Journal: Cancer immunology research

Article Title: Impact of scFv on Functionality and Safety of Third-Generation CD123 CAR T Cells.

doi: 10.1158/2326-6066.CIR-23-0548

Figure Lengend Snippet: Figure 1. Design and functional evaluation of five CD123 CAR constructs. A, Schematic diagram of CD123 CARs composed of the EF1α promotor, signal peptide (SP), scFv

Article Snippet: Briefly, recombinant human CD123 protein (R&D Systems, Cat# 301-R3) was tested at 100 nmol/L diluted by one-half at seven spots and the five antibodies at 20 μg/mL (Diaclone, Besançon).

Techniques: Functional Assay, Construct

Journal: Cancer immunology research

Article Title: Impact of scFv on Functionality and Safety of Third-Generation CD123 CAR T Cells.

doi: 10.1158/2326-6066.CIR-23-0548

Figure Lengend Snippet: Figure 4. Impact of IFNγ and TNFα on CD123 expression by endothelial cells. CAL1, HMEC1, or Daudi cells were co-cultured with C0 or CD123 CAR T cells at a 1:1 E:T ratio. Supernatants were collected after 24 hours of co-culture and evaluated by ELISA for (A) IFNγ and (B) TNFα. All CD123 CAR T cells had a statistically significant increase in IFNγ and TNFα secretion against CAL1 cells. C, C0 or CD123 CAR T cells and CAL1 cells were co-cultured in the upper chamber at a 1:1 E:T ratio, and HMEC1 was deposited in the lower chamber for 24 hours in a Transwell co-culture model. After tri-culture, HMEC1 cells were digested with trypsin to assess CD123 expression (D), determined via FC on HMEC1 cells. The RFI of CD123 on HMEC1 cells was determined by the quotient between the MFI of the isotype control and the MFI of the CD123 expression (n ¼ 3). E, CD123 expression RFI on HMEC1 cells after treatment with IFNγ, TNFα, and neutralizing specific antibodies for 24 hours. TNFα induced an increase in CD123 expression in endothelial cells. This expression returned to basal expression in the presence of antibodies blocking TNFα (n ¼ 4). All data shown as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001, one-way ANOVA.

Article Snippet: Briefly, recombinant human CD123 protein (R&D Systems, Cat# 301-R3) was tested at 100 nmol/L diluted by one-half at seven spots and the five antibodies at 20 μg/mL (Diaclone, Besançon).

Techniques: Expressing, Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Control, Blocking Assay

Journal: Cancer immunology research

Article Title: Impact of scFv on Functionality and Safety of Third-Generation CD123 CAR T Cells.

doi: 10.1158/2326-6066.CIR-23-0548

Figure Lengend Snippet: Figure 5. Antitumor response of CD123 CAR T cells against the BPDCN cell line (CAL1) and BPDCN PDX in vivo. A, Schematic representation of the xenograft model. NSG mice aged 6–8 weeks were irradiated (2.5 Gy) 1 day before 1.106 CAL1 luciferase+ i.v. injection into the tail vein. Three days after CAL1 engraftment, 5.106

Article Snippet: Briefly, recombinant human CD123 protein (R&D Systems, Cat# 301-R3) was tested at 100 nmol/L diluted by one-half at seven spots and the five antibodies at 20 μg/mL (Diaclone, Besançon).

Techniques: In Vivo, Irradiation, Luciferase, Injection

Journal: Cancer immunology research

Article Title: Impact of scFv on Functionality and Safety of Third-Generation CD123 CAR T Cells.

doi: 10.1158/2326-6066.CIR-23-0548

Figure Lengend Snippet: Figure 7. CD123 CAR T cells produced from BPDCN patient samples at diagnosis or relapse eliminate autologous blasts and the CAL1 cell line in vivo. A, CAR expression analyzed by FC using biotinylated CD123 protein at day 9 after T-cell transduction. B, CAL1 or BPDCN blasts taken from patients at diagnosis or relapse were co- cultured with autologous C0 or CAR #1 at a 1:1 E:T ratio. C, Schematic representation of the xenograft model. NSG mice aged 6–8 weeks were irradiated (2.5 Gy) 1 day before 1.106 CAL1-luc+ IV injection into the tail vein. Ten days after CAL1-luc+ engraftment, 5.106 of C0 or CAR #1 produced by the sample at diagnosis or relapse were injected (i.v.). D, Graft uptake was then assessed by bli during the experiment. Tumor engraftment was monitored by bli. E, The radiance of the bli signal was collected using IVIS Illumina III (ph/s/cm2/sr). As soon as the C0-treated mice died, the bli monitoring of the CAR #1–treated mice was stopped. Statistical significance was calculated with two-way ANOVA, multiple comparison test, ***, P < 0.001 (n ¼ 8 mice for the treated group). F, Survival curves of CAL1-luc+ mice untreated or treated by C0 or CAR #1. CAR #1–treated mice at diagnosis or relapse had significantly longer survival compared with C0-treated mice. Median survival: PBS, 20 days; C0 at diagnosis, 25 days; CAR #1 at diagnosis, 78.5 days; C0 at relapse, 23.5 days; CAR #1 at relapse, 90 days.

Article Snippet: Briefly, recombinant human CD123 protein (R&D Systems, Cat# 301-R3) was tested at 100 nmol/L diluted by one-half at seven spots and the five antibodies at 20 μg/mL (Diaclone, Besançon).

Techniques: Produced, Biomarker Discovery, In Vivo, Expressing, Transduction, Cell Culture, Irradiation, IV Injection, Injection, Comparison

Journal: Molecular Therapy

Article Title: CD123-Engager T Cells as a Novel Immunotherapeutic for Acute Myeloid Leukemia

doi: 10.1038/mt.2016.116

Figure Lengend Snippet: Generation of CD123-ENG T cells. (a) Schematic of retroviral vector encoding CD123-ENG and mOrange. (b,c) Representative FACS diagram and summary data (CD123-ENG T cells (n = 14), NT T cells (n = 6) of mOrange expression post-transduction. (d) A mouse F(ab')2 antibody was used to detect cell surface-bound CD123 T-cell ENG protein. mOrange-positive and -negative T cells stained positive (filled curve) for CD123 T-cell ENG in contrast to samples that were stained with isotype alone (open curve). NT T cells cultured without CD123-ENG T cells did not stain positive with the mouse F(ab')2 antibody, confirming specificity. (e) Detection of CD123 T-cell ENG protein in media of CD123-ENG and CD19-ENG T cells after 24 hours of culture (n = 4, performed in triplicates, box graph, whiskers: min, max, CD123-ENG versus CD19-ENG T cells P < 0.001).

Article Snippet: Human recombinant CD123 (R&D Systems) was plated at 10 ng/well on a 96-well non- tissue culture treated plate.

Techniques: Retroviral, Plasmid Preparation, Expressing, Transduction, Staining, Cell Culture

Journal: Molecular Therapy

Article Title: CD123-Engager T Cells as a Novel Immunotherapeutic for Acute Myeloid Leukemia

doi: 10.1038/mt.2016.116

Figure Lengend Snippet: CD123-ENG T cells have potent anti-acute myeloid leukemia activity in vivo. Antitumor activity of CD123-ENG T cells in MOLM-13 leukemia NSG xenograft model. MOLM-13.GFP.ffLuc-bearing mice received an i.v. dose of 1 × 107 CD123-ENG (n = 5) or CD19-ENG T cells (n = 5) on day 7 and 14 post-tumor cell injection. Untreated animals served as controls (n = 10). Tumor growth was followed by bioluminescence imaging. (a) Representative images of animals (day post-tumor cell injection is shown in the upper right corner of images). (b–d) Quantitative bioluminescence imaging results (dotted lines: individual mice; solid lines: median; radiance=photons/sec/cm2/sr). Starting day 3 post-first T-cell injection for CD123-ENG versus CD19-ENG T cells, and CD123-ENG T cells versus untreated: P < 0.001). (e) Kaplan-Meier survival curve (control versus CD123-ENG T cells: P = 0.0004; control versus CD19-ENG T cells: NS; CD123-ENG versus CD19-ENG T cells: P = 0.0015).

Article Snippet: Human recombinant CD123 (R&D Systems) was plated at 10 ng/well on a 96-well non- tissue culture treated plate.

Techniques: Activity Assay, In Vivo, Injection, Imaging, Control

Journal: Molecular Therapy

Article Title: CD123-Engager T Cells as a Novel Immunotherapeutic for Acute Myeloid Leukemia

doi: 10.1038/mt.2016.116

Figure Lengend Snippet: CD123-ENG T cells recognize and kill CD123-positive acute myeloid leukemia cells. (a, b) CD123-ENG or CD19-ENG T cells were cocultured with CD123-positive (K562-CD123, MV-4-11, MOLM-13, KG1a) or -negative (K562) cell lines. After 24 hours, (a) IFNγ or (b) IL-2 was determined by ELISA (n = 3–4, assay performed in duplicates; CD123-ENG versus CD19-ENG: *P < 0.05, **P < 0.01, ***P < 0.001). (C) Cytotoxicity assays were performed using CD123-ENG or CD19-ENG T cells as effectors and CD123-positive (K562-CD123, MV-4-11, KG1a) or -negative (K562) cell lines as targets at a E:T ratio of 10:1 (mean + SD; n = 4; assay was performed in triplicates, *P < 0.02, **P < 0.002).

Article Snippet: Human recombinant CD123 (R&D Systems) was plated at 10 ng/well on a 96-well non- tissue culture treated plate.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy

Article Title: CD123-Engager T Cells as a Novel Immunotherapeutic for Acute Myeloid Leukemia

doi: 10.1038/mt.2016.116

Figure Lengend Snippet: CD123-ENG T cells recognize HSPCs at high effector to target ratios. Bone marrow mononuclear cells (BMMCs) or cord blood mononuclear cells (CBMCs) were cultured of E:T ratios of 5:1, 1:1, and 1:10 with CD19-ENG or CD123-ENG T cells for 6 hours. Cells were plated in MethoCult media and CFUs were counted after 10–14 days. Box graph, whiskers: min, max. (a) BMMCs: n = 4; CD123-ENG versus CD19-ENG T cells: 5:1 P = 0.001, 1:1 P = ns, and 1:10 P = ns. (b) CBMCs: n = 4; CD123-ENG versus CD19- ENG T cells: 5:1 P < 0.001, 1:1 P < 0.001, and 1:10 P < 0.02.

Article Snippet: Human recombinant CD123 (R&D Systems) was plated at 10 ng/well on a 96-well non- tissue culture treated plate.

Techniques: Cell Culture

Journal: Molecular Therapy

Article Title: CD123-Engager T Cells as a Novel Immunotherapeutic for Acute Myeloid Leukemia

doi: 10.1038/mt.2016.116

Figure Lengend Snippet: CD123-ENG T cells activate bystander T cells and freshly isolated peripheral blood mononuclear cells (PBMCs) against CD123-positive target cells. (a) 1 × 106 MOLM-13.GFP.ffLuc cells were plated in the bottom well with or without 1 × 106 NT T cells. Control (NT, CD19-ENG) or CD123-ENG T cells were plated in the insert well at the indicated T-cell dose. Following 24-hour incubation, viable MOLM-13.GFP.ffLuc cells were quantified by luciferase assay (n = 3; assay was performed in duplicates; for CD123-ENG: 106 ENG-T cells in insert, 106 NT T cells in bottom well versus 106 ENG-T cells in insert, no NT T cells in bottom well: P < 0.005, 105 ENG-T cells in insert, 106 NT T cells in bottom well versus 106 ENG-T cells in insert, no NT T cells in bottom well: P < 0.05). (b) 1 × 106 MOLM-13.GFP.ffLuc cells were plated in the bottom well with or without 5 × 106 PBMCs. 1 × 106 autologous NT, CD19-ENG, or CD123-ENG T cells were plated in the insert well. Following 24-hour incubation, viable MOLM-13.GFP.ffLuc cells were quantified by luciferase assay (n=3; assay was performed in triplicates; for CD123-ENG: PBMCs versus no PBMCs: P < 0.02).

Article Snippet: Human recombinant CD123 (R&D Systems) was plated at 10 ng/well on a 96-well non- tissue culture treated plate.

Techniques: Isolation, Control, Incubation, Luciferase

Journal: Molecular Therapy

Article Title: CD123-Engager T Cells as a Novel Immunotherapeutic for Acute Myeloid Leukemia

doi: 10.1038/mt.2016.116

Figure Lengend Snippet: Primary AML cells are killed by CD123-ENG T cells in vitro. (a) FACS analysis of pediatric primary acute myeloid leukemia (AML) samples for CD123 expression. AML blasts,(CD33+, CD3-, CD19- (data not shown for CD19)) were analyzed for CD123 expression (filled curve: isotype control; open curve: CD123 MAb). (b) Primary AML samples were treated with CD19-ENG or CD123-ENG T cells at E:T ratios of 1:1 and 1:10 for 6 hours. Following coculture, cells were plated in MethoCult media and incubated for 10–14 days. Final cell counts are displayed as percentage of control; n = 3; CD19-ENG versus CD123-ENG T cells: *P < 0.05.

Article Snippet: Human recombinant CD123 (R&D Systems) was plated at 10 ng/well on a 96-well non- tissue culture treated plate.

Techniques: In Vitro, Expressing, Control, Incubation

Journal: Molecular Therapy

Article Title: CD123-Engager T Cells as a Novel Immunotherapeutic for Acute Myeloid Leukemia

doi: 10.1038/mt.2016.116

Figure Lengend Snippet: Antigen-specific persistence of CD123-ENG T cells in vivo. MOLM-13 bearing or control mice were injected I.V. with 1 × 107 CD123-ENG.mOrange T cells that were also genetically modified to express GFP.ffLuc (n = 5 per group). (a) Images of individual mice. (b) Quantitative bioluminescence imaging results (radiance = photons/sec/cm2/sr, mean and SD is plotted, *P < 0.001, **P < 0.0001). (c, d) On day 6 post-T-cell injection, mice were euthanized and spleens and bone marrows (both femurs) of all (5) acute myeloid leukemia (AML)-bearing mice and of two control mice that received CD123-ENG T cells were processed for FACS analysis. (c) Percentage of human T cells (CD45-positive, CD3-positive) gated on live cells (AML-bearing mice versus control mice: P = NS). (d) mOrange-positive cells (%) gated on human T cells (CD45-positive, CD3-positive); dotted line: mOrange-positive cells (%) in infused CD123-ENG T cells (AML- bearing mice versus control mice: P = NS; % mOrange positive cells in infused CD123-ENG T cells versus % mOrange-positive T cells on day 6 postinfusion: P = NS).

Article Snippet: Human recombinant CD123 (R&D Systems) was plated at 10 ng/well on a 96-well non- tissue culture treated plate.

Techniques: In Vivo, Control, Injection, Genetically Modified, Imaging

Journal: Molecular Therapy

Article Title: CD123-Engager T Cells as a Novel Immunotherapeutic for Acute Myeloid Leukemia

doi: 10.1038/mt.2016.116

Figure Lengend Snippet: CD20 gene-modified CD123-ENG T cells retain their effector function against CD123-positive acute myeloid leukemia and are effectively eliminated by rituximab. (a) Cytotoxicity assays were performed using CD123-ENG, CD20.CD123-ENG, or CD19-ENG T cells as effectors and KG1a as CD123-positive target. (n = 3; assay was performed in triplicates, P = 0.450, CD123-ENG versus CD20.CD123-ENG T cells). (b) MOLM-13.GFP.ffLuc-bearing mice received an i.v. dose of 1 × 107 CD20.CD123-ENG T cells on day 7 post-tumor cell injection. Quantitative bioluminescence imaging results (dotted lines: individual mice; solid line: median; radiance = photons/sec/cm2/sr). (c) CD20.CD123-ENG, CD123-ENG, or NT T cells were labeled with 51Chromium and treated with rituximab and/or complement in a standard cytotoxicity assay. (n = 4; assay was performed in triplicate; for CD20.CD123-ENG T cells: untreated versus rituximab and complement: P = 0.003). (d, e) CD20.CD123-ENG T cells were treated with rituximab, complement, or rituximab and complement, and cultured for 7 days. (D) FACS analysis for CD20-positive cells. Data is presented as percent cell death of CD20-positive cells 7 days post-treatment (n = 5; untreated, rituximab treated, complement treated vs rituximab and complement treated cells: P < 0.001). (e) Cytotoxicity assays using untreated CD123-ENG, CD19-ENG, CD20.CD123-ENG, or rituximab and complement treated CD20.CD123-ENG T-cell lines as effectors and KG1a cells as targets. n = 3; assay performed in triplicates; untreated versus treated CD20.CD123-ENG T cells: P < 0.001; untreated CD20.CD123-ENG T cells versus CD19-ENG T cells: P < 0.001; treated CD20.CD123-ENG T cells versus CD19-ENG T cells: P = ns for all E:T ratios tested. (f) Mice engrafted with MOLM-13 were treated with 3x106 CD20.CD123-ENG/GFP.ffLuc T cells on Day 7 post-leukemia injection. On Days 3–5 post-T-cell injection mice (n = 4) received 250 µg rituximab IP. Untreated mice (n = 3) served as controls. Quantitative bioluminescence imaging results (radiance = photons/sec/cm2/sr, mean and SD is plotted; P < 0.05 starting 2 days post-first dose of rituximab).

Article Snippet: Human recombinant CD123 (R&D Systems) was plated at 10 ng/well on a 96-well non- tissue culture treated plate.

Techniques: Modification, Injection, Imaging, Labeling, Cytotoxicity Assay, Cell Culture

Journal: Scientific Reports

Article Title: A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins

doi: 10.1038/s41598-024-82288-9

Figure Lengend Snippet: Representative SPR sensorgrams obtained by Biacore of antibody mAb I3RB18 Fab (I3RB119), and BsAbs binding to recombinant human antigen CD123 SP1 ( A ) and its truncated variant CD123 SP2 ( B ).

Article Snippet: Recombinant human CD123 SP1 ECD-His tag protein was obtained from R&D Systems (301-R3/CF, lot# LV081110A).

Techniques: Binding Assay, Recombinant, Variant Assay

Journal: Scientific Reports

Article Title: A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins

doi: 10.1038/s41598-024-82288-9

Figure Lengend Snippet: Biacore and MSD-CAT affinity data for the binding of anti-CD123 to human antigen-full length (CD123 SP1) and its truncated variant (CD123 SP2). For the SPR experiment recombinant CD123 SP1 or CD123 SP2 were used for complex formation. For the MSD-CAT experiments reported in the last column, complex formation was obtained by incubating antibodies or Fabs with cells expressing human CD123 SP1 or CD123 SP2, or cynomolgus full length CD123. Detection of antibody in the cell-antibody equilibrated mixture was performed by capturing free antibody in the supernatant with biotinylated recombinant CD123 SP1 or CD123 SP2 which has been previously captured on a streptavidin MSD-plate.

Article Snippet: Recombinant human CD123 SP1 ECD-His tag protein was obtained from R&D Systems (301-R3/CF, lot# LV081110A).

Techniques: Binding Assay, Variant Assay, Recombinant, Expressing

Journal: Scientific Reports

Article Title: A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins

doi: 10.1038/s41598-024-82288-9

Figure Lengend Snippet: Representative binding curves for MSD-CAT affinity analysis for cell surface expressed CD123, SP1 ( A ) and CD123 SP2 ( B ). The Y-axis shows % free anti-receptor molecules (BsAb, Fab or mAb) or CD123 negative control mAb (I3CB15) while the X-axis shows the concentration of cell surface antigen. Each curve represents a different but fixed concentration of mAb, bispecific or Fab as indicated.

Article Snippet: Recombinant human CD123 SP1 ECD-His tag protein was obtained from R&D Systems (301-R3/CF, lot# LV081110A).

Techniques: Binding Assay, Negative Control, Concentration Assay

Journal: Scientific Reports

Article Title: A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins

doi: 10.1038/s41598-024-82288-9

Figure Lengend Snippet: Binding curves for MSD-CAT affinity analysis for cyno CD123. The Y-axis shows % free anti-receptor molecules (mAb or its Fab) while the X-axis shows the concentration of cell surface antigen. Each curve represents a different but fixed concentration of mAb, or Fab as indicated. The figure also shows a plot of the control commercial antibody, 7G3, showing cross reactivity to human CD123 (SP1).

Article Snippet: Recombinant human CD123 SP1 ECD-His tag protein was obtained from R&D Systems (301-R3/CF, lot# LV081110A).

Techniques: Binding Assay, Concentration Assay, Control