Journal: Cancer letters

Article Title: Bispecific antibody CD73xEpCAM selectively inhibits the adenosine-mediated immunosuppressive activity of carcinoma-derived extracellular vesicles.

doi: 10.1016/j.canlet.2021.08.037

Figure Lengend Snippet: Fig. 2. BsAb CD73xEpCAM has dual binding specificity for CD73 and EpCAM. (A) Topology of expression plasmid pbsAb CD73xEpCAM-IgG2s. (B) Schematic depiction of bsAb CD73xEp CAM. (C) bsAb CD73xEpCAM (lane 1 and 2) or oleclumab (lane 3 and 4) (5 μg each) were separated by an SDS-PAGE gel using non-reducing (NR) and reducing (R) conditions. Under non- reducing conditions CD73xEpCAM migrated as a single protein band with an apparent molecular weight of 175 kDa (lane 1), which dropped to 75 kDa when separated under reducing condi tions (lane 2). This is in good agreement with the calculated molecular weight of 83 kDa for CD73xEpCAM monomer and the proposed disulfide-stabilized dimeric single chain composition of the native protein. Oleclumab showed the expected heterodimeric composition of heavy and light chain characteristic for conven tional antibodies (lane 3 and 4). The protein bands of bsAb CD73xEpCAM are indicated by solid black arrows and bands of oleclumab by open black ar rows. (D) Comparison of dose-dependent binding of bsAb CD73xEpCAM, bsAb CD73xMock and bsAb MockxEpCAM to CHO, CHO.EpCAM and CHO.CD73 cells, respectively. (E) Competitive binding assay in which bsAb CD73xEpCAM was pretreated with excess amounts of solu ble CD73 (sCD73), soluble EpCAM (sEpCAM) or a combination thereof (10 μg) prior to incubation with H292 can cer cells. Graphs D-E were analyzed by flow cytometry. All graphs represent mean ± SD. Statistical analysis in graphs E was performed using un-paired T test (****p < .0001). M = marker in graph C.

Article Snippet: CHO.CD73 cells stably expressing human CD73 were generated by lipofection (Fugene-HD, Promega) using plasmids containing cDNAs encoding human CD73 (Origene).

Techniques: Binding Assay, Expressing, Plasmid Preparation, SDS Page, Molecular Weight, Comparison, Competitive Binding Assay, Incubation, Flow Cytometry, Marker

Journal: Cancer letters

Article Title: Bispecific antibody CD73xEpCAM selectively inhibits the adenosine-mediated immunosuppressive activity of carcinoma-derived extracellular vesicles.

doi: 10.1016/j.canlet.2021.08.037

Figure Lengend Snippet: Fig. 1. CD73 and EpCAM are highly abundant on EVs. (A) Representative images of immunoblot anal ysis for presence of CD73, EpCAM, PD-L1, CD9, TSG101, calnexin and β-actin in cancer cells H292, OvCAR3, DLD1 and corresponding EVs. (B) CD73, EpCAM and TSG101 in EVs derived from parental H292, H292CD73−KO and H292EpCAM−KO cells, respectively. 20 μg protein of each sample (both cells and EVs) was loaded. Of note: The apparent differ ence in EpCAM signal in H292 cells between Fig. 1A and B is due to a reduction of exposure time during bioluminescent-based detection to prevent over exposure of the very high EpCAM signal from H292- derived EVs in Fig. 1B.

Article Snippet: CHO.CD73 cells stably expressing human CD73 were generated by lipofection (Fugene-HD, Promega) using plasmids containing cDNAs encoding human CD73 (Origene).

Techniques: Western Blot, Derivative Assay

Journal: Cancer letters

Article Title: Bispecific antibody CD73xEpCAM selectively inhibits the adenosine-mediated immunosuppressive activity of carcinoma-derived extracellular vesicles.

doi: 10.1016/j.canlet.2021.08.037

Figure Lengend Snippet: Fig. 3. BsAb CD73xEpCAM inhibits the enzyme activity of CD73 on cancer cells and EVs in an EpCAM-directed manner. (A–B) Increasing amounts of H292 and H292CD73−KO cancer cells or EVs were incubated with AMP (100 μM) and inorganic phosphate (Pi) produced by CD73- mediated hydrolysis of AMP was evaluated. (C–D) BsAb CD73xEpCAM, bsAb-controls or oleclumab (1 μg/ml) were added to H292 cancer cells or EVs and evaluated for capacity to inhibit the enzyme activity of CD73. Background levels of Pi in the absence of bsAb CD73xEpCAM was used to normalize CD73 inhibition to 0%. (E) Competitive CD73 enzyme inhibition assay in which bsAb CD73xEpCAM was pretreated with excess amounts of sEpCAM (10 μg) prior to incubation with H292-derived EVs and assessed for its ca pacity to inhibit the enzyme activity of CD73. (F) EpCAM- directed blockade of CD73 on H292-derived parental, EpCAM-KO and CD73-KO EVs using bsAb CD73xEpCAM (1 μg/ml). CD73-mediated hydrolysis of AMP into ADO was evaluated using a colorimetric malachite green-based Pi assay. All graphs represent mean ± SD. Statistical analysis in graphs C-D was performed using un-paired T test. Statistical analysis in graph E was performed using one-way ANOVA followed by a Tukey post-hoc test. Statistical analysis in graph F was performed using multiple T tests (*p < .05, **p < .01, ***p < .001, ****p < .0001).

Article Snippet: CHO.CD73 cells stably expressing human CD73 were generated by lipofection (Fugene-HD, Promega) using plasmids containing cDNAs encoding human CD73 (Origene).

Techniques: Activity Assay, Incubation, Produced, Inhibition, Enzyme Inhibition Assay, Derivative Assay

Journal: Cancer letters

Article Title: Bispecific antibody CD73xEpCAM selectively inhibits the adenosine-mediated immunosuppressive activity of carcinoma-derived extracellular vesicles.

doi: 10.1016/j.canlet.2021.08.037

Figure Lengend Snippet: Fig. 5. BsAb CD73xEpCAM restores the anticancer activity of EV-suppressed PBMCs. (A) PBMCs were cultured in the presence, or absence, of H292EVs or H292 CD73-KOEVs (50 μg/106 PBMCs) at 37 ◦C for 3 d. Next, PBMCs were incubated (or not) with AMP (100 μM) at 37 ◦C for 24 h. Subsequently, cytotoxic T (Effector) cells were stimulated and re-directed to kill EpCAM-expressing PC3M (Target) cancer cells using BIS-1 in an effector (E) to target (T) cell ratio of 4:1. Subsequently, effector and target cells were co- cultured for 2 d in the presence of a conditionally fluorescent caspase 3/8–488 probe. Live cell imaging technology was used to evaluate induction of apoptotic cancer cell death (caspase-3/8 activation, count per image) by taking pictures every 1.5 h at 10x magnification at 37 ◦C for 2 d. (B) PBMCs were incubated with H292EVs (50 μg/106 PBMCs) in the present or absence of bsAb CD73xEpCAM, bsAb controls or oleclumab (1 μg/ml) at 37 ◦C for 3 d. Subsequently, PBMCs were incubated (or not) with AMP (100 μM) at 37 ◦C for 24 h and re-directed using BIS-1 to kill PC3M cancer cells. Using live cell imag ing technology, apoptotic cell death was analyzed over time. (C) IFN-γ levels in culture supernatant of graph B were measured by ELISA. All graphs repre sent mean ± SD. Statistical analysis in graph C was performed using un-paired T test (****p < .0001). Ole = oleclumab in B and C.

Article Snippet: CHO.CD73 cells stably expressing human CD73 were generated by lipofection (Fugene-HD, Promega) using plasmids containing cDNAs encoding human CD73 (Origene).

Techniques: Activity Assay, Cell Culture, Incubation, Expressing, Live Cell Imaging, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Cancer letters

Article Title: Bispecific antibody CD73xEpCAM selectively inhibits the adenosine-mediated immunosuppressive activity of carcinoma-derived extracellular vesicles.

doi: 10.1016/j.canlet.2021.08.037

Figure Lengend Snippet: Fig. 6. BsAb CD73xEpCAM inhibits the enzyme activity of CD73 on cancer patient-derived EVs. (A) Characterization of colon carcinoma patient- derived EVs. (B) Representative immunoblotting im ages of the detection of CD73, EpCAM and TSG101 in cancer patient-derived EVs (20 μg). (C) Cancer patient-derived EVs were incubated with AMP (100 μM) and Pi produced by CD73-mediated hydrolysis of AMP was evaluated. (D) BsAb CD73xEpCAM, bsAb- controls or oleclumab (1 μg/ml) were added to EVs derived from patient # 6 and evaluated for capacity to inhibit the enzyme activity of CD73. Background levels of Pi present in the medium in the absence of bsAb CD73xEpCAM was used to normalize CD73 in hibition to 0%. All graphs represent mean ± SD. Statistical analysis in graph D was performed using un-paired T test (*p < .05, ***p < .001).

Article Snippet: CHO.CD73 cells stably expressing human CD73 were generated by lipofection (Fugene-HD, Promega) using plasmids containing cDNAs encoding human CD73 (Origene).

Techniques: Activity Assay, Derivative Assay, Western Blot, Incubation, Produced

Journal: Journal of Personalized Medicine

Article Title: Breast Cancer Cell-Derived Adenosine Enhances Generation and Suppressor Function of Human Adaptive Regulatory T Cells

doi: 10.3390/jpm11080754

Figure Lengend Snippet: Ectonucleotidase expression and function in MDA-MB231 versus MCF-7 cell lines. ( A ) Tumor cells were stained for CD39 and CD73 and analyzed by flow cytometry. One representative experiment of 3 performed is shown. ( B ) MDA-MB231 and MCF-7 tumor cell lines were incubated with exogenous ATP or AMP in the presence or absence of ARL67156, a CD39 inhibitor, or αβ-methylene-ADP, a CD73 inhibitor. Levels of generated adenosine were measured by mass spectrometry. Data are from 3 independent experiments and show means ± SD. ( C ) MDA-MB231 cells were treated with siRNA specific for CD73 or with scrambled siRNA as control for 24 h. Cells were layered on slides, and after fixation with methanol were stained with Abs specific for CD73. Tumor cells treated with the scrambled siRNA were positive for CD73, while those treated with CD73-specific siRNA were negative. (Mag × 400). ( D ) Flow cytometry analysis of MDA-MB231 cells treated with CD73-specific siRNA or scrambled siRNA for 24 h. Loss of CD73 expression in cells treated with CD73-specific siRNA is evident. Isotype control IgG is shown as a gray histogram. ( E ) Western blot showing a partial decrease in CD73 after 24, 48, and 72 h of treatment of MD-MB231 cells with CD73-specific siRNA. The densitometry readings in pixels were: empty vector = 127,942; siRNA 24 h = 57,708; siRNA 48 h = 12,748; and siRNA 72 h = 52,950. Results are representative of one experiment of 5 performed.

Article Snippet: CD73 was transfected into CD73neg BrCa cell lines using a human cDNA ORF clone purchased from Origene, Rockville.

Techniques: Expressing, Staining, Flow Cytometry, Incubation, Generated, Mass Spectrometry, Western Blot, Plasmid Preparation

Journal: Journal of Personalized Medicine

Article Title: Breast Cancer Cell-Derived Adenosine Enhances Generation and Suppressor Function of Human Adaptive Regulatory T Cells

doi: 10.3390/jpm11080754

Figure Lengend Snippet: Phenotypic characterization and ectonucleotidase expression and activity in generated Tr1. ( A ) Flow cytometry analysis of Tr1 generated in the presence of a CD73 + (TrlTU73 + ) or CD73 neg (Tr1TU73 neg ) tumor cell lines or exogenous CADO (6 μM). CD4 + CD25 neg T cells cultured for 10 d in the presence of IL-2 (150 IU/mL) but in the absence of tumor cells and DC served as reference cells for all co-cultures. ( B ) Flow cytometry analysis of Tr1TU73 + generated in the presence of MDA-MB231 cell line with and without the addition of αβ-methylene-ADP, a CD73 inhibitor, or in the presence of the MDA-MB231 cell line treated with CD73 siRNA. Data are from 10 independent experiments and show means ± SD. ( C ) CD39 and CD73 expression of Tr1TU73 + or Tr1TU73 neg was measured by flow cytometry. CD4 + CD25 neg T cells cultured for 10 d in the presence of IL-2 (150 IU/mL) served as control. ( D ) Tr1TU73 + or Tr1TU73 neg were plated in 96-well plates (25,000 cells/well) in serum-free medium with 10 μM of exogenous ATP. Unhydrolyzed ATP was measured after 30 min of incubation. The % hydrolysis was calculated based on a standard curve. ARL67156 (an ecto-ATPase inhibitor) was added to selected wells. Data (means ± SD) are from 10 independent experiments. ( E ) Adenosine production by Tr1TU73 + or Tr1TU73 neg was determined by mass spectrometry. In some experiments, α,β methylene ADP, a CD73 inhibitor, α,β methylene ADP or ARL67156, a CD39 inhibitor, was added. Data show one representative experiment of 3 performed. In ( A – E ), asterisks * indicate differences at p < 0.01 to p < 0.05.

Article Snippet: CD73 was transfected into CD73neg BrCa cell lines using a human cDNA ORF clone purchased from Origene, Rockville.

Techniques: Expressing, Activity Assay, Generated, Flow Cytometry, Cell Culture, Incubation, Mass Spectrometry

Journal: Journal of Personalized Medicine

Article Title: Breast Cancer Cell-Derived Adenosine Enhances Generation and Suppressor Function of Human Adaptive Regulatory T Cells

doi: 10.3390/jpm11080754

Figure Lengend Snippet: Phenotypic analysis of ectonucleotidase expressing tumor cells and Treg in tumor tissues of breast cancer patients. Breast cancer tissue biopsies from basal and luminal BrCa were examined using multicolor immunofluorescence and confocal microscopy. ( A ) Expression of CD73 in tumor cells of basal and luminal BrCa is shown in representative samples of 14 basal and 18 luminal individual tumor specimens examined (final Mag × 300). ( a , e ): DAPI staining; ( b , f ): cytokeratin staining; ( c , g ): CD73 staining; ( d , h ): merged images: tumor cells are red, CD73 + cells are green and CD73 + tumor cells appear yellow. Inserts in ( d , h ) show cells at Mag × 400. ( B , C ) CD4 + CD25 + CD73 + as well as FOXP3 + CD25 + CD39 + T cells are shown in sections of a representative tumor of 5 examined (Mag × 400). Sections are stained for CD4 + , CD25 + or CD4 + /CD25 + /CD73 + cells and FOXP3 + /CD25 + or FOXP3 + /CD25 + /CD39 + cells. In ( B ), CD4 + cells are green, CD25 + cells are red, CD4 + CD25 + cells are yellow, while CD4 + CD25 + CD73 + cells appear pseudo-blue. In ( C ), FOXP3 + cells are green, CD25 + cells are red, FOXP3 + CD25 + cells are yellow, and FOXP3 + CD25 + CD39 + cells appear pseudo-blue. Expression of ectonucleotidases on tumor-infiltrating Treg is higher in basal than luminal BrCa.

Article Snippet: CD73 was transfected into CD73neg BrCa cell lines using a human cDNA ORF clone purchased from Origene, Rockville.

Techniques: Expressing, Immunofluorescence, Confocal Microscopy, Staining