cd73 enzymatic activity inhibitor ab680  (MedChemExpress)

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Targeting CD73 limits tumor progression and enhances anti-tumor activity of anti-PD-1 therapy in intrahepatic cholangiocarcinoma

doi: 10.1007/s00432-024-05869-1

Figure Lengend Snippet: Elevated CD73 expression of malignant cells in response to ICBs immunotherapy. ( A ) Schematic representation of the treatment schedule for anti-PD-1 monotherapy in AKT/NICD-induced spontaneous murine iCCAs. ( B ) Representative gross images and statistical results from AKT/NICD-induced murine iCCAs that received indicated treatments (6 mice for IgG and 5mice for anti-PD-1 treatment, 10 mg/kg). ( C ) scRNA-seq analysis of GSE151530 showing 12 iCCA biopsies from 10 patients collected at baseline or during ICBs therapy (PD-1 or PD-L1/CTLA-4). UMAP plot showing single cells distinguished by cell types and cell origins from different biopsy time points. ( D ) Volcano plot showing differentially expressed genes in malignant cells before and after ICBs treatment. ( E ) 40 genes that were upregulated in single malignant cells following ICBs therapy were associated with poor prognosis in both FU-iCCA Cohort and ICGC Cohort. ( F ) The drug-gene interaction database (DGIdb) database was used to identify the potential drug targets among the indicated genes. ( G ) Violin plot showing the expression levels of CD73 in different cell types in iCCA from GSE125449. ( H ) Immunohistochemical staining images showing CD73 expression in AKT/NICD iCCAs that received IgG or anti-PD-1 treatment. ( I ) Analysis of T cell dysfunction signature scores by TIDE algorithm in patients with high or low CD73 expression in FU-iCCA cohort. ( J ) Expression levels of CD73 between Non-Responsive versus Responsive (NR vs. R) samples to anti-PD-1 monotherapy in RCC cohort. Kaplan-Meier survival curves of OS for patients grouped by CD73 mRNA expression level in FU-iCCA cohort ( K ) and ICGC cohort ( L ), as well as CD73 protein levels in CCA protein cohort ( M ). ** P < 0.01; OS, overall survival; iCCA, intrahepatic cholangiocarcinoma

Article Snippet: For CD73 enzymatic activity inhibitor AB680 (MedChemExpress) administration, the stocks were dissolved in 100% DMSO.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Targeting CD73 limits tumor progression and enhances anti-tumor activity of anti-PD-1 therapy in intrahepatic cholangiocarcinoma

doi: 10.1007/s00432-024-05869-1

Figure Lengend Snippet: ICBs therapy upregulates CD73 expression in iCCA cells through TNF-α/NF-κB signaling pathway. ( A ) Evaluation of the effect of TNF-α and IFN-γ on CD73 mRNA expression in human iCCA cells CCLP1 and 9810 as determined by RT-PCR assays. ( B ) Evaluation of the effect of IFN-γ on CD73 protein expression in iCCA cells as detected by WB assays. ( C ) WB assays evaluating the effect of different concentrations of TNF-α on CD73 protein expression, as well as the downstream NF-κB signaling pathway in iCCA cells. ( D ) WB assays evaluating the effect of TNF-α on CD73 expression and NF-κB signaling in iCCA cells, with or without NF-κB pathway inhibitor BAY11-7082. WB, western blot; * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For CD73 enzymatic activity inhibitor AB680 (MedChemExpress) administration, the stocks were dissolved in 100% DMSO.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Targeting CD73 limits tumor progression and enhances anti-tumor activity of anti-PD-1 therapy in intrahepatic cholangiocarcinoma

doi: 10.1007/s00432-024-05869-1

Figure Lengend Snippet: CD73 inhibitor AB680 synergizes with GC chemotherapy to inhibit CCLP1 tumor growth in vivo. ( A ) Establishment of the subcutaneous xenograft model with nude mice using sh-MOCK or CD73-knockdown CCLP1 cells. Growth curves ( B ), gross images ( C ), volumes and weight ( D ) of CCLP1 subcutaneous tumors from indicated groups. ( E ) Subcutaneous xenograft model with nude mice using vector or CD73-OE CCLP1 cells. Growth curves ( F ), gross images ( G ), volumes and weight ( H ) of CCLP1 subcutaneous tumors from indicated groups. ( I ) Subcutaneous CCLP1 tumor-bearing nude mice receiving PBS, Gem/Cis chemotherapy, CD73 inhibitor AB680, or combination therapy. Growth curves ( J ), gross images ( K ), volumes and weight ( L ) of CCLP1 tumors from indicated treatment groups. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For CD73 enzymatic activity inhibitor AB680 (MedChemExpress) administration, the stocks were dissolved in 100% DMSO.

Techniques: In Vivo, Knockdown, Plasmid Preparation

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Targeting CD73 limits tumor progression and enhances anti-tumor activity of anti-PD-1 therapy in intrahepatic cholangiocarcinoma

doi: 10.1007/s00432-024-05869-1

Figure Lengend Snippet: CD73 activates the AKT/GSK3β/β-catenin signaling pathway in iCCA cells via adenosine ( A ) Volcano plot showing differentially expressed genes in RBE cells transfected with shCD73 and shMOCK identified by RNA-seq analysis. ( B-C ) KEGG pathway analysis showing differential signaling pathways regulated by CD73 knockdown in RBE cells. ( D ) Gene set enrichment analysis (GSEA) showing that Hallmark gene sets Hypoxia, Inflammatory response, Glycolysis, Epithelial-mesenchymal transition pathways were enriched in shMOCK RBE cells compared with shCD73 cells. ( E ) Phosphorylation levels of AKT, GSK3β, and total β-catenin protein expression in the indicated iCCA cells as determined by WB assays. ( F ) Phosphorylation levels of AKT, GSK3β, and total β-catenin protein levels in CCLP1 cells treated with different concentrations of CD73 enzymatic activity inhibitor AB680 (left) or in 9810 cells under different concentrations of adenosine treatments (right) detected by WB assays

Article Snippet: For CD73 enzymatic activity inhibitor AB680 (MedChemExpress) administration, the stocks were dissolved in 100% DMSO.

Techniques: Transfection, RNA Sequencing Assay, Knockdown, Expressing, Activity Assay

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Targeting CD73 limits tumor progression and enhances anti-tumor activity of anti-PD-1 therapy in intrahepatic cholangiocarcinoma

doi: 10.1007/s00432-024-05869-1

Figure Lengend Snippet: CD73 inhibitor AB680 potentiates therapeutic efficacy of anti-PD-1 immunotherapy in murine iCCAs ( A ) Schematic representation of the schedule for anti-PD-1, CD73 inhibitor AB680, or combination therapy in AKT/NICD-induced murine iCCA model. ( B ) Body weight of tumor-bearing mice treated with vehicle + 10 mg/kg IgG, 10 mg/kg anti-PD-1, 10 mg/kg AB680, or the combination therapy. ( C ) Liver images from spontaneous iCCA models that received the indicated treatments (6 mice per group) at the indicated endpoint. ( D ) Representative IHC staining images for CK19 of liver sections from AKT/NICD-driven murine iCCAs from indicted groups. ( E ) Statistical analysis of liver weight to body weight ratios (LW/BW) and tumor nodule numbers in AKT-NICD injected mice * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For CD73 enzymatic activity inhibitor AB680 (MedChemExpress) administration, the stocks were dissolved in 100% DMSO.

Techniques: Immunohistochemistry, Injection

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Targeting CD73 limits tumor progression and enhances anti-tumor activity of anti-PD-1 therapy in intrahepatic cholangiocarcinoma

doi: 10.1007/s00432-024-05869-1

Figure Lengend Snippet: CD73 inhibition combined with anti-PD-1 therapy transforms the immune landscape of the TME ( A ) The heatmap showing the normalized expression of 42 surface and intracellular immune markers in all 33 immune-cell subclusters. ( B ) t-SNE plots of tumor-infiltrating B cells, macrophages, CD8 + T cells, CD4 + T cells, NK cells, neutrophils, and dendritic cells in the AKT/NICD-induced spontaneous murine iCCAs identified by CyTOF analysis. ( C ) t-SNE plots showing the major tumor-infiltrating immune cell populations from the four treatment groups ( n = 4 per group). ( D ) t-SNE plots of tumor-infiltrating immune cells colored by the relative expression of corresponding lineage markers. ( E ) Stacked bar plots showing proportions of tumor-infiltrating immune cell populations identified by CyTOF analysis in each group. ( F ) Quantification of tumor-infiltrating B cells, macrophages, CD8 + T cells, CD4 + T cells, NK cells, neutrophils, and dendritic cells in AKT/NICD-induced iCCAs given the indicated treatment, assessed by CyTOF. ( G ) The expression level of indicated markers in tumor-infiltrating CD45 + immune cells among the four groups. * P < 0.05, ** P < 0.01; TME, tumor microenvironment

Article Snippet: For CD73 enzymatic activity inhibitor AB680 (MedChemExpress) administration, the stocks were dissolved in 100% DMSO.

Techniques: Inhibition, Expressing

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Targeting CD73 limits tumor progression and enhances anti-tumor activity of anti-PD-1 therapy in intrahepatic cholangiocarcinoma

doi: 10.1007/s00432-024-05869-1

Figure Lengend Snippet: Schematic diagram depicting the rationale for combination therapy with CD73 inhibitor AB680 and PD-1 blockade

Article Snippet: For CD73 enzymatic activity inhibitor AB680 (MedChemExpress) administration, the stocks were dissolved in 100% DMSO.

Techniques:

Journal: mBio

Article Title: The small molecule CBR-5884 inhibits the Candida albicans phosphatidylserine synthase

doi: 10.1128/mbio.00633-24

Figure Lengend Snippet: A malachite-green-based nucleotidase-coupled assay measures the activity of purified Cho1 protein. ( A ) Blue native PAGE gel of the purified hexameric tag-free Cho1 protein. Purified Cho1 and protein ladder with known MW are indicated. The gel was stained with Coomassie Blue R-250. ( B ) Schematic representation of the malachite-green-based nucleotidase-couple assay. Cho1 synthesizes PS from CDP-DAG (cytidyldiphosphate-diacylglycerol) and serine. This releases PS and CMP (cytidylmonophosphate). The phosphate from CMP is cleaved by the nucleotidase CD73 to release inorganic phosphate, which can be bound by the malachite green reagent and measured colorimetrically at OD 620 . AB-680 is a potent inhibitor of CD73 and can, thus, inhibit the reaction. ( C ) OD 620 signal from the malachite green reagent that was added to the reaction (shown in B ) at different time points after the reaction started. Reactions were set up with the same conditions and stopped by adding malachite green at the time indicated. The dots represent the mean of four biological replicates, and the error bars are ±standard deviation (S.D.) values. ( D ) Inhibition of the nucleotidase-coupled assay by AB-680 is shown for a series of replicates in 384-well format and ( E ) is quantified for a total of 21 replicates. Statistics were conducted using one-way ANOVA using Tukey’s multiple comparisons test (ns, not significant; **** P < 0.0001).

Article Snippet: Equal volumes of either the selective CD73 nucleotidase inhibitor AB680 at a final concentration of 1 µM (MedChemExpress, cat# HY-125286) or DMSO were used as positive and negative controls, respectively.

Techniques: Activity Assay, Purification, Blue Native PAGE, Staining, Standard Deviation, Inhibition

Journal: Molecular Cancer Therapeutics

Article Title: Targeting CD73 with AB680 (Quemliclustat), a Novel and Potent Small-Molecule CD73 Inhibitor, Restores Immune Functionality and Facilitates Antitumor Immunity

doi: 10.1158/1535-7163.MCT-21-0802

Figure Lengend Snippet: CD73 expression is variable at the tissue and cellular level across and within cancer indications. A, Total CD73 mRNA expression in various cancer indications from TCGA. B, Quantification of frequency of CD73 + T cells and CD45 − cells in dissociated tumor biopsy samples from various indications. Each data point is an individual donor. Flow cytometry data pooled from at least two replicate experiments. Data represented as mean ± range. HNSC, head and neck squamous cell carcinoma; SKCM, skin cutaneous melanoma; CRC, colorectal carcinoma; NSCLC, non–small cell lung cancer; KIRC, kidney renal clear cell carcinoma. C and D, Fluorescent IHC staining for panCK (red), CD8 (magenta), and CD73 (teal) on two independent human colorectal cancer tumors. Pockets of panCK-positive cancer cells are outlined in yellow and marked “Tumor.” Yellow arrows denote CD73-positive epithelial cells in the normal adjacent tissue, and white arrows show CD73-positive CD8 T cells. CRC, colorectal carcinoma; HNSC, head and neck squamous cell carcinoma; KIRC, kidney renal clear cell carcinoma; NSCLC, non–small cell lung cancer; SKCM, skin cutaneous melanoma.

Article Snippet: CD73 inhibitor AB680 was synthesized by Arcus Biosciences as described previously ( ).

Techniques: Expressing, Flow Cytometry, Immunohistochemistry

Journal: Molecular Cancer Therapeutics

Article Title: Targeting CD73 with AB680 (Quemliclustat), a Novel and Potent Small-Molecule CD73 Inhibitor, Restores Immune Functionality and Facilitates Antitumor Immunity

doi: 10.1158/1535-7163.MCT-21-0802

Figure Lengend Snippet: The CD73 inhibitor AB680 rescues the inhibitory effects of adenosine generation on T-cell activation and function. A, Dose–response curve of AB680 in CD73 enzymatic activity assay to determine the IC 50 in isolated human CD8 + T cells. B, Left: Frequency of CD73 + on human naïve/central memory (T CM ; CD8 + CCR7 + ) and effector/effector memory (T EM ; CD8 + CCR7 − ) CD8 + T cells determined by flow cytometry. Right three panels: Total, naïve, or memory CD8 + T cells were activated with anti-CD2/CD3/CD28 beads in the presence of indicated concentrations of AMP (+10 μmol/L EHNA). Secreted IFNγ was measured after 48 hours. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001, Dunnett multiple comparisons test versus activation alone. Human CD4 + ( C and D ) and CD8 + ( E ) T cells were activated with anti-CD2/CD3/CD28 beads in the presence of AMP and EHNA ± AB680 (200 nmol/L). C, Proliferation was measured by cell trace violet after 96 hours and shown as representative histograms (left) and quantified for 4 donors (right). D and E, Secreted IFNγ (CD4 + and CD8 + T cells), IL2 (CD4 + T cells), and granzyme B (CD8 + T cells) was measured after 72 hours. For B–E, bar graphs with data points are individual donors pooled from multiple independent experiments. Paired t test; *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001. F, Left: mouse CD8 + T cells were isolated from splenocytes from either WT or CD73 −/− mice as indicated, and AMP hydrolysis was measured with AMP-Glo. ****, P < 0.0001, Sidak multiple comparisons test versus WT. Right: WT or CD73 −/− CD8 + T cells were activated with anti-CD3/CD28 beads + IL2 in the presence of 50 μmol/L of AMP + 2.5 μmol/L EHNA ± AB680 (200 nmol/L). Secretion of IFNγ was measured after 96 hours to determine T-cell activation. Results were repeated in an independent experiment with another CD73 inhibitor. *, P < 0.05; **, P < 0.01, Dunnett multiple comparisons test versus AMP alone.

Article Snippet: CD73 inhibitor AB680 was synthesized by Arcus Biosciences as described previously ( ).

Techniques: Activation Assay, Enzyme Activity Assay, Isolation, Flow Cytometry

Journal: Molecular Cancer Therapeutics

Article Title: Targeting CD73 with AB680 (Quemliclustat), a Novel and Potent Small-Molecule CD73 Inhibitor, Restores Immune Functionality and Facilitates Antitumor Immunity

doi: 10.1158/1535-7163.MCT-21-0802

Figure Lengend Snippet: Pharmacologic inhibition of CD73 enhances activation and cytolytic capabilities of tumor-specific T cells. A, Human CD4 + and CD8 + T cells from a healthy donor were transduced with a Neo-12 TCR and examined for CD73 expression and antigen specificity (dextramer staining) by flow cytometry. Greater than 95% of transduced cells expressed CD73, and 48.8% of CD4 + and 51.3% of CD8 + T cells expressed the transduced TCR as measured by dextramer staining. B, T cells from A were cocultured with K562 cells expressing HLA-A2 alone or HLA-A2 with the Neo12 epitope in the presence of AMP + EHNA ± AB680 for 72 hours. Frequency of CD25 + CD69 + T cells and secretion of IL2 (CD4 + T cells) and IFNγ (CD8 + T cells) were quantified. ***, P < 0.001; ****, P < 0.0001, Dunnett multiple comparisons test versus AMP alone. C, Representative flow plots displaying activated phenotype (CD44 + , CD25 + , CD62L hi ) of mouse OT-I CD8 + T cells and frequency of CD73 + cells on both activated OT-I CD8 + T cells and EG7.OVA tumor cells. Stained samples (red) overlaid on the isotype controls (blue). D, CTL killing assay showing time course (top) and 48 hours quantification (bottom) of tumor cell growth measured by the IncuCyte of red fluorescently labeled EG7.OVA cell confluence when cocultured with preactivated OT-I CD8 + T cells in the presence of 50 μmol/L AMP + 2.5 μmol/L EHNA ± 50 nmol/L AB680. Data presented as mean ± SEM and representative of two independent experiments. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001, Dunnett multiple comparisons test versus AMP alone.

Article Snippet: CD73 inhibitor AB680 was synthesized by Arcus Biosciences as described previously ( ).

Techniques: Inhibition, Activation Assay, Transduction, Expressing, Staining, Flow Cytometry, Labeling

Journal: Molecular Cancer Therapeutics

Article Title: Targeting CD73 with AB680 (Quemliclustat), a Novel and Potent Small-Molecule CD73 Inhibitor, Restores Immune Functionality and Facilitates Antitumor Immunity

doi: 10.1158/1535-7163.MCT-21-0802

Figure Lengend Snippet: Adenosine signaling results in a dominant suppression of T-cell activation in the presence of PD-1 blockade that can be restored by blocking CD73 with AB680. A – D , Human moDCs were cultured with CD4 + T cells in an allogeneic MLR for 96 hours in the presence of anti–PD-1, AMP and AB680 as indicated. A and B , Gene expression measured by quantitative RT-PCR from an MLR treated with IgG4 isotype (0.67 nmol/L), anti–PD-1 (0.67 nmol/L), AMP (100 μmol/L), and AB680 (100 nmol/L) as indicated for 72 hours. Data shown from one representative donor pair. Similar results obtained in another donor pair. C, Secretion of IFNγ was measured to determine T-cell functionality after 96 hours treatment with IgG4 isotype, anti–PD-1 (0.67 or 6.7 nmol/L), AMP (100 μmol/L) ± AB680 (100 nmol/L) as indicated. Data shown of IFNγ in one donor pair (left) and of normalized IFNγ of 16 donor pairs from three independent experiments (right). Each symbol represents a unique donor pairing. D, Secretion of IFNγ was measured after 96 hours treatment with IgG4 isotype or anti–PD-1 (0.67 or 6.7 nmol/L) ± the addition of AMP (100 μmol/L) and AB680 (100 nmol/L) at the beginning of the coculture or after 48 hours. Data shown of IFNγ in one donor pair (left) and normalized IFNγ of nine donor pairs from two independent experiments (right). Data represented as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, ( A – C ) Dunnett multiple comparisons test versus AMP + anti–PD-1. D, Sidak multiple comparisons test.

Article Snippet: CD73 inhibitor AB680 was synthesized by Arcus Biosciences as described previously ( ).

Techniques: Activation Assay, Blocking Assay, Cell Culture, Gene Expression, Quantitative RT-PCR

Journal: Molecular Cancer Therapeutics

Article Title: Targeting CD73 with AB680 (Quemliclustat), a Novel and Potent Small-Molecule CD73 Inhibitor, Restores Immune Functionality and Facilitates Antitumor Immunity

doi: 10.1158/1535-7163.MCT-21-0802

Figure Lengend Snippet: Inhibition of CD73 enzymatic activity in a syngeneic B16F10 tumor model enhances antitumoral immunity. A, AMPase activity was assessed on B16F10 tumor sections using a modified form of the Wachstein–Meisel method of enzyme histochemistry. Red arrows indicate regions with active AMP hydrolysis. Data repeated in independent experiment with related CD73 inhibitor AB1421. B, Tumors from B16F10 tumor-bearing mice examined by flow cytometry for CD73 expression on B16F10 cancer cells (CD45 − ) as well as T-cell subsets. n = 11, mean ± SEM. C, Dose-dependent inhibition of CD73 enzymatic activity by AB680 was quantified in B16F10 tumor homogenates using 13 C 5 -AMP hydrolysis method. Data presented as mean ± SD. D, Top: tumor growth curve showing C57Bl6/J mice implanted subcutaneously with B16F10 cells and subsequently treated with vehicle or AB680 at 10 mg/kg every day. Data representative of three independent experiments, *, P < 0.05, mixed effects analysis, Sidak multiple comparisons test for each timepoint. Bottom: percent of CD8 + T cells and the CD8 + T cell to Treg ratio within tumors from each treatment group were determined by flow cytometry and plotted. n = 9–15, mean ± SEM, *, P < 0.05, Student t test. E, Left: tumor growth and survival curve showing B16F10 tumor-bearing mice treated with anti–PD-1 (2.5 mg/kg) and AB680 (10 mg/kg) as indicated. Data representative of three independent experiments, ns = not significant, ***, P < 0.001. Tumor growth curve: mixed effects analysis, Tukey multiple comparisons test. Kaplan–Meier survival plot: multiple comparisons conducted using family-wise significance level of 5%. Right: percent of CD8 + T cells and the CD8 + T cell to Treg ratio within tumors from each treatment group were determined by flow cytometry and plotted. n = 9–15 mice, mean ± SEM. Tumor-infiltrating lyphocyte data repeated in independent experiment, *, P < 0.05; **, P < 0.01; ***, P < 0.001, Sidak multiple comparisons test, vehicle versus anti–PD-1, anti–PD-1 versus anti–PD-1/AB680, vehicle versus anti–PD-1/AB680.

Article Snippet: CD73 inhibitor AB680 was synthesized by Arcus Biosciences as described previously ( ).

Techniques: Inhibition, Activity Assay, Modification, Flow Cytometry, Expressing