Programmed Death Protein Ligand Research Articles

Discovery of Novel Small-Molecule-Based Potential PD-L1/EGFR Dual Inhibitors with High Druggability for Glioblastoma Immunotherapy.

Based on the close relationship between programmed death protein ligand 1 (PD-L1) and epidermal growth factor receptor (EGFR) in glioblastoma (GBM), we designed and synthesized a series of small molecules as potential dual inhibitors of EGFR and PD-L1. Among them, compound EP26 exhibited the highest inhibitory activity against EGFR (IC50 = 37.5 nM) and PD-1/PD-L1 interaction (IC50 = 1.77 μM). In addition, EP26 displayed superior in vitro antiproliferative activities and in vitro immunomodulatory effects by promoting U87MG cell death in a U87MG/Jurkat cell coculture model. Furthermore, EP26 possessed favorable pharmacokinetic properties (F = 22%) and inhibited tumor growth (TGI = 92.0%) in a GBM mouse model more effectively than Gefitinib (77.2%) and NP19 (82.8%). Moreover, EP26 increased CD4+ cells and CD8+ cells in tumor microenvironment. Collectively, these results suggest that EP26 represents the first small-molecule-based PD-L1/EGFR dual inhibitor deserving further investigation as an immunomodulating agent for cancer treatment.

Effects of Metformin on JNK Signaling Pathway and PD-L1 Expression in Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Metformin has been shown to have the potential to inhibit the proliferation of malignant cells. This study aimed to investigate the regulatory effect of metformin on the expression of programmed death protein ligand 1(PD-L1) and mechanisms in TNBC. Mouse breast cancer cell line 4T1 was co-cultured with metformin, and the effect of metformin on cell proliferation was detected by MTT assay. The effect of metformin on the expression of JNK, RSK2 and CREB was detected by MAPK pathway protein chip. BALB/c mice were inoculated with 4T1 cells with knockdown/overexpression of C-Jun N-terminal kinase (JNK), and administered with metformin. The weight of tumor tissue was observed at the end of the experiment. The expression of PD-L1 in tumor cells was observed by immunofluorescence staining and the level of INF-γwas quantitatively determined by ELISA. Metformin inhibited the viability of 4T1 cells and increased the phosphorylation of JNK to reduce the phosphorylation of RSK2 and CREB. Metformin and JNK knockdown reduced the expression of PD-L1 in tumor cells, but there was no significant difference in the weight of tumor tissue. Metformin can reduce the level of INF-γ in tumor tissues, but JNK has no effect. Metformin can inhibit the expression of PD-L1 in triple-negative breast cancer mice and improve the tumor microenvironment, but does not reduce the size of the tumor.

LRP1 induces anti-PD-1 resistance by modulating the DLL4-NOTCH2-CCL2 axis and redirecting M2-like macrophage polarisation in bladder cancer

The tumour microenvironment (TME) drives bladder cancer (BLCA) progression. Targeting the TME has emerged as a promising strategy for BLCA treatment in recent years. Furthermore, checkpoint blockade therapies are only beneficial for a minority of patients with BLCA, and drug resistance is a barrier to achieving significant clinical effects of anti-programmed cell death protein-1 (PD-1)/programmed death protein ligand-1 (PD-L1) therapy. In this study, higher low-density lipoprotein receptor-related protein 1 (LRP1) levels were related to a poorer prognosis for patients with various cancers, including those with higher grades and later stages of BLCA. Enrichment analysis demonstrated that LRP1 plays a role in the epithelial–mesenchymal transition (EMT), NOTCH signalling pathway, and ubiquitination. LRP1 knockdown in BLCA cells delayed BLCA progression both in vivo and in vitro. Furthermore, LRP1 knockdown suppressed EMT, reduced DLL4-NOTCH2 signalling activity, and downregulated M2-like macrophage polarisation. Patients with BLCA and higher LRP1 levels responded weakly to anti-PD-1 therapy in the IMvigor210 cohort. Moreover, LRP1 knockdown enhanced the therapeutic effects of anti-PD-1 in mice. Taken together, our findings suggest that LRP1 is a potential target for improving the efficacy of anti-PD-1/PD-L1 therapy by preventing EMT and M2-like macrophage polarisation by blocking the DLL4-NOTCH2 axis.

Prognostic implications of tumor-infiltrating lymphocytes within the tumor microenvironment in gastric cancer

Tumor-infiltrating lymphocytes (TILs) play a regulatory role in the tumor-associated immune response and are important in the prognosis and treatment response of several cancers. However, because of its heterogeneity, the prognostic value of TILs in gastric cancer (GC) is still controversial. Thus, this study aimed to investigate the association between the density of TILs and patients' outcomes in GC. Patients with gastric adenocarcinoma who underwent curative intent gastrectomy were retrospectively investigated. The groups for analysis were determined on the basis of TIL intensity and percentage of CD3+T-cell infiltration by immunohistochemical. Furthermore, Epstein-Barr virus (EBV), microsatellite instability (MSI), T-cell ratio of CD4 to CD8, and programmed death protein ligand 1 (PD-L1) status were evaluated. A total of 345 patients were enrolled: 124 patients with GCs (35.9%) were classified as the low-CD3+TIL group, and 221 patients with GCs (64.1%) were classified as the high-CD3+TIL group. Poorly differentiated histology (P=.014), EBV-positive status (P<.001), PD-L1-positive status (P=.001), and CD4<CD8 (P<.001) were associated with high-CD3+GC. There was no difference regarding MSI status, the degree of tumor invasion (pT), the presence of lymph node metastasis, and pTNM stage between low- and high-CD3+groups. In survival analysis, the high-CD3+group had better disease-free survival and overall survival rates than had the low-CD3+group (P=.055 and P=.041, respectively). In the multivariate analysis, total gastrectomy, lymph node metastasis, advanced pT stage, and low CD3+levels were independent factors related to worse survival. High CD3+TILs levels were significantly associated with improved survival and could serve as prognostic biomarkers in GC. In addition, CD3+T-cell infiltration was related to both EBV-positive and PD-L1-positive GC and may assist in the investigation of targets in immunotherapy.

Discovery of novel resorcinol biphenyl ether-based macrocyclic small molecules as PD-1/PD-L1 inhibitors with favorable pharmacokinetics for cancer immunotherapy

Programmed death protein 1 (PD-1)/programmed death protein ligand 1 (PD-L1) is one of the most promising immune checkpoints (ICs) in tumor immunology and has been actively pursued as a target for anticancer drug discovery. Based on our previous research in small molecule PD-1/PD-L1 modulators, we designed and synthesized a series of resorcinol biphenyl ether-bearing macrocyclic compounds and evaluated their anti-PD-1/PD-L1 activities. Among them, compound 8d exhibited the highest inhibitory activity against PD-1/PD-L1 interaction with IC50 of 259.7 nM in the homogenous time-resolved fluorescence (HTRF) assay. In addition, 8d displayed in vitro immunomodulatory effects by promoting HepG2 cell death in a HepG2/Jurkat cell co-culture model. Furthermore, 8d effectively inhibited tumor growth (TGI = 74.6% at 40 mg/kg) in a melanoma tumor model in mice without causing obvious toxicity. Moreover, 8d exhibited favorable pharmacokinetics [e.g. high stability, reasonable half-life, and good oral bioavailability (F = 21.5%)]. Finally, molecular modeling studies showed that 8d bound to PD-L1 with high affinity. These results suggest that 8d may serve as a starting point for further development of macrocyclic small molecule-based PD-1/PD-L1 inhibitors for cancer treatment.

5-methyladenosine regulators play a crucial role in development of chronic hypersensitivity pneumonitis and idiopathic pulmonary fibrosis

5-methyladenosine (m5C) modification regulates gene expression and biological functions in oncologic areas. However, the effect of m5C modification in chronic hypersensitivity pneumonitis (CHP) and idiopathic pulmonary fibrosis (IPF) remains unknown. Expression data for 12 significant m5C regulators were obtained from the interstitial lung disease dataset. Five candidate m5C regulators, namely tet methylcytosine dioxygenase 2, NOP2/Sun RNA methyltransferase 5, Y-box binding protein 1, tRNA aspartic acid methyltransferase 1, and NOP2/Sun RNA methyltransferase 3 were screened using random forest and nomogram models to predict risks of pulmonary fibrosis. Next, we applied the consensus clustering method to stratify the samples with different m5C patterns into two groups (cluster A and B). Finally, we calculated immune cell infiltration scores via single-sample gene set enrichment analysis, then compared immune cell infiltration, related functions as well as the expression of programmed cell death 1 (PD-1, PDCD1) and programmed death protein ligand-1 (PD-L1, CD274) between the two clusters. Principal component analysis of m5C-related scores across the 288 samples revealed that cluster A had higher immune-related expression than B. Notably, T helper cell (Th) 2 type cytokines and Th1 signatures were more abundant in clusters A and B, respectively. Our results suggest that m5C is associated with and plays a crucial role in development of pulmonary fibrosis. These m5C patterns could be potential biomarkers for identification of CHP and IPF, and guide future development of immunotherapy or other new drugs strategies for pulmonary fibrosis.

The expression and function of PD-L1 in CD133(+) human liver cancer stem-like cells

Objective: To investigate the expression of programmed death protein-ligand 1 (PD-L1) in liver cancer stem-like cells (LCSLC) and its effect on the characteristics of tumor stem cells and tumor biological function, to explore the upstream signaling pathway regulating PD-L1 expression in LCSLC and the downstream molecular mechanism of PD-L1 regulating stem cell characteristics, also tumor biological functions. Methods: HepG2 was cultured by sphere-formating method to obtain LCSLC. The expressions of CD133 and other stemness markers were detected by flow cytometry, western blot and real-time quantitative polymerase chain reaction (RT-qPCR) were used to detect the expressions of stemness markers and PD-L1. The biological functions of the LCSLC were tested by cell function assays, to confirm that the LCSLC has the characteristics of tumor stem cells. LCSLC was treated with cell signaling pathway inhibitors to identify relevant upstream signaling pathways mediating PD-L1 expression changes. The expression of PD-L1 in LCSLC was down regulated by small interfering RNA (siRNA), the expression of stem cell markers, tumor biological functions of LCSLC, and the changes of cell signaling pathways were detected. Results: Compared with HepG2 cells, the expression rate of CD133 in LCSLC was upregulated [(92.78±6.91)% and (1.40±1.77)%, P<0.001], the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were also higher than those in HepG2 cells (P<0.05), the number of sphere-formating cells increased on day 7 [(395.30±54.05) and (124.70±19.30), P=0.001], cell migration rate increased [(35.41±6.78)% and (10.89±4.34)%, P=0.006], the number of transmembrane cells increased [(75.77±10.85) and (20.00±7.94), P=0.002], the number of cloned cells increased [(120.00±29.51) and (62.67±16.77), P=0.043]. Cell cycle experiments showed that LCSLC had significantly more cells in the G(0)/G(1) phase than those in HepG2 [(54.89±3.27) and (32.36±1.50), P<0.001]. The tumor formation experiment of mice showed that the weight of transplanted tumor in LCSLC group was (1.32±0.17)g, the volume is (1 779.0±200.2) mm(3), were higher than those of HepG2 cell [(0.31±0.06)g and (645.6±154.9)mm(3), P<0.001]. The expression level of PD-L1 protein in LCSLC was 1.88±0.52 and mRNA expression level was 2.53±0.62, both of which were higher than those of HepG2 cells (P<0.05). The expression levels of phosphorylation signal transduction and transcription activation factor 3 (p-STAT3) and p-Akt in LCSLC were higher than those in HepG2 cells (P<0.05). After the expression of p-STAT3 and p-Akt was down-regulated by inhibitor treatment, the expression of PD-L1 was also down-regulated (P<0.05). In contrast, the expression level of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) in LCSLC was lower than that in HepG2 cells (P<0.01), there was no significant change in PD-L1 expression after down-regulated by inhibitor treatment (P>0.05). After the expression of PD-L1 was knockdown by siRNA, the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were decreased compared with those of siRNA-negative control (NC) group (P<0.05). The number of sphere-formating cells decreased [(45.33±12.01) and (282.00±29.21), P<0.001], the cell migration rate was lower than that in siRNA-NC group [(20.86±2.74)% and (46.73±15.43)%, P=0.046], the number of transmembrane cells decreased [(39.67±1.53) and (102.70±11.59), P=0.001], the number of cloned cells decreased [(57.67±14.57) and (120.70±15.04), P=0.007], the number of cells in G(0)/G(1) phase decreased [(37.68±2.51) and (57.27±0.92), P<0.001], the number of cells in S phase was more than that in siRNA-NC group [(30.78±0.52) and (15.52±0.83), P<0.001]. Tumor formation in mice showed that the tumor weight of shRNA-PD-L1 group was (0.47±0.12)g, the volume is (761.3±221.4)mm(3), were lower than those of shRNA-NC group [(1.57±0.45)g and (1 829.0±218.3)mm(3), P<0.001]. Meanwhile, the expression levels of p-STAT3 and p-Akt in siRNA-PD-L1 group were decreased (P<0.05), while the expression levels of p-ERK1/2 and β-catenin did not change significantly (P>0.05). Conclusion: Elevated PD-L1 expression in CD133(+) LCSLC is crucial to maintain stemness and promotes the tumor biological function of LCSLC.

Combinatory analysis of immune cell subsets and tumor-specific genetic variants predict clinical response to PD-1 blockade in patients with non-small cell lung cancer.

Immunotherapy by blocking programmed death protein-1 (PD-1) or programmed death protein-ligand1 (PD-L1) with antibodies (PD-1 blockade) has revolutionized treatment options for patients with non-small cell lung cancer (NSCLC). However, the benefit of immunotherapy is limited to a subset of patients. This study aimed to investigate the value of combining immune and genetic variables analyzed within 3-4 weeks after the start of PD-1 blockade therapy to predict long-term clinical response. Blood collected from patients with NSCLC were analyzed for changes in the frequency and concentration of immune cells using a clinical flow cytometry assay. Next-generation sequencing (NGS) was performed on DNA extracted from archival tumor biopsies of the same patients. Patients were categorized as clinical responders or non-responders based on the 9 months' assessment after the start of therapy. We report a significant increase in the post-treatment frequency of activated effector memory CD4+ and CD8+ T-cells compared with pre-treatment levels in the blood. Baseline frequencies of B cells but not NK cells, T cells, or regulatory T cells were associated with the clinical response to PD-1 blockade. NGS of tumor tissues identified pathogenic or likely pathogenic mutations in tumor protein P53, Kirsten rat sarcoma virus, Kelch-like ECH-associated protein 1, neurogenic locus notch homolog protein 1, and serine/threonine kinase 11, primarily in the responder group. Finally, multivariate analysis of combined immune and genetic factors but neither alone, could discriminate between responders and non-responders. Combined analyses of select immune cell subsets and genetic mutations could predict early clinical responses to immunotherapy in patients with NSCLC and after validation, can guide clinical precision medicine efforts.

PD-1/L1 inhibitor plus chemotherapy in the treatment of sarcomas.

There is an urgent clinical need for new therapeutic regimens for the effective treatment of advanced sarcomas. Accumulating evidence suggests that programmed death receptor-1/programmed death protein ligand-1 (PD-1/L1) inhibitors have synergistic effects with chemotherapy and have been approved for treatment of lung cancer, gastroesophageal cancer, and breast cancer. In this review, we reviewed the synergistic mechanism of PD-1/L1 inhibitors plus chemotherapy in the treatment of cancers, and the application of this combined regimen in several cancers, followed by a summary of the current evidence on the application of this combined regimen in the treatment of sarcomas as well as the main clinical trials currently underway. Based on the findings of this review, we believe that this combined approach will play an important role in the treatment of some subtypes of sarcomas in the future.

Partial advances in the diagnosis and treatment of gastrointestinal cancer.

Gastrointestinal cancers are difficult to be cured with a high recurrence rate accounting for more than 50% of global cancer-related morbidity and mortality.1 Many patients with gastrointestinal cancer are diagnosed at a late stage. Over the past few decades, basic and clinical research with new technologies have made significant progress in the diagnosis and treatment of gastrointestinal cancers, which significantly improved the quality of life and prolonged the survival of patients. Here, we briefly highlight several advances in diagnosing and treating gastrointestinal tumors, mainly gastric cancer and colorectal cancer from multiple perspectives. Early diagnosis is crucial for the treatment of gastrointestinal cancers. With the continuous advances in molecular biology, genomics, and epigenetics, individualized diagnosis of gastric cancer and colorectal cancer holds promise for basic research and clinical applications. Liquid biopsy, including detection of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), tumor-related extracellular vesicles (exosomes and microvesicles), tumor-educated platelets, proteins as well as metabolites in a range of bodily fluids offers a cost-efficient and noninvasive approach to screening tumor and monitor relapse and response to treatment.2 CTCs are tumor cells in peripheral blood, falling off from the solid tumor focus (primary focus or metastatic focus) due to spontaneous or diagnostic and treatment procedures.3 Most of the CTCs undergo apoptosis or phagocytosis and are engulfed by immune cells after entering the peripheral blood; only a few can escape and invade a distant organ to form metastatic foci, increasing the risk of death in patients with gastrointestinal cancers.3 Recent studies have shown that CTCs are heterogenic and can proliferate in vivo and in vitro. These, together with the findings from the single-cell molecular analysis, have provided unique insights into the biology of cancer metastasis and therapeutic response. ctDNA may reflect tumor-specific abnormalities, and analysis of ctDNA can be applied in the diagnosis, therapeutic response, and prognosis of cancer patients. The mutations in specific genes have been detected in the plasma of patients with several types of gastrointestinal cancers, suggesting that ctDNA may be a possible biomarker of gastrointestinal cancers. The minimal residual disease (MRD) is a microscopic focus of treatment-insensitive tumor cells or the early recurrence of tumors.4 Hence, precise evaluation of MRD is especially significant during or after the treatment of gastrointestinal tumors.5 The detection of ctDNA and CTCs can help identify and explain the nature of MRD and may provide a new strategy for the prevention and treatment of tumor metastasis.3, 5 Nevertheless, ctDNA fragments usually have a short half-life, and currently, there are no convincing cut-off values to discriminate the boundary between high and low ctDNA concentrations. Therefore, the clinical value in the detection of ctDNA has not been gained wide recognition yet.6 Another attractive lipid biopsy item is exosomes derived from cancer cells which can promote tumor cell invasion and metastasis by mediating the interaction between tumor cells and the intercellular matrix; exosomes are also closely related to epithelial-mesenchymal transformation (EMT), tumor angiogenesis, and chemotherapeutic drug resistance.7 The Food and Drug Administration (FDA) has approved several single-gene and multigene tests as complementary diagnoses for cancer-specific molecular targeted therapy, marking the widespread clinical use of liquid biopsies, especially in patients with advanced cancer.2, 7 AI, especially machine learning and deep learning, has opened up new avenues for scientific and clinical research. With increasingly multidimensional data generated in daily treatment, AI can aid clinicians to form a personalized perspective on patients' treatment paths and ultimately guide clinical decision-making. These decisions rely on integrating different and complex data streams, including clinical manifestations, patient history, pathological results, genomics, medical imaging, and matching these data with the conclusions of the growing scientific literature. Early data and computational limitations often require the simplification of unstructured patient data, such as medical images and biopsies, to a set of discrete observations of the degree of disease that humans can understand. A prominent example of this simplification is the cancer staging system, most notably the TNM classification of the American Joint Commission on Cancer (AJCC).8 AI has been used to improve the accuracy of liquid biopsy analysis of the data from CTC and ctDNA tests, which may facilitate its integration into clinical workflows in the future. For example, machine learning has been applied to identify CTCs in cancer patients,9 analyze ctDNA for cancer detection and location, comprehensive multigroup analysis, liquid biopsy tests and other clinical genetics, metabolomics, immunology, microbiology, and steady-state data to guide treatment decisions. Currently, the FDA has approved a few AI applications for oncology indications and many applications are underway. There is a great interest in simplifying the gap between the development of AI and clinical transformation. Consequently, the FDA is designing specific guidelines for AI and machine learning for the approved clinical applications.10 The prospect of AI is bright, but there are still many challenges in successfully integrating AI into clinical oncology, it may be only suitable for some particular cancer problems and its specific value may stem from specific cases in clinics.11 Future studies should analyze the prospects, successes, and failures of AI as well as translate them clinically on a case-by-case basis. The further development of AI applications in cancer diagnosis and treatment may benefit the clinical management of gastrointestinal cancers. In 2018, the European Society for Medical Oncology (ESMO) and oncologists from Asian countries revised the guidelines 2016 version, based on scientific evidence, for better management of gastric cancer. The main change in the 2019 ESMO metastatic gastric cancer guidelines updated immunotherapy as a treatment for gastric cancer.12 According to the tumor immunoediting theory, immune checkpoint inhibitors (ICIs) have become effective strategies for the treatment of gastrointestinal tumors. The most commonly used ICIs include monoclonal antibodies programmed cell death protein-1 (PD-1)/programmed death protein ligand-1 (PD-L1) and cytotoxic T-lymphocyte-associated protein-4 (CTLA-4). In addition, other immunotherapies are available, including adoptive T cell therapies (ACT), such as adoptive transfer of chimeric antigen receptor (CAR)- and T cell receptor (TCR)-T cells, as well as bulk tumor-infiltrating lymphocyte (TIL) therapy. Unfortunately, the response rates of immunotherapy are not satisfactory, and some patients may lose their therapeutic effects after a period of treatment. Moreover, gastrointestinal cancers usually have a low response rate to immunotherapies because cancer cells may evade the immune response, contributing to immune tolerance.13 Further exploration of the mechanisms by which cancer cells are resistant to immunotherapies may provide a basis for the development of new promising immunotherapies for gastrointestinal cancers.14 A previous study has revealed that the gut microbiota, a complex community of trillions of microorganisms, plays a crucial role in immunotherapeutic responses in patients with gastrointestinal cancer.15 Besides physiological functions, the gut microbiota can regulate oncogenesis and resistance to antitumor therapies. During antitumor therapies, many medicines are commonly used for reducing adverse events and these medicines include antidiabetics, aspirin, proton pump inhibitors, psychotropic drugs, and nonsteroidal anti-inflammatory drugs, and analgesics such as opioids. Interestingly, treatment with these drugs can significantly modulate the composition of gut microbiota. Hence, understanding the regulatory effect of gut microbiota on antitumor immunotherapies may help improve their therapeutic efficacy in gastrointestinal cancers. Nanoparticles represent a promising strategy to enhance efficacy and reduce the toxicity of immunotherapy. In the context of treatment of colorectal cancer, nanoparticles can be used to transmit various substances with the potential to affect immune cells and their immunoreactivity within the tumor immune microenvironment, targeting the immune system, and activating multiple immune cells including, but not limited to, T cells.16 Besides tumor cells, there are several types of immune cells, endothelial cells, fibroblasts, extracellular matrix as well as others in the TME of gastrointestinal tumors. These components in TME are the key factors affecting tumor initiation, progression, and immune responses. The balance between cellular and humoral components as well as various inflammatory responses in the TME is critical for tumor growth. Cellular immunotherapy and pharmacological immunotherapy can modulate the TME to enhance tumor immunogenicity and antitumor immune responses to limit tumor growth. However, the efficacy of immunotherapies, such as ICIs, is impaired by the dynamic changes in the TME, which may induce acquired resistance in gastrointestinal cancers. Thus, further understanding of the dynamic changes in the TME may help develop new therapeutic strategies for improvement of immunotherapies and prevention of therapeutic resistance in gastrointestinal cancers. To determine the interaction between TME and tumor cells, further exploration of the mechanism underlying immune escape and immune tolerance, and effectively reshaping the TME are the leading solutions for cancer immunotherapies. Bioprinting of 3D cell culture models can help monitor complex cell behaviors under physical simulation conditions, modulate multiple types of cells in an easy-to-handle system, and study cell interaction in real-time.17, 18 With improved access to clinical tissues and better technologies to visualize cells in situ and dissect heterocellular signaling, the use of bioprinting to understand the TME holds great promise to address translational research questions. Furthermore, the 3D cell culture models are valuable for the development of anticancer drugs. Personalized cancer models can also be used to screen personalized antitumor drugs and promote basic cancer research, drug development, and precise medicines. Despite the attractiveness of 3D bioprinting, there are several technical challenges in the printing platforms, cells, and materials used to build 3D models because of the inherent challenge of comprehensively replicating the cellular behaviors and structural complexity of natural tissues. Emerging biomarkers, particularly ctDNA, CTCs, and exosomes, are increasingly driving diagnosis advances, allowing for the early identification and dynamic monitoring of gastrointestinal cancers. Apart from the other useful treatment modalities (such as endoscopic therapy, surgery, radiotherapy, chemotherapy, and targeted therapy), immunotherapy, with its unique and valuable benefits, has made significant progress in the management of gastrointestinal cancers, which is our primary focus area. Concomitant nano-based therapeutics represent a promising strategy to improve the efficacy of immunotherapy. Fortunately, the collaboration in nonmedical disciplines, such as AI, 3D bioprinting, and materials science, can enable in-depth characterization of the TME, improving the diagnosis and treatment of gastrointestinal cancers and fostering multidisciplinary treatment strategies in the future. However, limitations are yet undeniable, which implies that new advances are often accompanied by formidable challenges to overcome. Kai Li and Zhenning Wang conceptualized and designed the study. Mingguang Ju drafted the first version of the manuscript. All authors read and edited the manuscript. The authors declare no conflict of interest. None. Data from the current study are available from the first author upon reasonable request.

Induction chemoimmunotherapy followed by CD8+ immune cell-based patient selection for chemotherapy-free radioimmunotherapy in locally advanced head and neck cancer

PurposeThe first aim of the trial is to study feasibility of combined programmed death protein ligand 1/cytotoxic T-lymphocyte-associated protein 4 inhibition concomitant to radiotherapy. In addition, efficacy of the entire...

A Novel Small Cyclic Peptide-Based 68Ga-Radiotracer for Positron Emission Tomography Imaging of PD-L1 Expression in Tumors.

In the tumor microenvironment, programmed death protein 1 and programmed death protein ligand 1 (PD-L1) signaling pathways help tumors escape the immune system. We designed a gallium-68 (68Ga)-labeled small-molecule peptide-targeting PD-L1 and used positron emission tomography/computed tomography (PET/CT) to detect and dynamically monitor the expression level of PD-L1 in tumors. S-Cyclo(ETSK)-SF-NH2 (SETSKSF) is a cyclic peptide inhibitor comprising seven amino-acid residues. We connected it with the chelating agent DOTA, labeled DOTA-SETSKSF, with the short half-life nuclide Ga-68, and measured the stability of 68Ga-2,2',2″-(10-(2-((S)-1-((3S,6S,9S,18S)-18-((S)-1-((S)-1-amino-1-oxo-3-henylpropan-2-ylamino)-3-hydroxy-1-oxopropan-2-ylcarbamoyl)-6-((R)-1-hydroxyethyl)-3-(hydroxymethyl)-2,5,8,12-tetraoxo-1,4,7,13-tetraazacyclooctadecan-9-ylamino)-3-ydroxy-1-oxopropan-2-ylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (68Ga-DOTA-SETSKSF) in normal saline (NS), phosphate-buffered saline (PBS), and fetal bovine serum (FBS) in vitro. We conducted the 68Ga-DOTA-SETSKSF affinity test, cell-specific uptake experiments, time-combined experiments, western blotting, and laser confocal experiments to confirm the expression and localization of PD-L1 at the cell level and determine the uptake. Biodistribution and imaging experiments were performed using the H1975, B16F10, and A549 tumor models. 68Ga-DOTA-SETSKSF was successfully synthesized, and the radiochemical purity was >99% after purification. The in vitro stability of 68Ga-DOTA-SETSKSF was >95% in NS, PBS, and FBS at 37 °C after 4 h of incubation. Cell-binding experiments confirmed that 68Ga-DOTA-SETSKSF exhibited high uptake in H1975 tumors with high PD-L1 expression and low uptake in A549 tumors with low PD-L1 expression. The clear half-life (T1/2) of 68Ga-DOTA-SETSKSF from the blood was 14.48 ± 3.26 min. The percentages of the injected dose per gram of tissue (%ID/g) for H1975 and A549 tumors were 5.29 ± 0.21 and 0.89 ± 0.10 at 1 h after injection, respectively. The H1975 tumor-to-muscle and tumor-to-blood ratios were 41.79 ± 5.81 and 4.75 ± 0.19 at 4 h, respectively. Apart from the H1975 tumor, the kidney and the bladder showed high accumulation because 68Ga-DOTA-SETSKSF was excreted through the urinary system. PET/CT images showed high accumulation of 68Ga-DOTA-SETSKSF in H1975 tumors and low uptake in A549 tumors, which was consistent with the results of biodistribution experiments. 68Ga-DOTA-SETSKSF is convenient to prepare, has high stability, can be used to monitor the expression of PD-L1, and has an extremely high clinical application value.

Lynch syndrome-associated epithelial ovarian cancer and its immunological profile

IntroductionLynch syndrome is a multi-tumor syndrome characterized by mismatch repair deficiency (MMR-d), microsatellite instability (MSI), and increased tumor-infiltrating lymphocytes (TILs) making these tumors candidates for treatment with immune checkpoint inhibitors. However, response may depend on tumor-induced immune evasion mechanisms, e.g. loss of Beta-2-Microglobulin (B2M) or upregulation of programmed death protein ligand 1 (PD-L1). We investigated the immune response and B2M and PD-L1 expression in Lynch syndrome-associated ovarian cancers. MethodsWe successfully analyzed 30 Lynch syndrome-associated epithelial ovarian cancers collected through the Danish Hereditary Non-Polyposis Colorectal Cancer (HNPCC) register. MMR-d, MSI, immune response (CD3, CD8, and CD68), and immune evasion mechanisms (B2M and PD-L1) were investigated. Statistical associations between these markers were evaluated in addition to survival in relation to B2M/PD-L1. ResultsOf the 29 evaluable tumors, 27 were MMR-d (93.1%). Likewise of 26 evaluable tumors, 14 were MSI (53.8%). MMR-d/MMR-proficiency associated with MSI/MSS in 60.0%. Half of the ovarian tumors presented with high levels of TILs. Loss of B2M expression was observed in 46.7% of the tumors, while expression of PD-L1 was seen in 28.0% of the cases. There was no association between B2M/PD-L1 and MSI/TILs/survival. Loss of B2M was often seen in tumors with low TILs (p = 0.056 or p = 0.059 for CD3 and CD8 positive cells, respectively). ConclusionMMR-d, MSI, and TILs are also seen in Lynch syndrome-associated ovarian cancers making these potential candidates for checkpoint-based immunotherapy. The clinical impact from immune evasion through loss of B2M needs to be investigated further in larger cohorts.

Synthesis and pharmacological evaluation of novel resorcinol biphenyl ether analogs as small molecule inhibitors of PD-1/PD-L1 with benign toxicity profiles for cancer treatment

Programmed death protein 1 (PD-1)/programmed death protein ligand 1 (PD-L1) pathway is one of the most actively pursued targets in cancer immunotherapy. In a continuation of our research interest in this pathway, we synthesized and evaluated the pharmacological activities of a series of resorcinol biphenyl ether analogs as small molecule PD-1/PD-L1 inhibitors for cancer treatment. Among the 27 newly synthesized compounds, CH1 was found to have the highest inhibitory effect against PD-1/PDL-1 with an IC50 value of 56.58 nM in the HTRF (homogenous time-resolved fluorescence) assay. In addition, CH1 dose-dependently promoted HepG2 cell death in a co-culture model of HepG2/hPD-L1 and Jurkat T cells. Furthermore, molecular modeling study indicated that CH1 binds with high affinity to the binding interface of PD-L1. Moreover, CH1 effectively inhibited tumor growth (TGI of 76.4% at 90 mg/kg) in an immune checkpoint humanized mouse model with no obvious toxicity. Finally, CH1 did not cause in vivo cardiotoxicity and bone marrow suppression (myelosuppression) to BALB/c mice. Taken together, these results suggest that CH1 deserves further investigation as a potent and safe PD-1/PDL-1 inhibitor for cancer treatment.

A combination of PD‑1/PD‑L1 inhibitors: The prospect of overcoming the weakness of tumor immunotherapy (Review)

Programmed cell death protein-1 (PD-1)/programmed death protein ligand-1 (PD-L1) inhibitors for treatment of a various types of cancers have revolutionized cancer immunotherapy. However, PD-1/PD-L1 inhibitors are associated with a low response rate and are only effective on a small number of patients with cancer. Development of an anti-PD-1/PD-L1 sensitizer for improving response rate and effectiveness of immunotherapy is a challenge. The present study reviews the synergistic effects of PD-1/PD-L1 inhibitor with oncolytic virus, tumor vaccine, molecular targeted drugs, immunotherapy, chemotherapy, radiotherapy, intestinal flora and traditional Chinese medicine, to provide information for development of effective combination therapies.

PD-L1 and VEGFR-2 expression in synchronous metastatic renal cell carcinoma treated with targeted therapy following cytoreductive nephrectomy

ObjectivesFew studies have independently investigated the population of patients with synchronous metastatic renal cell carcinoma (smRCC). In this study, we evaluated programmed death protein-ligand 1 (PD-L1) and vascular endothelial growth factor receptor 2 (VEGFR-2) expression in primary tumor tissue of smRCC. MethodsA total of 96 patients with smRCC who were treated with cytoreductive nephrectomy followed by targeted therapy from January 2006 to January 2013 were identified. PD-L1 and VEGFR-2 expression were evaluated by immunohistochemistry. Kaplan-Meier and Cox methods were used for analysis. ResultsPD-L1 and VEGFR-2 protein immunopositivity were observed in 39.6% (38 of 96) and 58.3% (56 of 96) of patients, respectively. A significant correlation was detected between VEGFR-2 and PD-L1 expression (P = 0.030). Based on PD-L1 and VEGFR-2 expression, patients with intermediate-risk disease (n = 63) were divided into 4 subgroups including patients who were PD-L1 (+) VEGFR-2 (+) (n = 21), PD-L1 (+) VEGFR-2 (−) (n = 11), PD-L1 (−) VEGFR-2 (+) (n = 15) and PD-L1 (−) VEGFR-2 (−) (n = 16). Compared to the PD-L1 (−) VEGFR-2 (+), PD-L1 (+) VEGFR-2 (+) and PD-L1 (−) VEGFR-2 (−) groups, patients in the PD-L1 (+) VEGFR-2 (−) group had shorter progression-free survival (median, 9.0 vs. 20.0, 16.0 and 15.5 months, P < 0.05) and overall survival (median, 14.0 vs. 33.0, 24.0 and 26.5 months, P < 0.05). ConclusionsIntermediate-risk smRCC patients with PD-L1-positive and VEGFR-2-negative expression who were treated with targeted therapy following cytoreductive nephrectomy had poor prognoses. We suggest that other treatments beyond sunitinib or sorafenib may be explored in this subgroup.

Gated Resonance Energy Transfer (gRET) Controlled by Programmed Death Protein Ligand 1.

The resonance energy transfer (RET) between an excited fluorescent probe molecule and a plasmonic nanoparticle (AuNP) has been investigated to evaluate the effect of protein molecules on the RET efficiency. We have found that the energy transfer to a functionalized AuNP can be modulated by a sub-monolayer film of programmed death-ligand 1 (PD-L1) protein. The interactions of PD-L1 with AuNP@Cit involve incorporation of the protein in AuNP shell and formation of a submonolayer adsorption film with voids enabling gated surface plasmon resonance energy transfer (SPRET). A model of the gated-RET system based on the protein size, estimated using Fisher–Polikarpov–Craievich density approximation, has been developed and can be utilized for other proteins, with minimum data requirement, as well. The value of the equilibrium constant KL determined for the Langmuir isotherm is high: KL = 1.27 × 108 M−1, enabling highly sensitive control of the gated-RET by PD-L1. Thus, with the gated-RET technique, one can determine PD-L1 within the dynamic range, extending from 1.2 to 50 nM. Moreover, we have found that the Gibbs free energy for PD-L1 binding to AuNP@Cit is −46.26 kJ/mol (−11.05 kcal/mol), indicating a strong adsorption with supramolecular interactions. The proposed gated-RET system, with the fluorescence intensity of the fluorophore probe molecule modulated by plasmonic quenching with AuNP and shielding of energy transfer by the adsorbed PD-L1 can be further developed for determination of PD-L1 in pharmaceutical formulations for immune checkpoint control in cancer therapy.

CD19-targeting fusion protein combined with PD1 antibody enhances anti-tumor immunity in mouse models

ABSTRACT In our previous studies, using a B cell vaccine (scFv-Her2), the targeting of tumor-associated antigen Her2 (human epidermal growth factor receptor-2) to B cells via the anti-CD19 single chain variable fragment (scFv) was shown to augment tumor-specific immunity, which enhanced tumor control in the prophylactic and therapeutic setting. However, the fusion protein displayed limited activity against established tumors, and local relapses often occurred following scFv-Her2 treatment, indicating that scFv-Her2-induced responses are inadequate to maintain anti-tumor immunity. In this study, targeting the IV region (D4) of the extracellular region of Her2 to B cells via CD19 molecules (scFv-Her2D4) was found to enhance IFN-γ-producing-CD8+ T cell infiltration in tumor tissues and reduced the number of tumor-infiltrating myeloid-derived suppressor cells (MDSCs). However, negative co-stimulatory molecules such as programmed cell death protein-1 (PD-1), CD160, and LAG-3 on T cells and programmed death protein ligand-1 (PD-L1) on tumor cells were upregulated in the tumor microenvironment after scFv-Her2D4 treatment. Further, anti-PD1 administration enhanced the efficacy of scFv-Her2D4 and anti-tumor immunity, as evidenced by the reversal of tumor-infiltrating CD8+ T cell exhaustion and the reduction of MDSCs and Treg cells, which suppress T cells and alter the tumor immune microenvironment. Moreover, combining this with anti-PD1 antibodies promoted complete tumor rejection. Our data provide evidence of a close interaction among tumor vaccines, T cells, and the PD-L1/PD-1 axis and establish a basis for the rational design of combination therapy with immune modulators and tumor vaccine therapy.

332P - Analyse the association between adverse events (AEs) and survival in patients treated with immune checkpoint inhibitors (ICIs)

BackgroundICIs with a programmed death protein 1 (PD-1) or programmed death protein ligand 1 (PD-L1) antibodies have shown survival benefits for various metastatic cancer patients. However, some patients treated with ICIs may suffer from AEs. We investigated the association between AEs and survival in metastatic cancer patients treated with PD-1/PD-L1 inhibitors. MethodsWe retrospectively reviewed medical records of metastatic cancer patients treated with pembrolizumab, nivolumab or atezolizumab from April 2016 to June 2019 for AEs, progression-free survival (PFS), and overall survival (OS). Kaplan-Meier curve was used to estimate survival probability and the Cox proportional hazard model was used to estimate hazard ratio (HR) and 95% confidence interval (CI) between each interesting group. ResultsWe enrolled 101 patients. Primary cancers were urothelial carcinoma (n = 52), lung cancer (n = 42), hepatocellular carcinoma (n = 5), and cholangiocarcinoma (n = 2). Atezolizumab was the most common prescribed agents (n = 69), followed by pembrolizumab (n = 29) and nivolumab (n = 3). The AEs happened more frequently were associated with lung (n = 25), skin (n = 24), endocrine (n = 17), and liver (n = 11). Median time for these AEs happened was 1.4 months. Median OS (mOS) for these patients was 16.9 months and median PFS (mPFS) was 9.1 months. Among various AEs, patients with skin AE had significantly longer PFS and OS compared to those without skin AE (mPFS: 11.9 months vs. 4.9 months, p = 0.0016 ; mOS: 16.4 months vs. 10.3 months, p = 0.0022). In comparison with those without endocrine AE, the occurrence of endocrine AE was associated with an improved PFS (mPFS: 19.2 months vs. 6.0 months, p = 0.0476 ) and a trend toward to OS (mOS: 19.4 months vs. 10.9 months, p = 0.1731 ). There were no survival difference between patients who experienced lung or liver AEs. ConclusionsIn metastatic cancer patients treated with anti-PD-1/PD-L1 ICIs, development of skin or endocrine AEs may indicate improved survival time. The patients with AEs involved visceral organ, like lung or liver, have no better survival time. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

New insight on the correlation of metabolic status on 18F-FDG PET/CT with immune marker expression in patients with non-small cell lung cancer.

Metabolic information obtained through 18F-flurodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) is used to evaluate malignancy by calculating the glucose uptake rate, and these parameters play important roles in determining the prognosis of non-small cell lung cancer (NSCLC). The expression of immune-related markers in tumor tissue reflects the immune status in the tumor microenvironment. However, there is lack of reports on the association between metabolic variables and intra-tumor immune markers. Herein, we investigate the correlation between metabolic status on 18F-FDG PET/CT and intra-tumor immunomarkers' expression in NSCLC patients. From April 2008 to August 2014, 763 patients were enrolled in the analysis to investigate the role of maximum standardized uptake value (SUVmax) in lung cancer. One hundred twenty-two tumor specimens were analyzed by immunohistochemistry (IHC) to intra-tumor immune cells and programmed death protein ligand 1(PD-L1) expression on tumor cells. The correlation between metabolic variables and the expression of tissue immune markers were analyzed. SUVmax values have significant variations in different epidermal growth factor receptor (EGFR) statuses (wild type vs mutant type), high/low neutrophil-to-lymphocyte ratio (NLR) groups, and high/low platelets-to-lymphocyte ratio (PLR) groups (p < 0.001, p < 0.001, p = 0.003, respectively). SUVmax was an independent prognostic factor in lung cancer patients (p = 0.013). IHC demonstrated a statistically significant correlation between SUVmax and the expression of CD8 tumor-infiltrating lymphocytes (p = 0.015), CD163 tumor-associated macrophages (TAMs) (p = 0.003), and Foxp3-regulatory T cells (Tregs) (p = 0.004), as well as PD-1 and PD-L1 (p = 0.003 and p = 0.012, respectively). With respect to patient outcomes, disease stage, BMI, SUVmax, metabolic tumor volume (MTV), TLG (tumor lesion glycolysis), CD163-TAMs, CD11c-dendritic cells (DCs), PD-L1, and Tregs showed a statistically significant correlation with progression-free survival (PFS) (p < 0.001, 0.023, < 0.001, 0.007, 0.005, 0.004, 0.008, 0.048, and 0.014, respectively), and disease stage, SUVmax, MTV, TLG, CD163-TAMs, CD11c-DCs, and PD-L1 showed a statistically significant correlation with overall survival (OS) (p < 0.001, < 0.001, 0.014, 0.012, < 0.001, 0.001, and < 0.001, respectively). This study revealed an association between metabolic variable and immune cell expression in the tumor microenvironment and suggests that SUVmax on 18F-FDG PET/CT could be a potential predictor for selecting candidates for immunotherapy.