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: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Anti-CD47 Monoclonal Antibody Therapy Reduces Ischemia-Reperfusion Injury of Renal Allografts in a Porcine Model of Donation after Cardiac Death

doi: 10.1111/ajt.14567

Figure Lengend Snippet: (A) The CD47mAb was strongly bound to the renal tissue immediately after flushing. (B) CD47mAb binding to glomeruli could be detected on day 5 after transplant. (These staining were performed on grafts from different animals.)

Article Snippet: Just prior to implantation, the grafts were flushed either with control (n=4) or a humanized CD47mAb (clone anti-CD47 649, Tioma Therapeutics, Inc, St. Louis, n=4) at a dosage of 10 mg per 250–300 g kidney.

Techniques: Binding Assay, Staining

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Anti-CD47 Monoclonal Antibody Therapy Reduces Ischemia-Reperfusion Injury of Renal Allografts in a Porcine Model of Donation after Cardiac Death

doi: 10.1111/ajt.14567

Figure Lengend Snippet: Graft blood flow prior (0s) and post reperfusions at 1s, 15s, 30s, and 60s were studied using an in vivo imaging system (n=2/group). (A) Representative perfusion using ICG fluorescence imaging are shown at time points 0, 16 and 60 seconds after release of clamps (see Figure S1 for additional images). (B) The fluorescence density values of the region of interest (ROI) were extracted to evaluate reperfusion characteristics of the grafts. CD47mAb-treated grafts had more uniform and greater average tissue perfusion as compared to control organs. (C) The repeated measure two-way ANOVA shows that Time (p<0.001), Treatment (p=0.002), and Interaction (p<0.001) terms are all significant, indicating florescent density differed by treatment and over time.

Article Snippet: Just prior to implantation, the grafts were flushed either with control (n=4) or a humanized CD47mAb (clone anti-CD47 649, Tioma Therapeutics, Inc, St. Louis, n=4) at a dosage of 10 mg per 250–300 g kidney.

Techniques: In Vivo Imaging, Fluorescence, Imaging

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Anti-CD47 Monoclonal Antibody Therapy Reduces Ischemia-Reperfusion Injury of Renal Allografts in a Porcine Model of Donation after Cardiac Death

doi: 10.1111/ajt.14567

Figure Lengend Snippet: (A) Serum creatinine, blood urea nitrogen (BUN), and phosphorus were significantly decreased, while the calcium levels were significantly increased in the CD47mAb-treated group than that in the control group. (B) Histological study of pig kidney grafts at day 5 post-KTx indicated that CD47mAb treated grafts had significantly less evidence of acute tubular injury (ATI) compared with the control group. (C) Animals with CD47mAb treated grafts had significantly earlier urination than that in control (n=4/group, CD47mAb treatment versus control, *p<0.05 or **p<0.01, respectively).

Article Snippet: Just prior to implantation, the grafts were flushed either with control (n=4) or a humanized CD47mAb (clone anti-CD47 649, Tioma Therapeutics, Inc, St. Louis, n=4) at a dosage of 10 mg per 250–300 g kidney.

Techniques:

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Anti-CD47 Monoclonal Antibody Therapy Reduces Ischemia-Reperfusion Injury of Renal Allografts in a Porcine Model of Donation after Cardiac Death

doi: 10.1111/ajt.14567

Figure Lengend Snippet: (A) Revealed by qRT-PCR, CD47mAb-treated organs showed significantly less expression in each of these oxidative injury-related genes of superoxide dismutase-1 (sod-1), glutathione peroxidase-1 (gpx-1), thioredoxin (txn), except heme oxygenase-1 (hmox-1) compared to control. (B) Immunofluorescence staining revealed a decreased expression of Voltage-dependent anion-selective channel protein 1 (VDAC-1) and poly (ADP-ribose) polymerase (PARP) in the CD47mAb-treated tissues than in control. (C) Western blotting and (D) densitometry analysis also support that the protein levels of VDAC-1 and PARP are decreased in the grafts treated with CD47mAb compared with control (n=4/group, *p<0.05, ** p<0.01, or *** p<0.001, respectively).

Article Snippet: Just prior to implantation, the grafts were flushed either with control (n=4) or a humanized CD47mAb (clone anti-CD47 649, Tioma Therapeutics, Inc, St. Louis, n=4) at a dosage of 10 mg per 250–300 g kidney.

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Western Blot

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Anti-CD47 Monoclonal Antibody Therapy Reduces Ischemia-Reperfusion Injury of Renal Allografts in a Porcine Model of Donation after Cardiac Death

doi: 10.1111/ajt.14567

Figure Lengend Snippet: (A) qRT-PCR showed the relative mRNA levels of il-2, il-6, tgf-b, and inf-g were significantly lower in the CD47mAb treated renal allografts than that in control at day 5 after kidney transplantation. (B) Immunofluorescence staining indicated the CD4+ and CD8+ cells infiltration were significantly decreased in the CD47mAb treated renal allografts than that in the control (n=4/group, *p<0.05 or ** p<0.01, respectively).

Article Snippet: Just prior to implantation, the grafts were flushed either with control (n=4) or a humanized CD47mAb (clone anti-CD47 649, Tioma Therapeutics, Inc, St. Louis, n=4) at a dosage of 10 mg per 250–300 g kidney.

Techniques: Quantitative RT-PCR, Transplantation Assay, Immunofluorescence, Staining

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Anti-CD47 Monoclonal Antibody Therapy Reduces Ischemia-Reperfusion Injury of Renal Allografts in a Porcine Model of Donation after Cardiac Death

doi: 10.1111/ajt.14567

Figure Lengend Snippet: (A) Immunofluorescence staining showed a reduction of matrix metalloproteinase-2 (MMP-2) and MMP-9 in CD47mAb treated renal allografts than that in control. Western blotting (B) and densitometry analysis (C) confirmed the decreasing expression of MMP-2 and MMP-9 in renal grafts treated with CD47mAb compared to control (n=4/group, *p<0.05 or ** p<0.01, respectively).

Article Snippet: Just prior to implantation, the grafts were flushed either with control (n=4) or a humanized CD47mAb (clone anti-CD47 649, Tioma Therapeutics, Inc, St. Louis, n=4) at a dosage of 10 mg per 250–300 g kidney.

Techniques: Immunofluorescence, Staining, Western Blot, Expressing

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Anti-CD47 Monoclonal Antibody Therapy Reduces Ischemia-Reperfusion Injury of Renal Allografts in a Porcine Model of Donation after Cardiac Death

doi: 10.1111/ajt.14567

Figure Lengend Snippet: (A) Immunofluorescence staining showed a decrease of Bcl-2-associated X protein (BAX) and Caspase-3 in the CD47mAb treated renal allografts compared to control. Western blotting (B) and densitometry analysis (C) confirmed the decreasing expression of BAX and Caspase-3 in renal grafts treated with CD47mAb than that in control (n=4/group, *p<0.05 or ** p<0.01, respectively).

Article Snippet: Just prior to implantation, the grafts were flushed either with control (n=4) or a humanized CD47mAb (clone anti-CD47 649, Tioma Therapeutics, Inc, St. Louis, n=4) at a dosage of 10 mg per 250–300 g kidney.

Techniques: Immunofluorescence, Staining, Western Blot, Expressing

Journal: Journal of Hematology & Oncology

Article Title: Targeting CD47 for cancer immunotherapy

doi: 10.1186/s13045-021-01197-w

Figure Lengend Snippet: CD47-SIRPα

Article Snippet: ZL-1201 , IgG4 , Zai Lab , Anti-human CD47 mAb , NCT04257617 , Recruiting , Advanced Malignancies , Phase 1 , 66 , 11-May-20.

Techniques:

Journal: Journal of Hematology & Oncology

Article Title: Targeting CD47 for cancer immunotherapy

doi: 10.1186/s13045-021-01197-w

Figure Lengend Snippet: Blockades to CD47-SIRPα signal pathway

Article Snippet: ZL-1201 , IgG4 , Zai Lab , Anti-human CD47 mAb , NCT04257617 , Recruiting , Advanced Malignancies , Phase 1 , 66 , 11-May-20.

Techniques:

Journal: Journal of Hematology & Oncology

Article Title: Targeting CD47 for cancer immunotherapy

doi: 10.1186/s13045-021-01197-w

Figure Lengend Snippet: CD47-related clinical trials registered in US national clinical trials registry (NCT) system www.clinicaltrials.gov

Article Snippet: ZL-1201 , IgG4 , Zai Lab , Anti-human CD47 mAb , NCT04257617 , Recruiting , Advanced Malignancies , Phase 1 , 66 , 11-May-20.

Techniques: Clinical Proteomics, Recombinant, Transformation Assay

Journal: Journal of Hematology & Oncology

Article Title: Targeting CD47 for cancer immunotherapy

doi: 10.1186/s13045-021-01197-w

Figure Lengend Snippet: CD47-related clinical trials registered in the China drug trials registry (CDT) system www.chinadrugtrials.org.cn

Article Snippet: ZL-1201 , IgG4 , Zai Lab , Anti-human CD47 mAb , NCT04257617 , Recruiting , Advanced Malignancies , Phase 1 , 66 , 11-May-20.

Techniques: Clinical Proteomics

Journal: Research

Article Title: Development and Characterization of Nanobody-Derived CD47 Theranostic Pairs in Solid Tumors

doi: 10.34133/research.0077

Figure Lengend Snippet: CD47 was highly expressed on SKOV-3 ovarian and LS174T colorectal cancer cells, as well as in the No. 490 gastric cancer PDX models. (A) Flow cytometry histogram of SKOV-3 and LS174T cells incubated with 2 μg/ml MCA-911 and a second antibody, or second antibody only. (B) Immunohistochemical images of 3 representative CD47-positive tumors. B6H12 was used as the primary antibody for staining.

Article Snippet: All the cells were washed with sterile PBS and stained with a purified mouse anti-human CD47 mAb (MCA-911, Clone BRIC126; Bio-Rad), followed by washing and incubation of 5 μg/ml of Alexa Fluor 488-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories).

Techniques: Flow Cytometry, Incubation, Immunohistochemical staining, Staining

Journal: Research

Article Title: Development and Characterization of Nanobody-Derived CD47 Theranostic Pairs in Solid Tumors

doi: 10.34133/research.0077

Figure Lengend Snippet: Surface plasmon resonance studies showing the association and dissociation kinetics of C2 (A and C) and ABDC2 (B and D) interacting with recombinant human (A and B) and mouse (C and D) CD47 proteins. ABDC2 also showed high binding affinities to human serum albumin (HSA) (E) and murine serum albumin (MSA) (F). RU, response units.

Article Snippet: All the cells were washed with sterile PBS and stained with a purified mouse anti-human CD47 mAb (MCA-911, Clone BRIC126; Bio-Rad), followed by washing and incubation of 5 μg/ml of Alexa Fluor 488-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories).

Techniques: SPR Assay, Recombinant, Binding Assay

Journal: Blood

Article Title: Targeted inhibition of CD47-SIRPα requires Fc-FcγR interactions to maximize activity in T-cell lymphomas

doi: 10.1182/blood.2019001744

Figure Lengend Snippet: Human CD47 protein expression is heterogeneous, whereas MHC class I expression is homogeneous across TCL. (A) CD47 expression on normal peripheral blood-derived CD3+ T cells from healthy donors and TCL lines was determined by flow cytometry. CD47 surface density was plotted after mean fluorescence intensity was normalized for cell size. Each point represents a different healthy donor (n = 10) or TCL line (ALCL = 9 [DL-40, FEPD, KI-JK, L82, MAC2A, Karpas 299, SR-786, SU-DHL-1, SUP-M2], CTCL = 4 [HH, HuT-78, MJ, Myla], NKTCL = 3 [MTA, KHYG-1, SNK6], γδTCL [Karpas 384], HSTCL [DERL-2], PTCL-NOS = 2 [OCI-Ly12, SMZ-1], ATLL = 1 [HuT-102], and NKLGL = 1 [NKL]) or BCL line (Burkitt [Raji, Daudi], DLBCL [SU-DHL-2, SU-DHL-5, and Mantle cell [JVM-2]). **P = .0021 by 2-sided Welch t test. Normalized mean expression for each population was: CD3+ T cells 69.85 (range, 47.1-93.5) and TCL lines 176.9 (range, 20.9-694.8). (B) Resting CD3+ T cells from 2 additional donors (D#1, D#2) and TCL whole cell lysates were immunoblotted with the indicated antibodies. (C) Differential CD47 expression measured by RNA-seq was batch corrected and plotted. Three healthy donors were used for naïve, effector, and follicular helper T-cell groups. Cell lines: ALK+ ALCL = 3 (KI-JK, L82, SR-786), ALK- ALCL = 3 (MAC2A, OCI-Ly13.2, FEPD), PTCL-NOS = 2 (OCI-Ly12, SMZ-1), CTCL = 3 (HH, HuT-78, Myla), NK/TCL = 2 (KHYG-1, MTA). PDXs: ALK+ ALCL = 2 (WCTL-91953, WCTL-81162), AITL = 3 (DFTL-94393, DFTL-78024, DFTL-47880), HSTL = 1 (DFTL-81777), ATLL = 1 (DFTL-69579), T-PLL = 1 (DFTL-28776), and NK/TCL = 1 (DFTL-85005). (D) Representative images of patient lymph nodes stained for H&E, CD47 showing low and high H-scores and for MHC class I. Scale bar: 0.05 mm. (E) Histological analysis of CD47 expression in TCL patient-derived primary samples in the TMA (other: CTCL = 3, ATLL = 5, NK/TCL = 3, T-PLL = 1, subcutaneous panniculitis-like TCL = 2, γ/Δ TCL = 2, TCL unclassifiable = 3, and T-cell lymphoblastic lymphoma = 2). (F) PFS comparison between quartile groups by H-score (log-rank, P = .43). (G) PFS comparison between quartile groups by percent CD47+ lymphoma cells (log-rank, P = .38). H&E, hematoxylin and eosin.

Article Snippet: Transduced cell lines were cultured for 5 to 7 days in RPMI1640 supplemented with 20% fetal calf serum and l -glutamine before depletion of CD47-expressing cells using an anti-CD47 mAb (B6H12.2, ATCC) and magnetic beads coated with sheep anti-mouse IgG (Dynabeads, Life Technologies, Carlsbad, CA).

Techniques: Expressing, Derivative Assay, Flow Cytometry, Fluorescence, RNA Sequencing, Staining, Comparison

Journal: Blood

Article Title: Targeted inhibition of CD47-SIRPα requires Fc-FcγR interactions to maximize activity in T-cell lymphomas

doi: 10.1182/blood.2019001744

Figure Lengend Snippet: Therapy with anti-CD47 antibody SRF231 induces durable responses in TCL PDX and murine models in vivo. (A-F) Tumor burden and organ weights of mice engrafted with the HSTCL PDX DFTL-81777 and treated with hIgG4 isotype control or anti-CD47 antibody. (G-N) Tumor burden in blood and BM (n = 3 per antibody group). *P < .05; **P < .01; ***P < .001, ****P < .0001 by 2-sided Welch t test. Kaplan-Meier survival analyses (n = 3-6 per antibody group) by log-rank test. Arrows indicate start (day 0) and stop (day 10 for models 81777 and 22685) and start (day 0) and stop (day 4 for model 85005) of treatment. (O) Kaplan-Meier survival analysis of ITK-SYK xenograft model treated with murine anti-CD47 antibody, MIAP410, or with mIgG1κ isotype control, MOPC-21 (n = 3 per antibody group) log-rank test. BM, bone marrow.

Article Snippet: Transduced cell lines were cultured for 5 to 7 days in RPMI1640 supplemented with 20% fetal calf serum and l -glutamine before depletion of CD47-expressing cells using an anti-CD47 mAb (B6H12.2, ATCC) and magnetic beads coated with sheep anti-mouse IgG (Dynabeads, Life Technologies, Carlsbad, CA).

Techniques: In Vivo, Control

Journal: Blood

Article Title: Targeted inhibition of CD47-SIRPα requires Fc-FcγR interactions to maximize activity in T-cell lymphomas

doi: 10.1182/blood.2019001744

Figure Lengend Snippet: Anti-CD47 antibody efficacy is macrophage-mediated in TCL PDX. (A) Burden at involved sites for each mouse. P values by 2-sided Welch t test. (B) Flow plots of Gr-1+ neutrophils in the peripheral blood of mice before and after treatment with anti-Ly6G Ab. (C) Flow plots of F4/80+ macrophages in the peripheral blood of mice before and after treatment with clodronate. PBS, phosphate-buffered saline.

Article Snippet: Transduced cell lines were cultured for 5 to 7 days in RPMI1640 supplemented with 20% fetal calf serum and l -glutamine before depletion of CD47-expressing cells using an anti-CD47 mAb (B6H12.2, ATCC) and magnetic beads coated with sheep anti-mouse IgG (Dynabeads, Life Technologies, Carlsbad, CA).

Techniques: Saline

Journal: Blood

Article Title: Targeted inhibition of CD47-SIRPα requires Fc-FcγR interactions to maximize activity in T-cell lymphomas

doi: 10.1182/blood.2019001744

Figure Lengend Snippet: Anti-human CD47 monoclonal antibodies, B6H12 and SRF231 induce phagocytosis of TCL cells over nonmalignant T cells in vitro. (A) B6H12 induced phagocytosis of peripheral blood CD3+ T cells from 4 healthy donors using mBMDM and hMDM in comparison with isotype control (mIgG1κ) are presented. The human anti-CD20 mAb rituximab (Rit) and anti-CD52 mAb alemtuzumab (Alem) both with hIgG1 isotype were used as negative and positive controls, respectively. (B, C) B6H12-mIgG1κ and SRF231 mediated phagocytosis of HuT-78, Myla, and HH (cutaneous T-cell lymphoma) and SUP-M2, MAC2A, Karpas 299 (K-299) (peripheral T-cell lymphoma) cells compared with isotype controls (mIgG1κ and hIgG4). Fold change in CFSE+ of CD11b+ mBMDMs and of CD14+ hMDMs relative to the isotype control (mIgG1κ for B6H12-mIgG1κ, hIgG4 for SRF231, and hIgG1 for B6H12-hIgG1) for that cell line. Alemtuzumab was used as a positive control for induction of ADCP for CD52-expressing HuT-78 cell line and as a negative control for other lines. B6H12-mIgG1κ and B6H12-hIgG1 mediated phagocytosis of Myla and MAC2A relative to isotype controls is plotted for direct comparison between antibody isotypes. (D) Flow plots of mBMDM-engulfed CFSE+ tumor cells with SRF231 and hIgG4 isotype control upon incubation of tumor cells ex vivo harvested from PDXs (ALK+ ALCL [WCTL-81162], T-PLL [DFTL-28776], AITL [DFTL-47880], and PTCL-NOS [DFTL-84867]).

Article Snippet: Transduced cell lines were cultured for 5 to 7 days in RPMI1640 supplemented with 20% fetal calf serum and l -glutamine before depletion of CD47-expressing cells using an anti-CD47 mAb (B6H12.2, ATCC) and magnetic beads coated with sheep anti-mouse IgG (Dynabeads, Life Technologies, Carlsbad, CA).

Techniques: Bioprocessing, In Vitro, Comparison, Control, Positive Control, Expressing, Negative Control, Incubation, Ex Vivo

Journal: Blood

Article Title: Targeted inhibition of CD47-SIRPα requires Fc-FcγR interactions to maximize activity in T-cell lymphomas

doi: 10.1182/blood.2019001744

Figure Lengend Snippet: Anti-CD47 monoclonal antibodies exert minimal apoptosis, no CDC, but affect ADCC. (A) TCL cells were incubated with the indicated antibodies at 10 µg/mL or 10 μM staurosporine (positive control) for 2 h and the percentage of live cells was quantified by flow cytometry. (B, C) CDC assay with 30% murine and human complement was performed in triplicate and % PI+ cells are reported. Rituximab and Raji cells were used as positive controls, whereas trastuzumab was used as negative control. (D) ADCC reporter assay measuring luminescence with murine FcγRIIIA-expressing Jurkat cells was performed in triplicate at an effector:target ratio of 25:1 with indicated antibodies. Human anti-CD3 mAb (OKT3) was used as positive control along with its isotype control, mIgG2aκ. Fold induction is relative to isotype control (mIgG1κ for B6H12 and mIgG2aκ for OKT3). Shown are mean values of technical triplicates with error bars representing SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001 by 2-sided Welch t-test. (E) CFSE-labeled TCL cells were incubated with human-derived NK cells in the presence of indicated antibodies for 4 hours. Percentage of propidium iodide positive (% PI pos) (of CFSE+) cells was determined by flow cytometry and plotted. Rituximab and Raji cells were used as positive controls whereas trastuzumab was used as negative control. Shown are mean values of technical triplicates with error bars representing SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001 by 2-sided Welch t test. SEM, standard error of the mean.

Article Snippet: Transduced cell lines were cultured for 5 to 7 days in RPMI1640 supplemented with 20% fetal calf serum and l -glutamine before depletion of CD47-expressing cells using an anti-CD47 mAb (B6H12.2, ATCC) and magnetic beads coated with sheep anti-mouse IgG (Dynabeads, Life Technologies, Carlsbad, CA).

Techniques: Bioprocessing, Incubation, Positive Control, Flow Cytometry, CDC Assay, Negative Control, Reporter Assay, Expressing, Control, Labeling, Derivative Assay

Journal: Blood

Article Title: Targeted inhibition of CD47-SIRPα requires Fc-FcγR interactions to maximize activity in T-cell lymphomas

doi: 10.1182/blood.2019001744

Figure Lengend Snippet: Anti-CD47 antibody increases phagocytosis through FcγR-independent and FcγR-dependent mechanisms and is not Mac-1 dependent. (A) CFSE+ TCL cells were incubated in vitro with B6H12 and F(ab′)2 fragment of B6H12 or isotype control in the presence of mBMDMs and hMDMs. (B) Same as panel A, using full-length and F(ab′)2 fragments of 2D3 and MIAP410. (C) CFSE+ TCL cells were incubated with WT or Fcer1g−/− (KO) mBMDMs in the presence of the indicated antibodies. Shown are mean values of technical triplicates with error bars representing SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001 by 2-sided Welch t test. (D-E) CFSE+ TCL cells were incubated with WT or Mac1−/− (KO) mBMDMs in the presence of the indicated antibodies or F(ab′)2 fragment of B6H12. A representative experiment performed in triplicate with error bars representing SEM is shown. (F) On the left, phagocytosis of CFSE-labeled CTCL cells, Myla and HH by hMDMs in the presence of various antibodies including Moga at concentrations of 5 µg/mL is plotted. On the right, percent PI positive of the CFSE-labeled Myla and HH cells is plotted after culture with human NK cells in the presence of various antibodies at concentrations of 10 µg/mL. Shown are mean values of technical triplicates with error bars representing SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001 by 2-sided Welch t test. (G) Phagocytosis of Myla, HuT-78, MAC2A, and K-299 cells mediated by full-length B6H12-mIgG1κ, anti-HLA A,B,C mAb, W6/32 and their F(ab′)2 portions alone and in combination relative to isotype controls at 10 µg/mL (mIgG1κ for B6H12-mIgG1κ and mIgG2aκ for W6/32). Percentage of CFSE+ macrophages (of CD14+ hMDMs) is plotted. Shown are mean values of technical triplicates ± SEM. *P < .05; **P < .01; ***P < .001, ****P < .0001 by 2-sided Welch t test. KO, knockout; ns, not significant.

Article Snippet: Transduced cell lines were cultured for 5 to 7 days in RPMI1640 supplemented with 20% fetal calf serum and l -glutamine before depletion of CD47-expressing cells using an anti-CD47 mAb (B6H12.2, ATCC) and magnetic beads coated with sheep anti-mouse IgG (Dynabeads, Life Technologies, Carlsbad, CA).

Techniques: Incubation, In Vitro, Control, Labeling, Knock-Out