Immunotherapy-based combination strategies for treatment of EGFR-TKI-resistant non-small-cell lung cancer

Lung cancer is one of the most widely studied malignancies worldwide [1] and can be classified as non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) on the basis of its histopathological character. NSCLC accounts for approximately 80% of all lung cancer cases [2]. Around 75% of patients are found to have invasion and distant metastasis due to the lack of effective early screening and diagnosis and the 5-year survival rate is less than 15% [3]. The management of advanced NSCLC has recently attracted extensive attention and EGFR-tyrosine kinase inhibitors (TKIs) are considered the standard first-line therapy for patients with NSCLC with EGFR mutations [4,5], in light of significantly improved objective response rates (ORR; 67.0 vs 40.8%) and median progression-free survival (PFS; 10.9 months vs 7.4 months) compared with traditional chemotherapy [6]. Unfortunately, the majority of patients will exhibit disease progression within one year of EGFR-TKI treatment [7]. A meta-analysis found that osimertinib noticeably improved PFS, ORR and disease control rate (DCR), especially in T790M-positive NSCLC compared with other EGFR-TKIs or chemotherapy. Despite the dramatic response to osimertinib, most T790M-positive cases develop resistance within 9–14 months [8]. EGFR-TKI resistance remains a crucial unmet need and the optimal strategy is not yet clearly defined after treatment failure of EGFR-TKIs. Nivolumab and pembrolizumab, a PD-1 antibody, have been authorized for advanced NSCLC for their superior efficacy and tolerable toxicity over traditional chemotherapy by the US FDA [9–11]. However, only about 20% of patients with NSCLC respond to PD-1/PD-L1 monotherapy [12]. The expression of PD-L1 on tumors cells can be upregulated by EGFR activation [13]. EGFR-TKIs were also found to have the potential to affect the tumor microenvironment (TME) [14–16], providing a theoretical basis for the application of immune checkpoint inhibitors (ICIs) in NSCLC with poor efficacy of targeted therapy coupled with upregulated PD-L1 expression. Multiple intensive clinical studies are currently underway to improve the clinical benefit for such patients. The current review provides insights into the recent development of a combination strategy based on immunotherapy for EGFR-TKI-resistant NSCLC.

EGFR mutation & PD-L1 expression

Researchers have examined the relationship between EGFR mutation status and the expression of PD-L1 with controversial results. Multiple preclinical studies suggest that PD-L1-positivity in patients with EGFR mutant NSCLC is higher than in those with wild-type EGFR mutations and PD-1 antibody prolonged survival in a nude mouse model [17–19]. Conversely, a negative or insignificant correlation was reported in this setting and PD-L1 was highly expressed in EGFR-wild-type NSCLC with a higher sensitivity to immunotherapy [20–23]. A variety of factors, such as immunohistochemistry (IHC) scoring criteria for PD-L1 in different clinical studies, tumor heterogeneity, baseline characteristics of patients as well as tumor stage and previous treatment before detection may explain such differences between experimental studies [24]. Much work remains to identify the heterogeneity between them. Recent studies indicate that cell-intrinsic EGFR signaling through the induction of T-cell apoptosis and promotion of immune escape in EGFR-mutated NSCLC directly or indirectly upregulated the PD-L1 expression in tumor cells, thus enhancing sensitivity to PD-1/PD-L1 blockade [25]. This regulatory mechanism was first found by Akbay and colleagues in 2013 [26] and has recently attracted attention from scholars. Downregulation of PI3K, AKT and mTOR were confirmed to have the potential to reduce the PD-L1 expression in recent research [27,28], which suggests that EGFR mutations may participate in the regulation of PD-L1 expression by activating the PI3K/AKT/mTOR signaling pathway [29]. Furthermore, a study showed that EGF stimulation, exon 19 deletions and L858R mutations also led to the activation of EGFR and induced PD-L1 expression, which may occur through the mechanism of the p-ERK1/2/p-c-Jun pathway [30]. NF-κB, an important downstream pathway of EGFR activation, can be activated by the tumor suppressor gene and cytokines produced by the inflammatory microenvironment. A study found that subunits of NF-κB could bind to the PD-L1 promoter in NSCLC cells and directly regulate PD-L1 transcription in the form of a complex [31]. In addition, the expression of PD-L1 decreased with silencing of the subunits of NF-κB in tumor cell lines with EGFR mutation, proving that this type of regulation requires the involvement of NF-κB [32]. That the IL-6/JAK/STAT3 pathway participated in the activation of the EGFR signaling pathway had been previously revealed [33]. STAT3, the important regulatory molecule in the downstream signaling pathway of EGFR, can bind to the promoter region of PD-L1 and promote its transcription. Further activation of the EGFR pathway can stimulate the secretion of IL-6 and then activate the JAK/STAT3 signaling pathway [34,35], indicating that the EGFR pathway can upregulate PD-L1 expression through the mechanism of the IL-6/JAK/STAT3 pathway. This hypothesis was subsequently verified in the research by Zhang and colleagues [36]. The MEK/ERK signaling pathway in the MAPK pathway is usually activated by abnormal amplification or mutation of the RTK, and the MEK/ERK pathway exerts a regulatory role in PD-L1 expression in EGFR-mutated NSCLC cells [37,38]. Ota et al. further revealed that both the EML4-ALK fusion gene and mutant EGFR upregulated PD-L1 expression by activating the PI3K-AKT and MEK-ERK signaling pathways in NSCLC [37]. Knowledge of the regulatory mechanisms contributes to the development of innovative therapeutic options to overcome the limited clinical response to EGFR-TKIs. However, the specific molecular mechanism has not been firmly established and additional investigation is required. Figure 1 described the possible mechanisms by which EGFR regulates the expression of PD-L1.

Correlation between PD-L1 expression & EGFR-TKIs

The epidemiological correlation between PD-L1 expression and activation of EGFR in NSCLC has been observed in many studies. However, the relationship between PD-L1 expression and EGFR-TKIs and the potential mechanism of resistance to this treatment requires more research. Immunophenotypes have the potential to influence the effectiveness of targeted therapy. Recent studies have assessed the relationship between PD-L1 expression and sensitivity to EGFR-TKIs and found that high expression of PD-L1 exhibited poor clinical efficacy characterized by shortened ORR (35.7 vs 67.3%, p = 0.002) and PFS (3.8 vs 9.5 months, p < 0.001) respectively, especially for patients with NSCLC with primary resistance [39], To better explain this suboptimal effect, Lin et al. explored the impact of TME on EGFR-TKIs and found that patients who completed EGFR-TKI therapy exhibited increased infiltration of CD8⁺ T cells and dendritic cells (DCs) and decreased Foxp3+ Treg cells in the early stage. However, these changes disappeared at the late stage of treatment, and myeloid-derived suppressor cells (MDSCs) continued to increase throughout treatment [40–42]. Notably, Isomoto et al. retrospectively analyzed the TME of 138 patients with EGFR-mutated NSCLC with EGFR-TKI resistance by non-T790M and found lower tumor mutational burden (TMB) FOXP3+ Tregs and higher PD-L1 expression level as well as CD8⁺ tumor-infiltrating lymphocytes (TILs) densities, which generally confer a promising TME for ICI treatment [16]. Gainor et al. also observed a 20% increase in PD-L1 expression in NSCLC samples with sensitized EGFR mutation [43]. A similar result was found in erlotinib-resistant individuals; the expression of PD-L1 was consistently at a high level [44]. Inomata et al. found that the proportion of PD-L1 strong positive in T790M-negative patients was greater than in those with positive-T790M (p = 0.0565) and those who developed acquired resistance mediated by non-T790M mechanism also showed a superior PFS during subsequent immunotherapy than those with positive-T790M (16.5 vs 8.6 months, p = 0.001) [45]. A subsequent retrospective study also reported a better PFS in patients with a high expression of PD-L1 after treatment with EGFR-TKIs (7.1 vs.1.7 months, p = 0.0033) [46]. Such dynamic changes associated with EGFR-TKI agents indicate that a subset of patients with high-PD-L1 NSCLC who failed treatment with EGFR-TKIs might be more suitable for immunotherapy. Notably, a higher positive rate (TPS: ≥1%) of PD-L1 in a subset of patients with NSCLC harboring EGFR activation was also demonstrated to be associated with primary resistance to EGFR-TKIs when compared with those with lower PD-L1 expression (45.5 vs 12.3%, p < 0.001) by the mechanism upregulating the expression of YAP1, the transforming growth factor/Drosophila mothers against decapentaplegic (TGF-Smad) signaling pathway and continuous activation of the extracellular regulated ERK pathway by the PD-L1/BAG-1 axis [13,40,47–49]. EGFR-TKIs were also found to downregulate the PD-L1 expression of tumor cells through the IL-6/JAK/STAT3 pathway and Hippo/YAP signaling pathway [36,50]. Such studies suggest that in addition to targeting the EGFR signaling pathway to attenuate tumor proliferation and improve survival, downregulation of PD-L1 expression and consequent stimulation of anti-tumor immune effects also have the potential to overcome EGFR-TKI resistance in NSCLC with strong PD-L1 expression, especially for those negative for T790M. Knowledge of identified primary or acquired resistance mechanisms continues to accumulate, motivating the development of diverse new therapies to overcome EGFR-TKI resistance.

Immunotherapy monotherapy

Research on the curative efficacy of immunotherapy in EGFR-TKI-resistant NSCLC has yielded controversial results and the effect of ICIs in patients who failed EGFR-TKIs has not been clarified. Regardless of the dramatic success of immunotherapies in some patients with lung adenocarcinoma, patients with EGFR-mutated NSCLC continue to be excluded from some critical phase III randomized controlled trials (RCTs) of first-line immunotherapy due to suboptimal response [51,52]. The activity of immunotherapy in patients with PD-L1-positive EGFR-mutant NSCLC has been described in previous clinical trials. The results from the subgroup of the Checkmate057 trial, which analyzed 82 patients with EGFR-mutated NSCLC who completed nivolumab after the failure of EGFR-TKIs, indicated no dramatic response to nivolumab compared with docetaxel (HR: 1.18, 95% CI: 0.69–2.00) in terms of PFS and overall survival (OS) benefit [53]. The KEYNOTE010 trial aimed to evaluate second-line treatment with pembrolizumab and docetaxel for PD-L1 positive NSCLC (TPS ≥1%). In a subgroup of 86 advanced patients previously treated with EGFR-TKIs, no significant OS benefit was demonstrated compared with the docetaxel (HR: 0.88, 95% CI: 0.45–1.70) [54]. Similarly, the latest results of the WJOG8515L trial presented at the American Society of Clinical Oncology (ASCO) conference in 2021 indicated that the therapeutic effect of nivolumab was much inferior to chemotherapy (ORR: 9.6 vs 36%; DCR: 38.4 vs 76%; median PFS [mPFS]: 1.7 months vs 5.6 months) regardless of PD-L1 expression [55]. Consistently with previous results from the recent POPLAR and OAK trials, patients with EGFR-mutated NSCLC with previous EGFR-TKI treatment derived no OS benefit from PD-1 inhibitors. It is crucial to underly the mechanisms of this unfavorable effectiveness with immunotherapy in the EGFR-mutated subset. TME, consisting of many indicators such as PD-L1 expression, TMB and Tregs has been revealed to influence the response of immunotherapy in patients with EGFR-positive NSCLC [40,56]. Preliminary studies showed that EGFR-signaling cancer cells may overexpress immune suppressors, such as TGF-β and IL-10 cytokines, which may directly restrain the capacity of natural killer (NK) cells, further inhibit the recruitment of effector CD8⁺ T cells and facilitate Treg infiltration, finally leading to the proliferation of tumor cells [57,58]. High levels of PD-L1 expression and TMB are considered promising predictive biomarkers for immunological therapy efficacy based on recent studies [59,60]. EGFR mutations were strongly associated with T-cell depletion and dormancy immunophenotypes (high expression of CD3, low expression of Ki67 and granase B) compared with wild-type EGFR, which may result in impaired TIL-mediated anti-tumor effects [61].

Evidence also indicates that CD73 is overexpressed in EGFR-mutated NSCLC, which can split ATP into adenosine and act on A2a/A2b receptors to exert an immunosuppressive effect. It can also negatively regulate the function of DCs and NK cells, polarize macrophages to M2 and inhibit the anti-tumor response mediated by T cells, thus enhancing immune escape, metastasis and the proliferation of tumors and activating immunosuppressive effect by activating Treg cells and MDSCs [62,63]. In addition, Gainor et al. found only 11.6% of EGFR-TKI-progressed tumors had concurrent high levels of PD-L1 expression and CD8⁺ TILs [43]. The little available evidence may, to some extent, explain the discouraging clinical response to ICIs among NSCLC patients with sensitizing EGFR mutations [64,65]. Despite these unfavorable subgroup analyses in RCTs, modest but promising efficacy was initially verified in some preclinical studies. The ATLANTIC study explored the efficacy and safety of durvalumab as third-line treatment in EGFR-positive NSCLC pre-treated with EGFR-TKIs (PD-L1 expression ≥25%), and encouraging clinical data were observed in terms of the ORR (12.2%, 95% CI: 5.7–21.8) [66]. Jia et al. investigated dynamic TME responding to EGFR-TKIs using a murine model and the results showed that tumor cells with the EGFR mutation responded to EGFR-TKIs as early as the first day, accompanied by significant tumor shrinkage. However, no significant change in the anti-tumor effect was observed compared with the untreated group after long-term EGFR-TKI infiltration of CD8⁺ T cells and macrophages were significantly lower in EGFR-TKI-resistant cells [25].

EGFR-mutated NSCLC represents a heterogeneous subgroup of patients. Recent studies have found that different subtypes of EGFR mutations derive a differential clinical advantage from ICIs [67]. Patients with EGFR L858R mutation showed a high TMB and significantly improved ORR compared with those with EGFR exon 19 deletion [68]. Insertions in exon 20 (Ex20ins) of EGFR were also related to greater benefit with comparable PFS and ORR to immunotherapy compared with those with common EGFR and HER2 mutations (PFS: 4.0 vs 1.9 vs 1.9 months; ORR: 19 vs 0 vs 9%; p < 0.05), which may be partially explained by relatively higher PD-L1 expression in individuals with Ex20ins mutation [69]. EGFR mutated individuals usually establish an immunosuppressive noninflammatory TME leading to resistance to immunotherapy. Nevertheless, patients with EGFR mutations are not forbidden from immunotherapy; highly selected patients with high expression of immunological biomarkers (PD-L1 and TMB) are more likely to obtain benefits from immunotherapy. The discouraging preliminary clinical results emerging from early-phase trials warrant additional long-term follow-up and further investigation requires carefully estimating the effects and safety profile of ICIs in EGFR-TKI-resistant NSCLC, including specific duration of treatment (DOR), and subtype of EGFR mutations. In the near future, outcomes from numerous ongoing clinical trials evaluating immunotherapy for EGFR-TKI-resistant populations will be reported.

EGFR-TKI–immunotherapy combination treatment

Targeted therapies may reduce tumor-mediated immunosuppression by eliminating the production of tumorigenic inflammation and suppressing immunosuppressive cell types indicating a synergistic effect with immunotherapies that enhances T-cell immune response [70,71]. Preclinical studies also found that EGFR-TKIs were indirectly capable of inhibiting the function of Treg cells through the EGFR/GSK-3/Foxp3 axis and strengthening the anti-tumor effect by inducing tumor-specific T cells to release inflammatory cytokines such as IFN-γ, thereby significantly increasing peripheral NK cells and IFN-γ, and the level of IL-6 decreased after EGFR-TKIs [30,72]. Short-term exposure to low-dose EGFR-TKIs (e.g., erlotinib) can make tumor cells tend toward an epithelial phenotype and enhance the recognition and lysis of tumors modulated by antigen-specific T cells and NK cells [73]. Recent studies also showed that activation of the EGFR pathway may inhibit immune responses in murine melanoma models by activating Treg cells or decreasing T-cell chemotactic levels [40], while gefitinib is capable of reversing the immunosuppression effect by upregulating the major receptors (NKG2D) expression and enhancing immune recognition of NK cells, especially for those resistant to common mutations (EGFR L858R + T790M) [74]. The indirect and remarkable enhancement of anti-tumor immunity after EGFR-TKIs reverses immunosuppression in the TME and makes it possible for combinations of targeted therapy and immunotherapies. Immunotherapy combined with EGFR-TKIs is under scrutiny in current clinical studies, and the effectiveness and feasibility of this exploratory regime remain to be confirmed. The subgroup of KEYNOTE-012 analysis indicated that the combination of pembrolizumab and erlotinib provided better clinical benefits for EGFR-TKI-naive NSCLC individuals regarding moderate ORR (41.7 vs 14.3%) and prolonged mPFS (19.5 months vs 1.4 months) without dose-limiting toxicity or grade 5 adverse events (AEs) compared with those treated with pembrolizumab and gefitinib [75]. However, the efficacy of the combination therapy requires clarification with further phase II clinical trial data due to lack of an adequate sample population.

Recently, the simultaneous introduction of various strategy profiles into standard practice has led to clinical challenges regarding optimal treatment sequencing and unintended serious toxicities. The CAURAL trial compared osimertinib with durvalumab in EGFR-TKI pretreated participants with T790M positive mutation. The results showed no historically improved ORR (64 vs 80%), DOR (17.5 vs 21.4 months) or DCR (93 vs 100%) in the combined treatment group compared with those treated with osimertinib monotherapy and the trial was terminated early due to the high incidence of interstitial lung disease (38%) [76]. Several concerns have been raised regarding the potential high incidence of AEs associated with immunotherapy combined with EGFR-TKIs in patients who progressed on EGFR-TKIs. A meta-analysis of PD-1/PD-L1 blockade in combination with EGFR-TKIs showed an ORR of 55% (95% CI: 28–83%), the incidence of grade 3–5 drug-related AEs (DRAEs) was 41% (95% CI: 20–62%), of which the most common incidence was pneumonia at 3% (95% CI: 1–7%), and drug-related mortality was 1% (95% CI: 3–6%) [77]. Similar safety profiles were also reported in 13 patients with EGFR-TKI-naive NSCLC with positive EGFR included in the CheckMate370 (NCT02574078) phase II/III RCT of nivolumab and crizotinib, showing that only five cases (38%) had achieved partial response (PR), and the strategy was ultimately discontinued due to severe hepatotoxicity (38%) [78]. Consistently with previous reports, nivolumab in combination with erlotinib was associated with 19% grade 3/4 toxicity [79] and the incidence of grade 3/4 elevation of liver enzymes in combination with duvalizumab was high (40–70%). Moreover, 39% of the patients who received atezolizumab and erlotinib experienced grade 3/4 immune-related AEs (irAEs) [71]. Despite a promising ORR (70%) for EGFR-TKI-naive patients from the combination of durvalumab and osimertinib in the TATTON trial, more attention should be paid to the high incidence of grade 3/4 irAEs (34% of the enrolled patients developed interstitial pneumonia) [80]. Results from the current study showed that 15% (6/41) of all patients receiving sequential osimertinib with PD-1/PD-L1 inhibitors developed severe irAEs. In contrast, no severe irAEs were observed in those who received osimertinib sequentially with PD-1/PD-L1 inhibitors (0/29) or PD-1/PD-L1 inhibitors sequentially with other EGFR-TKIs (afatinib or erlotinib, 0/27) [81]. The results highlight the urgent need for careful consideration in the selection of initial treatment for patients with NSCLC, particularly for osimertinib in patients who have recently been treated with PD-1/PD-L1 inhibitors. Most studies suggest that the joint application of PD-1/PD-L1 inhibitors and EGFR-TKIs can improve clinical ORR and prolong PFS. However, there were also many serious AEs, especially interstitial pneumonia. Given the challenge of the potential toxicity of combination therapy, which resulted in the early closure of some studies (and the results from these trials are preliminary and unfavorable), the optimal sequence, time interval and dosage of administration remain unclear. At present, multiple phase I clinical trials are intensively investigating the efficacy and safety of this combined therapy, including NCT01454102, NCT02039674 and NCT02013219, which are underway to estimate the combination of EGFR-TKIs and nivolumab, pembrolizumab and atezolizumab, respectively. Table 1 shows relevant clinical trials that assess the combination of anti-PD-1/PD-L1 therapy and EGFR-TKIs in patients with NSCLC resistant to EGFR-TKIs.

Chemotherapy-immunotherapy combination treatment

The standard first-line treatment of pembrolizumab in combination with platinum-based chemotherapy is currently approved for patients with nonsquamous cell carcinoma without EGFR/AKL mutations by the US FDA [82]. Numerous studies have suggested that patients with NSCLC with actionable mutations have a poor response to ICIs [43,83], and immunotherapy in combination with chemotherapy is more applicable to NSCLC patients with PD-L1 expression ≥50% [84–86]. Nevertheless, research on cytotoxic chemotherapeutic agents that may affect the immune microenvironment in terms of PD-L1 expression, TMB and activity of anti-tumor immunity has been elucidated [87]. Studies have shown that chemotherapeutic agents, such as gemcitabine, paclitaxel and carboplatin, can induce the upregulation of PD-L1 by activating the NF-κB signaling pathway [88]. In addition, the immunomodulatory property of chemotherapy can expose or release neoantigen molecules in tumor cells and transform them into immunogenic cells [89]. This process eventually results in a series of immune reactions, such as the activating of DCs through toll-like receptors (TLRs), the enhancement of effector T cells, reduction of Treg and MDSCs, activation of mature DC cells and recruitment of related antigen-presenting cells to kill tumors and facilitate tumor immune response [90–92]. Remarkably, chemotherapy contributes to significant activation of anti-tumor immunity and an ICI-based strategy may extend this synergistic effect [93]. A pooled analysis of three RCTs (KEYNOTE-021, KEYNOTE-189 and KEYNOTE-407) showed that combined pembrolizumab and standard-care chemotherapy considerably improved OS (HR: 0.63, 95% CI: 0.50–0.79) and PFS (HR: 0.68; 95% CI: 0.56–0.83) over chemotherapy alone with manageable AEs [94]. Additionally, exposure to single-agent chemotherapy after treatment with EGFR-TKIs yielded improved response rates compared with pre-immunotherapy in patients with advanced NSCLC [95]. It is thus hypothesized that chemotherapy in tandem with immunotherapy may improve the anti-tumor response. The preliminary results of a single-arm, multicenter, phase II study of toripalimab and carboplatin/pemetrexed in EGFR-positive patients who failed prior EGFR-TKIs were presented at the CSCO conference in 2019. A total of 40 eligible patients were treated with toripalimab (a PD-1 inhibitor) and pemetrexed/carboplatin for four or six cycles (every 3 weeks), followed by a maintenance combination regimen until disease progression. The encouraging results showed that 20 patients achieved PR (50%), 15 patients achieved stable disease (SD; 37.5%), the ORR was 50%, DCR reached 87.5%, median PFS was 7.0 months and the mPFS of PD-L1 positive patients reached 8.2 months. The results indicated that the combination regimen was superior to chemotherapy alone both in PFS and ORR in the PD-L1 positive subset [96]. Subsequently, the clinical data from the EGFR-positive (n = 124) subgroup of the IMPOWER150 trial showed that atezolizumab plus bevacizumab plus carboplatin plus chemotherapy (ABCP) had historically increased median OS compared with bevacizumab plus carboplatin plus paclitaxel (BCP; 29.4 months vs 18.1 months, HR: 0.6, 95% CI: 0.31–1.14), and the group with baseline liver metastasis also achieved parallel results [97]. Successful results from research to date support the potential of the ABCP regimen as an innovative therapeutic option for post-line treatment after failure on EGFR-TKIs.

The application of consolidation therapy with durvalumab after concurrent chemoradiotherapy (CRT) in unresectable stage III NSCLC remains controversial. A retrospective analysis explored the PFS with durvalumab in this setting and no improvements were observed compared with those who completed CRT alone. Patients experienced a high frequency of severe irAEs, numerically similar to the subset of analysis from the PACIFIC trial [98]. To extend survival benefit and attenuate the occurrence of resistance to targeted therapy, more and more clinical trials are in the process of investigating the feasibility of chemotherapy and immunotherapy combination treatment [99]. The ongoing clinical trials KEYNOTE-789 (NCT03515837), CheckMate722 (NCT02864251) and Orient-31 (NCT03802240) will explore the response to ICIs plus traditional chemotherapy after progression on EGFR-TKIs. Table 2 summarizes relevant clinical trials that evaluate the combination of ICIs and chemotherapy in patients with NSCLC after EGFR-TKI treatment failure.

Antiangiogenic-immunotherapy combination treatment

VEGF, a highly specific provascular endothelial cell growth factor, promotes the movement, proliferation and division of vascular endothelial cells [100], increases vascular permeability and has proangiogenic activity [101]. It can inhibit the maturation of DCs and the response of effector T cells and increases the infiltration of MDSCs, Tregs and monocytes/macrophages, contributing to the infiltration of various immunosuppressive cells into tumor tissues and exerting an immunosuppressive role, which may be stimulated by EGFR/ALK signaling [102]. The relationship between VEGF and its receptors and the immune system is complex. Antiangiogenic drugs can activate the immune system and immunotherapy can also combat angiogenesis [103–105], providing a novel and powerful rationale for the combination of anti-VEGF and ICI therapies. Previous in vitro experiments revealed that a combination of low-dose apatinib and PD-L1 blockade showed an obvious anti-tumor effect in a syngeneic lung cancer mouse model [106]. Wu et al. [107] also showed that anti-PD-1 in combination with endostar (antivascular drug) can remarkably attenuate tumor growth by inhibiting the accumulation of MDSCs in a mice model. Interestingly, recent evidence suggested that the combined strategy of PD-1-based immunotherapy and antiangiogenic drugs (e.g., endostar) synergistically stimulate the immune response of tumor cells and dramatically enhance the anti-tumor effect by suppressing the common PI3KAKT/mTOR pathway in an LLC mouse model [107]. These preclinical studies corroborated the complementary therapeutic effects of this combination. Additionally, the synergistic benefit of the combination of antiangiogenic therapy with ICIs has been reported in an observational study [108]. As early as the ASCO conference, 2019, results on the combination of camrelizumab and apatinib have been announced with a 30.8% ORR and 5.9 months median PFS [109]. A recent study of 69 patients with NSCLC receiving anti-PD-L1 agents (nivolumab or pembrolizumab) in combination with antiangiogenic therapy are presented and the short-term outcomes include a moderate ORR of 31.9% (95% CI: 20.6–43.2%), and a median PFS of 8.37 months (95% CI: 1.0–9.5) [110]. Albina et al. [111] reported that the PFS of a female patient resistant to erlotinib and osimertinib was significantly improved after combination therapy consisting of carboplatin, paclitaxel, atezolizumab and bevacizumab. However, it is unclear whether a combination of anti-PD-1 antibody and antiangiogenic therapy can bring more survival benefits for the EGFR-TKI-resistant NSCLC population. A subsequent clinical case reported a stage IA lung adenocarcinoma patient with exon 19 deletion mutations after first-line chemotherapy failure who then received gefitinib treatment. After the failure of gefitinib, the patient received immunotherapy in combination with antiangiogenic agents (sintilimab + bevacizumab). Radiological imaging subsequently demonstrated a reduced adrenal metastatic lesion 1 month later, and the PFS was 6 months [112]. The available evidence suggests that a combination of antiangiogenic therapy and immunotherapy displays promising efficacy in EGFR-TKI-resistant NSCLC individuals, particularly in those with high VEGF levels, and more clinical trials are required to confirm these synergistic effects. Table 3 shows relevant clinical trials assessing combined treatment with ICIs and antiangiogenic agents in patients with NSCLC.

Combination CTLA-4 inhibitor-immunotherapy treatment

PD-1, an immune suppressor transmembrane protein, is found to be not only widely expressed in activated T-lymphocytes, B-lymphocytes, NK cells, monocytes and other immune cells, but also in some tumor cell lines or tumor cell surfaces [113]. It can also exert an important role in promoting tumor immune escape and tumor cell growth by combining with its ligand, PD-L1 [114]. Similar to PD-1, CTLA-4 is another coinhibitory molecule of the B7 family that can relieve the immunosuppressive effect of Treg cells in the TME by blocking the binding of CTLA-4 and B7 molecules on Treg cells, and strengthen the activity of effector T cells [115]. In contrast to the fact that CTLA-4 principally restricts T-cell response in the early immune response in lymphoid tissues, PD-1 mainly inhibits T-cell response in late peripheral tissues, which can contribute effector T cells to long-lasting anti-tumor activity by blocking the binding of PD-L1 of tumor cells to B7-1 on the surface of T cells [116,117]. The response to a single-agent with CTLA-4 inhibitors or PD-1/PD-L1 blockade remains unsatisfactory compared with the combined regime [118]. The joint blocking of PD-1 and CTLA-4 increased tumor infiltration by CD8⁺ T cells and CD4⁺ T cells, as well as the ratio of CD8⁺ and Treg cells, which has been recognized as a complementary mechanism of ICIs [119]. Therefore, it is hypothesized that the combined immunotherapies of CTLA-4 inhibitors and anti-PD1/PD-L1 agents may generate a coadjutant anti-tumor effect compared with single-agent treatment through affecting the complementary pathways in the cancer-immunity cycle [120,121].

The combination of nivolumab and ipilimumab was approved as applicable for advanced unresectable melanoma [122]. Subsequently, the synergistic effect was verified in a subset population with positive EGFR mutation in the CheckMate-012 trial, and the ORR of such dual immunotherapy with nivolumab and ipilimumab reached 50% [123]. Moreover, the activity of nivolumab plus low-dose ipilimumab as first-line standard care for 288 untreated patients with advanced NSCLC was evaluated in the CheckMate 568 trial [124]. Effective outcomes emerging from the study includ greater ORR in those with PD-L1 expression ≥1% than lower PD-L1 expression (41 vs 15%) [124] and superior PFS in patients with higher TMB than those with low TMB (7.1 vs 2.6 months). In this study, only 29% of patients exhibited grade 3/4 irAEs [124]. Furthermore, the KEYNOTE-021 trial evaluated the combination of pembrolizumab (2 mg/kg) and ipilimumab (1 mg/kg) in previously treated advanced NSCLC with a considerable ORR (30%), although there was no statistical significance (p = 0.0858) [125]. The combined agents of durvalumab (1 mg/kg) and tremelimumab (1 mg/kg), to a great extent, improved OS in an Asian advanced NSCLC population compared with chemotherapy (17.7 vs 10.6 months, p < 0.05), and clinical benefits have been revealed in the post hoc analysis of the MYSTIC trial (multicenter, open-label, phase III) [126]. The aforementioned critical clinical model of combined CTLA-4 and PD-1/PD-L1 inhibitors enhanced the anti-tumor activity of EGFR-TKI-resistant NSCLC, especially for those with higher TMB and PD-L1expression. Although, to date, there have been limited trials on anti-PD-1 and CTLA-4 immunotherapy in EGFR-TKI-resistant NSCLC, which may be attributed to previous cases in which immunotherapy may have a limited effect against NSCLC with EGFR mutations. Therefore, future studies should verify the safety and efficacy of dual immunosuppressive agents in NSCLC with EGFR-TKI resistance. Ongoing phase III trials are evaluating nivolumab plus ipilimumab and durvalumab plus tremelimumab in patients with previously untreated NSCLC [127]. Table 4 summarizes clinical trials that evaluate the combined regime of PD-1/PD-L1 and CTLA-4 inhibitors in patients with advanced NSCLC.

Conclusion

Collectively, this review pointed out that an immunotherapy-based combination regimen might become another selective therapy after the failure of targeted drug therapy by analyzing the close relationship between EGFR mutation and PD-L1 expression status, as well as the dynamic changes of TME of before and after treatment of EGFR-TKIs. At present, drug resistance of EGFR-TKIs is being continuously studied in depth. Meanwhile, it is particularly necessary to explore the resistance mechanism to targeted therapy, additional potential targets, and the development of effective biomarkers to screen the advantaged populations receiving immunotherapy for the realization of precision therapy in the future. Therefore, more research results are required to provide the best-individualized treatment for EGFR-TKI-resistant patients.

Future perspective

EGFR-TKIs targeting EGFR signaling pathways have brought favorable survival benefits to the population with EGFR mutation, but most people inevitably develop drug resistance within about one year of treatment. The lack of available treatment options makes the management of such patients challenging. Thus, it is urgent and necessary to investigate innovative strategies. Recently, ICIs have yielded dramatic clinical responses for patients with NSCLC. However, preclinical data showed that EGFR-positive NSCLC patients have a suboptimal response to immunotherapy. However, current clinical experiences of immunotherapy in patients with EGFR-TKI-resistant NSCLC remain absent, and the results of different clinical trials are controversial (Table 5). Given the potential advantages of ICIs in the EGFR-TKI-resistant population, it is vital to understand the correlation between tumor immune-mediated treatment-related biomarkers and tumor drivers in NSCLC. The limited efficacy of immunotherapy in patients with EGFR-positive NSCLC has been described in early-phase trials and different subtypes of EGFR mutations, different administration sequence, time and dosage influence the effect of immunotherapy in this setting, indicating that the strict selection of a high-standard population would be conducive to improving the outcomes of immunotherapy.

Although the response to ICIs in EGFR-mutant individuals was worse in those with wild-type EGFR mutation, the high expression of PD-L1 in such patients resistant to EGFR-TKIs still demonstrated a better response to ani-PD-1/PD-L1 agents. The effect of EGFR-TKIs in combination with ICIs is currently being investigated in a large number of clinical trials, most of which have shown no significantly enhance clinical activity and result in a limitation of further active investigation due to the unexpectedly high incidence of irAEs. The inspiring results from the recent IMpower 150 trial made the chemoimmunotherapy regimen a possible therapeutic alternative for EGFR-TKI-resistant patients. At present, the drug resistance of EGFR-TKIs is being studied in depth. It is particularly necessary to explore the resistance mechanism to targeted therapy, additional potential targets and the development of effective biomarkers to screen the advantaged populations receiving immunotherapy for the realization of precision therapy in the future.

In this review, we analyzed preclinical and clinical studies and concluded the following highly selective populations may benefit from immunotherapy: high expression of PD-L1 and TMB; EGFR L858R, G719X, EX20 insertion mutation and T790M negative; primary resistance to EGFR-TKIs; a long-term treatment duration of EGFR-TKIs and CD73 overexpression after resistance to EGFR-TKIs. Therefore, more research is required to provide the best-individualized treatment for EGFR-TKI-resistant patients. Further experiments confirm the synergistic anti-tumor effect of the combinational strategy of EGFR-TKIs and ICIs. This review summarized the four immunotherapy combinations for patients with NSCLC who fail EGFR-TKIs, mainly focused on immunotherapy-based combination therapy: immunotherapy in combination with EGFR-TKIs; immunotherapy in combination with chemotherapy; immunotherapy in combination with antiangiogenic agents and immunotherapy in combination with CTLA-4 inhibitors (Figure 2). Most of the clinical data are preliminary and immature, and more investigations and additional long-term follow-ups are required. Dramatic success from the ABCP regimen makes it a potentially promising therapy for those who fail EGFR-TKIs. It is hypothesized that a combination of multiple anti-tumor drugs will be employed to overcome EGFR-TKI resistance in the future. Better clinical outcomes with manageable adverse reactions may be achieved in the ongoing combinatorial clinical trials.