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The ability of Natural Killer (NK) cells to kill tumor targets has been extensively studied in various hematological malignancies. However, NK cell therapy directed against solid tumors is still in early development. Epidermal Growth Factor Receptor (EGFR) targeted therapies using monoclonal antibodies (mAbs) such as cetuximab and panitumumab are widely used for the treatment of metastatic colorectal cancer (mCRC). Still, the clinical efficacy of this treatment is hampered by mutations in RAS gene, allowing tumors to escape from anti-EGFR mAb therapy. It is well established that NK cells kill tumor cells by natural cytotoxicity and can in addition be activated upon binding of IgG1 mAbs through Fc receptors (CD16/FcγRIIIa) on their surface, thereby mediating antibody dependent cellular cytotoxicity (ADCC). In the current study, activated Peripheral Blood NK cells (PBNK) were combined with anti-EGFR mAbs to study their effect on the killing of EGFR+/- cancer cell lines, including those with RAS mutations. In vitro cytotoxicity experiments using colon cancer primary tumors and cell lines COLO320, Caco-2, SW620, SW480 and HT-29, demonstrated that PBNK cells are cytotoxic for a range of tumor cells, regardless of EGFR, RAS or BRAF status and at low E:T ratios. Cetuximab enhanced the cytotoxic activity of NK cells on EGFR+ tumor cells (either RASwt, RASmut or BRAFmut) in a CD16 dependent manner, whereas it could not increase the killing of EGFR- COLO320. Our study provides a rationale to strengthen NK cell immunotherapy through a combination with cetuximab for RAS and BRAF mutant mCRC patients.

γδ T-cells directly recognize and kill transformed cells independently of HLA-antigen presentation, which makes them a highly promising effector cell compartment for cancer immunotherapy. Novel γδ T-cell-based immunotherapies, primarily focusing on the two major γδ T-cell subtypes that infiltrate tumors ( i.e. Vδ1 and Vδ2), are being developed. The Vδ1 T-cell subset is enriched in tissues and contains both effector T-cells as well as regulatory T-cells with tumor-promoting potential. Vδ2 T-cells, in contrast, are enriched in circulation and consist of a large, relatively homogeneous, pro-inflammatory effector T-cell subset. Healthy individuals typically harbor in the order of 50-500 million Vγ9Vδ2 T-cells in the peripheral blood alone (1-10% of the total CD3 + T-cell population), which can rapidly expand upon stimulation. The Vγ9Vδ2 T-cell receptor senses intracellular phosphorylated metabolites, which accumulate in cancer cells as a result of mevalonate pathway dysregulation or upon pharmaceutical intervention. Early clinical studies investigating the therapeutic potential of Vγ9Vδ2 T-cells were based on either ex vivo expansion and adoptive transfer or their systemic activation with aminobisphosphonates or synthetic phosphoantigens, either alone or combined with low dose IL-2. Immune-related adverse events (irAE) were generally but the clinical efficacy of these approaches provided overall limited benefit. In recent years, critical advances have renewed the excitement for the potential of Vγ9Vδ2 T-cells in cancer immunotherapy. Here, we review γδ T-cell-based therapeutic strategies and discuss the prospects of those currently evaluated in clinical studies in cancer patients as well as future therapies that might arise from current promising pre-clinical results.

Immune checkpoint blockade (ICB) has changed the therapeutic landscape of oncology but its impact is limited by primary or secondary resistance. ICB resistance has been related to a lack of T cells infiltrating into the tumor. Strategies to overcome this hurdle have so far focused on the tumor microenvironment, but have mostly overlooked the role of tumor-draining lymph nodes (TDLN). Whereas for CTLA-4 blockade TDLN have long since been implicated due to its perceived mechanism-of-action involving T cell priming, only recently has evidence been emerging showing TDLN to be vital for the efficacy of PD-1 blockade as well. TDLN are targeted by developing tumors to create an immune suppressed pre-metastatic niche which can lead to priming of dysfunctional antitumor T cells. In this review, we will discuss the evidence that therapeutic targeting of TDLN may ensure sufficient antitumor T cell activation and subsequent tumor infiltration to facilitate effective ICB. Indeed, waves of tumor-specific, proliferating stem cell-like, or progenitor exhausted T cells, either newly primed or reinvigorated in TDLN, are vital for PD-1 blockade efficacy. Both tumor-derived migratory dendritic cell (DC) subsets and DC subsets residing in TDLN, and an interplay between them, have been implicated in the induction of these T cells, their imprinting for homing and subsequent tumor control. We propose that therapeutic approaches, involving local delivery of immune modulatory agents for optimal access to TDLN, aimed at overcoming hampered DC activation, will enable ICB by promoting T cell recruitment to the tumor, both in early and in advanced stages of cancer.

BackgroundDespite the notable success of programmed death ligand 1 (PD-L1)/programmed death 1 (PD-1) immune checkpoint blockade in cancer, resistance remains a substantial challenge. Combining immune checkpoint blockade with direct targeting of effector T cells to tumor cells might improve outcome for a broader spectrum of patients with cancer. Phosphoantigen-responsive Vγ9Vδ2-T cells are potent immune cells that play a pivotal role in tumor immunosurveillance and can coordinate downstream immune activity. Here, we explored whether a Vδ2 bispecific T cell engager (bsTCE) directed against PD-L1 could combine lysis of PD-L1+ tumor cells with PD-L1 immune checkpoint blockade.MethodsPD-L1 specific single domain antibodies (VHHs) were tested for binding to PD-L1 and their ability to interfere with PD-1 binding and function. One PD-L1 VHH was selected for fusion to a Vδ2-T cell receptor specific VHH (PD-L1xVδ2 bsTCE) and tested for its ability to activate Vγ9Vδ2-T cells and lyse melanoma cell lines, as well as patient-derived renal cell carcinoma (RCC) and metastatic melanoma cells. These patient-derived tumor suspensions were also used to explore effects on CD4+ and CD8+ T cells and myeloid cells. Infiltration of Vγ9Vδ2-T cells and tumor kill was tested in a three-dimensional tumor spheroid melanoma model.ResultsA PD-L1xVδ2 bsTCE was generated and shown to block PD-1 binding resulting in the release of PD-1+ cells from PD-L1 mediated inhibition. The PD-L1xVδ2 bsTCE also mediated robust Vγ9Vδ2-T cell activation, efficient lysis of RCC and melanoma cell lines and/or patient-derived tumor cells, and infiltration of Vγ9Vδ2-T cells into a three-dimensional melanoma spheroid model. Of interest, exposure of co-cultures of Vγ9Vδ2-T cells and patient-derived tumor suspensions to the PD-L1xVδ2 bsTCE resulted in upregulation of activation markers on tumor-infiltrated CD4+ and CD8+ T cells and lysis of PD-L1+ myeloid cells with a shift in the myeloid compartment from macrophage-like cells to more mature dendritic cells with costimulatory molecule expression.ConclusionsA PD-L1xVδ2 bsTCE was generated that acts as PD-1/PD-L1 immune checkpoint inhibitor, enhances Vγ9Vδ2-T cell activation, infiltration and tumor lysis and reshapes the tumor microenvironment towards a more proinflammatory state. By targeting both PD-L1 expressing tumor and myeloid cells, it addresses key challenges of current therapies and thereby offers a promising novel therapeutic strategy.

Programmed death-ligand 1 (PD-L1) is expressed in various immune cells and tumor cells, and is able to bind to PD-1 on T lymphocytes, thereby inhibiting their function. At present, the PD-1/PD-L1 axis is a major immunotherapeutic target for checkpoint inhibition in various cancer types, but information on the clinical significance of PD-L1 expression in cervical cancer is largely lacking. Here, we studied PD-L1 expression in paraffin-embedded samples from two cohorts of patients with cervical cancer: primary tumor samples from cohort I (squamous cell carcinoma, n =156 and adenocarcinoma, n =49) and primary and paired metastatic tumor samples from cohort II (squamous cell carcinoma, n =96 and adenocarcinoma, n =31). Squamous cell carcinomas were more frequently positive for PD-L1 and also contained more PD-L1-positive tumor-associated macrophages as compared with adenocarcinomas (both P <0.001). PD-L1-positive tumor-associated macrophages were found to express CD163 and/or CD14 by triple fluorescent immunohistochemistry, demonstrating an M2-like phenotype. Interestingly, disease-free survival ( P =0.022) and disease-specific survival ( P =0.046) were significantly poorer in squamous cell carcinoma patients with diffuse PD-L1 expression as compared with patients with marginal PD-L1 expression (i.e., on the interface between tumor and stroma) in primary tumors. Disease-specific survival was significantly worse in adenocarcinoma patients with PD-L1-positive tumor-associated macrophages compared with adenocarcinoma patients without PD-L1-positive tumor-associated macrophages ( P =0.014). No differences in PD-L1 expression between primary tumors and paired metastatic lymph nodes were detected. However, PD-L1-positive immune cells were found in greater abundance around the metastatic tumors as compared with the paired primary tumors ( P =0.001 for squamous cell carcinoma and P =0.041 for adenocarcinoma). These findings point to a key role of PD-L1 in immune escape of cervical cancer, and provide a rationale for therapeutic targeting of the PD-1/PD-L1 pathway.

BackgroundTumor-draining lymph nodes (TDLN) play a key role in inducing and promoting antitumor immunity. TDLN are commonly situated near the primary tumor and are therefore often exposed to therapeutic radiation. The impact of induction therapies comprising concurrent immunotherapy and radiation on TDLN is poorly understood. We studied the immune-modulating effects in TDLN of patients with T3-4N0-2 non-small cell lung cancer (NSCLC) using a combination of spatial transcriptomics and immunohistochemical analyses.MethodsThis observational cohort study collected TDLN from 2 groups: (1) patients from the INCREASE trial who were treated with neoadjuvant ipilimumab/nivolumab (IPI/NIVO) plus chemoradiotherapy (CRT) (n=25) and (2) a matched control cohort of patients with NSCLC who had neoadjuvant CRT only (n=25). TDLN were classified based on the cumulative dose of radiation received, categorized as low (≤5 Gy), intermediate (20–30 Gy), or high dose (50–60 Gy). The TDLN were subjected to duplex immunohistochemistry of CD8/Ki67, PD-1/FOXP3, and CD8/cleaved caspase-3. On a subset of TDLN, additional GeoMx spatial transcriptomics profiling of CD8/Ki67+T cell hotspots was carried out.ResultsAfter addition of IPI/NIVO to CRT in the INCREASE cohort, resected TDLN showed robust type I immune responses and increased levels of CD8 and regulatory T cells in irradiated TDLN when compared with the control cohort. These immune responses were observed across all radiation dose groups, with the most pronounced effects in high dose TDLN. Significant changes in extracellular matrix and macrophage-associated gene signatures, indicating elevated fibrosis and prolonged inflammation, were observed in the TDLN with high radiation exposure, changes which were partially alleviated by immunotherapy.ConclusionIn the INCREASE trial, neoadjuvant IPI/NIVO combined with CRT for patients with T3–4N0–2 NSCLC elicited enhanced immune responses in TDLN despite exposure to high dose radiation. Even though radiation-induced fibrosis was evident in high dose TDLN, the immune responses were not diminished when compared with low dose TDLN. These findings underscore the resilience of TDLN immunological function under intense radiation exposure.

Background Stereotactic ablative radiotherapy (SABR) has emerged as a new treatment option for patients with oligometastatic disease. SABR delivers precise, high-dose, hypofractionated radiotherapy, and achieves excellent rates of local control for primary tumors or metastases. A recent randomized phase II trial evaluated SABR in a group of patients with a small burden of oligometastatic disease (mostly with 1–3 metastatic lesions), and found that SABR was associated with benefits in progression-free survival and overall survival. The goal of this phase III trial is to assess the impact of SABR in patients with 4–10 metastatic cancer lesions. Methods One hundred and fifty-nine patients will be randomized in a 1:2 ratio between the control arm (consisting of standard of care palliative-intent treatments), and the SABR arm (consisting of standard of care treatment + SABR to all sites of known disease). Randomization will be stratified by two factors: histology (Group 1: prostate, breast, or renal; Group 2: all others), and type of pre-specified systemic therapy (Group 1: immunotherapy/targeted; Group 2: cytotoxic; Group 3: observation). SABR is to be completed within 2 weeks, allowing for rapid initiation of systemic therapy. Recommended SABR doses are 20 Gy in 1 fraction, 30 Gy in 3 fractions, or 35 Gy in 5 fractions, chosen to minimize risks of toxicity. The primary endpoint is overall survival, and secondary endpoints include progression-free survival, time to development of new metastatic lesions, quality of life, and toxicity. Translational endpoints include assessment of circulating tumor cells, cell-free DNA, and tumor tissue as prognostic and predictive markers, including assessment of immunological predictors of response and long-term survival. Discussion This study will provide an assessment of the impact of SABR on clinical outcomes and quality of life, to determine if long-term survival can be achieved for selected patients with 4–10 oligometastatic lesions. Trial registration Clinicaltrials.gov identifier: NCT03721341 . Date of registration: October 26, 2018.

Noncoding regulatory microRNAs (miRNAs) of cellular and viral origin control gene expression by repressing the translation of mRNAs into protein. Interestingly, miRNAs are secreted actively through small vesicles called \"exosomes\" that protect them from degradation by RNases, suggesting that these miRNAs may function outside the cell in which they were produced. Here we demonstrate that miRNAs secreted by EBV-infected cells are transferred to and act in uninfected recipient cells. Using a quantitative RT-PCR approach, we demonstrate that mature EBV-encoded miRNAs are secreted by EBV-infected B cells through exosomes. These EBV-miRNAs are functional because internalization of exosomes by MoDC results in a dose-dependent, miRNA-mediated repression of confirmed EBV target genes, including CXCL11/ITAC, an immunoregulatory gene down-regulated in primary EBV-associated lymphomas. We demonstrate that throughout coculture of EBV-infected B cells EBV-miRNAs accumulate in noninfected neighboring MoDC and show that this accumulation is mediated by transfer of exosomes. Thus, the exogenous EBV-miRNAs transferred through exosomes are delivered to subcellular sites of gene repression in recipient cells. Finally, we show in peripheral blood mononuclear cells from patients with increased EBV load that, although EBV DNA is restricted to the circulating B-cell population, EBV BART miRNAs are present in both B-cell and non-B-cell fractions, suggestive of miRNA transfer. Taken together our findings are consistent with miRNA-mediated gene silencing as a potential mechanism of intercellular communication between cells of the immune system that may be exploited by the persistent human γ-herpesvirus EBV.

Natural killer (NK) cells are critical immune effector cells in the fight against cancer. As NK cells in cancer patients are highly dysfunctional and reduced in number, adoptive transfer of large numbers of cytolytic NK cells and their potential to induce relevant antitumor responses are widely explored in cancer immunotherapy. Early studies from autologous NK cells have failed to demonstrate significant clinical benefit. In this review, the clinical benefits of adoptively transferred allogeneic NK cells in a transplant and non-transplant setting are compared and discussed in the context of relevant NK cell platforms that are being developed and optimized by various biotech industries with a special focus on augmenting NK cell functions.

Lymph nodes draining the primary tumor are essential for the initiation of an effective anti-tumor T-cell immune response. However, cancer-derived immune suppressive factors render the tumor-draining lymph nodes (TDLN) immune compromised, enabling tumors to invade and metastasize. Unraveling the different mechanisms underlying this immune escape will inform therapeutic intervention strategies to halt tumor spread in early clinical stages. Here, we review our findings from translational studies in melanoma, breast, and cervical cancer and discuss clinical opportunities for local immune modulation of TDLN in each of these indications.