EGFR-mediated tumor immunoescape: The imbalance between phosphorylated STAT1 and phosphorylated STAT3

The epidermal growth factor receptor (EGFR) supports the escape of malignant cells from immunosurveillance by inhibiting the activation of signal transducer and activator of transcription 1 (STAT1) while promoting that of STAT3. We have recently demonstrated that protein tyrosine phosphatase, non-receptor type 11 (PTNP11, best known as SHP2), a phosphatase that operates downstream of EGFR, is responsible for the dephosphorylation of active STAT1 and for the inhibition of the antigen-processing machinery (APM), hence favoring tumor immunoescape. Thus, EGFR signaling may skew the tumor microenvironment to suppress cellular immune responses

STAT1 contributes to HLA class I upregulation and CTL reactivity after anti-EGFR mAb cetuximab therapy in head and neck cancer patients

Squamous cell carcinoma of head and neck (HNSCC) cells express low HLA class I and antigen processing machinery (APM) components, such as transporter TAP-1/2, which is associated with the reduced sensitivity to cytotoxic T lymphocyte (CTL) mediated lysis. Epidermal growth factor receptor (EGFR) is overexpressed in HNSCC and is associated with the poor prognosis. FDA approved anti-EGFR blockade mAb cetuximab inhibits HNSCC proliferation, and induces EGFR-specific CTL. However, the molecular mechanism(s) underlying the EGFR-specific CTL recognition of HNSCC in the therapeutic efficacy of anti-EGFR mAb is still emerging. We show that cetuximab or EGFR knockdown enhanced expression of HLA class I antigens, which is associated with the EGFR expression level on HNSCC. These findings were validated in a prospective trial of neoadjuvant cetuximab therapy. Interestingly, upregulation of HLA-B/C alleles were more pronounced than HLA-A alleles after cetuximab or EGFR knockdown treatment. EGFR signaling blockade or EGFR depletion also enhanced IFN gamma receptor (IFNAR) on HNSCC and augmented induction of HLA class I and TAP-1/2 caused by IFN gamma treatment. Cetuximab or EGFR knockdown enhanced the level of HLA class I, STAT-1, TAP-1/2 in a STAT-1+/+ cell line but not in STAT-1-/- cell line, documenting the STAT-1 dependence of this effect. We also found that Src homology domain-containing phosphatase 2 (SHP-2), which is downstream of EGFR and also overexpressed in SCCHN, can suppress the immunostimulatory effect of cetuximab treatment on HLA class I/STAT-1 upregulation, and dual targeting of EGFR and SHP-2 co-operates in the most efficient reversal of immune escape phenotype. In addition, cetuximab-based EGFR inhibition and SHP-2 depletion enhanced the recognition of HNSCC cells by EGFR 853-861 specific CTL, and enhanced surface presentation of non-EGFR TA, such as MAGE-3 271-279 , indicating that a broad tumor antigen repertoire is processed and presented by HLA/APM upregulation. These findings elucidate a novel immune escape mechanism associated with EGFR signaling through STAT1 suppression and the reversal with cetuximab, which may provide additional targets for on-going mAb-based immunotherapy

Anti-PD-1 mAb pre-radiotherapy (RT) loading dose and fractionated RT induce better tumor-specific immunity and tumor shrinkage than sequential administration in an HPV+ head and neck cancer model

Radiotherapy (RT) is a standard therapeutic strategy in the treatment in head and neck cancer (HNC), but many patients still experience recurrence and metastasis. Interestingly, radiotherapy (RT) may also induce immunomodulatory effects. Given the recent, exciting responses seen using anti-PD-1 (programmed death-1) checkpoint blockade immunotherapy in recurrent/metastatic disease, including HNC, we evaluated the combination of RT with anti-PD-1 therapy in a pre-clinical mouse model of locally advanced, untreated HPV-positive HNC. We compared utilizing PD-1 blockade before, during or after RT, as well as whether a single large faction (12Gy) or multiple smaller RT doses (2 Gy X 10 fractions) confers optimal antitumor immune responses and tumor shrinkage. We observed that fractionated doses of RT induced the highest PD-L1 (programmed death-ligand 1) expression on HNC cells in vitro and in treated mice. A loading dose of anti-PD-1 mAb therapy prior to RT appeared to be important for best therapeutic outcome, with greatest tumor response and tumor-specific immunity using PD-1 Ab loading dose than sequential administration of anti-PD-1 mAb after fractionated RT (p < 0.0001). Expression and intensity of PD-1 receptor expression on circulating T cells differentially impacted the T cell phenotype and anti-tumor outcome, with loss of PD-1(high) exhausted T cells during the best tumor response (p < 0.05). The combination of fractionated RT + anti-PD-1 Ab optimally upregulated the frequency of HPV E7 tumor antigen-specific T cells (p < 0.05). This study may facilitate strategies required for the combination of RT and immune checkpoint inhibitor in clinical trials, enabling more effective clinical activity and biomarker evaluation

PD-1 is a marker of activation on tumor infiltrating NK cells in head and neck cancer

Co-inhibitory immune checkpoint receptors have become important targets for cancer immunotherapy. Programmed death 1 (PD-1) has been well-characterized on T cells in many cancer types, including head and neck cancer (HNC), for its ability to mediate activation and eventually T cell exhaustion in the tumor microenvironment. However, PD-1 expression on NK cells, which are crucial innate immune effector cells against cancer, remains largely undefined. In the setting of HNC, NK cells mediate lysis of EGFR-overexpressing tumor targets via cetuximab-mediated antibody dependent cytotoxicity (ADCC). Indeed, cetuximab has shown to be clinically effective but only to a modest extent. Therefore, it is necessary to investigate how cetuximab modulates activation of immune effector cell infiltrates in the tumor microenvironment in order to improve or extend its therapeutic efficacy. We hypothesized that expression of PD-1 per se on NK cells may constitute a marker of a chronically activated phenotype, which is suppressed only after ligation by its cognate ligand programmed death ligand-1 (PD-L1). Thus, tumor cell-expressing PD-L1 may present as a crucial mediator of immunosuppression in the tumor microenvironment decreasing cytotoxicity of cetuximab activated PD-1 expressing NK cells. Herein, using The Cancer Genome Atlas (TCGA) data for 500 HNC patients' tumors, we found that PD-1 expression correlates with NK activation markers. Indeed, HNC patients also exhibit higher levels of circulating and tumor infiltrating PD-1+ NK cells, and neoadjuvant cetuximab treatment increased this frequency in vitro and in vivo in a prospective Phase II trial. In addition, anti-PD-1 mAb nivolumab enhanced cetuximab mediated NK cell activation and HNC cell lysis. Therefore, blocking PD-L1/PD-1 axis may be a useful approach to reverse immune evasion of HNC tumors to cetuximab therapy by reversing NK cell dysfunction

Anti-EGFR targeted monoclonal antibody isotype influences anti-tumor immunity in head and neck cancer patients

EGFR is frequently overexpressed on several cancers, and two targeted antibodies are FDA approved but differ by isotype. Cetuximab (IgG1 isotype) has been shown to be effective at both inhibiting downstream signaling of EGFR and activating anti-tumor, cellular immune mechanisms. While panitumumab (IgG2 isotype) can inhibit downstream EGFR signaling similar to cetuximab, panitumumab might also induce antibody-dependent cell cytotoxicity (ADCC) or adaptive immunity. We sought to investigate the cellular immunity specifically activated by cetuximab or panitumumab showing that both mAb primarily activate NK cells, although cetuximab was significantly more potent than panitumumab. We also observed that although panitumumab may activate monocytes through the CD32 (FcγRIIa) receptor, neither mAb activated monocytes sufficiently to mediate ADCC. Cetuximab enhanced DC maturation to a greater extent than panitumumab, corresponding with improved cross presentation of tumor antigen by cetuximab compared with panitumumab. Indeed, improved adaptive immune responses with increased EGFR-specific cytotoxic CD8+ T cells were present in patients treated with cetuximab compared to those treated with panitumumab. These results suggest that although panitumumab effectively inhibits EGFR signaling to a similar extent as cetuximab, it is less effective at mediating anti-tumor, cellular immune mechanisms which may be crucial for effective therapy for HNSCC

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Activating and inhibitory receptors and their role in Natural Killer cell function

291-299Last decade has seen a significant advancement in our understanding of the Natural Killer (NK) cell biology and function. Several receptors present on NK cells have been identified, which are involved in either their activation or inhibition. Similarly, a large number of interacting ligands have been identified on the target cells that upon interaction transmit either activating or inhibitory signals. This review is oriented towards understanding the role of these receptors on the NK cells, which are considered as the first line of defense