Overcoming immunotherapy resistance in non-small cell lung cancer (NSCLC) - novel approaches and future outlook

Immune checkpoints (IC) play a central role in negative regulation of T cell reactivity and their inhibition via monoclonal antibodies can unleash T cell-triggered antitumor immune responses. The best studied IC are PD-1 and cytotoxic T lymphocyte antigen 4 (CTLA-4). PD-1 is broadly expressed on CD8⁺ T lymphocytes, regulatory T cells (Treg) and natural killer (NK) cells and modulates T cell activity via interaction with its ligand (PD-L1) in the TME (Fig. 2). CTLA-4 is expressed on CD8⁺ and CD4⁺ T lymphocytes and Treg and regulates early naïve T cell activation in secondary lymphoid organs [3, 4]. Other IC are constantly being discovered and under investigation for their clinical utility as druggable IC (e.g. TIM-3, LAG-3 or TIGIT).

Somatic mutations in the cancer genome, such as in DNA repair genes including mismatch repair (MMR), homologous recombination (HR) or polymerase epsilon (POLE) increase tumor mutational and neoantigen burden, which has been linked to greater TIL density and enhanced ICI efficacy [17‐19]. This observation is clinically underscored as mutagen-driven cancer types (e.g. melanoma, NSCLC) typically show high initial ICI responses [17]. Moreover, components of the major histocompatibility complex I (MHC I) such as B2M are often downregulated (Fig. 2), hence curbing neo-epitope presentation to T cells [20]. Antigen presentation pathways can also be inactivated through mutations (e.g. B2M is mutated or deleted in about 5% of lung cancers) [21] and also other pathway members are inactivated [22]. Importantly, IO may increase the frequency of such mutations [19, 23, 24] suggesting an active immune-editing of cells failing to present neo-epitopes.

Concerning TMB as predictive biomarker of ICI response, clinical trials report divergent results, possibly due to technical issues with TMB assessment (e.g. use of inhomogenous cut-off values) [25]. On the one hand, high TMB was the strongest variable linked to benefit of combined PD-1 plus CTLA-4 blockade in NSCLC and TMB was independent of PD-L1 expression [26]. Accordingly, pembrolizumab was recently FDA-approved in TMB^high advanced solid cancers (≥10 mutations/megabase) in response to results from KEYNOTE-158. In contrast, in the complex multi-arm CheckMate227 trial testing ipilimumab plus nivolumab versus chemotherapy or nivolumab plus chemotherapy in NSCLC, neither TMB nor PD-L1 expression could segregate therapy responsiveness [27]. Concerning CTLA4-specific biomarkers, different genomic signatures were correlated with enhanced clinical outcome [28, 29], however none have been translated into clinical practice yet.

Cancer cells can overexpress PD-L1 upon type I interferon (IFN I) stimulation [30] to evade cytotoxic immune responses. Immune cells, including Treg, myeloid-derived suppressor cells (MDSC), dendritic cells (DC) and TEC can similarly upregulate PD-L1 upon inflammatory signals (especially by IFNs) fostering an immunosuppressive TME [31]. Interestingly, myeloid cells show markedly higher PD-L1 expression than cancer cells or lymphocytes (Fig. 2) and especially extra-tumoral PD-L1 expressing myeloid cells, e.g. in tumor draining lymph nodes, might be essential for ICI response [31]. A preclinical study demonstrated that myeloid progenitors that accumulate during cancer-driven emergency myelopoiesis (in bone marrow, spleen and tumor site) show both PD-L1 and particularly prominent PD-1 expression. Selective deletion of myeloid-specific PD-1 by targeting the Pdcd1 gene effectively suppressed tumor growth in several tumor models by mediating antitumor immunity (enhanced T effector memory cells) despite preserved T cell-specific PD-1 expression. These data underline the important role of myeloid-intrinsic effects in regulating anti-tumor immunity [32].

Clearly, PD-L1 expression is necessary to achieve adequate responses to PD-1/PD-L1 blockade and numerous studies associated high tumor cell PD-L1 expression with better outcomes to anti-PD-1/PD-L1 monotherapy in NSCLC. Controversially, some patients with very low or even absent PD-L1 expression show durable responses [33], an observation currently lacking a mechanistic explanation see 2.4.1. Besides cancer cells, also PD-L1 positive immune cells may exert a predictive value. In the Impower110 trial, presence of PD-L1 positive TIL significantly associated with enhanced OS in patients treated with atezolizumab [34]. These results are in line with other tumor entities (e.g. bladder and breast cancer).

Tumor-associated macrophages (TAM) are an abundant cell type within the TME and despite growing research, their role in cancer progression remains ambiguous. Along a functional scale, TAM polarize to either M1 or M2 phenotypes in response to environmental cues, including metabolic changes (e.g. cyclic hypoxia, nitric oxide) [36, 37]. The classically activated M1 phenotype is stimulated upon type 1 T helper cell (Th1)-produced IFN-γ or Toll-like receptor (TLR) ligands such as microbiota-derived lipopolysaccharide (LPS) and is characterized by phagocytic, cytotoxic and antigen-presenting functions and secretion of pro-inflammatory cytokines (e.g. TNFα, IL-1β, IL-6) [36, 38]. Alternatively, the M2 phenotype expands in response to Th2-derived IL-4 and IL-13 [39], but cancer cell-derived macrophage-colony stimulating factor (M-CSF) also promotes M2 polarization by binding CSF1 receptor (CSF1-R). M2 macrophages express anti-inflammatory cytokines (e.g. IL-10, CCL22, CCL18) and low levels of IL-12, thereby exerting anti-inflammatory, angiogenic and pro-tumoral effects [36]. Impeding M2 polarization to promote anti-tumor immune responses has gained clinical interest (e.g. CSF1 inhibition) and also preclinical studies of genetic TAM reprogramming are promising [40, 41].

Cancer associated fibroblasts (CAF) constitute one of the most prominent, yet highly heterogenous components of the TME. They express a variety of molecular markers, e.g. α-SMA, S100A4, FAP, PDGFRα/β, none of which, however, is unique for the fibroblast lineage. Next to immune cells CAFs have emerged as important mediators of the complex stroma-tumor interactions, promoting local immunosuppression and orchestrating immune cell trafficking [42]. CAFs may express PD-L1 (e.g. upon IFN-γ) (Fig. 2) but may also promote PD-L1 expression on tumor cells via cytokine secretion (e.g. CXCL5, CXCL2) [43]. Further knowledge on CAF functionality might unveil insights in IO sensitivity.

Tumor endothelial cells (TEC) have immunomodulatory functions by controlling immune cell transmigration, lymphocyte activation and function. They hold a “sentinel” role in detecting foreign antigens as antigen (cross)-presenting cells, though this has been studied extensively in non-malignant inflammation and less in TEC [44, 45]. TEC are strategically positioned at the blood–TME interface, serving as “immune gatekeepers” by controlling immune cell trafficking. In NSCLC, TEC may express PD-L1 (Fig. 2) and downregulate inflammatory pathways (e.g. antigen presentation, chemotaxis, immune cell homing) [2]. On the single cell level, Goveia et al. identified distinct lung TEC subpopulations carrying the transcriptome signature of HEVs and semi-professional APCs, suggesting a role in tumor immune surveillance. Specific TEC subtypes were associated with prognosis and response to anti-angiogenic therapy [46].

It remains to be answered why some patients attain sustained durable IO therapy response while others evolve primary or secondary resistance. The mode of action is definitely multifactorial and includes intrinsic (e.g. cell signaling, immune recognition, gene expression, DNA damage response) and extrinsic (e.g. T cell activation, neo-angiogenesis) mechanisms [47]. The following sections briefly address relevant resistance mechanisms, many of which are already used as targets of novel therapeutic strategies as to overcome resistance (see Fig. 1).

IC co-inhibition, by expanding the anti-PD-1 or PD-L1 backbone with a second ICI has been one of the first strategies to overcome IO resistance and most clinical experience has been gathered with combinational CTLA-4 inhibitor. The observed synergistic effect of PD-1/CTLA-4 inhibitors likely depends on the distinct patterns of PD-1 and CTLA4 in immune activation, as PD1 blockade inhibits peripheral and CTLA4-blockade central tolerance see 2.1, 3.