Journal: Journal for immunotherapy of cancer

Article Title: Bispecific antibody CD73xEGFR more selectively inhibits the CD73/adenosine immune checkpoint on cancer cells and concurrently counteracts pro-oncogenic activities of CD73 and EGFR.

doi: 10.1136/jitc-2023-006837

Figure Lengend Snippet: Figure 1 bsAb CD73xEGFR has dual binding specificity for CD73 and EGFR. (A) Dose-dependent binding of bsAb CD73xEGFR and bsAb CD73xMock to CHO, CHO.hEGFR and CHO.hCD73, respectively. (B) Competitive binding assay in which bsAb CD73xEGFR (1 µg/mL) was pre-incubated with excess amounts of soluble human CD73 (s.hCD73), EGFR- competing bsAb MockxEGFR or a combination thereof and then evaluated for binding to SK-BR-3 cancer cells. (C) Binding of bsAb CD73xEGFR and bsAb CD73xMock (both 1 µg/mL) to parental cancer cells (FaDu, H292, A549, H1650) and corresponding EGFR-KO variants thereof. All experiments were analyzed by flow-cytometry. Graphs A–B (representative graphs): n=3 (two technical repeats), graph C: n=3 (three technical replicates). All graphs represent mean±SD. Statistical analysis in B was performed using one-way analysis of variance followed up by Tukey post hoc test (**p<0.01, ***p<0.001). bsAb, bispecific antibody; CHO, Chinese hamster ovary; KO, knockout; MFI, mean fluorescent intensity.

Article Snippet: CHO.CD73 cells stably expressing human CD73 were generated by lipofection (FuGENE- HD, Promega) of a plasmid containing complementary DNA (cDNA) encoding human CD73 (OriGene).

Techniques: Binding Assay, Competitive Binding Assay, Incubation, Flow Cytometry, Knock-Out

Journal: Journal for immunotherapy of cancer

Article Title: Bispecific antibody CD73xEGFR more selectively inhibits the CD73/adenosine immune checkpoint on cancer cells and concurrently counteracts pro-oncogenic activities of CD73 and EGFR.

doi: 10.1136/jitc-2023-006837

Figure Lengend Snippet: Figure 2 bsAb CD73xEGFR induces rapid co-internalization and subsequent prolonged displacement of both CD73 and EGFR from the cancer cell surface. Residual (A) CD73 and (B) EGFR membrane presence on H292 cells after treatment with bsAb CD73xEGFR or controls (0.01–10 µg/mL) for 24 hours. Residual (C) CD73 and (D) EGFR membrane presence on H292 cells after treatment with bsAb CD73xEGFR or controls (1 µg/mL) for 1, 4, 24, 48, 72 and 96 hours. (E) Residual CD73 and EGFR membrane presence after treatment of primary patient-derived ovarian and colon cancer cells with bsAb CD73xEGFR or controls (1 µg/mL) for 24 hours. (F) Mean fluorescent intensity (MFI) of H292 cells after incubation with bsAb CD73xEGFR or controls (1 µg/mL) labeled with pH-sensitive dye CypHer5E for 10, 60, 240 and 360 min. (G) Percentage of annexin-Vpos/PIpos H292 cancer cells after incubation for 24 hours with bsAb CD73xEGFR or controls (1 µg/mL) in the presence of an anti-human IgG-Fab fragment labeled with a saporin-based toxin. (H) Residual cell surface EGFR-expression levels plotted against residual cell surface CD73-expression levels of a panel of 10 cancer cell lines after treatment with bsAb CD73xEGFR or oleclumab (both 1 µg/mL) for 24 hours. All experiments were analyzed by flow-cytometry. Graphs A–D: n=4 (two technical replicates), graph E: n=1 (four technical replicates), graph F (representative graph): n=3 (two technical replicates), graph G: n=2 (three technical replicates), graph H: n=3 (two technical replicates). All graphs represent mean±SD. Statistical analysis in E (based on group- means) and G was performed using unpaired t-test (*p<0.05, **p<0.01). Linear regression was used to calculate the regression coefficient in graph H. bsAb, bispecific antibody.

Article Snippet: CHO.CD73 cells stably expressing human CD73 were generated by lipofection (FuGENE- HD, Promega) of a plasmid containing complementary DNA (cDNA) encoding human CD73 (OriGene).

Techniques: Membrane, Derivative Assay, Incubation, Labeling, Expressing, Flow Cytometry

Journal: Journal for immunotherapy of cancer

Article Title: Bispecific antibody CD73xEGFR more selectively inhibits the CD73/adenosine immune checkpoint on cancer cells and concurrently counteracts pro-oncogenic activities of CD73 and EGFR.

doi: 10.1136/jitc-2023-006837

Figure Lengend Snippet: Figure 3 bsAb CD73xEGFR potently inhibits the enzyme activity of CD73 in an EGFR-directed manner. (A) Percentage of inhibition CD73 enzyme activity on H292 cells after treatment with bsAb CD73xEGFR or controls (0.01–10 µg/mL) for 24 hours. (B) Percentage of inhibition CD73 enzyme activity on H292, OvCAR3, and H322 cancer cell lines or on (C) primary patient- derived cancer cells after treatment with bsAb CD73xEGFR or controls (1 µg/mL) for 24 hours. (D) Competitive CD73 enzyme inhibition assay in which H292 cells were pretreated with excess amounts EGFR-competing bsAb MockxEGFR prior to incubation with bsAb CD73xEGFR (0.01–2 µg/mL). (E) Percentage of inhibition CD73 enzyme activity on A549 parental and EGFR-KO cells after treatment with bsAb CD73xEGFR or bsAb CD73xMock (both 1 µg/mL). CD73-mediated hydrolysis of AMP into adenosine was evaluated using a colorimetric malachite green-based inorganic phosphate assay. Graphs A–B: n=4 (two technical replicates), graph C: n=1 (four technical replicates), graph D–E: n=3 (two technical replicates). All graphs represent mean±SD. Statistical analysis in B and C was performed using unpaired t-test (based on group-means) (**p<0.01). bsAb, bispecific antibody; KO, knockout.

Article Snippet: CHO.CD73 cells stably expressing human CD73 were generated by lipofection (FuGENE- HD, Promega) of a plasmid containing complementary DNA (cDNA) encoding human CD73 (OriGene).

Techniques: Activity Assay, Inhibition, Derivative Assay, Enzyme Inhibition Assay, Incubation, Knock-Out

Journal: Journal for immunotherapy of cancer

Article Title: Bispecific antibody CD73xEGFR more selectively inhibits the CD73/adenosine immune checkpoint on cancer cells and concurrently counteracts pro-oncogenic activities of CD73 and EGFR.

doi: 10.1136/jitc-2023-006837

Figure Lengend Snippet: Figure 6 bsAb CD73xEGFR inhibits the proliferative and migratory capacity of cancer cells. (A) Proliferation assay in which H292 and corresponding CD73-KO cancer cells were incubated with bsAb CD73xEGFR or controls (1 µg/mL) and then evaluated using the RTCA xCELLigence instrument. Readout is indicated as cell‐index, which is an arbitrary unit for attachment of adherent cells and cell proliferation measured at 37°C every 15 min. (B) Proliferation assay in which a panel of cell lines (H292, H322, FaDu, and OvCAR3) was incubated with bsAb CD73xEGFR or controls (1 µg/mL). The percentage of cell confluence was evaluated after 72 hours. (C) Representative images of H292 cell colonies after treatment with bsAb CD73xEGFR (1 µg/mL) or indicated controls at 37°C for 14 days. (D, E) Percentage of OvCAR3 cell colonies after treatment with bsAb CD73xEGFR or controls (0.01–10 µg/mL) at 37°C for 14 days. Subsequently, number and area of OvCAR3 cell colonies were calculated using ImageJ software. (F) Representative images of H292 cells, seeded in a culture plate equipped with a stopper, which enabled the formation of a cell-free detection zone, and incubated with bsAb CD73xEGFR or controls (1 µg/mL) at 37°C for 72 hours. (G) Similar, closure of cell-free detection zone (migration) evaluated over time using live cell imaging technology by taking pictures at 4× magnification at 37°C every 0.5 hours for 3 days. (H) Athymic nude mice (Crl:NU(NCr)-Foxnnu) were inoculated subcutaneous in the flank with human H292-luc2 non-small lung cancer cells and injected I.P. with bsAb CD73xEGFR or controls (7.5 mg/kg) on days 7, 10, and 17. (I) Representative bioluminescent images of bsAb CD73xEGFR or control treated mice on day 28 post tumor injection. (J) Mean tumor volume (mm3) of each nine animals per group determined by caliper measurements. Graph A–B: n=3 (two technical replicates), Graph D–E: n=3 (two technical replicates), graph G: n=3 (two technical replicates). All graphs represent mean±SD. Statistical analysis in B and in J (at 28 days) was performed using one-way analysis of variance followed up by Tukey post hoc test (*p<0.05, **p<0.01). APCP, adenosine 5'-(α,β-methylene)diphosphate; bsAb, bispecific antibody; I.P., intraperitoneal; KO, knockout.

Article Snippet: CHO.CD73 cells stably expressing human CD73 were generated by lipofection (FuGENE- HD, Promega) of a plasmid containing complementary DNA (cDNA) encoding human CD73 (OriGene).

Techniques: Proliferation Assay, Incubation, Software, Migration, Live Cell Imaging, Injection, Control, Knock-Out

Journal: Journal for immunotherapy of cancer

Article Title: Bispecific antibody CD73xEGFR more selectively inhibits the CD73/adenosine immune checkpoint on cancer cells and concurrently counteracts pro-oncogenic activities of CD73 and EGFR.

doi: 10.1136/jitc-2023-006837

Figure Lengend Snippet: Figure 7 bsAb CD73xEGFR sensitizes cancer cells towards chemotherapeutic agents and radiation. Cell confluence of H292 cancer cells treated (or not) with bsAb CD73xEGFR (1 µg/mL) or controls in the presence (or absence) of (A/B) doxorubicin (50 nM), (B) cisplatin (1 µg/mL), taxol (50 nM) or 5FU (50 µg/mL) and then evaluated using live cell imaging by taking pictures at 4× magnification every 6 hours at 37°C for 6 days. (C) Percentage of H292 or (D) corresponding CD73-KO cell colonies after treatment with bsAb CD73xEGFR (1 µg/mL) or controls, irradiated (0.5, 1, 1.5, and 2 Gy) and then incubated at 37°C for 14 days. Cell colonies were calculated using ImageJ software. (E) IC50 values (Gy) calculated for graph C. All graphs: n=3 (two technical replicates). All graphs represent mean±SD. Statistical analysis in B was performed using unpaired t-test (*p<0.05, **p<0.01, ***p<0.001). bsAb, bispecific antibody; KO, knockout.

Article Snippet: CHO.CD73 cells stably expressing human CD73 were generated by lipofection (FuGENE- HD, Promega) of a plasmid containing complementary DNA (cDNA) encoding human CD73 (OriGene).

Techniques: Live Cell Imaging, Irradiation, Incubation, Software, Knock-Out

Journal: Frontiers in Pharmacology

Article Title: Nucleotide Analog ARL67156 as a Lead Structure for the Development of CD39 and Dual CD39/CD73 Ectonucleotidase Inhibitors

doi: 10.3389/fphar.2020.01294

Figure Lengend Snippet: Consecutive hydrolysis of ATP to adenosine by cleaving the terminal phosphate group and releasing inorganic phosphate (P i ), catalyzed by the enzymes CD39 and CD73.

Article Snippet: The cDNAs of the human enzymes NPP1, NPP3, NPP5, CD38 and CD73 (Genbank accession no. NM_006258, NM_005021, NM_021572, NM_ 001775, and NM_002526, respectively) were obtained from Origene (Rockville, USA).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Nucleotide Analog ARL67156 as a Lead Structure for the Development of CD39 and Dual CD39/CD73 Ectonucleotidase Inhibitors

doi: 10.3389/fphar.2020.01294

Figure Lengend Snippet: Inhibition of selected ecto-nucleotidases by ARL67156 (I) and analogs 31 and 33 ^a .

Article Snippet: The cDNAs of the human enzymes NPP1, NPP3, NPP5, CD38 and CD73 (Genbank accession no. NM_006258, NM_005021, NM_021572, NM_ 001775, and NM_002526, respectively) were obtained from Origene (Rockville, USA).

Techniques: Inhibition

Journal: Frontiers in Pharmacology

Article Title: Nucleotide Analog ARL67156 as a Lead Structure for the Development of CD39 and Dual CD39/CD73 Ectonucleotidase Inhibitors

doi: 10.3389/fphar.2020.01294

Figure Lengend Snippet: Comparison of (A) the putative substrate binding site of human CD39 (pale cyan) and (B) the substrate binding site of human CD73 (gray). Important amino acids are shown; positively and negatively charged amino acids are highlighted by blue and green boxes, respectively. Oxygen atoms are colored in red, nitrogen atoms in blue, sulfur atoms in yellow, calcium atom in green, and zinc atoms in dark gray.

Article Snippet: The cDNAs of the human enzymes NPP1, NPP3, NPP5, CD38 and CD73 (Genbank accession no. NM_006258, NM_005021, NM_021572, NM_ 001775, and NM_002526, respectively) were obtained from Origene (Rockville, USA).

Techniques: Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Nucleotide Analog ARL67156 as a Lead Structure for the Development of CD39 and Dual CD39/CD73 Ectonucleotidase Inhibitors

doi: 10.3389/fphar.2020.01294

Figure Lengend Snippet: Docked poses of ARL67156 (I) and derivatives in the substrate binding pocket of human CD73 based on an X-ray structure (PDB ID: 6s7f). (A) Binding pose of the co-crystallized inhibitor PSB-12379 (orange); (B) binding pose of I (green); (C) binding pose of 31 (marine blue), and (D) binding pose of 37 (magenta). Important amino acids are colored in orange and shown in line representation. The two zinc ions in the substrate binding site are represented as blue spheres. Oxygen atoms are colored in red and nitrogen atoms in blue.

Article Snippet: The cDNAs of the human enzymes NPP1, NPP3, NPP5, CD38 and CD73 (Genbank accession no. NM_006258, NM_005021, NM_021572, NM_ 001775, and NM_002526, respectively) were obtained from Origene (Rockville, USA).

Techniques: Binding Assay