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