NK92-CD16 cells are cytotoxic to non-small cell lung cancer cell lines that have acquired resistance to tyrosine kinase inhibitors.

BACKGROUND: Treatment with tyrosine kinase inhibitors (TKIs) has improved the outcomes for patients with non-small cell lung cancer (NSCLC) harboring targetable driver mutations. However, acquired resistance to TKIs invariably develops within approximately 1 year of treatment by various mechanisms, including gatekeeper mutations, alternative pathway activation and histological transformations. Because immunotherapy is an option for patients with drug-resistant cancers, we generated several TKI-resistant NSCLC cell lines in vitro, and then evaluated the cytotoxicity of NK92-CD16 cells to these resistant cells. MATERIALS AND METHODS: TKI-resistant NSCLC cells (H3122CR1, H3122LR1, H3122CR1LR1, PC-9GR, PC-9ER, EBC-CR1 and EBC-CR2) were established from NCI-H3122 (EML4-ALK fusion), PC-9 (EGFR exon19 deletion) and EBC-1 (MET amplification) after continuous exposure to crizotinib, ceritinib, gefitinib, erlotinib and capmatinib. Expression of ligands for natural killer (NK) cell receptors and total EGFR were analyzed using flow cytometry. NK cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) using anti-EGFR monoclonal antibody (mAb) cetuximab were measured using NK92-CD16 as effectors and detected using the 51Chromium-release assay. RESULTS: We found that NK92-CD16 cells preferentially killed TKI-resistant NSCLC cells when compared with their parental NSCLC cells. Mechanistically, intracellular adhesion molecule 1 (ICAM-1) was up-regulated in the TKI-resistant NSCLC cells and patients' tumors, and the ICAM-1 up-regulated cancer cells lines were less susceptible to NK cytotoxicity by blocking ICAM-1. Moreover, NK92-CD16 cell-induced cytotoxicity toward TKI-resistant NSCLC cells was enhanced in the presence of cetuximab, an EGFR-targeting mAb. CONCLUSION: These data suggest that combinational treatment with NK cell-based immunotherapy and cetuximab may be promising for patients with TKI-resistant NSCLC.

Monalizumab efficacy correlates with HLA-E surface expression and NK cell activity in head and neck squamous carcinoma cell lines.

PURPOSE: NKG2A, an inhibitory receptor expressed on NK cells and T cells, leads to immune evasion by binding to HLA-E expressed on cancer cells. Here, we investigated the relationship between HLA-E surface expression on head and neck squamous cell carcinoma (HNSCC) cell lines and the efficacy of monalizumab, an NKG2A inhibitor, in promoting NK cell activity. METHODS: Six HNSCC cell lines were used as target cells. After exposure to IFN- γ, HLA-E surface expression on HNSCC cell lines was measured by flow cytometry. Peripheral blood mononuclear cells (PBMCs) from healthy donors and isolated NK cells were used as effector cells. NK cells were stimulated by treatment with IL-2 and IL-15 for 5 days, and NK cell-induced cytotoxicity was analyzed by CD107a degranulation and 51Cr release assays. RESULTS: We confirmed that HLA-E expression was increased by IFN-γ secreted by NK cells and that HLA-E expression was different for each cell line upon exposure to IFN-γ. Cell lines with high HLA-E expression showed stronger inhibition of NK cell cytotoxicity, and efficacy of monalizumab was high. Combination with cetuximab increased the efficacy of monalizumab. In addition, stimulation of isolated NK cells with IL-2 and IL-15 increased the efficacy of monalizumab, even in the HLA-E low groups. CONCLUSION: Monalizumab efficacy was correlated with HLA-E surface expression and was enhanced when NK cell activity was increased by cetuximab or cytokines. These results suggest that monalizumab may be potent against HLA-E-positive tumors and that monalizumab efficacy could be improved by promoting NK cell activity.

Angiopoietin-2-Dependent Spatial Vascular Destabilization Promotes T-cell Exclusion and Limits Immunotherapy in Melanoma.

T-cell position in the tumor microenvironment determines the probability of target encounter and tumor killing. CD8+ T-cell exclusion from the tumor parenchyma is associated with poor response to immunotherapy, and yet the biology that underpins this distinct pattern remains unclear. Here we show that the vascular destabilizing factor angiopoietin-2 (ANGPT2) causes compromised vascular integrity in the tumor periphery, leading to impaired T-cell infiltration to the tumor core. The spatial regulation of ANGPT2 in whole tumor cross-sections was analyzed in conjunction with T-cell distribution, vascular integrity, and response to immunotherapy in syngeneic murine melanoma models. T-cell exclusion was associated with ANGPT2 upregulation and elevated vascular leakage at the periphery of human and murine melanomas. Both pharmacologic and genetic blockade of ANGPT2 promoted CD8+ T-cell infiltration into the tumor core, exerting antitumor effects. Importantly, the reversal of T-cell exclusion following ANGPT2 blockade not only enhanced response to anti-PD-1 immune checkpoint blockade therapy in immunogenic, therapy-responsive mouse melanomas, but it also rendered nonresponsive tumors susceptible to immunotherapy. Therapeutic response after ANGPT2 blockade, driven by improved CD8+ T-cell infiltration to the tumor core, coincided with spatial TIE2 signaling activation and increased vascular integrity at the tumor periphery where endothelial expression of adhesion molecules was reduced. These data highlight ANGPT2/TIE2 signaling as a key mediator of T-cell exclusion and a promising target to potentiate immune checkpoint blockade efficacy in melanoma. SIGNIFICANCE: ANGPT2 limits the efficacy of immunotherapy by inducing vascular destabilization at the tumor periphery to promote T-cell exclusion.

Angiopoietin-2 blockade suppresses growth of liver metastases from pancreatic neuroendocrine tumors by promoting T cell recruitment.

Improving the management of metastasis in pancreatic neuroendocrine tumors (PanNETs) is critical, as nearly half of patients with PanNETs present with liver metastases, and this accounts for the majority of patient mortality. We identified angiopoietin-2 (ANGPT2) as one of the most upregulated angiogenic factors in RNA-Seq data from human PanNET liver metastases and found that higher ANGPT2 expression correlated with poor survival rates. Immunohistochemical staining revealed that ANGPT2 was localized to the endothelial cells of blood vessels in PanNET liver metastases. We observed an association between the upregulation of endothelial ANGPT2 and liver metastatic progression in both patients and transgenic mouse models of PanNETs. In human and mouse PanNET liver metastases, ANGPT2 upregulation coincided with poor T cell infiltration, indicative of an immunosuppressive tumor microenvironment. Notably, both pharmacologic inhibition and genetic deletion of ANGPT2 in PanNET mouse models slowed the growth of PanNET liver metastases. Furthermore, pharmacologic inhibition of ANGPT2 promoted T cell infiltration and activation in liver metastases, improving the survival of mice with metastatic PanNETs. These changes were accompanied by reduced plasma leakage and improved vascular integrity in metastases. Together, these findings suggest that ANGPT2 blockade may be an effective strategy for promoting T cell infiltration and immunostimulatory reprogramming to reduce the growth of liver metastases in PanNETs.

Blockade of CD47 enhances the antitumor effect of macrophages in renal cell carcinoma through trogocytosis.

Immune checkpoint inhibitors and vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR TKIs) are mainstream treatments for renal cell carcinoma (RCC). Both T cells and macrophages infiltrate the tumor microenvironment of RCC. CD47, an immune checkpoint of macrophages, transmits the "don't eat me" signal to macrophages. We propose a novel therapeutic strategy that activates the antitumor effect of macrophages. We found that CD47 was expressed in patients with RCC, and high CD47 expression was indicative of worse overall survival in datasets from The Cancer Genome Atlas. We observed that CD47-blocking antibodies enhanced the antitumor effect of macrophages against human RCC cell lines. Trogocytosis, rather than phagocytosis, occurred and was promoted by increased cell-to-cell contact between macrophages and RCC cells. Trogocytosis induced by CD47 blockade occurred in the presence of CD11b integrin signaling in macrophages and was augmented when RCC cells were exposed to VEGFR TKIs, except for sunitinib. In conclusion, this study presents evidence that anti-CD47 blocking antibodies improve the antitumor effect of macrophages in RCC. In combination with VEGFR TKIs, CD47 blockade is a potential therapeutic strategy for patients with RCC.

Acquired Resistance to Third-Generation EGFR Tyrosine Kinase Inhibitors in Patients With De Novo EGFRT790M-Mutant NSCLC.

INTRODUCTION: EGFRT790M mostly exists subclonally and is acquired as the most common mechanism of resistance to EGFR tyrosine kinase inhibitors (TKIs). Nevertheless, because de novo EGFRT790M-mutant NSCLC is rare, little is known on acquired resistance mechanisms to third-generation EGFR TKIs. METHODS: Acquired resistance mechanisms were analyzed using tumor and plasma samples before and after third-generation EGFR TKI treatment in four patients with de novo EGFRT790M-mutant NSCLC. Genetic alterations were analyzed by whole-exome sequencing, targeted sequencing, fluorescence in situ hybridization, and droplet digital PCR. MTORL1433S, confirmed for oncogenicity using the Ba/F3 system, was reproduced in H1975 cell lines using CRISPR/Cas9-RNP. RESULTS: Of seven patients with NSCLC with de novo EGFRT790M/L858R mutation, four (LC1-4) who received third-generation EGFR TKIs acquired resistance after achieving a partial response (median = 27 mo, range: 17-48 mo). Novel MTORL1433S and EGFRC797S/L798I mutations in cis, MET amplification, and EGFRC797S mutation were identified as acquired resistance mechanisms to third-generation EGFR TKIs. The MTORL1433S mutation was oncogenic in Ba/F3 models and revealed resistance to osimertinib through AKT signaling activation in NCI-H1975 cells harboring the MTORL1433S mutation edited by CRISPR/Cas9 (half-maximal inhibitory concentration, 800 ± 67 nM). Osimertinib in combination with mTOR inhibitors abrogated acquired resistance to osimertinib. CONCLUSIONS: Activation of bypass pathways and the EGFRC797S or EGFRC797S/L798I mutation were identified as acquired resistance mechanisms to third-generation EGFR TKIs in patients with NSCLC with de novo EGFRT790M mutation. In addition, MTORL1433S- and EGFRL858R/T790M-mutant NSCLC cells were sensitive to osimertinib plus mTOR inhibitors.

Anti-tumor effects of NK cells and anti-PD-L1 antibody with antibody-dependent cellular cytotoxicity in PD-L1-positive cancer cell lines.

BACKGROUND: Although programmed cell death-1/programmed death-ligand 1 (PD-L1) inhibitors show remarkable antitumor activity, a large portion of patients with cancer, even those with high PD-L1-expressing tumors, do not respond to their effects. Most PD-L1 inhibitors contain modified fragment crystallizable region (Fc) receptor binding sites to prevent antibody-dependent cellular cytotoxicity (ADCC) against PD-L1-expressing non-tumor cells. However, natural killer (NK) cells have specific antitumor activity in the presence of tumor-targeting antibody through ADCC, which could enhance NK cell-induced cytotoxicity. We evaluated the antitumor efficacy of ADCC via anti-PD-L1 monoclonal antibodies (mAbs) and NK cells against several PD-L1-positive cancer cell lines. METHODS: Various cancer cell lines were used as target cell lines. Surface PD-L1 expression was analyzed by flow cytometry. IMC-001 and anti-hPD-L1-hIgG1 were tested as anti-PD-L1 mAbs with ADCC and atezolizumab as an anti-PD-L1 mAb without ADCC. NK cell cytotoxicity was measured by 51Cr-release assay and CD107a degranulation assay. Also, live cell imaging was performed to evaluate cytotoxicity in a single-cell level. NK-92-CD16 (CD16-transduced NK-92 cell line) and peripheral blood mononuclear cells from healthy donors, respectively, were used as an effector cell. FcγRIIIa (CD16a)-V158F genotyping was performed for healthy donors. RESULTS: We demonstrated that the cytotoxicity of NK-92-CD16 cells toward PD-L1-positive cancer cell lines was significantly enhanced in the presence of anti-PD-L1 mAb with ADCC. We also noted a significant increase in primary human NK cell cytotoxicity against PD-L1-positive human cancer cells when cocultured with anti-PD-L1 mAb with ADCC. Moreover, NK cells expressing a FCGR3A high-affinity genotype displayed higher anti-PD-L1 mAb-mediated ADCC lysis of tumor cells than donors with a low-affinity genotype. CONCLUSION: These results suggest that NK cells induce an ADCC response in combination with anti-PD-L1 mAbs, which helps promote ADCC antitumor activity against PD-L1-positive tumors. This study provides support for NK cell immunotherapy against high PD-L1-expressing tumors in combination with ADCC through anti-PD-L1 mAbs.

Inhibition of MEK with trametinib enhances the efficacy of anti-PD-L1 inhibitor by regulating anti-tumor immunity in head and neck squamous cell carcinoma.

Major histocompatibility complex (MHC) class I downregulation is the primary immune evasion mechanism associated with failure in anti-PD-1/PD-L1 blockade therapies for cancer. Here, we examined the role of MEK signaling pathway inhibition in head and neck squamous cell carcinoma (HNSCC) both in vitro and in vivo. We found that trametinib, a small molecule inhibitor of MEK, significantly enhanced MHC class I and PD-L1 expression in human HNSCC cell lines, and this occurred via STAT3 activation. Trametinib also further upregulated the increase in CXCL9 and CXCL10 expression caused by IFN-γ in HNSCC cells, which is associated with T cell infiltration in tumor tissues. Finally, we evaluated the therapeutic efficacy of trametinib combined with an anti-PD-L1 monoclonal antibody in vivo, using SCCVII mouse syngeneic tumor model for HNSCC. While neither PD-L1 blockade nor trametinib treatment alone affected tumor growth, the combined therapy significantly delayed tumor growth. Our results indicate that in the combined therapy trametinib increases CD8+ T cell infiltration in the tumor site and upregulates antigen presentation, and this may be associated with enhanced PD-L1 blockade efficacy. Furthermore, our results suggest that this combination would therapeutically benefit patients with HNSCC.