Journal: Oncoimmunology

Article Title: Bendamustine with total body irradiation conditioning yields tolerant T-cells while preserving T-cell-dependent graft-versus-leukemia

doi: 10.1080/2162402X.2020.1758011

Figure Lengend Snippet: BEN-TBI does not result in appreciable donor T-cell phenotypic differences post-transplant when compared to CY-TBI. (a–c) BALB/c recipient mice received 40 mg/kg BEN iv or 200 mg/kg CY ip on day −2, 400 cGy TBI on day −1, and 10 7 TCD-BM from naïve C57BL/6 mice with 3 × 10 6 CellTrace Violet-stained tT from naïve BoyJ mice on day 0. Blood and spleen were collected on day +3. (a) % donor T-cells (CD45.1+) was determined by flow cytometry. Using CBCs determined by HemaVet analysis, absolute number of donor T-cells was calculated. (b) After gating on CD45.1+ cells (representing donor T-cells), CellTrace Violet dilution was analyzed using ModFit software to determine proliferation index. Representative CellTrace Violet dilution is shown. (c) Within the CD45.1+ gate, cells were stratified by CellTrace high (non- proliferative) and CellTrace low (proliferative) and CD25 and CD47 expression were analyzed by flow cytometry. Pooled data from 2 experiments with line at mean are shown, n = 6–7 mice/group. * p < .05 , ** p < .01 . (d-g) BALB/c recipient mice received 40 mg/kg BEN iv or 200 mg/kg CY ip on day −2, 400 cGy TBI on day −1, and 10 7 BM with 3 × 10 6 SC from naïve C57BL/6 mice on day 0. Peripheral blood was collected on days +7, +14, +21, +35, and +70 and stained for CD8, CD4, Tbet, GATA3, RORγt, CD134, CD278, PD-1, TIM3, CTLA-4, and CD272. CBCs were determined and used to calculate absolute cell numbers. Average absolute numbers of cells per μL of blood are shown with SEM. Representative flow plots from day +7 with fluorescence minus one (FMO) controls are shown (e). (d) Pooled data from 4 experiments are shown, n = 19 mice/group. (e-g) Pooled data from 2 experiments are shown, n = 10 mice/group. ** p < .01.

Article Snippet: Antibodies used were anti-mouse H2kb PerCP-eFluor710 (clone AF6-88.5.5.3; 46–5958), CD8α PE-Cy7 (53–6.7; 25–0081), CD4 APC (RM4-5; 50-148-54), FoxP3 APC (FJK-16s; 17–5773), CD45.1 APC (A20; 17–0453), GATA3 PE-Cy7 (TWAJ; 25–9966), CD134 PE-Cy7 (OX-86; 25–1341), FoxP3 PE (150D/E4; 12–4774), CD69 PE-Cy5 (H1.2F3; 15–0691), RORγt APC (AFKJS-9; 17–6988), IFN gamma PE (XMG1.2, 12–7311) (Thermo Fisher Scientific), CD45.1 PE-CF594 (A20; 562452), CD3ε PE-CF594 (145–2 C11; 562286), CD44 BB515 (IM7; 565941), CD44 BV510 (IM7; 563114) (BD Biosciences), CD47 PE-Vio770 (REA170; 130-102-383), TIM-3 VioBright FITC (REA602; 130-109-449), TIM-3 PE (REA602; 130-118-563), CD278 VioGreen (REA192; 130-100-739), CD272 PE (REA224; 130-102-689), CD4 APC-Vio770 (GK1.5; 130-102-786), CD4 VioGreen (GK1.5; 130-102-444) (Miltenyi Biotec), Tbet PE-Dazzle594 (4B10; 644828), CTLA-4 PE-Dazzle594 (UC10-4B9; 106318), CD8α Brilliant Violet 421 (53–6.7; 100738), CCR7 PE-Cy5 (4B12; 120114), PD-1 APC (29F.1A12; 135210), CD25 AlexaFluor700 (PC61; 102024), and TNF-α Brilliant Violet 510 (MP6-XT22, 506339) (Biolegend).

Techniques: Staining, Flow Cytometry, Software, Expressing, Fluorescence

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Surface CD47 and CRT expression in EGFR wild-type and mutant NSCLC cells. Surface CD47 (A) and ecto-CRT protein expression (B) shown as geometric MFI in a panel of six different NSCLC cell lines. Each histogram represents the mean (± SD) of three to five independent experiments. Comparisons made by ANOVA with Fisher's post hoc multiple comparison analysis. ### p < 0.03, ## p < 0.01, # p < 0.0005. Below each histogram, a matrix table where all p values resulting from post hoc analysis are reported. Expression levels of CD47 (C) and CRT mRNA (D) in 226 untreated primary NSCL adenocarcinomas (GEO accession number GSE31210 ). Middle lines in box plots represent the medians and whiskers represent 5–95% CI ( ### p < 0.03, Kruskal-Wallis test).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Expressing, Mutagenesis, Comparison

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Modulation by gefitinib of surface CD47 and CRT expression in EGFR wild-type and mutant NSCLC cells. Flow cytometric profiles of surface CD47 (A) and ecto-CRT expression (B) on DMSO-treated (CTRL, gray lines) and gefitinib-treated (GEF, red lines) NSCLC cells. Histograms show the mean (± SD) of fold changes of CD47 (C) and ecto-CRT (D) geometric MFI, relative to DMSO-treated controls ( N = 3–5, * p < 0.05, ** p < 0.01 paired two-tailed Student's t -test).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Expressing, Mutagenesis, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Gefitinib-induced CD47 down-regulation promotes tumor cell phagocytosis by dendritic cells. Representative flow cytometric analyses and mean ± SD ( N = 4 independent healthy donors) of phagocytic activity of monocyte-derived dendritic cells (see Methods) against PC9 (A,B) , HCC827 (C,D) , and H1975 cells (E,F) treated with DMSO (CTRL) or gefitinib (GEF) as indicated. Cancer cells exposed to the drug for 48 h were labeled with DiO tracer and then co-cultured with dendritic cells for 2 h at a 1:1 ratio. Phagocytosis assays were also run at 4°C as controls. Histograms represent the percentages of positive cells for both CD11c and DiO tracer relative to total dendritic cells (* p < 0.05, n.s., not significant, paired two-tailed Student's t -test).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Activity Assay, Derivative Assay, Labeling, Cell Culture, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Blocking of CD47 on tumor cells induces phagocytosis by dendritic cells. Dendritic cells were co-cultured with DiO tracer-labeled HCC827 (A) and H1975 (B) cancer cells in the presence of IgG isotype control or anti-CD47 mAb as indicated. Shown is the mean (± SD, N = 3 independent healthy donors) percentage increase of CD11c/DiO tracer double positive cells, relative to dendritic cells co-cultured with DMSO-treated tumor cells ( # p < 0.05, ## p < 0.01, ANOVA with Fisher's post hoc analysis).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Blocking Assay, Cell Culture, Labeling, Control

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Expression levels of surface CD47 increase in cancer cells acquiring resistance to gefitinib and inhibit tumor cell phagocytosis by dendritic cells. Surface CD47 (A) and ecto-CRT expression (B) in gefitinib-sensitive PC9 and HCC827 (gray lines) and resistant PC9GR and HCC827GR (green lines) cell lines. Representative flow cytometric histograms (left) and mean (± SD, N = 3–5) fold changes of treatment-resistant over sensitive cells (right). (C) Representative flow cytometric histogram plots (left) and mean (± SD) fold changes (right) of surface CD47 levels in resistant cell lines treated with DMSO (CTRL) or gefitinib (GEF) as indicated. Acquisition of resistance to gefitinib abolished drug-induced CD47 down-regulation in PC9GR (* p < 0.05, ** p < 0.01, n.s., not significant, paired two-tailed Student's t -test). (D) Mean ± SD ( N = 3 independent healthy donors) of phagocytic activity of monocyte-derived dendritic cells against PC9GR cells in the absence or presence of gefitinib treatment, performed at 4°C as control and at 37°C. Histograms represent the percentages of positive cells for both CD11c and DiO tracer relative to total dendritic cells (paired two-tailed Student's t -test. n.s., not significant). (E) Dendritic cells were co-cultured with gefitinib-treated, DiO tracer-labeled PC9GR cells in the presence of IgG isotype control or anti-CD47 mAb. Shown is the mean ± SD ( N = 3 independent healthy donors) percent change of CD11c + /DiO + tracer double positive dendritic cells, relative to dendritic cells co-cultured with DMSO-treated tumor cells ( ## p < 0.01, ANOVA with Fisher's post hoc analysis).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Expressing, Two Tailed Test, Activity Assay, Derivative Assay, Control, Cell Culture, Labeling

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: CD47 is overexpressed on malignant lymphocytes in mycosis fungoides (MF) tumors. a A representative image of a skin involved by MF demonstrates intense CD47 staining on atypical TOX + malignant cells. HE, hematoxylin and eosin (20x). b Targeted single-cell RNA transcriptomics as tSNE plots of concatenated tumors from three patients with MF tumors (n = 287 cells total). Clusters called by recursive dendrogram split and annotated from preferentially expressed genes. Tcm, T cell central memory; Tem, T cell effecor memory; DC, dendritic cells. c The intensity of CD47 expression (anti-CD47 antibody-oligo conjugate; AbSeq) over the various cell population defined in Fig. 1b. d Statistical analysis of expression of CD47 (molecules per cells) in different cell populations. *, p < 0.05; ***, p < 0.001 e CD47 expression on CD3 + TOX + MBL2 cells. Flow cytometry of a cell suspension from a primary cell culture. Grey tinted area, an isotype control. Red tinted area, anti-CD47 antibody. f Tumor growth curves of CD47hi WT MBL2 (WT) and CD47 KO MBL2 (CD47 KO) after implantation in B6.SJL mice. n = 5 mice in each group. g Representative imaging of mice 10 days after implantation of CD47hi WT MBL2 (WT) or CD47 KO MBL2 (KO) cells demonstrating large ulcerated tumor in WT, while KO mouse exhibited medium-size tumor without ulceration

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques: Staining, Expressing, Flow Cytometry, Suspension, Cell Culture, Control, Imaging

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: Anti-CD47 therapy is efficient in controlling the malignant lymphoma growth in a murine model of mycosis fungoides (MF). a Design of anti-CD47 experiments (I.P., intraperitoneal). b Tumor thickness after MBL2 implantation during treatment with anti-CD47 antibody or irrelevant IgG control (n = 5 mice per group). c Percentage of TOX + tumor cells in the inflammatory infiltrate of auricular skin at day 24 after implantation. The percentage of caspase-3 + cells is indicated in black (n = 5 mice per group) (Tx, treatment). d Percentage of F4/80 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). e Percentage of MHC class II + cells among F4/80 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). f Percentage of TNF-α + cells among F4/80 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). g Percentage of NK1.1 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). h Percentage of IFN-γ + cells among NK1.1 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). i Percentage of CD62L + NKG2A-cells among NK1.1. + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). j Schematic of experimental design. k Representative images of mice with tumors after treatment with IFN-α, anti-CD47, or combination anti-CD47 + IFN-α. l The percentage of malignant cells (TOX +) in the TME on day 24 after implantation of MBL2 (n = 5 mice per group). m Representative flow cytometry of CD107a and IFN-γ NK cells (gated on NK1.1 + cells) during therapy with anti-CD47 antibody. n Quantification of CD107a + IFN-γ + NK cells and CD107a + IFN-γ-NK cells (n = 5 mice per group). o The cytotoxic assay of NK cells derived from splenocytes of treated mice co-cultured with MBL2 cells at the indicated effector to target cell ratios

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques: Control, Flow Cytometry, Derivative Assay, Cell Culture

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: The effect of anti-CD47 therapy is mediated by cytotoxic NK cells and does not depend on IFN-γ. a Schematic of NK1.1 depletion experiment. b A representative flow showing the percentage of NK cells in non-depleted and NK-depleted mice prior to therapy. c Representative images of mice with tumors 14 days after MBL2 implantation with and without NK1.1 depletion prior to therapy initiation. D The percentage of malignant cells (CD3 + TOX + cells) per ear in non-depleted and NK-depleted mice 24 days after MBL2 implantation. N = 5 mice per group. ***, p < 0.001 e Tumor thickness 24 days after MBL2 implantation in mice treated with irrelevant IgG or anti-CD47 antibody (n = 5 mice per group). f Schematic of experimental design for IFN-γ KO mice. g The volume of lymph nodes 24 days after MBL2 implantation in mice treated with anti-CD47 antibody (n = 3 mice per group). *,p < 0.05; **,p < 0.01; ****,p < 0.0001; ns, non-significant

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques:

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: The anti-CD47 therapy is accompanied by an influx of NK cells in the TME in patients with relapsed/refractory mycosis fungoides (MF). a Representative images of high and low CD56 number in the dermal infiltrate of tumor MF. b Inverse correlation of the number of CD56 cells per 100 dermal lymphocytes and histoscore of CD47 on the epidermotropic malignant lymphocytes (n = 17, r = – 0.48, p < 0.05). c Representative images of a patient treated with six intra-tumoral injections of 10 mg TTI-621 demonstrate a significant reduction of all tumors and plaques after treatment (I, injected tumor; C, control non-injected tumor). d Multispectral fluorescent immunohistochemistry of CD3, CD56, TOX, and DAPI staining of representative tumors before and after treatment with TTI-621 (60X). e Percentage of malignant cells (CD3 + CD4 + TOX +) and NK cells (CD3-CD56 +) in the TME (n = 11 patients; 4 responders and 7 non-responders). f tSNE analysis of cellular composition in patients with cutaneous T cell lymphoma who had responded to therapy with TTI-621 (n = 4 patients), showing eight distinct clusters of NK cells. g Phenotypic characterization of eight distinct clusters of NK cells depending on the percentage of markers. h Grouping of eight NK cell clusters in two groups based on high vs. dim CD56 expression. i Changes in CD56high NK cells vs. CD56dim NK cells after intra-tumoral injection of TTI-621 (2 weeks of therapy) in patients who responded to TTI-621 (n = 4). j Percentage of NK cells before and after six intra-tumoral injections of TTI-621 (2 weeks of therapy) in patients who responded to TTI-621 (n = 4).. *,p < 0.05; **,p < 0.01

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques: Injection, Control, Immunohistochemistry, Staining, Expressing

Journal: Advanced Science

Article Title: Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

doi: 10.1002/advs.202206912

Figure Lengend Snippet: Preparation and characterization of anti‐CD47‐PCM@NP. A) Hydrodynamic size and zeta potential of CM vesicles, PLGA cores (NP), PCM@NP, and anti‐CD47‐PCM@NP. Data are means ± SD ( n = 3). B) Colocalization of NP/C6 (green) with DiD‐PCM (red), and the colocalization of FITC‐antibody (green) with DiD‐PCM@NP (red), both assessed by confocal laser scanning microscope (CLSM) (scale bar = 5 µm). C) Transmission electron micrographs of (a) NP, (b) CM vesicle, (c) PCM@NP, (d) Anti‐CD47‐PCM@NP, and (e) multiple anti‐CD47‐PCM@NP. All scale bars = 100 nm. D) SDS‐PAGE protein analysis of NP, PCM@NP, CM vesicles, and cancer cell lysate. Samples were tested at equal protein concentrations. CD47 protein and membrane‐specific protein on the cancer cell membrane were efficiently retained on the extracted membrane vesicles and the PCM@NP, detected by western blot. E) Determination of the antibody labeled by PE loaded on the surface of anti‐CD47‐PCM@NP by flow nanoanalyzer. F) The binding affinity of the antibody to the CM vesicles by surface plasmon resonance (SPR).

Article Snippet: The cells were diluted at 1× 10 6 cells per mL in a culture medium and incubated with 1.0 mg mL −1 of a PE anti‐human/mouse/rat CD47 antibody (E‐AB‐F1016D, Elabscience) or anti‐Neu/ErbB2/HER2 Antibody (SC‐7301, Santa Cruz Biotechnology) and CoraLite488‐conjugated Affinipure coat anti‐mouse IgG(H+L) (SA00013‐1, Proteintech) for 30 min at 4 °C.

Techniques: Zeta Potential Analyzer, Laser-Scanning Microscopy, Transmission Assay, SDS Page, Membrane, Western Blot, Labeling, Binding Assay, SPR Assay

Journal: Advanced Science

Article Title: Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

doi: 10.1002/advs.202206912

Figure Lengend Snippet: Anti‐CD47‐PCM@NP effectively distinguished target cells from CD47‐expressing nontarget cells in vitro through indirect active targeting. A) Anti‐CD47‐PCM@NP avoided the blocking of CD47 on RBC and subsequent phagocytosis thus circumvented the on‐target toxicity of free anti‐CD47 towards RBC through the INTACT strategy. Scale bar = 50 µm. B,C) PCM@NP and anti‐CD47‐PCM@NP efficiently escaped the capture by B) macrophages with enhanced and parallel cellular uptake by C) target 4T1 cells, measured by flow cytometry. The antibody selectively dissociated from the carrier PCM@NP at the presence of 4T1 cells with high expression of D) CD47 in contrast to E) CD47 −/− 4T1 cells, shown by colocalization images and distribution map of PE‐antibody (red) and PCM@NP/C6 (green). Scale bar = 20 µm (multi‐cell images), 2 µm (single‐cell images). F) The schematic diagram of the microfluidic device. The tumor cells were cultured in the cavity of the microfluidic chip till adherence, and then exposed to flowing anti‐CD47 or anti‐CD47‐PCM@NP, and fluorescent images were captured at predetermined time points. G) Free anti‐CD47 sufficiently bound to the surface of 4T1 cells with high expression of CD47 (a). The antibody dissociated from PCM@NP at the presence of 4T1 cells (b), in contrast with CD47 −/− 4T1 group (c) (antibody labeled with FITC, green. PCM@NP labeled with DiD, red). Scale bar = 10 µm. Original movies are shown in Movie S1 (Supporting Information) (a), Movie S2 (Supporting Information) (b), and Movie S3 (Supporting Information) (c), respectively. Data are presented as mean ± SD ( n = 3). (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; NS represents non‐significance).

Article Snippet: The cells were diluted at 1× 10 6 cells per mL in a culture medium and incubated with 1.0 mg mL −1 of a PE anti‐human/mouse/rat CD47 antibody (E‐AB‐F1016D, Elabscience) or anti‐Neu/ErbB2/HER2 Antibody (SC‐7301, Santa Cruz Biotechnology) and CoraLite488‐conjugated Affinipure coat anti‐mouse IgG(H+L) (SA00013‐1, Proteintech) for 30 min at 4 °C.

Techniques: Expressing, In Vitro, Blocking Assay, Flow Cytometry, Cell Culture, Labeling

Journal: Advanced Science

Article Title: Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

doi: 10.1002/advs.202206912

Figure Lengend Snippet: Evaluation of the biological functions of anti‐CD47‐PCM@NP in vivo. A) In vivo and ex vivo targeting ability of anti‐CD47‐PCM@NP and anti‐CD47 in tumor‐bearing mice models determined by live imaging. B) The semiquantitative analysis of the ratio of fluorescence intensity (tumor/liver) of ex vivo imaging. C) In vivo biodistribution of coumarin 6 (C6)‐labeled formulations in tumor‐bearing mice models at (a) 2 h, (b) 4 h, (c) 8 h, and (d) 12 h after i.v. injection of NP/C6, PCM@NP/C6 and anti‐CD47‐PCM@NP/C6. Data are presented as mean ± SD ( n = 3) (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; NS represents non‐significance).

Article Snippet: The cells were diluted at 1× 10 6 cells per mL in a culture medium and incubated with 1.0 mg mL −1 of a PE anti‐human/mouse/rat CD47 antibody (E‐AB‐F1016D, Elabscience) or anti‐Neu/ErbB2/HER2 Antibody (SC‐7301, Santa Cruz Biotechnology) and CoraLite488‐conjugated Affinipure coat anti‐mouse IgG(H+L) (SA00013‐1, Proteintech) for 30 min at 4 °C.

Techniques: In Vivo, Ex Vivo, Imaging, Fluorescence, Labeling, Injection

Journal: Advanced Science

Article Title: Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

doi: 10.1002/advs.202206912

Figure Lengend Snippet: Antitumor efficacy of anti‐CD47‐PCM@NP and mechanistic investigation by CyTOF analysis. A) Representative images and phagocytic index of C57BL/6 bone marrow‐derived macrophages (BMDM) phagocytosing tumor cells following treatment with PCM@NP, anti‐CD47, and anti‐CD47‐PCM@NP. Scale bar = 50 µm. B) Timeline of the anti‐tumor efficacy study on tumor‐bearing mice (red arrows indicate intravenous administrations), and average tumor growth curves and picture of tumor tissues after the treatment. C) Individual tumor growth curves in each group. D) viSNE plot of intratumoral cells in tumor tissues after treatment with saline, PCM@NP, anti‐CD47, anti‐CD47‐PCM@NP and all groups merged. E) Heat map of the surface molecule and functional molecule expression of different subsets of immune cells in tumor tissues from all groups merged. F) tSNE visualization of all samples with the expression of CD4 and CD8a respectively. G) Percentage of cells in each cluster after treatment from each group. Data represented as mean ± SD ( n = 6). (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; NS represents nonsignificance).

Article Snippet: The cells were diluted at 1× 10 6 cells per mL in a culture medium and incubated with 1.0 mg mL −1 of a PE anti‐human/mouse/rat CD47 antibody (E‐AB‐F1016D, Elabscience) or anti‐Neu/ErbB2/HER2 Antibody (SC‐7301, Santa Cruz Biotechnology) and CoraLite488‐conjugated Affinipure coat anti‐mouse IgG(H+L) (SA00013‐1, Proteintech) for 30 min at 4 °C.

Techniques: Derivative Assay, Saline, Functional Assay, Expressing

Journal: Advanced Science

Article Title: Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

doi: 10.1002/advs.202206912

Figure Lengend Snippet: The INTACT strategy efficiently delivers antibodies to tumors with reduced in vivo toxicity. A) Anti‐CD47‐PCM@NP exhibited no significant influence on red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), and platelet (PLT). Data represented as mean ± SD ( n = 3). B–D) Anti‐CD47‐PCM@NP relieved the occurrence of fungal infection during antitumor treatment. B) Experimental timeline and treatments in tumor‐bearing mice (arrows indicate intravenous administrations). At day 14, mice were infected with C. albicans via tail vein injection. C) Colony‐forming units (CFU) on day 7 in the kidneys of infected mouse models ( n = 6). D) The survival rates of infected mice with different treatments ( n = 12). Data represented as mean ± SD. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; NS represents non‐significance).

Article Snippet: The cells were diluted at 1× 10 6 cells per mL in a culture medium and incubated with 1.0 mg mL −1 of a PE anti‐human/mouse/rat CD47 antibody (E‐AB‐F1016D, Elabscience) or anti‐Neu/ErbB2/HER2 Antibody (SC‐7301, Santa Cruz Biotechnology) and CoraLite488‐conjugated Affinipure coat anti‐mouse IgG(H+L) (SA00013‐1, Proteintech) for 30 min at 4 °C.

Techniques: In Vivo, Infection, Injection

Journal: Advanced Science

Article Title: Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

doi: 10.1002/advs.202206912

Figure Lengend Snippet: The INTACT strategy is adaptive to multiple antibody‐based systems. A) Relative tumor volume growth with anti‐CD47‐PCM@NP/PTX treatment ( n = 6). B–G) The INTACT therapy refined the targeting precision of ADC. B) The diagram of ADC construction: Anti‐CD47 was modified with streptavidin and conjugated with DM1 via the crosslinker BMCC‐biotin. C) The conjugation of ADC was confirmed with SDS‐PAGE. D) Experimental timeline for the anti‐tumor efficacy study and hematology assessments of ADC‐PCM@NP (red arrows indicate intravenous administrations). E) Average tumor growth curves, and picture of the tumor tissues after the treatment ( n = 6). F) Individual tumor growth curves in each group ( n = 6). G) Hematology assessments of red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), and platelet (PLT) ( n = 3). Data represented as mean ± SD. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; NS represents non‐significance).

Article Snippet: The cells were diluted at 1× 10 6 cells per mL in a culture medium and incubated with 1.0 mg mL −1 of a PE anti‐human/mouse/rat CD47 antibody (E‐AB‐F1016D, Elabscience) or anti‐Neu/ErbB2/HER2 Antibody (SC‐7301, Santa Cruz Biotechnology) and CoraLite488‐conjugated Affinipure coat anti‐mouse IgG(H+L) (SA00013‐1, Proteintech) for 30 min at 4 °C.

Techniques: Modification, Conjugation Assay, SDS Page

Journal: Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society

Article Title: CD47 Blockade Reduces Ischemia/Reperfusion Injury and Improves Survival in a Rat Liver Transplantation Model

doi: 10.1002/lt.24059

Figure Lengend Snippet: Western blot analysis of CD47 and TSP-1 expression levels (A,B) during cold ischemia and (C,D) 48 hours after OLT. A significant elevation of CD47 protein with little change in TSP-1 expression after cold ischemia was noted after 18 hours of cold ischemia in comparison with livers that were flushed and then immediately frozen. The expression of both CD47 and TSP-1 protein was reduced 48 hours after OLT in CD47mAb400-treated recipients versus the IgG group (P < 0.05).

Article Snippet: After hepatectomy, the donor liver was flushed via the portal vein with 10 mL of cold (4 °C) normal saline, which was followed by 10 mL of cold University of Wisconsin (UW) solution containing either a mouse anti-CD47 monoclonal antibody, CD47mAb400 (Vasculox, Inc., St. Louis, MO), or an isotype matched-control mouse immunoglobulin G 2a (IgG2a) at a dose of 1 μg/g liver weight.

Techniques: Western Blot, Expressing