Fig 1: Levels of serum CD73 activity within 3 months therapy with anti-PD-1 agents. A, Levels of serum CD73 activity at time points pretreatment (0) and on-treatment (3 months) in all patients analyzed (n=60). B, Comparison of serum CD73 activity in patients with or without clinical benefits to nivolumab or pembrolizumab at time points pretreatment (0) and on-treatment (3 months). Mean±SD is shown. P values were calculated with the two-way analysis of variance (B). CR, complete response; PD-1, programmed cell death protein 1; PD, progressive disease; Pi, inorganic phosphate; PR, partial response; SD, stable disease.
Fig 2: Prognostic value of soluble CD73 in patients with metastatic melanoma. A, Kaplan-Meier curves estimate overall survival of all patients receiving nivolumab or pembrolizumab or (B) nivolumab plus ipilimumab according to baseline serum activity of CD73. CD73 high (red line) is >38.8 pmol/min/mg protein and CD73 low is <38.8 pmol/min/mg protein (blue line). P values were calculated using the log-rank test.
Fig 3: Prognostic value of soluble CD73 in subgroups of patients with metastatic melanoma receiving anti-PD-1 therapy alone. Groups of patients aged <65 years (A) or female (B) or with elevated serum lactate dehydrogenase (LDH) levels (C) or with BRAF mutations (D) or with bone metastases (E) or with brain metastases (F) and high CD73 activity (>38.8 pmol/min/mg protein) (red line) have worse overall survival compared with patients with low CD73 activity (<38.8 pmol/min/mg protein). P values were calculated using the log-rank test. ULN, upper limit of normal.
Fig 4: The level of CD73 enzymatic activity in serum discriminates patients with metastatic melanoma from healthy donors. A, AMPase activity in serum samples from patients with melanoma (MP, n=546) and healthy donors (HD; n=96), measured with malachite green assay kit. B, The AMP hydrolysis in samples of MPs is significantly reduced in presence of the selective CD73 inhibitor, APCP (100 µM) (n=71). C, Percentage of hydrolysis of 15N AMP by multiple reaction monitoring mass spectrometry in samples of MPs (n=13) in presence of APCP (100 µM) or anti-human CD73 monoclonal antibody (clone 7G2) (5 µg/mL) or vehicle (Ctrl). D, ELISA of CD73 in serum of MPs (n=83) and HD (n=38). E, Receiving operating characteristic curve analysis for CD73 protein expression and CD73 enzymatic activity in MPs compared with healthy donors. Mean±SD is shown. P values are from two-sided Mann-Whitney test (A, B, D) or one-way analysis of variance test (C). APCP, 5’-α,β-methylene diphosphate; AUC, area under the curve; Pi, inorganic phosphate.
Fig 5: CD73 was downregulated on DNT cells from TNs compared to HCs. Flow cytometry analysis of CD39 and CD73 expression was performed on PBMCs collected from HCs and different TNs groups. (A) Representative flow data showed the expression of CD39 and CD73 gated on DN T cells from HCs and different TNs groups. (B) Scatter plots of the percentage of CD73+, CD39+ and CD39+ CD73+ DN T cells from HCs and different TNs groups (n=22-66 each group). P values were obtained by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. (C) Representative flow data showed the expression of CD39 and CD73 gated on activated Treg cells from HCs and different TNs groups. (D) Scatter plots of the percentage of CD73+, CD39+and CD39+ CD73+ activated Treg cells from HCs and different TNs groups (n=21-66 each group). P values were obtained by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. *P<0.05, **P<0.01, ***P<0.001.
Supplier Page from Abcam for Human CD73 ELISA Kit (NT5E)