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: 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