, Inc., and Sanofi) is a monoclonal IgG4 antibody against PD-1. The FDA and EMA approved Libtayo monotherapy (if PD-L1 expression is ≥ 50% of tumor cells) or in combination with chemotherapy (PD-L1 1-49%) as first-line therapy for adult NSCLC patients who express PD-L1 (in ≥50% tumor cells), with no EGFR, ALK or ROS1 aberrations, and who have locally advanced or metastatic NSCLC and are not candidates for definitive chemoradiation therapy.

Unfortunately, PD-1 inhibitor treatment can cause severe side effects of immune origin - immune-related adverse events (irAEs), such as hepatitis, colitis, and skin disorders. However, irAEs have been reported to be associated with good clinical outcomes while causing treatment discontinuation and death (154–157).

Atezolizumab (Tecentriq, Genentech, Inc.) is a monoclonal human IgG1 antibody. In, 2016, the FDA approved Atezolizumab as a therapy for patients with metastatic NSCLC who have experienced disease progression during or following platinum-containing chemotherapy, and if their tumor has EGFR or ALK gene abnormalities. Tecentriq is currently used in combination with Avastin (bevacizumab), paclitaxel and carboplatin (chemotherapy), for the initial (first-line) treatment of individuals with metastatic NSCLC with no EGFR or ALK genomic tumor aberrations. Additionally, it is used as monotherapy for first-line treatment of adult patients with metastatic NSCLC whose tumors have a PD-L1 expression in ≥ 50% tumor cells or ≥ 10% tumor-infiltrating immune cells and lacks EGFR or ALK genomic tumor aberrations. In, 2021, the FDA approved Tencentriq as an adjuvant treatment for patients with stage II to IIIA NSCLC whose tumors have PD-L1 expression on ≥ 1% of tumor cells, as determined by an FDA-approved VENTANA PD-L1 (SP263) assay (Ventana Medical Systems, Inc.), following resection and platinum-based chemotherapy.

Durvalumab (Imfinzi, AstraZeneca Inc.) is a monoclonal anti-PD-L1 IgG1 kappa antibody that was approved in, 2018 by the FDA and EMA for treating patients with stage III NSCLC whose tumors cannot be surgically removed and whose cancer has not progressed after chemotherapy and radiation treatment. Nowadays, 141 clinical trials are testing Durvulumab in other stages of NSCLC individually or in combination with other drugs (source:ClinicalTrials.gov) (158).

Avelumab (Bavencio., EMD Serono, Inc. and Pfizer, Inc.) is a human monoclonal IgG1 antibody, which has been approved for the treatment of metastatic Merkel cell carcinoma and is used in therapy against advanced urothelial carcinoma. Currently, 10 ongoing clinical trials are testing Avelumab in NSCLC patients (source: ClinicalTrials.gov). To evaluate PD-L1, Dako PD-L1 IHC 22C3 pharmDx assay and Dako PD-L1 IHC 73-10 assay are used as quantitative evaluators (159).

The effectiveness of anti-PD-1/PD-L1 treatment is tightly connected with glycosylation of PD-1/PD-L1, which is crucial for proper PD-1 and PD-L1 binding. Therapeutic antibodies such as Avelumab, Atezolizumab and Durvalumab recognize the glycosylated form of PD-L1 in contrast to diagnostic antibodies which favor the non-glycosylated form of PD-L1 (160). Most commercially available antibodies are produced to recognize antigens or recombinant proteins that are expressed in bacterial or other hosts; however, this does not provide the proper glycosylation present in mammalian cells. Removing the glycosylation moieties may help to overcome the steric hindrance and enable more accurate stratification of patients. Lee et al. have demonstrated that the diagnostic antibody clone -8, which is widely used to detect PD-L1 expression and to evaluate formalin-fixed paraffin-embedded (FFPE) tissue samples, has limited recognition of heavily glycosylated PD-L1 forms, which may result in false negative scoring of patients (161). PNGase-treated lung cells showed increased immunofluorescence signal (detected by ELISA assay) compared to untreated samples. In 14% of FFPE tissue samples, PD-L1 TPS was increased from 1% to more than 49% after deglycosylation. This may indicate that certain evaluations using IHC 28-8 may bring false-negative results. Thus, selecting the appropriate PD-L1 diagnostic strategy may affect the therapeutic strategy for the patients and their chances of survival (161).

Ipilimumab (Yervoy, Bristol-Myers Squibb Company) is a human CTLA-4-blocking antibody approved by the FDA and EMA in, 2020. It is used in combination with nivolumab and 2 cycles of platinum-based chemotherapy for first-line treatment of metastatic NSCLC in adults whose tumors express PD-L1 (≥1%) and have no EGFR and ALK mutations (ClinicalTrials.gov Identifier: NCT02658890).

Tremelimumab (Imjudo, AstraZeneca, Inc.) is a human monoclonal antibody, approved by the FDA and EMA, that blocks the activity of CTLA-4 and thus activates the anti-tumor immune response. In September, 2021, AstraZeneca showed that combinatory treatment of Imfinzi (Durvalumab) and Tremelimumab when added to platinum-based chemotherapy, improved overall survival (OS) and progression-free survival compared to chemotherapy alone in first-line treatment of patients with metastatic NSCLC (162).