Preclinical development of novel PD-L1 tracers and first-in-human study of [68Ga]Ga-NOTA-RW102 in patients with lung cancers.

BACKGROUND: The programmed cell death protein-1 (PD-1)/programmed death receptor ligand 1 (PD-L1) axis critically facilitates cancer cells' immune evasion. Antibody therapeutics targeting the PD-1/PD-L1 axis have shown remarkable efficacy in various tumors. Immuno-positron emission tomography (ImmunoPET) imaging of PD-L1 expression may help reshape solid tumors' immunotherapy landscape. METHODS: By immunizing an alpaca with recombinant human PD-L1, three clones of the variable domain of the heavy chain of heavy-chain only antibody (VHH) were screened, and RW102 with high binding affinity was selected for further studies. ABDRW102, a VHH derivative, was further engineered by fusing RW102 with the albumin binder ABD035. Based on the two targeting vectors, four PD-L1-specific tracers ([68Ga]Ga-NOTA-RW102, [68Ga]Ga-NOTA-ABDRW102, [64Cu]Cu-NOTA-ABDRW102, and [89Zr]Zr-DFO-ABDRW102) with different circulation times were developed. The diagnostic efficacies were thoroughly evaluated in preclinical solid tumor models, followed by a first-in-human translational investigation of [68Ga]Ga-NOTA-RW102 in patients with non-small cell lung cancer (NSCLC). RESULTS: While RW102 has a high binding affinity to PD-L1 with an excellent KD value of 15.29 pM, ABDRW102 simultaneously binds to human PD-L1 and human serum albumin with an excellent KD value of 3.71 pM and 3.38 pM, respectively. Radiotracers derived from RW102 and ABDRW102 have different in vivo circulation times. In preclinical studies, [68Ga]Ga-NOTA-RW102 immunoPET imaging allowed same-day annotation of differential PD-L1 expression with specificity, while [64Cu]Cu-NOTA-ABDRW102 and [89Zr]Zr-DFO-ABDRW102 enabled longitudinal visualization of PD-L1. More importantly, a pilot clinical trial shows the safety and diagnostic value of [68Ga]Ga-NOTA-RW102 immunoPET imaging in patients with NSCLCs and its potential to predict immune-related adverse effects following PD-L1-targeted immunotherapies. CONCLUSIONS: We developed and validated a series of PD-L1-targeted tracers. Initial preclinical and clinical evidence indicates that immunoPET imaging with [68Ga]Ga-NOTA-RW102 holds promise in visualizing differential PD-L1 expression, selecting patients for PD-L1-targeted immunotherapies, and monitoring immune-related adverse effects in patients receiving PD-L1-targeted treatments. TRIAL REGISTRATION NUMBER: NCT06165874.

Rigosertib promotes anti-tumor immunity via autophagic degradation of PD-L1 in colorectal cancer cells.

Rigosertib (RGS) is a benzyl styryl sulfone which exhibits impressive cytotoxicity in cancer cells. However, its modulating effect on tumor immune microenvironment remains elusive. In our experiments, compared with immunodeficient mouse model, increased tumor growth arrest and robust anti-tumor immunity were observed in RGS-treated colorectal cancer (CRC) isograft tumors in immunocompetent mice. Intriguingly, RGS markedly down-regulated programmed cell death ligand 1 (PD-L1) expression in both vivo and in vitro. Meanwhile, RGS increased autophagic vacuole number in CRC cells as seen by transmission electron microscopy and immunofluorescence. Moreover, increased LC3-II level and tandem-mRFP- GFP- LC3 labeled vacuole accumulation demonstrated RGS-induced autophagic flux. Mechanistically, it is the activation of AMP-activated protein kinase-UNC-51-like kinase 1 (AMPK-ULK1) axis, rather than the canonical mTOR signaling pathway, that plays a pivotal role in RGS-induced autophagy. AMPK-ULK1 dependent autophagy inhibition, by either short interfering RNA or chemical inhibitors, blocked RGS-induced PD-L1 degradation. Finally, RGS exhibited synergistic anti-tumor activity with cytotoxic T-lymphocyte-associated protein 4 monoclonal antibody in the CRC isograft model. Furthermore, apart from the immunomodulatory effect, we also confirmed the direct cytotoxicity of RGS in inducing mitochondria-related apoptosis. Altogether, considering its PD-L1 inhibitory and cytotoxic effects, RGS could be a promising drug for CRC therapy.

Bullous pemphigoid associated with anti-programmed cell death protein 1 and anti-programmed cell death ligand 1 therapy: A case series of 13 patients.

We present a case series of 13 patients, the first Australian single-centre study of bullous pemphigoid (BP) associated with immune checkpoint inhibitors (ICI): cytotoxic T-lymphocyte antigen (CTLA4) and programmed cell death receptor (PD1) inhibitors. All our patients achieved adequate control of BP with a combination of treatments including oral prednisolone, intravenous immunoglobulin, rituximab and omalizumab. The majority of patients ceased or interrupted immunotherapy treatment upon diagnosis of BP and greater tumour progression was seen in the cohort who ceased immunotherapy.

PD-1/PD-L1 inhibitor ameliorates silica-induced pulmonary fibrosis by maintaining systemic immune homeostasis.

Pulmonary fibrosis induced by silica particles is defined as silicosis, which is an incurable disease. The pathogenesis of silicosis is not completely clear, but it's certain that immune system dysfunction is closely related to it. Immune checkpoint inhibitors (ICIs) are emerging immunotherapeutic agents that mainly target adaptive immune cells, and there is abundant evidence that ICIs are of great value in cancer treatment. However, whether these attractive agents can be implemented in silicosis treatment is unclear. In this study, we explored the efficacy of small molecule inhibitors targeted PD-1/PD-L1 and CTLA-4 on silica-induced pulmonary fibrosis in mice. ICIs were injected intraperitoneally into mice that received silica instillation twice a week. The mice were sacrificed 7 and 28 days after the injection. The lungs, spleen, hilar lymph nodes, thymus, and peripheral blood of mice were collected and subjected to histological examination, flow cytometry analysis, and mRNA and protein quantification. Our results demonstrated that silica exposure caused damage to multiple immune organs in mice, leading to an imbalance in systemic immune homeostasis. Specifically, proportions and subtypes of T and B cells were significantly altered, and the expressions of PD-1, PD-L1 and CTLA-4 were abnormal on these cells. Both PD-1/PD-L1 and CTLA-4 inhibitor administration modulated silica-induced immune system disruption, however, only PD-1/PD-L1 signaling inhibition showed significant amelioration of silicosis. Our findings confirmed for the first time the potential value of ICIs for the treatment of silica-induced pulmonary fibrosis, and this may provide new ideas for the treatment of other fibrosis-related diseases.

D-mannose facilitates immunotherapy and radiotherapy of triple-negative breast cancer via degradation of PD-L1.

Breast cancer is the most frequent malignancy in women worldwide, and triple-negative breast cancer (TNBC) patients have the worst prognosis and highest risk of recurrence. The therapeutic strategies for TNBC are limited. It is urgent to develop new methods to enhance the efficacy of TNBC treatment. Previous studies demonstrated that D-mannose, a hexose, can enhance chemotherapy in cancer and suppress the immunopathology of autoimmune diseases. Here, we show that D-mannose can significantly facilitate TNBC treatment via degradation of PD-L1. Specifically, D-mannose can activate AMP-activated protein kinase (AMPK) to phosphorylate PD-L1 at S195, which leads to abnormal glycosylation and proteasomal degradation of PD-L1. D-mannose-mediated PD-L1 degradation promotes T cell activation and T cell killing of tumor cells. The combination of D-mannose and PD-1 blockade therapy dramatically inhibits TNBC growth and extends the lifespan of tumor-bearing mice. Moreover, D-mannose-induced PD-L1 degradation also results in messenger RNA destabilization of DNA damage repair-related genes, thereby sensitizing breast cancer cells to ionizing radiation (IR) treatment and facilitating radiotherapy of TNBC in mice. Of note, the effective level of D-mannose can be easily achieved by oral administration in mice. Our study unveils a mechanism by which D-mannose targets PD-L1 for degradation and provides methods to facilitate immunotherapy and radiotherapy in TNBC. This function of D-mannose may be useful for clinical treatment of TNBC.

Vitamin D can reduce severity in COVID-19 through regulation of PD-L1.

COVID-19 is a highly contagious viral infection that has killed millions of people around the world. The most important diagnostic feature of COVID-19 is lymphocyte depletion, particularly the depletion of T cells. In COVID-19 infections, there is a link between destruction of T cells and increased expression of inhibitory immune checkpoint molecules (PD-1/PD-L1) on T cell surfaces. It was shown that PD-1/PD-L1 levels increase in severely COVID-19 infected individuals. Higher proinflammatory cytokine levels cause increased PD-1/PD-L1 expression. In severe COVID-19, higher proinflammatory cytokine levels may increase PD-1/PD-L1. Vitamin-D is an important immune regulator. It is known that the numbers of CD4+ and CD8+ T lymphocytes decrease in vitamin D deficiency while vitamin D supplementation increases CD + 4 lymphocytes. Vitamin D can increase regulatory T cell (Treg) activity. Vitamin D also has a diminishing effect on proinflammatory cytokines. In severe COVID-19 cases, vitamin D supplementation may inhibit the increase of PD-L1 expression through reducing proinflammatory cytokine levels. Thus, vitamin D supplementation could eliminate the suppressive effect of PD-L1 on CD4+ and CD8+ T cells, preventing lymphopenia and reducing disease severity and mortality in patients infected with COVID-19. Besides, vitamin D supplementation can reduce inflammation by increasing Treg activity. The aim of this letter is to discuss the functions of inhibitory immune checkpoint molecules and their effects on dysfunction and depletion of T-cells as well as to explain the possible modulatory effect of vitamin D on these checkpoints and T cells.

Induction Immunotherapy Followed by Thoracic Radiation without Chemotherapy in Unresectable Stage III Non-Small Cell Lung Cancer: A Case Series.

OBJECTIVE: Although concurrent chemoradiation has been the standard of care for unresectable stage III non-small cell lung cancer (NSCLC) due to increased survival and decreased disease progression, patients with poor performance status cannot tolerate chemotherapy toxicity well. Durvalumab, an immune checkpoint inhibitor targeting the programmed death receptor-1 (PD-1) / programmed death-ligand 1 (PD-L1) axis, demonstrated efficacy as maintenance therapy after definitive chemoradiation. However, the role of immunotherapy in those who cannot tolerate chemoradiation is unclear. METHODS: This retrospective case series reports adult patients with PD-L1-expressing stage III NSCLC diagnosed at Parkview Cancer Institute from 2019-2021 and treated initially with pembrolizumab followed by sequential consolidation chest radiation (CXRT) without cytotoxic chemotherapy. RESULTS: Four cases of stage IIIA squamous cell carcinoma were disease-controlled by this approach, with two partial and one complete response. One case of stage IIIC adenocarcinoma had progressive disease with brain metastasis prior to CXRT. CONCLUSION: This case series suggests that pembrolizumab with sequential CXRT may be beneficial for stage III NSCLC patients with high PD-L1 expression, but additional studies are needed to confirm this hypothesis.

Combination of epigenetic regulation with gene therapy-mediated immune checkpoint blockade induces anti-tumour effects and immune response in vivo.

Immunotherapy has become a powerful cancer treatment, but only a small fraction of patients have achieved durable benefits due to the immune escape mechanism. In this study, epigenetic regulation is combined with gene therapy-mediated immune checkpoint blockade to relieve this immune escape mechanism. PPD (i.e., mPEG-b-PLG/PEI-RT3/DNA) is developed to mediate plasmid-encoding shPD-L1 delivery by introducing multiple interactions (i.e., electrostatic, hydrogen bonding, and hydrophobic interactions) and polyproline II (PPII)-helix conformation, which downregulates PD-L1 expression on tumour cells to relieve the immunosuppression of T cells. Zebularine (abbreviated as Zeb), a DNA methyltransferase inhibitor (DNMTi), is used for the epigenetic regulation of the tumour immune microenvironment, thus inducing DC maturation and MHC I molecule expression to enhance antigen presentation. PPD plus Zeb combination therapy initiates a systemic anti-tumour immune response and effectively prevents tumour relapse and metastasis by generating durable immune memory. This strategy provides a scheme for tumour treatment and the inhibition of relapse and metastasis.

Metronomic delivery of orally available pemetrexed-incorporated colloidal dispersions for boosting tumor-specific immunity.

In this study, we developed oral pemetrexed (PMX) for metronomic dosing to enhance antitumor immunity. PMX was electrostatically complexed with positively charged lysine-linked deoxycholic acid (DL) as an intestinal permeation enhancer, forming PMX/DL, to enhance its intestinal permeability. PMX/DL was also incorporated into a colloidal dispersion (CD) comprised of the block copolymer of poly(ethylene oxide) and poly(propylene oxide), and caprylocaproyl macrogol-8 glycerides (PMX/DL-CD). CD-containing PMX/DL complex in a 1:1 molar ratio [PMX/DL(1:1)-CD] showed 4.66- and 7.19-fold greater permeability than free PMX through the Caco-2 cell monolayer and rat intestine, respectively. This resulted in a 282% improvement in oral bioavailability in rats. In addition, low-dose metronomic PMX led to more immunogenic cell death in CT26.CL25 cells compared to high PMX concentrations at the maximum tolerated dose. In CT26.CL25 tumor-bearing mice, oral metronomic PMX/DL-CD elicited greater antitumor immunity not only by enhancing the number of tumor-infiltrating lymphocytes but also by suppressing T cell functions. Oral PMX/DL-CD substantially increased programmed cell death protein ligand-1 (PD-L1) expression on tumor cells compared to the control and PMX-IV groups. This increased antitumor efficacy in combination with anti-programmed cell death protein-1 (aPD-1) antibody in terms of tumor rejection and immunological memory compared to the combination of PMX-IV and aPD-1. These results suggest that oral metronomic scheduling of PMX/DL-CD in combination with immunotherapy has synergistic antitumor effects.

Synergy of nanodiamond-doxorubicin conjugates and PD-L1 blockade effectively turns tumor-associated macrophages against tumor cells.

BACKGROUND: Tumor-associated macrophages (TAMs) are the most abundant stromal cells in the tumor microenvironment. Turning the TAMs against their host tumor cells is an intriguing therapeutic strategy particularly attractive for patients with immunologically "cold" tumors. This concept was mechanistically demonstrated on in vitro human and murine lung cancer cells and their corresponding TAM models through combinatorial use of nanodiamond-doxorubicin conjugates (Nano-DOX) and a PD-L1 blocking agent BMS-1. Nano-DOX are an agent previously proved to be able to stimulate tumor cells' immunogenicity and thereby reactivate the TAMs into the anti-tumor M1 phenotype. RESULTS: Nano-DOX were first shown to stimulate the tumor cells and the TAMs to release the cytokine HMGB1 which, regardless of its source, acted through the RAGE/NF-κB pathway to induce PD-L1 in the tumor cells and PD-L1/PD-1 in the TAMs. Interestingly, Nano-DOX also induced NF-κB-dependent RAGE expression in the tumor cells and thus reinforced HMGB1's action thereon. Then, BMS-1 was shown to enhance Nano-DOX-stimulated M1-type activation of TAMs both by blocking Nano-DOX-induced PD-L1 in the TAMs and by blocking tumor cell PD-L1 ligation with TAM PD-1. The TAMs with enhanced M1-type repolarization both killed the tumor cells and suppressed their growth. BMS-1 could also potentiate Nano-DOX's action to suppress tumor cell growth via blocking of Nano-DOX-induced PD-L1 therein. Finally, Nano-DOX and BMS-1 achieved synergistic therapeutic efficacy against in vivo tumor grafts in a TAM-dependent manner. CONCLUSIONS: PD-L1/PD-1 upregulation mediated by autocrine and paracrine activation of the HMGB1/RAGE/NF-κB signaling is a key response of lung cancer cells and their TAMs to stress, which can be induced by Nano-DOX. Blockade of Nano-DOX-induced PD-L1, both in the cancer cells and the TAMs, achieves enhanced activation of TAM-mediated anti-tumor response.

Arsenic sulfide reverses cisplatin resistance in non-small cell lung cancer in vitro and in vivo through targeting PD-L1.

BACKGROUND: Recent studies have found that programmed death ligand 1 (PD-L1) might be involved in chemotherapy resistance in non-small cell lung cancer (NSCLC). Arsenic sulfide (As4 S4 ) has been recognized to have antitumor activities and enhance the cytotoxic effect of chemotherapy drugs. In this study, we aimed to verify the relationship between PD-L1 and cisplatin (DDP) resistance and identify whether As4 S4 could reverse DDP resistance through targeting PD-L1 in NSCLC. METHODS: The effect of As4 S4 and DDP on cell proliferation and apoptosis was investigated in NSCLC cell lines. The expression of p53 and PD-L1 proteins was measured by western blotting analysis. The levels of miR-34a-5p, miR-34a-3p and PD-L1 in cells were measured by real-time qPCR analysis. Mouse xenograft models were established by inoculation with A549/DDP (DDP-resistant) cells. RESULTS: Depletion of PD-L1 inhibited DDP resistance in A549/DDP and H1299/DDP cells. As4 S4 was capable of sensitizing A549/DDP cells to DDP by enhancing apoptosis. As4 S4 upregulated p53 expression and downregulated PD-L1 expression in A549/DDP cells. As4 S4 increased miR-34a-5p level in A549/DDP cells. Inhibition of p53 by PFT-α partially restored the levels of PD-L1 and miR-34a-5p. Pretreatment with PFT-α suppressed the apoptosis rate induced by cotreatment of As4 S4 and DDP in A549/DDP cells. Cotreatment of DDP and As4 S4 notably reduced the tumor size when compared with DDP treatment alone in vivo. CONCLUSIONS: Upregulation of PD-L1 was correlated with DDP resistance in NSCLC cells. Mechanistic analyses indicated that As4 S4 might sensitize NSCLC cells to DDP through targeting p53/miR-34a-5p/PD-L1 axis.

Clinical Response to Immunotherapy Targeting Programmed Cell Death Receptor 1/Programmed Cell Death Ligand 1 in Patients With Treatment-Resistant Microsatellite Stable Colorectal Cancer With and Without Liver Metastases.

Importance: Microsatellite stable (MSS) metastatic colorectal cancer has been historically characterized as resistant to immunotherapy. Recent studies have demonstrated limited clinical activity of programmed cell death receptor 1/programmed death ligand 1 (PD-1/PD-L1) targeting in MSS metastatic colorectal cancer. The association of metastatic disease in the liver with treatment response has not been fully investigated. Objective: To investigate the association of liver metastases with response to PD-1/PD-L1-targeting therapy in MSS metastatic colorectal cancer. Design, Setting, and Participants: This single-center retrospective cohort study evaluated clinical responses to PD-1- or PD-L1-targeting therapy, with or without other investigational agents, in patients with MSS metastatic colorectal cancer and disease progression after standard of care therapy from January 1, 2014, to December 31, 2020. Main Outcomes and Measures: Objective response rate (ORR) and progression-free survival (PFS), measured from initiation of PD-1/PD-L1-targeting therapy. Results: Ninety-five patients with MSS metastatic colorectal cancer were identified (54 men [56.8%]; median age, 55 [interquartile range (IQR), 49-64] years). The overall ORR was 8.4% (8 of 95 patients). Eight of 41 patients without liver metastases achieved an ORR of 19.5%, and no response was observed in 54 patients with liver metastases. The disease control rate was 58.5% (24 of 41) in patients without liver metastasis and 1.9% (1 of 54) in patients with liver metastasis. Patients without liver metastases at the time of PD-1/PD-L1-targeting treatment had a superior median PFS compared with patients with liver metastases (4.0 [IQR, 2.0-7.5] vs 1.5 [IQR, 1.0-2.0] months; P < .001). In addition, median PFS was 5.5 (IQR, 2.0-11.5) months for patients without any prior or current liver involvement at the time of PD-1/PD-L1-targeting treatment initiation. Using a multivariate Cox regression model correcting for Eastern Cooperative Oncology Group status, primary tumor location, RAS and BRAF status, tumor mutation burden, and metastatic sites, liver metastases was the variable with the most significant association with faster progression after PD-1/PD-L1 treatment inhibition (hazard ratio, 7.00; 95% CI, 3.18-15.42; P < .001). Conclusions and Relevance: Findings of this cohort study suggest that patients with MSS metastatic colorectal cancer and without liver metastases may derive clinical benefits from checkpoint inhibitors, whereas the presence of liver metastases was associated with resistance. Further prospective studies are needed to investigate PD-1/PD-L1 inhibitors in patients with MSS metastatic colorectal cancer without liver metastases.

Peptide vaccine-conjugated mesoporous carriers synergize with immunogenic cell death and PD-L1 blockade for amplified immunotherapy of metastatic spinal.

The clinical treatment of metastatic spinal tumor remains a huge challenge owing to the intrinsic limitations of the existing methods. Programmed cell death protein 1 (PD1)/programmed cell death ligand 1 (PD-L1) pathway blockade has been explored as a promising immunotherapeutic strategy; however, their inhibition has a low response rate, leading to the minimal cytotoxic T cell infiltration. To ameliorate the immunosuppressive microenvironment of intractable tumor and further boost the efficacy of immunotherapy, we report an all-round mesoporous nanocarrier composed of an upconverting nanoparticle core and a large-pore mesoporous silica shell (UCMS) that is simultaneously loaded with photosensitizer molecules, the IDO-derived peptide vaccine AL-9, and PD-L1 inhibitor. The IDO-derived peptide can be recognized by the dendritic cells and presented to CD8+ cytotoxic T cells, thereby enhancing the immune response and promoting the killing of the IDO-expressed tumor cells. Meanwhile, the near-infrared (NIR) activated photodynamic therapy (PDT) could induce immunogenic cell death (ICD), which promotes the effector T-cell infiltration. By combining the PDT-elicited ICD, peptide vaccine and immune checkpoint blockade, the designed UCMS@Pep-aPDL1 successfully potentiated local and systemic antitumor immunity and reduced the progression of metastatic foci, demonstrating a synergistic strategy for cancer immunotherapy.

Carbon nanotubes (CNT)-loaded ginsenosides Rb3 suppresses the PD-1/PD-L1 pathway in triple-negative breast cancer.

Carbon nanotubes (CNTs), as advanced nanotechnology with specific properties and structures, have presented practical drug delivery properties. Ginsenoside Rg3 is a component of puffed ginseng and demonstrates anti-cancer activities. To explore the effect of CNTs-loaded Rg3 (Rg3-CNT) on the PD-1/PD-L1 signaling and the development of triple-negative breast cancer (TNBC). Our data revealed that Rg3 inhibited the cell viability of TNBC cells, in which Rg3-CNT further enhanced this effect in the system. Similarly, the colony formation of TNBC cells was decreased by Rg3, while Rg3-CNT could reinforce its effect in the cells. Besides, the treatment of Rg3 induced apoptosis of TNBC cells, in which Rg3-CNT treatment further increased the phenotype in the cells. Remarkably, Rg3-CNT, but not Rg3, attenuated PD-L1 expression in TNBC cells. Rg3-CNT decreased the PD-L1 upregulation induced by interferon-γ (IFN-γ) in breast cancer cells. Importantly, Rg3-CNT was able to reduce PD-1 expression in activated T cells. Specifically, Rg3-CNT reduced the PD-1/PD-L1 axis in a T cell/triple-negative TNBC cell co-culture system. Moreover, the levels of IFN-γ, interleukins-2 (IL-2), interleukins-9 (IL-9), interleukins-10 (IL-10), interleukins-22 (IL-22), and interleukins-23 (IL-23) were significantly stimulated in the activated T cells, while the treatment of Rg3-CNT could reverse these phenotypes in the cells. Rg3-CNT attenuated the TNBC cell growth in vivo. The Rg3-CNT improved the anti-cancer effect of Rg3 toward TNBC by inhibiting the PD-1/PD-L1 axis. Our finding provides new insights into the mechanism by which Rg3-CNT attenuates the development of TNBC. Rg3-CNT may be applied as the potential therapeutic strategy for immunotherapy of TNBC.

Targeted PD-L1 PLGA/liposomes-mediated luteolin therapy for effective liver cancer cell treatment.

Stealth PLGA/Liposome nanoparticles (NPs) modified with tumor-targeting PD-L1 antibody for systemic delivery of luteolin for liver cancer were prepared. The morphologies and therapeutic effects of luteolin-loaded PD-L1 targeted stealth PLGA/Liposomes (L-PD-SP/Ls) in vitro were analyzed. Functional L-PD-P/L NPs composed of PLGA, DOPC and DSPE-PEG display low cell cytoxicity in HepG2 cells, and has more cell uptake ability than P/Ls NPs. L-PD-SP/Ls was more effective in inhibiting HepG2 cell proliferation than free luteolin in solution (p < 0.05) and luteolin-loaded P/Ls (p < 0.05). Compared with the cell control group and the non-PD-L1 targeted group, the mediated effect of PD-L1 can significantly enhance the uptake of drugs by cells, and L-PD-SP/Ls can significantly reduce the expression of Bcl-2 and increase the level of LDH in cells. Our findings collectively support the utility of PD-L1-targeted P/L NPs as a potentially effective drug delivery system.

PD-L1 expression in gastric cancer: interchangeability of 22C3 and 28-8 pharmDx assays for responses to immunotherapy.

Recent clinical trials have shown the promising therapeutic effects of pembrolizumab and nivolumab in patients with advanced gastric cancer. Currently, the programmed death ligand-1 (PD-L1) 22C3 pharmDx assay is the only companion diagnostic assay for assessing the safety and effectiveness of pembrolizumab. The purpose of this study was to compare 22C3 pharmDx and 28-8 pharmDx, a complementary diagnostic assay for nivolumab, in gastric cancer. In this study, 22C3 and 28-8 pharmDx assays were performed on the same formalin-fixed, paraffin-embedded tissue blocks of gastric adenocarcinoma clinical samples (n = 55). The concordance rate was evaluated using combined positive score (CPS) cutoffs of 1, 10, and 50. PD-L1 positivity with CPS ≥ 1 was 45.5% using the 22C3 pharmDx assay and 49.1% using the 28-8 pharmDx assay. At a CPS cutoff of 1, the overall percentage agreement was 96.4%. The positive and negative percentage agreements were 93.3% and 100%, respectively. All cases positive for PD-L1 using the 22C3 pharmDx assay were also positive using the 28-8 pharmDx assay. At a CPS cutoff of 10, the overall percentage agreement was 96.4%. At a CPS cutoff of 50, the two assays exhibited 100% concordance. Nonspecific cytoplasmic staining in the background tissues and tumor cells was often observed in the 28-8 pharmDx assay. When the results of the two assays were matched for response to immunotherapy, the overall response rate was higher in patients with a PD-L1 CPS ≥ 1 than in PD-L1-negative patients (22C3 pharmDx, P = 0.001; 28-8 pharmDx, P = 0.002). In conclusion, PD-L1 22C3 and 28-8 pharmDx assays were highly comparable at CPS cutoffs of 1, 10, and 50 in gastric cancer. These results provide evidence for the potential interchangeability of the two PD-L1 assays in gastric cancer.

Ex vivo modelling of PD-1/PD-L1 immune checkpoint blockade under acute, chronic, and exhaustion-like conditions of T-cell stimulation.

Blockade of PD-1/PD-L1 interactions is proving an exciting, durable therapeutic modality in a range of cancers whereby T cells are released from checkpoint inhibition to revive their inherent anti-tumour activity. Here we have studied various ways to model ex vivo T cell function in order to compare the impact of the clinically utilised anti-PD-1 antibody, pembrolizumab (Keytruda) on the activation of human T cells: focussing on the release of pro-inflammatory IFNγ and anti-inflammatory IL-10 to assess functionality. Firstly, we investigated the actions of pembrolizumab in an acute model of T-cell activation with either immature or mature allogeneic dendritic cells (DCs); pembrolizumab enhanced IFNγ and IL-10 release from purified CD4+ T-cells in the majority of donors with a bias towards pro-inflammatory cytokine release. Next, we modelled the impact of pembrolizumab in settings of more chronic T-cell activation. In a 7-day antigen-specific response to EBV peptides, the presence of pembrolizumab resulted in a relatively modest increase in both IFNγ and IL-10 release. Where pembrolizumab was assessed against long-term stimulated CD4+ cells that had up-regulated the exhaustion markers TIM-3 and PD-1, there was a highly effective enhancement of the otherwise exhausted response to allogeneic DCs with respect to IFNγ production. By contrast, the restoration of IL-10 production was considerably more limited. Finally, to assess a direct clinical relevance we investigated the consequence of PD-1/PD-L1 blockade in the disease setting of dissociated cells from lung and colon carcinomas responding to allogeneic DCs: here, pembrolizumab once more enhanced IFNγ production from the majority of tumour preparations whereas, again, the increase in IL-10 release was modest at best. In conclusion, we have shown that the contribution of PD-1-revealed by using a canonical blocking antibody to interrupt its interaction with PD-L1-to the production of an exemplar pro- and anti-inflammatory cytokine, respectively, depends in magnitude and ratio on the particular stimulation setting and activation status of the target T cell. We have identified a number of in vitro assays with response profiles that mimic features of dissociated cell populations from primary tumours thereby indicating these represent disease-relevant functional assays for the screening of immune checkpoint inhibitors in current and future development. Such in vitro assays may also support patient stratification of those likely to respond to immuno-oncology therapies in the wider population.

PD-L1 positively regulates MET phosphorylation through inhibiting PTP1B.

Increasing bodies of evidence support the involvement of tumor-intrinsic action in PD-L1-mediated cancer progression. However, the mechanisms underlying the tumor-intrinsic function of PD-L1 are less well understood. In the present study, we found a positive correlation between PD-L1 expression and MET phosphorylation in lung cancer and melanoma cell lines. PD-L1 inhibition led to a decrease in MET phosphorylation, while PD-L1 induction by IFN-γ resulted in a PD-L1-dependent increase of MET phosphorylation both in vitro and in vivo. The results indicated that MET phosphorylation can be positively regulated by PD-L1. Furthermore, we identified PTP1B as a mediator contributing to the regulation of MET phosphorylation by PD-L1. In agreement with the induction of MET phosphorylation by PD-L1, inhibition of PD-L1 caused reduced phosphorylation of ERKs, a known downstream kinase of MET, and inhibited cell proliferation. Collectively, the present study demonstrated for the first time that the MET pathway, as a downstream of PD-L1, contributed to its tumor-intrinsic effect, and provided a novel mechanistic explanation for the tumor-intrinsic function of PD-L1 and a rationale for the combination of immunotherapy and MET-targeted therapy in cancer treatment.

Targeting PIM1-Mediated Metabolism in Myeloid Suppressor Cells to Treat Cancer.

There is a strong correlation between myeloid-derived suppressor cells (MDSC) and resistance to immune checkpoint blockade (ICB), but the detailed mechanisms underlying this correlation are largely unknown. Using single-cell RNA sequencing analysis in a bilateral tumor model, we found that immunosuppressive myeloid cells with characteristics of fatty acid oxidative metabolism dominate the immune-cell landscape in ICB-resistant subjects. In addition, we uncovered a previously underappreciated role of a serine/threonine kinase, PIM1, in regulating lipid oxidative metabolism via PPARγ-mediated activities. Enforced PPARγ expression sufficiently rescued metabolic and functional defects of Pim1 -/- MDSCs. Consistent with this, pharmacologic inhibition of PIM kinase by AZD1208 treatment significantly disrupted the myeloid cell-mediated immunosuppressive microenvironment and unleashed CD8+ T-cell-mediated antitumor immunity, which enhanced PD-L1 blockade in preclinical cancer models. PIM kinase inhibition also sensitized nonresponders to PD-L1 blockade by selectively targeting suppressive myeloid cells. Overall, we have identified PIM1 as a metabolic modulator in MDSCs that is associated with ICB resistance and can be therapeutically targeted to overcome ICB resistance.