Incorporating Immune-Checkpoint Inhibitors into Systemic Therapy of NSCLC  Stéphane Champiat, MD, Ecaterina Ileana, MD, Giuseppe Giaccone, MD, PhD, Benjamin Besse, MD, PhD, Giannis Mountzios, MD, PhD, Alexander Eggermont, MD, PhD, Jean-Charles Soria, MD, PhD  Journal of Thoracic Oncology  Volume 9, Issue 2, Pages 144-153 (February 2014) DOI: 10.1097/JTO.0000000000000074 Copyright © 2014 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1 Expected effect of the combinatorial strategy of immune-checkpoint blockade with conventional therapies. Targeted therapies can induce impressive regressions in molecularly defined subsets of patients. Nevertheless, virtually all patients treated with targeted therapies will ultimately develop progressive disease by treatment resistance. Immune-checkpoint inhibitors can induce long-lasting disease control in a small proportion of responders. Combination strategies might lead to a synergistic activity and improve efficacy for a higher frequency of patients (adapted from Ribas et al., CCR 18: 336–341, 2012). Journal of Thoracic Oncology 2014 9, 144-153DOI: (10.1097/JTO.0000000000000074) Copyright © 2014 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2 NSCLC tumor immunology and modulation by conventional therapies. Many reports suggest a role of the immune system in NSCLC.50 Higher numbers of TILs are strongly associated with improved survival, irrespective of NSCLC subtype.51,52 To develop efficient antitumor immune responses, multiple steps are required and in NSCLC many of them can be affected. First, tumor cells can be recognized and eliminated by the innate immune system, thanks to the NK cells. To escape such attack, NSCLC can induce down-regulation of NK ligands such as NKG2D or avoid Fas-induced apoptosis through reduction of Fas receptor cell surface expression.53,54 Subsequently, the immune system develops an adaptive immune response, mediated by CTLs. For that purpose, antigen presenting cells capture tumor antigens and process them into MHC to prime T cells in a regional lymph node. NSCLC tumors display reduced expression of MHC I, and thereby avoid stimulation of the adaptive immune response.55,56 After priming, T cells are released into the circulation and migrate into the tumor tissue until they find their specific tumor antigen to become activated and differentiate into effector T cells. This step requires a combination of signals from both the T-cell receptor and several costimulatory/coinhibitory molecules. For activation, T cells must avoid negative regulatory signals from the immune checkpoints such as PD-1/PD-L1. Around 50% of NSCLC tumors are reported to express PD-L1.4,5 Ultimately, the induced antitumor immune response must be able to overcome immunosuppressive factors present in the tumor microenvironment such as TRegs or immunosuppressive cytokines (e.g., IL-10 or TGFß). Remarkably, NSCLCs have higher infiltration of TRegs compared with normal lung tissue, and higher proportion of TRegs have been associated with increased risk of recurrence.57,58 Moreover, the microenvironment of NSCLC displays immunosuppressive characteristics with production of IL-10 and TGF-ß that can directly inhibit T-cell activation, proliferation, and differentiation.59 Interestingly, the SCC subtype seems to have a more specific immunologic profile. Compared with nonsquamous subtypes, SCC are more frequently expressing known tumor-specific antigens such as NY-ESO-1 or MAGE antigens, and a higher CTLs/TRegs tumor infiltration ratio has been described.56,60,61 NSCLC, non–small-cell lung cancer; NK, natural killer; PD-L1, programmed death 1 protein; Tregs, T regulatory cells; SCC, squamous-cell carcinoma; DC, dendritic cell; CTLA4, cytotoxic T-lymphocyte–associated antigen 4; MDSC, myeloid-derived suppressor cells; MHC, major histocompatibility complex. Journal of Thoracic Oncology 2014 9, 144-153DOI: (10.1097/JTO.0000000000000074) Copyright © 2014 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3 Left panel, Molecular pathways underlying T cell activation. TCR and CD28 signaling Peptides are presented on the surface via the MHC molecules, for recognition and binding by the TCR. TCR intracellular signaling is initiated after phosphorylation of immunoreceptor tyrosine–based activation motifs in the CD3 cytoplasmic domains. This phosphorylation leads to recruitment of the kinase Zap70 (70 kDa Zeta–chain associated protein) via its tandem SH2 domains. One of the key Zap70 substrates responsible for TCR signal transmission is LAT. LAT is able to recruit directly or indirectly several key signaling intermediates from the Ras/MAPK, PI3K and PKCθ signaling pathways.62 The initial signal of the TCR is insufficient to generate an immune response and an additional costimulatory signal is required, like with the CD28 receptor. CD28 is located on the T-cell surface and recognizes CD80/CD86 present on the APC. CD28 is able to recruit protein kinase signaling molecules through its proximal intracellular domain (YMNM and PYAP motifs) to costimulate several major signaling pathways including PI3K/AKT and Ras/MAPK and activate subsequent distal molecules: mTOR, glucose transporter type 1, nuclear factor of activated T cells, NF-κB, Bcl2, and Bcl-XL.63 This collectively leads to T-cell proliferation, cytokine production, and increased survival. Center panel, CTLA4 coinhibitory signaling. Initially, CTLA4 receptors are primarily inactive and remain complexed within the intracellular compartment. TCR activation induces up-regulation of CTLA4 and enhances the exocytosis of CTLA4-containing vesicles, resulting in an increase in CTLA4 surface expression. CTLA4 cytoplasmic region motif VKM associates with SHP2 and serine/threonine PP2A. SHP2 and PP2A inhibit proximal TCR signaling through dephosphorylation of the TCR–CD3ζ complex, of LAT and of Zap70, resulting in blocking of cell-cycle progression and cytokine production.64 CTLA4 was also reported to inhibit ERK and JNK phosphorylation. Right panel, PD-1 coinhibitory signaling. Additional regulatory molecules, including PD-1, are also important in limiting T-cell activation. The cytoplasmic domain of PD-1 contains an immunoreceptor tyrosine–based inhibition motif and an ITSM.65 Both motifs are phosphorylated after interaction with B7-H1 or B7-dendritic cell. Once phosphorylated, the ITSM motif recruits SHP2 and SHP1. PD-1 colocalizes with TCR microclusters and induces dephosphorylation of CD3ζ, Zap70, and PKCθ. PD-1 is also reported to inhibit RAS and its downstream targets ERK1 and ERK2 through an SHP1- and SHP2-independent mechanism.66 This signaling results in limiting T-cell activation and expansion. Both PD-1 and CTLA4 signaling inhibit Akt activation; however, PD-1 ligation inhibits a more upstream membrane proximal step by blockingPI3K activation through SHP1. By contrast, signaling by CTLA4 preserves PI3K activity, allowing expression of certain genes such as Bcl-xL, but inhibits Akt directly by activation of the phosphatase PP2A. MHC, major histocompatibility complex; SH2, Src-homology 2; LAT, linker for activation of T cells; PKCθ, protein kinase Cθ; APC, antigen presenting cells; P13K, phosphatidylinositol-3-kinase; CTLA4, cytotoxic T-lymphocyte–associated antigen 4; SHP2, SH2 domain–containing tyrosine phosphatase 2; PP2A, protein phosphatase 2A; ITSM, immunoreceptor tyrosine–based switch motif; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor-κΒ; PD-L1, programmed death 1 protein. Journal of Thoracic Oncology 2014 9, 144-153DOI: (10.1097/JTO.0000000000000074) Copyright © 2014 International Association for the Study of Lung Cancer Terms and Conditions