PD-L1 checkpoint blockade promotes regulatory T cell activity that underlies therapy resistance.

Despite the clinical success of immune checkpoint blockade (ICB), in certain cancer types, most patients with cancer do not respond well. Furthermore, in patients for whom ICB is initially successful, this is often short-lived because of the development of resistance to ICB. The mechanisms underlying primary or secondary ICB resistance are incompletely understood. Here, we identified preferential activation and enhanced suppressive capacity of regulatory T cells (Treg cells) in αPD-L1 therapy-resistant solid tumor-bearing mice. Treg cell depletion reversed resistance to αPD-L1 with concomitant expansion of effector T cells. Moreover, we found that tumor-infiltrating Treg cells in human patients with skin cancer, and in patients with non-small cell lung cancer, up-regulated a suppressive transcriptional gene program after ICB treatment, which correlated with lack of treatment response. αPD-1/PD-L1-induced PD-1+ Treg cell activation was also seen in peripheral blood of patients with lung cancer and mesothelioma, especially in nonresponders. Together, these data reveal that treatment with αPD-1 and αPD-L1 unleashes the immunosuppressive role of Treg cells, resulting in therapy resistance, suggesting that Treg cell targeting is an important adjunct strategy to enhance therapeutic efficacy.

Combination of PD-1/PD-L1 checkpoint inhibition and dendritic cell therapy in mice models and in patients with mesothelioma.

Immunotherapy with anti-PD1/PD-L1 is effective in only a subgroup of patients with malignant pleural mesothelioma (MPM). We investigated the efficacy of a combination of anti-PD1/PD-L1 and dendritic cell (DC) therapy to optimally induce effective anti-tumor immunity in MPM in both humans and mice. Data of nine MPM patients treated with DC therapy and sequential anti-PD1 treatment were collected and analyzed for progression-free survival (PFS) and overall survival (OS). Survival and T-cell responses were monitored in AC29 mesothelioma-bearing mice treated concurrently with the combination therapy; additionally, the role of the tumor-draining lymph node (TDLN) was investigated. The combination therapy resulted in a median OS and PFS of 17.7 and 8.0 months, respectively. Grade 3 to 4 treatment-related adverse events had not been reported. Survival of the mesothelioma-bearing mice treated with the combination therapy was longer than that of untreated mice, and coincided with improved T-cell activation in peripheral blood and less T-cell exhaustion in end stage tumors. Comparable results were obtained when solely the TDLN was targeted. We concluded that this combination therapy is safe and shows promising OS and PFS. The murine data support that PD-L1 treatment may reinvigorate the T-cell responses induced by DC therapy, which may primarily be the result of TDLN targeting.

Immunomodulatory Effects of Stereotactic Body Radiotherapy and Vaccination with Heat-Killed Mycobacterium Obuense (IMM-101) in Patients with Locally Advanced Pancreatic Cancer.

BACKGROUND: Patients with locally advanced pancreatic cancer (LAPC) are treated with chemotherapy. In selected cases, stereotactic body radiotherapy (SBRT) can be added to the regimen. We hypothesized that adding an adjuvant containing a heat-killed mycobacterium (IMM-101) to SBRT may lead to beneficial immuno-modulatory effects, thereby improving survival. This study aims to investigate the safety of adding IMM-101 to SBRT and to investigate the immuno-modulatory effects of the combination treatment in the peripheral blood of LAPC patients. METHODS: LAPC patients were treated with SBRT (40 Gy) and six intradermal vaccinations of one milligram IMM-101. The primary endpoint was an observed toxicity rate of grade 4 or higher. Targeted gene-expression profiling and multicolor flow cytometry were performed for longitudinal immune-monitoring of the peripheral blood. RESULTS: Twenty patients received study treatment. No treatment-related adverse events of grade 4 or higher occurred. SBRT/IMM-101 treatment induced a transient decrease in different lymphocyte subsets and an increase in CD14+CD16-CD11b+HLA-DRlow myeloid-derived suppressor cells. Importantly, treatment significantly increased activated ICOS+, HLA-DR+ and Ki67+PD1+ T and NK cell frequencies. This was not accompanied by increased levels of most inhibitory markers, such as TIM-3 and LAG-3. CONCLUSIONS: Combination therapy with SBRT and a heat-killed mycobacterium vaccine was safe and had an immune-stimulatory effect.

Lurbinectedin shows clinical activity and immune-modulatory functions in patients with pre-treated small cell lung cancer and malignant pleural mesothelioma.

PURPOSE: Lurbinectedin is a promising new drug being investigated in pre-treated patients with small cell lung cancer (SCLC) or malignant pleural mesothelioma (MPM). Its clinical activity in the real-world setting has not been investigated yet. PATIENTS AND METHODS: Clinical data of patients with SCLC and MPM who were treated with lurbinectedin were prospectively collected. Comprehensive immune cell profiling by flow cytometry was performed on screening and treating peripheral blood samples. RESULTS: A total of 95 patients (43 SCLC and 52 MPM) were treated, mostly as ≥3-line of therapy. In the SCLC cohort, a median progression-free survival (mPFS) was 1.5 months (95% CI: 1.4-3.0), and median overall survival was 7.0 months (95% CI: 4.7-not reached). Objective radiological response and disease control rate after 12 weeks were 16% and 28%, respectively. In the MPM cohort, median progression-free survival was 2.8 months (95% CI: 1.4-4.2), and median overall survival was 7.2 months (95% CI: 5.9-not reached). Disease control rate after 12 weeks was 29%, whereas no partial responses were registered. No new safety signals were observed. Lurbinectedin treatment was significantly associated with the depletion of circulating classical monocytes, which correlated with a better PFS in patients with SCLC. Lurbinectedin increased the proliferation of CD4+ and CD8+ T cells (SCLC) and natural killer and natural killer T cells (SCLC and MPM) and altered co-stimulatory and co-inhibitory receptor expression on circulating lymphocytes. CONCLUSION: Lurbinectedin has a manageable safety profile and shows clinical activity in pre-treated patients with SCLC and MPM. Its immune-modulatory functions make lurbinectedin a potential platform for immunotherapy combinations.

Low-Dose JAK3 Inhibition Improves Antitumor T-Cell Immunity and Immunotherapy Efficacy.

Terminal T-cell exhaustion poses a significant barrier to effective anticancer immunotherapy efficacy, with current drugs aimed at reversing exhaustion being limited. Recent investigations into the molecular drivers of T-cell exhaustion have led to the identification of chronic IL2 receptor (IL2R)-STAT5 pathway signaling in mediating T-cell exhaustion. We targeted the key downstream IL2R-intermediate JAK 3 using a clinically relevant highly specific JAK3-inhibitor (JAK3i; PF-06651600) that potently inhibited STAT5-phosphorylation in vitro. Whereas pulsed high-dose JAK3i administration inhibited antitumor T-cell effector function, low-dose chronic JAK3i significantly improved T-cell responses and decreased tumor load in mouse models of solid cancer. Low-dose JAK3i combined with cellular and peptide vaccine strategies further decreased tumor load compared with both monotherapies alone. Collectively, these results identify JAK3 as a novel and promising target for combination immunotherapy.

Immune monitoring in mesothelioma patients identifies novel immune-modulatory functions of gemcitabine associating with clinical response.

BACKGROUND: Gemcitabine is a frequently used chemotherapeutic agent but its effects on the immune system are incompletely understood. Recently, the randomized NVALT19-trial revealed that maintenance gemcitabine after first-line chemotherapy significantly prolonged progression-free survival (PFS) compared to best supportive care (BSC) in malignant mesothelioma. Whether these effects are paralleled by changes in circulating immune cell subsets is currently unknown. These analyses could offer improved mechanistic insights into the effects of gemcitabine on the host and guide development of effective combination therapies in mesothelioma. METHODS: We stained peripheral blood mononuclear cells (PBMCs) and myeloid-derived suppressor cells (MDSCs) at baseline and 3 weeks following start of gemcitabine or BSC treatment in a subgroup of mesothelioma patients included in the NVALT19-trial. In total, 24 paired samples including both MDSCs and PBMCs were included. We performed multicolour flow-cytometry to assess co-inhibitory and-stimulatory receptor- and cytokine expression and matched these parameters with PFS and OS. FINDINGS: Gemcitabine treatment was significantly associated with an increased NK-cell- and decreased T-regulatory cell proliferation whereas the opposite occurred in control patients. Furthermore, myeloid-derived suppressor cells (MDSCs) frequencies were lower in gemcitabine-treated patients and this correlated with increased T-cell proliferation following treatment. Whereas gemcitabine variably altered co-inhibitory receptor expression, co-stimulatory molecules including ICOS, CD28 and HLA-DR were uniformly increased across CD4+ T-helper, CD8+ T- and NK-cells. Although preliminary in nature, the increase in NK-cell proliferation and PD-1 expression in T cells following gemcitabine treatment was associated with improved PFS and OS. INTERPRETATION: Gemcitabine treatment was associated with widespread effects on circulating immune cells of mesothelioma patients with responding patients displaying increased NK-cell and PD-1 + T-cell proliferation. These exploratory data provide a platform for future on treatment-biomarker development and novel combination treatment strategies.

The PD-1/PD-L1-Checkpoint Restrains T cell Immunity in Tumor-Draining Lymph Nodes.

PD-1/PD-L1-checkpoint blockade therapy is generally thought to relieve tumor cell-mediated suppression in the tumor microenvironment but PD-L1 is also expressed on non-tumor macrophages and conventional dendritic cells (cDCs). Here we show in mouse tumor models that tumor-draining lymph nodes (TDLNs) are enriched for tumor-specific PD-1+ T cells which closely associate with PD-L1+ cDCs. TDLN-targeted PD-L1-blockade induces enhanced anti-tumor T cell immunity by seeding the tumor site with progenitor-exhausted T cells, resulting in improved tumor control. Moreover, we show that abundant PD-1/PD-L1-interactions in TDLNs of nonmetastatic melanoma patients, but not those in corresponding tumors, associate with early distant disease recurrence. These findings point at a critical role for PD-L1 expression in TDLNs in governing systemic anti-tumor immunity, identifying high-risk patient groups amendable to adjuvant PD-1/PD-L1-blockade therapy.

Dendritic cell vaccination and CD40-agonist combination therapy licenses T cell-dependent antitumor immunity in a pancreatic carcinoma murine model.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is notoriously resistant to treatment including checkpoint-blockade immunotherapy. We hypothesized that a bimodal treatment approach consisting of dendritic cell (DC) vaccination to prime tumor-specific T cells, and a strategy to reprogram the desmoplastic tumor microenvironment (TME) would be needed to break tolerance to these pancreatic cancers. As a proof-of-concept, we investigated the efficacy of combined DC vaccination with CD40-agonistic antibodies in a poorly immunogenic murine model of PDAC. Based on the rationale that mesothelioma and pancreatic cancer share a number of tumor associated antigens, the DCs were loaded with either pancreatic or mesothelioma tumor lysates. METHODS: Immune-competent mice with subcutaneously or orthotopically growing KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) PDAC tumors were vaccinated with syngeneic bone marrow-derived DCs loaded with either pancreatic cancer (KPC) or mesothelioma (AE17) lysate and consequently treated with FGK45 (CD40 agonist). Tumor progression was monitored and immune responses in TME and lymphoid organs were analyzed using multicolor flow cytometry and NanoString analyzes. RESULTS: Mesothelioma-lysate loaded DCs generated cross-reactive tumor-antigen-specific T-cell responses to pancreatic cancer and induced delayed tumor outgrowth when provided as prophylactic vaccine. In established disease, combination with stimulating CD40 antibody was necessary to improve survival, while anti-CD40 alone was ineffective. Extensive analysis of the TME showed that anti-CD40 monotherapy did improve CD8 +T cell infiltration, but these essential effector cells displayed hallmarks of exhaustion, including PD-1, TIM-3 and NKG2A. Combination therapy induced a strong change in tumor transcriptome and mitigated the expression of inhibitory markers on CD8 +T cells. CONCLUSION: These results demonstrate the potency of DC therapy in combination with CD40-stimulation for the treatment of pancreatic cancer and provide directions for near future clinical trials.