Journal: bioRxiv

Article Title: Inhibitory CD200-receptor signaling is rewired by type I interferon

doi: 10.1101/2020.02.06.933739

Figure Lengend Snippet: (A-D) Phosphorylation of pErk (n=16, from 8 experiments), p-Akt473 (n=14, from 7 experiments), p-Akt308 (n=14, from 7 experiments) and p-rpS6 (n=7, from 7 experiments) in U937-CD200R-GFP (Blue) or CD200RΔ-GFP (Gray) cells after 1h stimulation with anti-CD200R (filled histogram) or isotype control (open histogram) as determined by intracellular flow cytometry. Significance was determined by a t test with Welch correction. The data are normalized to isotype stimulated cells within experiments. See supplemental figure 1A for expression of CD200R and CD200RΔ and gating strategy. ( E-H ) The effect of removal of Dok2 or RasGAP, using two different guide RNA per target, on the inhibitory capacity of CD200R-GFP on ( E ) p-Erk (n=8, from 4 experiments), ( F ) IL-8 secretion (n=4, from 4 experiments), ( G ) p-Akt473 (n=13, from 7 experiments) and ( H ) p-Akt308 (n=13, from 7 experiments). CD200R was ligated with an agonistic antibody or isotype control on U937-CD200R-GFP polyclonal lines deficient for Dok2 or RasGAP. Within experiments, data were normalized to the parental cell line and tested for significance by one-way ANOVA with Holm-Sidak’s correction. Data are obtained in at least 4 independent experiments. Boxplots: median with 25 and 75 percentiles and bars represent minimum and maximum values. ( I ) A model of CD200R signaling. CD200R-signaling bifurcates at the level of Dok2. Dok2 is required for inhibition of Akt and recruitment of RasGAP. Subsequently, RasGAP inhibits Ras/Erk signaling.

Article Snippet: For CD200R-ligation experiments, 10μg/ml culture grade anti-CD200R or isotype control; or 3μg/ml CD200his and CD200Rhis (both Sino Biologicals) were coated onto MaxiSORB (NUNC) plates at 4°C overnight in PBS.

Techniques: Flow Cytometry, Expressing, Inhibition

Journal: bioRxiv

Article Title: Inhibitory CD200-receptor signaling is rewired by type I interferon

doi: 10.1101/2020.02.06.933739

Figure Lengend Snippet: ( A ) Phosphorylation of p-Akt308, p-Akt473, p-Erk, and p-S6 after 1h stimulation of CD200R with agonistic-antibodies or isotype control in CD200R-U937-cells that over-express full-length RasGAP/p120 or RasGAP 1-455 . Data were normalized to isotype-stimulated cells. Significance was determined by a t test with Welch correction (n=6). ( B ) Caspase activity in fresh and IFNα stimulated PBMC as measured by CaspGlo 3/7 assay. Significance was determined by a t test (n=4). ( C, D ) Western blot analysis of RasGAP and Dok-2 protein expression and cleavage in fresh (Fr.) or 16h IFNα stimulated cells. Blots are lysates of (C) CD14 + monocytes and (D) PBMC cultures. >: full length RasGAP/p120; <: published RasGAP cleavage fragment . H3: Histone 3. HSP: heat shock protein 90. ( E ) Quantification of . Data represent the amount full length RasGAP/p120 or the amount of cleaved RasGAP as percentage of signal in the whole lane. Significance was determined by paired-t test.

Article Snippet: For CD200R-ligation experiments, 10μg/ml culture grade anti-CD200R or isotype control; or 3μg/ml CD200his and CD200Rhis (both Sino Biologicals) were coated onto MaxiSORB (NUNC) plates at 4°C overnight in PBS.

Techniques: Activity Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: Inhibitory CD200-receptor signaling is rewired by type I interferon

doi: 10.1101/2020.02.06.933739

Figure Lengend Snippet: ( A ) Cytokine secretion by moDC stimulated with R848 for 6h in presence of control, Akt-inhibitor AktVIII or Mek-inhibitor U0126. IL-1β: n=5, IL-8: n=8. Significance was determined by one-sample t test. ( B, C ) Phosphorylation of p-Akt473 and p-rpS6 in HC moDC. Fresh replated (n=8, 3 samples without IFN-counterpart) or IFNα-stimulated (16h, n=5, paired with fresh samples) moDCs were seeded on plates coated with control (recombinant human CD200R1, rhCD200R1) or recombinant human CD200 (rhCD200). After 2h pre-incubation, the moDC were stimulated with 1µg/ml R848 for 30 minutes. Phosphorylation was assessed by flow cytometry. Depicted is the ratio of phosphorylation in CD200-stimulated conditions over control stimulated conditions. Significance between the effect of CD200R on fresh and IFNα stimulated cells was determined by t test. Significance of the effect of CD200R in medium or IFNα stimulated cells was determined by one-sample t test. ( D, E ) mRNA (n=11) and protein (n=6) data on R848-induced IFNG or IFNγ production in PBMC. Donors with blue lines are matched between mRNA and protein data. Significance was determined by Wilcoxon test. ND = not detectable. ( F ) The ratio of R848 induced IFNG mRNA production of PBMC stimulated with anti-CD200R over PBMC stimulated with isotype. IFNG mRNA production was measured in healthy control PBMC that were used fresh after isolation (Fr., n=14), or have been cultured for 16h in medium (Med, n=20), 10 (n=8), 100 (n=8) or 1000 (n=20) U/ml of IFNα prior to ligation of CD200R with an agonistic antibody and stimulation of TLR7/8 with R848 for 4h. Significance was determined by a paired t test with Welch correction.

Article Snippet: For CD200R-ligation experiments, 10μg/ml culture grade anti-CD200R or isotype control; or 3μg/ml CD200his and CD200Rhis (both Sino Biologicals) were coated onto MaxiSORB (NUNC) plates at 4°C overnight in PBS.

Techniques: Recombinant, Incubation, Flow Cytometry, Isolation, Cell Culture, Ligation

Journal: bioRxiv

Article Title: Inhibitory CD200-receptor signaling is rewired by type I interferon

doi: 10.1101/2020.02.06.933739

Figure Lengend Snippet: ( A, B ) IFNG mRNA production relative to GAPDH (A) or B2M (B) by healthy control and SLE patients’ PBMC in two cohorts. PBMC were seeded on an isotype control (open circles) or an agonistic anti-CD200R antibody (filled circles) prior to stimulation with R848 for 4h. Each connected dot represents one patient/control. The color of the connecting arrow indicates: inhibition by CD200R ligation (blue, >15% inhibition compared to isotype control); potentiation by CD200R ligation (red, >15% potentiation compared to isotype control); or no effect of CD200R ligation (grey, <15 % inhibition or <15% potentiation compared to isotype control). ( C ) Ratio of R848-induced IFNG mRNA production from data in and , grouped to HC, SLE patients without lupus nephritis (LN-) and patients with (a history of) lupus nephritis (LN+). Significance was determined with a one-way ANOVA.

Article Snippet: For CD200R-ligation experiments, 10μg/ml culture grade anti-CD200R or isotype control; or 3μg/ml CD200his and CD200Rhis (both Sino Biologicals) were coated onto MaxiSORB (NUNC) plates at 4°C overnight in PBS.

Techniques: Inhibition, Ligation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis

doi: 10.3389/fcell.2021.745120

Figure Lengend Snippet: Expression of ARID1A and CD47 and their relationship in GC tissues. (A,B) Representative IHC images of ARID1A staining, ARID1A-preserved expression (A) , ARID1A-loss expression (B) . (C,D) Representative IHC images of CD47 staining, CD47 high expression (C) , CD47 low expression (D) . (E) Relationship between loss/preserved expression of ARID1A and CD47 expression ( n = 154). (F) Relationship between mutated/wild-type expression of ARD1A and CD47 expression (data from TCGA, n = 375). * p < 0.05, *** p < 0.001.

Article Snippet: In brief, equal amounts of cells were harvested, were Fc-blocked, and were incubated with anti-CD47 antibody (PE) (cat. 12283-MM07-P; SinoBiological) or isotype control for 30 min at room temperature.

Techniques: Expressing, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis

doi: 10.3389/fcell.2021.745120

Figure Lengend Snippet: The correlation between ARID1A status and CD47 expression and clinicopathological characteristics.

Article Snippet: In brief, equal amounts of cells were harvested, were Fc-blocked, and were incubated with anti-CD47 antibody (PE) (cat. 12283-MM07-P; SinoBiological) or isotype control for 30 min at room temperature.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis

doi: 10.3389/fcell.2021.745120

Figure Lengend Snippet: Loss of ARID1A increases CD47 expression. (A,B) Protein and mRNA expression levels of ARID1A and CD47 among various GC cell lines. (C–E) Knockdown of ARID1A significantly increased total and membranous CD47 protein levels, and CD47 mRNA level measured by western blot, flow cytometry and qRT-PCR in GC cell lines. (F) Schematic illustration of the relative positions of qPCR probes to putative CD47 promoter region for ChIP-qPCR experiments. (G) ARID1A binding to the promoter region of CD47 was determined by ChIP-qPCR in BGC-823 and MKN28 cells, respectively. *** p < 0.001, **** p < 0.0001.

Article Snippet: In brief, equal amounts of cells were harvested, were Fc-blocked, and were incubated with anti-CD47 antibody (PE) (cat. 12283-MM07-P; SinoBiological) or isotype control for 30 min at room temperature.

Techniques: Expressing, Western Blot, Flow Cytometry, Quantitative RT-PCR, Binding Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis

doi: 10.3389/fcell.2021.745120

Figure Lengend Snippet: Kaplan–Meier curves under various conditions. (A,B) Survival curves according to ARID1A status or CD47 expression ( n = 154). (C) Survival curves according to ARID1A status combined with CD47 expression level ( n = 154). (D–G) Survival curves according to ARID1A status or CD47 expression with HER2 positive or negative.

Article Snippet: In brief, equal amounts of cells were harvested, were Fc-blocked, and were incubated with anti-CD47 antibody (PE) (cat. 12283-MM07-P; SinoBiological) or isotype control for 30 min at room temperature.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis

doi: 10.3389/fcell.2021.745120

Figure Lengend Snippet: Characterization of tumor-infiltrating immune cells based on ARID1A and CD47 expression. (A) Estimation of tumor-infiltrating immune cells in different subgroups stratified by ARID1A status and CD47 expression. (B) Multiplex immunofluorescent images illustrate GC tissues with ARID1A loss CD47 high or ARID1A preserved CD47 low expression that express CD11c, CD163, and Foxp3. (C) Quantitative analysis of the densities of immune markers between ARID1A loss CD47 high and ARID1A preserved CD47 low GC tissues. Error bars represent mean with SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In brief, equal amounts of cells were harvested, were Fc-blocked, and were incubated with anti-CD47 antibody (PE) (cat. 12283-MM07-P; SinoBiological) or isotype control for 30 min at room temperature.

Techniques: Expressing, Multiplex Assay

Journal: Autophagy

Article Title: Autophagy-based unconventional secretion of HMGB1 by keratinocytes plays a pivotal role in psoriatic skin inflammation

doi: 10.1080/15548627.2020.1725381

Figure Lengend Snippet: Autophagy-related proteins are functionally active in psoriatic keratinocytes. (A) Expression of the autophagy marker BECN1 in the epidermis of psoriatic patients and normal subjects. Representative immunostaining images of skin (left) and quantification of staining intensity in the epidermis (right). Scale bar: 50 μm. n = 12/group. (B) A significant correlation was found between the average epidermal BECN1 intensities in (A) and the Baker scores of psoriatic lesions (r and p values obtained with Spearman’s rank correlation test). (C and D) Expression of the indicated autophagy markers in the epidermis of psoriatic patients and normal subjects. (C) Representative ATG5 immunostaining images (left) and quantification of staining intensity in the epidermis (right), scale bar: 50 μm, n = 6/group. (D) Representative immunofluorescence of LC3 immunostaining images (top) and quantification of LC3 puncta in the epidermis (bottom, more than 200 epidermal cells were counted each specimen), scale bar: overviews (50 μm) and insets (10 μm), n = 7/group. (E) Representative electron microscopic images of keratinocytes of lesional psoriatic skin and healthy skin (top) and quantification of autophagic vacuoles (bottom), more than 10 cells were counted from each specimen. Black arrow, autophagic vacuoles; N, nucleus. Scale bar: 1 µm. (F-I) Expression of autophagy markers in the back skin of the IMQ mouse model (F and H) and the ear skin of the Krt14-Vegfa transgenic mouse model (G and I) at the indicated time points (d: day; mo: month), n = 5/group. (F and G) Representative immunoblots of indicated proteins from skin lysates. (H and I) Quantification of immunohistochemical staining intensity of BECN1 in the epidermis. (J-M) Skin autophagy was detected in an IMQ-induced psoriasis mouse model (J and L) and a Krt14-Vegfa transgenic mouse model of psoriasis (K and M) after treatment with therapeutic, recombinant IL23R-FC (0.5 or 1 mg/kg) or cyclosporin A (CsA; 20 or 50 mg/kg), n = 5/group. (J and K) Representative immunoblots of BECN1, LC3A/B-II and ATG5 from skin lysates. (L and M) Quantification of immunohistochemical staining intensity of BECN1 in the epidermis. ACTB was detected as a loading control in (F, G, J and K). Mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Two-tailed Student’s T-test (A and C-E). One-way ANOVA (H, I, L and M). All the data are representative of three independent experiments

Article Snippet: The coverslips were stimulated with M5 combination for 48 h, and the coverslips were fixed in cold methanol-acetone (1:1) for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, blocked with 5% BSA in PBS for 30 min, and then incubated overnight at 4°C with primary antibodies against LC3A/B (1:200 dilution) and LAMP1 (Sino Biological, 11215-MM07; 1:100 dilution).

Techniques: Expressing, Marker, Immunostaining, Staining, Immunofluorescence, Transgenic Assay, Western Blot, Immunohistochemical staining, Recombinant, Two Tailed Test

Journal: Autophagy

Article Title: Autophagy-based unconventional secretion of HMGB1 by keratinocytes plays a pivotal role in psoriatic skin inflammation

doi: 10.1080/15548627.2020.1725381

Figure Lengend Snippet: The activation of MAPK signaling pathways is involved in autophagy in psoriasiform keratinocytes. (A and B) Representative LC3A/B-II immunoblots from HaCaT cells (A) and NHEK cells (B) stimulated with or without M5 for the indicated times. (C) EGFP-LC3-transfected HaCaT cells were stimulated with or without M5 for the indicated times, n = 3/group. Representative fluorescence images (left) and quantification of EGFP-LC3 puncta in keratinocytes (right, more than 50 cells were counted each condition). Scale bar: 10 μm. (D) Representative electron microscopic images of NHEKs treated with or without M5 for 48 h (top). Black arrow, autophagic vacuoles; N, nucleus; scale bar: 2 μm. Quantification of autophagic vacuoles (bottom, more than 20 cells were counted for each condition), n = 5/group. (E and F) Representative images of LC3A/B and lysosomal marker LAMP1 immunostaining in HaCaT cells (E) and NHEK cells (F) stimulated with or without M5 (left). Scale bar: 10 μm. Pearson’s colocalization coefficient for LC3A/B and LAMP1 (right), n = 5-6/group. (G) NHEKs were pre-treated with or without 3-methyladenine (3-MA; 4 h, 10 mM), bafilomycin A1 (BAF; 4 h, 200 nM) or wortmannin (WOR; 4 h, 100 nM), and then stimulated with or without M5 for 48 h. Immunoblot analysis of the LC3A/B-II levels. (H) Representative immunoblots of MAPK proteins in HaCaT cells stimulated with or without M5 in a time-dependent manner. (I) Representative immunoblots of MAPKs (MAPK8/9/10, MAPK1/3 and MAPK14) for HaCaT cells that were transiently transfected with MAPKs (MAPK8/9/10, MAPK1/3 and MAPK14) siRNA or control siRNA for 48 h. (J and K) HaCaT cells were transiently transfected with MAPKs (MAPK8/9/10, MAPK1/3 and MAPK14) siRNA or control siRNA for 48 h, and were then treated with M5 for 48 h. Representative LC3A/B-II immunoblots (J) and representative images of LC3A/B and lysosomal marker LAMP1 immunostaining (K, left), scale bar: 10 μm. Pearson’s colocalization coefficient for LC3A/B and LAMP1 (K, right), n = 5/group. ACTB was detected as a loading control (A, B and G-J). Mean ± SD. **P < 0.01; ***P < 0.001. Two-tailed Student’s T-test (C-F). One-way ANOVA (K). All the data are representative of three independent experiments

Article Snippet: The coverslips were stimulated with M5 combination for 48 h, and the coverslips were fixed in cold methanol-acetone (1:1) for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, blocked with 5% BSA in PBS for 30 min, and then incubated overnight at 4°C with primary antibodies against LC3A/B (1:200 dilution) and LAMP1 (Sino Biological, 11215-MM07; 1:100 dilution).

Techniques: Activation Assay, Western Blot, Transfection, Fluorescence, Marker, Immunostaining, Two Tailed Test

Journal: Autophagy

Article Title: Autophagy-based unconventional secretion of HMGB1 by keratinocytes plays a pivotal role in psoriatic skin inflammation

doi: 10.1080/15548627.2020.1725381

Figure Lengend Snippet: HMGB1, IL1B, and IL18 secretion are regulated by autophagy in psoriasiform keratinocytes dependent on GORASP2. (A and B) NHEK cells were pre-treated with or without 3-methyladenine (3-MA; 4 h, 10 mM), bafilomycin A1 (BAF; 4 h, 200 nM) or wortmannin (WOR; 4 h, 100 nM), and stimulated with or without M5 for 48 h, n = 3/group. (A) ELISA assessed the levels of HMGB1, IL18 and IL1B in the culture medium. (B) The LDH release assay determined cytotoxicity. (C) qRT-PCR analysis of the GORASP2 knockdown levels in HaCaT cells after transfection with GORASP2 shRNA or vector control, n = 3/group. (D-G) Ctrl shRNA KCs and GORASP2 shRNA KCs were stimulated with or without M5 for 24 h or 48 h, n = 3/group. (D) Representative LC3A/B-II immunoblots for KCs after treatment with M5 for 48 h, ACTB was used as a loading control. (E) Analysis of HMGB1, IL18 and IL1B levels in the culture medium by ELISA at 48 h. (F) The RNA levels of chemokines and antimicrobial peptides were assessed by qRT-PCR at 24 h. (G) Analysis of CXCL8 levels in the culture medium by ELISA at 48 h. Mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA (A-C, E-G). All the data are representative of three independent experiments

Article Snippet: The coverslips were stimulated with M5 combination for 48 h, and the coverslips were fixed in cold methanol-acetone (1:1) for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, blocked with 5% BSA in PBS for 30 min, and then incubated overnight at 4°C with primary antibodies against LC3A/B (1:200 dilution) and LAMP1 (Sino Biological, 11215-MM07; 1:100 dilution).

Techniques: Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Quantitative RT-PCR, Transfection, shRNA, Plasmid Preparation, Western Blot

Journal: Autophagy

Article Title: Autophagy-based unconventional secretion of HMGB1 by keratinocytes plays a pivotal role in psoriatic skin inflammation

doi: 10.1080/15548627.2020.1725381

Figure Lengend Snippet: Autophagy-based unconventional secretion of HMGB1 in psoriasiform KCs. (A) NHEK cells were stimulated with M5 for 24 h in the absence or presence of HMGB1-neutralizing antibody (anti-HMGB1,10 µg/mL), IL18- or IL1B-neutralizing antibody (anti-IL18,10 µg/mL; anti-IL1B, 10 µg/mL). The indicated genes levels were assayed by qRT-PCR, n = 3/group. (B) ELISA analysis of HMGB1 levels in cell culture supernatants of Krt14+/+-Atg5f/f and Krt14Cre/+-atg5f/f primary murine keratinocytes treated with M5 for 48 h, n = 3/group. (C) ELISA analysis of HMGB1 levels in serum from Krt14+/+-Atg5f/f and Krt14Cre/+-atg5f/f mice treated with IMQ for 5 d, n = 3/group. (D) NHEKs were pre-treated with or without 3-methyladenine (3-MA; 4 h, 10 mM), bafilomycin A1 (BAF; 4 h, 200 nM) or wortmannin (WOR; 4 h, 100 nM), and stimulated with or without M5 for 24 h. The HMGB1 gene expression levels were assayed by qRT-PCR, n = 3/group. (E) Ctrl shRNA HaCaT cells and GORASP2 shRNA HaCaT cells were stimulated with or without M5 for 24 h. HMGB1 gene expression levels were assayed by qRT-PCR, n = 3/group. (F-H) HaCaT cells were stimulated with M5 for 24 h or 48 h in the absence or presence of monensin (10 μM, 3 h) or FLI-06 (10 μM, 3 h), the blockers of Golgi transport in conventional secretion pathway. n = 3/group. (F) Representative immunoblots of HMGB1 in cell lysates at 48 h. (G) Analysis of HMGB1 levels in the culture medium by ELISA at 48 h. (H) qRT-PCR analysis of HMGB1 expression at 24 h. (I) HMGB1-EBFP2-transfected HaCaT cells were treated with M5, and representative confocal images of immunofluorescence of LC3A/B and LAMP1 are shown. Scale bars: 10 μm. (J) Line tracings, analysis of fluorescence signal intensity from images in (I), scale bars: 2 μm. (K) Pearson’s colocalization coefficient for HMGB1 and LC3A/B, HMGB1 and LAMP1. (L and M) Representative immunoblots of the indicated proteins in the cell lysates of NHEKs that were pretreated with or without 3-MA (4 h, 10 mM), BAF (4 h, 200 nM), and stimulated with or without M5 for 48 h. ACTB was used as a loading control (F, L and M). Mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA (A-E, G and H), Two-tailed Student’s T-test (K). All the data are representative of three independent experiments

Article Snippet: The coverslips were stimulated with M5 combination for 48 h, and the coverslips were fixed in cold methanol-acetone (1:1) for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, blocked with 5% BSA in PBS for 30 min, and then incubated overnight at 4°C with primary antibodies against LC3A/B (1:200 dilution) and LAMP1 (Sino Biological, 11215-MM07; 1:100 dilution).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing, shRNA, Western Blot, Transfection, Two Tailed Test

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: 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: Antibodies: Fluorescein isothiocyanate (FITC)-labeled-anti-CD73 (clone-MM07, Sino Biological), Allophycocyanin (APC)-labeled-anti-EGFR (clone-528, Santa Cruz), FITC-labeled-annexin-V (ImmunoTools), goat-anti-human-IgG-APC (SouthernBiotech), sheep-anti-human-CD73 (R&D), rabbit-anti-human-EGFR (Abcam), rabbit-anti-human-HER2 (R&D), rabbit-anti-human/mouse-β-Actin (Abcam), rabbit-anti-sheep-IgG (Thermo Fisher), goat-anti-rabbit-IgG (Dako), anti-CD4 (ab183685, Abcam), anti-CD8 (98941, Cell Signaling Technology), anti-FoxP3 (ab99964; Abcam), anti-F4/80 (ab240946, Abcam), anti-CD206 (ab64693, Abcam), anti-CD11c (ab52632; Abcam).

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: 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-V pos /PI pos 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: Antibodies: Fluorescein isothiocyanate (FITC)-labeled-anti-CD73 (clone-MM07, Sino Biological), Allophycocyanin (APC)-labeled-anti-EGFR (clone-528, Santa Cruz), FITC-labeled-annexin-V (ImmunoTools), goat-anti-human-IgG-APC (SouthernBiotech), sheep-anti-human-CD73 (R&D), rabbit-anti-human-EGFR (Abcam), rabbit-anti-human-HER2 (R&D), rabbit-anti-human/mouse-β-Actin (Abcam), rabbit-anti-sheep-IgG (Thermo Fisher), goat-anti-rabbit-IgG (Dako), anti-CD4 (ab183685, Abcam), anti-CD8 (98941, Cell Signaling Technology), anti-FoxP3 (ab99964; Abcam), anti-F4/80 (ab240946, Abcam), anti-CD206 (ab64693, Abcam), anti-CD11c (ab52632; Abcam).

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: 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: Antibodies: Fluorescein isothiocyanate (FITC)-labeled-anti-CD73 (clone-MM07, Sino Biological), Allophycocyanin (APC)-labeled-anti-EGFR (clone-528, Santa Cruz), FITC-labeled-annexin-V (ImmunoTools), goat-anti-human-IgG-APC (SouthernBiotech), sheep-anti-human-CD73 (R&D), rabbit-anti-human-EGFR (Abcam), rabbit-anti-human-HER2 (R&D), rabbit-anti-human/mouse-β-Actin (Abcam), rabbit-anti-sheep-IgG (Thermo Fisher), goat-anti-rabbit-IgG (Dako), anti-CD4 (ab183685, Abcam), anti-CD8 (98941, Cell Signaling Technology), anti-FoxP3 (ab99964; Abcam), anti-F4/80 (ab240946, Abcam), anti-CD206 (ab64693, Abcam), anti-CD11c (ab52632; Abcam).

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: 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)-Foxn nu ) 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 (mm 3 ) 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: Antibodies: Fluorescein isothiocyanate (FITC)-labeled-anti-CD73 (clone-MM07, Sino Biological), Allophycocyanin (APC)-labeled-anti-EGFR (clone-528, Santa Cruz), FITC-labeled-annexin-V (ImmunoTools), goat-anti-human-IgG-APC (SouthernBiotech), sheep-anti-human-CD73 (R&D), rabbit-anti-human-EGFR (Abcam), rabbit-anti-human-HER2 (R&D), rabbit-anti-human/mouse-β-Actin (Abcam), rabbit-anti-sheep-IgG (Thermo Fisher), goat-anti-rabbit-IgG (Dako), anti-CD4 (ab183685, Abcam), anti-CD8 (98941, Cell Signaling Technology), anti-FoxP3 (ab99964; Abcam), anti-F4/80 (ab240946, Abcam), anti-CD206 (ab64693, Abcam), anti-CD11c (ab52632; Abcam).

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: 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) IC 50 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: Antibodies: Fluorescein isothiocyanate (FITC)-labeled-anti-CD73 (clone-MM07, Sino Biological), Allophycocyanin (APC)-labeled-anti-EGFR (clone-528, Santa Cruz), FITC-labeled-annexin-V (ImmunoTools), goat-anti-human-IgG-APC (SouthernBiotech), sheep-anti-human-CD73 (R&D), rabbit-anti-human-EGFR (Abcam), rabbit-anti-human-HER2 (R&D), rabbit-anti-human/mouse-β-Actin (Abcam), rabbit-anti-sheep-IgG (Thermo Fisher), goat-anti-rabbit-IgG (Dako), anti-CD4 (ab183685, Abcam), anti-CD8 (98941, Cell Signaling Technology), anti-FoxP3 (ab99964; Abcam), anti-F4/80 (ab240946, Abcam), anti-CD206 (ab64693, Abcam), anti-CD11c (ab52632; Abcam).

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