anti-cd123 antibody csl362  (CSL Limited)

Journal: The Journal of Experimental Medicine

Article Title: Epitope-engineered human hematopoietic stem cells are shielded from CD123-targeted immunotherapy

doi: 10.1084/jem.20231235

Figure Lengend Snippet: Rational design of human CD123 protein variants to shield from targeted immunotherapy. (A) Crystal structure of the CD123-CSL362 complex in open conformation (PDB ID: 4JZJ ). CD123 is shown as ribbons. The CSL362 antibody variable domain is shown as a white surface. CD123 amino acid residues involved in IL-3 binding are highlighted as lines and in light blue. (B) Per-residue relative solvent accessibility (RSA) computed on the CSL362-free (solid line) and CSL362-bound (dashed line) states based on the x-ray structure of the CD123–CSL362 complex (PDB ID: 4JZJ ). RSA data are shown for the NTD. The CSL362 epitope region is highlighted in blue. (C) Amino acid residues at the interface of the CD123–CSL362 complex are highlighted as lines and sticks. Side chain–mediated intermolecular contacts are shown as dashed black lines. (D and E) The predicted ΔE mutational landscape of CD123 is shown as a heatmap for the full ECD (residue range 20–305, x axis; D) and selected amino acid positions: E51, S59, and R84 (E). Heatmap color ranges from yellow (ΔE < 0, predicted damaging) to blue (ΔE ≥ 0, predicted neutral or beneficial). (F) Selected amino acid variants at residues E51, S59, and R84 are sorted by decreasing ΔE values.

Article Snippet: Antibody CSL362 hIgG1 (captured ligand) was captured by Anti-Human Fc capture biosensor (AHC; Sartorius, PN: 18-5060) for 300 s at 0.5 µg/ml.

Techniques: Binding Assay, Residue, Solvent

Journal: The Journal of Experimental Medicine

Article Title: Epitope-engineered human hematopoietic stem cells are shielded from CD123-targeted immunotherapy

doi: 10.1084/jem.20231235

Figure Lengend Snippet: Preserved expression of engineered CD123 variants despite abolished binding to the mAb MIRG123. (A and B) Flow cytometry plots (A) and summarizing bar graph (B) showing binding of the anti-human CD123 antibody MIRG123 (biosimilar of CSL362) and the control clone 6H6 to wt CD123 and its 28 variants stably expressed in HEK-293 cells. Variants were categorized based on the dual staining to MIRG123 and 6H6 as non-binding (blue, <1% dual staining), weak (orange, 1–20%), or strong (red, >20%) binding variants. Control conditions (gray) are HEK-293 cells stably expressing wt CD123 (HEK-CD123) and non-transduced HEK-293 cells (HEK). Error bars: mean (SD). Data in A are representative of three independent experiments summarized in B.

Article Snippet: Antibody CSL362 hIgG1 (captured ligand) was captured by Anti-Human Fc capture biosensor (AHC; Sartorius, PN: 18-5060) for 300 s at 0.5 µg/ml.

Techniques: Expressing, Binding Assay, Flow Cytometry, Stable Transfection, Staining

Journal: The Journal of Experimental Medicine

Article Title: Epitope-engineered human hematopoietic stem cells are shielded from CD123-targeted immunotherapy

doi: 10.1084/jem.20231235

Figure Lengend Snippet: Cells expressing engineered CD123 variants are shielded from multiple targeted immunotherapy modalities in vitro. (A) Schematic to assess shielding of CD123 expressing cells from three targeted immunotherapies (ADCC, TCE, CAR T cell) in vitro. (B) MIRG123-induced ADCC measured by the luminescence of the effector cell line Jurkat/FcγRIIIa/NFAT-Luc following co-culture with HEK, HEK-CD123, or the CD123 variants. The luminescence signal normalized to the culture with HEK-CD123 (top dashed line). Data of two independent experiments. (C–F) 3-d co-culture of effector T cells and HEK-293 expressing CD123 and its variants with and without CSL362/OKT3-TCE (TCE). Data represent five independent donors and experiments with two technical replicates per group. (C) Representative images after 3-d co-culture with HEK, HEK-CD123, or E51K with and without TCE. White arrows indicate cell clustering. Scale bar: 100 μm. (D) Specific TCE-mediated killing of HEK-293 cells or its variants. (E) Representative flow cytometry plots indicating CD69-expression in effector T cells without (top) and with (bottom) TCE after 3 d co-cultures with HEK, HEK-CD123, and the variant E51K. (F) Frequency of CD69-expressing CD3 + effector T cells after 3 d. (G–J) Human 123CAR T cells were co-cultured with the target cells HEK, HEK-CD123, or its variants for 24 h. Control T cells were electroporated with an HDRT, Cas9 protein but no gRNA. Data are from three independent donors, each with two technical replicates. (G) Representative microscopy images after 1 d with white arrows indicating cell clustering. Scale bar: 100 μm. (H) Specific killing of target cells measured by flow cytometry at day 1 of co-culture. (I and J) Representative FACS plots (I) and (J) summary of CD69 + 123CAR T cells either alone (effector T cells) or in the presence of HEK, HEK-CD123, or all CD123 variants after 24 h co-culture. The data are normalized to % CD69 + cells in the presence of HEK target cells. (B–J) Error bars: mean (SD).

Article Snippet: Antibody CSL362 hIgG1 (captured ligand) was captured by Anti-Human Fc capture biosensor (AHC; Sartorius, PN: 18-5060) for 300 s at 0.5 µg/ml.

Techniques: Expressing, In Vitro, Co-Culture Assay, Flow Cytometry, Variant Assay, Cell Culture, Microscopy

Journal: The Journal of Experimental Medicine

Article Title: Epitope-engineered human hematopoietic stem cells are shielded from CD123-targeted immunotherapy

doi: 10.1084/jem.20231235

Figure Lengend Snippet: TCE-mediated effector T cell activation. (A–D) 72 h co-culture of human effector T cells with HEK, HEK-CD123, and CD123 variants (effector-to-target ratio = 10:1) in the presence of the CSL362/OKT3-TCE (300 ng/ml). Percentage of CD69 + cells within total (A), gated CD4 + (B), and CD8 + (C) effector T cells with (purple) and without (gray) TCE. Data are from five independent donors and experiments with two technical replicates per group. (D) IFNγ secretion was measured by ELISA in co-culture supernatants after 72 h. (A–D) Data are from four blood donors and experiments with two technical replicates per group. Error bars: mean (SD).

Article Snippet: Antibody CSL362 hIgG1 (captured ligand) was captured by Anti-Human Fc capture biosensor (AHC; Sartorius, PN: 18-5060) for 300 s at 0.5 µg/ml.

Techniques: Activation Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Epitope-engineered human hematopoietic stem cells are shielded from CD123-targeted immunotherapy

doi: 10.1084/jem.20231235

Figure Lengend Snippet: Biophysical characterization of selected CD123 protein variants. (A) Binding of CSL362 to the recombinant ECD of CD123 wt (left) and CD123 E51T (right) at increasing concentrations of CSL362 measured by BLI. (B) Binding levels of CD123 wt and its variants at different concentrations to captured CSL362 at 280 s. CD123 wt reaches its saturation to CSL362 at 50 nM, therefore higher concentrations were not measured. (C) Binding levels of CD123 wt and its variants to the captured antibody 6H6 (normalized to a loading level of 6H6) at 250 s. (D) Binding levels of IL-3 to biotinylated CD123 wt and variants (normalized to loading levels of biotinylated CD123 wt and its variants) at 250 s. (E) Thermal unfolding (relative fluorescence unit, RFU) of CD123 wt and variants measured by DSF with increasing temperatures. (A–E) Representative data are from two experiments with two technical replicates.

Article Snippet: Antibody CSL362 hIgG1 (captured ligand) was captured by Anti-Human Fc capture biosensor (AHC; Sartorius, PN: 18-5060) for 300 s at 0.5 µg/ml.

Techniques: Binding Assay, Recombinant, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: Epitope-engineered human hematopoietic stem cells are shielded from CD123-targeted immunotherapy

doi: 10.1084/jem.20231235

Figure Lengend Snippet: HSPCs expressing CD123 variants E51K and E51T are functional, differentiate normally in vitro, and display a good safety profile. (A–M) Characterization of non-virally CRISPR/Cas9-edited human CD34 + HSPCs. (A) Representative flow cytometry plots showing binding (%) of the anti-human CD123 antibody clones 6H6 and MIRG123 to edited CD34 + HSPCs 5 d after EP. EP (cells electroporated with Cas9 protein only); KO RNP (EP with RNP only); wt, KO, E51K, and E51T variants (electroporated with respective HDRT). In flow cytometry, cells double-positive for MIRG123 and 6H6 were defined as "wt," whereas MIRG123 − 6H6 − are indicated as “KO” although they include intended CD123 KO cells as well as cells naturally not expressing CD123. The MIRG123 − 6H6 + cell population is labeled as KI. Representative plots of five independent experiments each performed with different donors. (B) Frequency of ki cells (MIRG123 − 6H6 + ) 2 and 5 d after EP. Data from eight individual donors (each a color) were performed in six independent experiments with two to four technical replicates. (C) Quantification of the ki population in LT-HSCs (CD34 + CD38 − CD90 + CD45RA − ), multipotent progenitor 1 (MPP1; CD34 + CD38 − CD90 − CD45RA − ) and MPP2 (CD34 + CD38 − CD90 − CD45RA + ). (D) Representative Amplicon-NGS sequencing of the targeted CD123 locus at 2, 5, and 9 d after editing of control (EP), wt template, KO template, E51K, and E51T conditions. Data from four different experiments performed with different donors were pooled. (E) Representative FACS plots of CD123 stained with 6H6 and MIRG123 (left) and histograms of phosphorylated STAT5 (right) upon exposure to IL-3 in non-virally edited HSPCs. Color-coding in FACS plots and histogram is identical. Data are representative of four independent experiments. (F) In vitro differentiation of CD123-engineered HSPCs assessed by the number of colony-forming units (erythroid: E, myeloid: M). CFU were scored using STEMVision based on morphological characteristics. A representative experiment of two independent biological replicates performed in duplicates. (G) Allele frequency of the CD123-engineered HSPCs in a minimum of 38 colonies. (H) Frequency of GlyA + and CD33 + non-virally edited HSPCs cultured in high cytokine medium with or without CSL362-ADC for 14 d. Myeloid lineage: CD33 + , erythroid lineage: GlyA + . Data are from three experiments performed in triplicates. (I) Frequency of 6H6 + CD123-positive cells in the CD33 + , CD14 + , or CD15 + subsets. A representative experiment of two independent experiments performed in triplicates. (J) Computational off-target prediction. HBB and VEGFA site 2 as benchmarking gRNAs. On-target (OnT), off-target (OT1-6). (K) DISCOVER-Seq in KO RNP edited HSPCs. On-target (OnT), off-target (OT7-17). (L) rhAMPSeq validation of computational prediction and DISCOVER-Seq analysis. Shown are the editing rates as percentage of indels detected at the on-target (OnT) site and at the 17 off-target (OT) sites. Blue circles: Edited category comprises samples treated with gRNA for CD123 (KO RNP, KO template, E51K, and E51T). Red triangles: Unedited category comprises samples not treated with gRNA for CD123 (HSC, EP). Data are from one experiment performed with six samples generated in six independent editing experiments with cells from different donors. (M) CAST-Seq in unedited (left) and KO RNP edited HSPCs (center). Coverage plots of the on-target site in CD123 indicate large inversions (pink line) and deletions (orange line), respectively. Circos plot is used to illustrate chromosomal translocations. Data are from one experiment performed with two independent samples. Error bars: mean (SD).

Article Snippet: Antibody CSL362 hIgG1 (captured ligand) was captured by Anti-Human Fc capture biosensor (AHC; Sartorius, PN: 18-5060) for 300 s at 0.5 µg/ml.

Techniques: Expressing, Functional Assay, In Vitro, CRISPR, Flow Cytometry, Binding Assay, Clone Assay, Labeling, Amplification, Sequencing, Staining, Cell Culture, Generated

Journal: The Journal of Experimental Medicine

Article Title: Epitope-engineered human hematopoietic stem cells are shielded from CD123-targeted immunotherapy

doi: 10.1084/jem.20231235

Figure Lengend Snippet: Engineered HSPCs enable tumor-selective CD123 immunotherapy. (A and B) Non-virally edited HSPCs co-cultured with MOLM-14 (CTV-labeled) and control T cells or 123CAR for 3 d. EP are electroporated but non-edited HSPCs. (A) Representative dot plots indicating MOLM-14 cells and non-virally edited CD34 + HSPCs on day 3 of co-culture (left) and the proportion thereof quantified using absolute cell numbers relative to control T cells (right). (B) FACS plots illustrating wt or ki HSPCs by their binding to the mAb 6H6 at the end of the co-culture (left), and proportion of 6H6 + cells based on absolute counts. (A and B) Data of one out of two independent experiments performed in triplicate. (C) 2-d co-culture of AML cells MOLM-14-mCherry, OCI-AML2-mCherry, OCI-AML3-mCherry, and PDX (CTV-labeled) with control T cells or 123CAR. Data of one experiment performed in triplicate. (D and E) Non-virally edited HSPCs co-cultured with MOLM-14 (CTV-labeled) and autologous T cells with or without CSL362/OKT3-TCE (100 ng/ml) for 3 d. Control conditions (EP) are electroporated but non-edited HSPCs. (D) Representative dot plot (left) and proportion (right) of MOLM14 cells and CD34 + HSPCs in different conditions on day 3 of co-culture with or without TCE. Representative data are from one out of three independent experiments with two different donors performed in triplicate. (E) FACS plot (left) and percentage (right) of edited HSPCs (6H6 + cells) at the end of the co-culture with or without TCE. One of two independent experiments, each performed in triplicate, are shown. (F) Sanger sequencing chromatogram of FACS-sorted 6H6 + and 6H6 − HSPCs on day 3 of co-culture with autologous T cells and CSL362/OKT3-TCE. Blue boxes: silent mutations; red boxes: E51K and E51T amino acid substitutions. Data are from one experiment. (G and H) Non-virally edited HSPCs co-cultured with MOLM-14 (CTV-labeled) with or without CSL362-ADC (10 nM) for 3 d. EP are electroporated but genetically not modified HSPCs. (G) Representative dot plot (left) and fraction (right) of MOLM14 cells and CD34 + HSPCs in different conditions on day 3 of co-culture with CSL362-ADC. Representative data are from one out of three independent experiments with two individual donors were performed in triplicate. (H) Flow cytometry data (left) and summary (right) showing the percentage of edited 6H6 + HSPCs at the end of the co-culture. Data are from one out of three individual experiments performed in triplicate. Error bars: mean (SD).

Article Snippet: Antibody CSL362 hIgG1 (captured ligand) was captured by Anti-Human Fc capture biosensor (AHC; Sartorius, PN: 18-5060) for 300 s at 0.5 µg/ml.

Techniques: Cell Culture, Labeling, Co-Culture Assay, Binding Assay, Sequencing, Modification, Flow Cytometry

Journal: iScience

Article Title: CSL362 potently and specifically depletes pDCs in vitro and ablates SLE-immune complex-induced IFN responses

doi: 10.1016/j.isci.2023.107173

Figure Lengend Snippet: Characterization of cell subsets, CD123 expression and CSL362 mediated cell depletion in SLE patient peripheral blood and matched healthy controls Whole blood from 32 healthy donors (HD) and 33 SLE patients was analyzed by flow cytometry to determine absolute counts and expression of CD123. (A) SLE patients have significantly fewer pDCs, basophils, mDCs, CD4 T-cells, CD8 T-cells, NKT-cells, naive B-cells, memory B-cells, monocytes and NK cells. (B) pDCs and basophils express the highest levels of CD123, mDCs express an intermediate amount while all other cell types have a mean receptor expression of less than 1500 ABC. (C) PBMC from 19 to 29 SLE donors and 31–34 healthy controls were cultured with CSL362 (1 μg/mL) for 20 h before the proportion of each cell type was determined by flow cytometry and normalized to no antibody control. (D) PBMC cultures from 6 healthy controls and 7 SLE patients were cultured with CSL362 (0.0005–3 μg/mL) for 20 h before the proportion of pDCs was determined by flow cytometry and normalized to no antibody control. ABC = Antibodies bound per cell as determined by BD quantibrite beads. Bars depict mean ± SEM (Mann-Whitney test ∗∗∗∗p ≤ 0.0001, ∗∗∗p ≤ 0.001, ∗∗p ≤ 0.01, ∗p ≤ 0.05 and n.s p > 0.05. See also Figures S1 and .

Article Snippet: CSL362 (anti-human CD123) , CSL Limited, Reference: Busfield et al. , CSL362.

Techniques: Expressing, Flow Cytometry, Cell Culture, Control, MANN-WHITNEY

Journal: iScience

Article Title: CSL362 potently and specifically depletes pDCs in vitro and ablates SLE-immune complex-induced IFN responses

doi: 10.1016/j.isci.2023.107173

Figure Lengend Snippet: Effects of CSL362 on IFNα production and gene expression induced by TLR ligands, serum or purified IgG from SLE patients PBMC from 8 SLE donors were treated with 1 μg/mL CSL362 or isotype control for 20 h before the cells were resuspended in various stimuli (0.25 μM CpGc, 10 μg/mL LPS, HD Ig + NCL, SLE Ig + NCL, SLE sera + NCL or HD sera + NCL) and cultured for 24 h. (A) Supernatant was then collected and analyzed for IFNα production by ELISA. IFNα levels for each donor PBMC culture treated with isotype control (circles) or CSL362 (triangles) after stimulation are shown. (B) RNA was also extracted from the cultured cells and analyzed by RNA-seq. Graph shows the IFN gene score for each treatment (isotype control circles, CSL362 triangles) and stimulation determined as the average log2 fold change compared to control (HD PBMC unstimulated and treated with isotype control) of 11 IFN genes ( IFI44L, IFIT1, IFIT3, IRF7, ISG15, MX1, MX2, OAS1, OAS2, SERPING1 and XAF1 ). (C) The genes differentially expressed (absolute log2FC >1, FDR<0.05) between PBMCs stimulated with SLE immune complexes (SLE Ig + NCL) in the presence of CSL362 or isotype control were subjected to pathway analysis with Ingenuity (IPA). The top 5 altered processes, ranked by p value of overlap, are shown. (D) Heatmap of the expression changes of TLR9, TLR4 and TLR3 genes in response to the different stimuli with and without CSL362 treatment. PI:C columns show the expression of n = 3 SLE donors only, stimulated with 10 μg/mL poly I:C (PI:C) all other columns show n = 8 SLE donors. NCL = Necrotic Cell Lysates used at 0.1 mg/mL. See also Figure S3 .

Article Snippet: CSL362 (anti-human CD123) , CSL Limited, Reference: Busfield et al. , CSL362.

Techniques: Gene Expression, Purification, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Expressing

Journal: iScience

Article Title: CSL362 potently and specifically depletes pDCs in vitro and ablates SLE-immune complex-induced IFN responses

doi: 10.1016/j.isci.2023.107173

Figure Lengend Snippet: CSL362 reduces production of TLR9-induced proteins including proteins that are significantly elevated in SLE patients sera (A) PBMC from 9 healthy controls and 8 SLE patients were incubated in media alone (untreated; UT) or treated with 1 μg/mL CSL362 or isotype control for 20 h before wells were stimulated with 0.5 μM CpGc for 24 h. Supernatant was collected and assayed for various soluble proteins. Heatmap shows proteins that were significantly upregulated by CpGc stimulation when normalized to unstimulated control (fold change >2 and FDR <0.05). (B) Sera was collected from 33 SLE patients and 34 HD and analyzed for levels of 119 soluble proteins, 27 of which were found to be upregulated in SLE patient sera (see also Table S3 ). The IFN inducible proteins MCP-2/CCL8, IP-10/CXCL10, ITAC/CXCL11 and MIP-3β/CCL19 were among those significantly upregulated in the sera of SLE patients (Benjamini and Hockberg method, fold change >1.5 and adj p value <0.05).

Article Snippet: CSL362 (anti-human CD123) , CSL Limited, Reference: Busfield et al. , CSL362.

Techniques: Incubation, Control

Journal: iScience

Article Title: CSL362 potently and specifically depletes pDCs in vitro and ablates SLE-immune complex-induced IFN responses

doi: 10.1016/j.isci.2023.107173

Figure Lengend Snippet:

Article Snippet: CSL362 (anti-human CD123) , CSL Limited, Reference: Busfield et al. , CSL362.

Techniques: Control, Recombinant, Lysis, Enzyme-linked Immunosorbent Assay, Software

Journal: Blood Cancer Journal

Article Title: Humanized anti-CD123 antibody facilitates NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) of Hodgkin lymphoma targets via ARF6/PLD-1

doi: 10.1038/s41408-018-0168-2

Figure Lengend Snippet: a NK-92, haNK, and primary NK cells were tested for CD16 expression by flow cytometry. Gating for CD56-positive cells and CD16 expression on the each of the NK cells was compared to a murine isotype control. Additionally, HL cell lines (KM-H2, L-428, and L-540), NK cells (NK-92, haNK, and primary NK cells), and the K562 control cell line were tested for cell surface CD123. b Results of the HL cell lines as (%) expression. c Relative variation of the median fluorescence intensity (RMFI) to a murine isotype control in all cells

Article Snippet: The fully humanized IgG1 anti-CD123 mAb [CSL362 or JNJ 56022473] was kindly provided by Janssen Research and Development, LLC.

Techniques: Expressing, Flow Cytometry, Control, Fluorescence

Journal: Blood Cancer Journal

Article Title: Humanized anti-CD123 antibody facilitates NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) of Hodgkin lymphoma targets via ARF6/PLD-1

doi: 10.1038/s41408-018-0168-2

Figure Lengend Snippet: a NK-92 dose-dependent killing efficacy of 3 HL cell lines (KM-H2, L-428, and L-540) using the 4-h standard chromium release assay (CRA). b At 5:1 effector-to-target (E:T) ratio, the killing efficacy of haNK cells against the three HL cell lines and the K562 control cell line is compared. c Analysis of CSL362-binding capacity to CD123-positive HL cells and to CD123-negative control cells. Using flow cytometry, a secondary fluorescein isothiocyanate-conjugated anti-human Fc monoclonal antibody (mAb) was added after preincubation of HL cells with increasing doses of CSL362. d In a 4-h CRA, CSL362-mediated ADCC was studied using haNK cells against CD123-positive HL cells at a 5:1 E:T ratio (KM-H2 and L-428). Negative ADCC controls. e CD123-negative target cells. f The murine-Fc anti-CD123 mAb (7G3) was used instead of CSL362 and NK-92 cells (CD16-negative) as effector cells

Article Snippet: The fully humanized IgG1 anti-CD123 mAb [CSL362 or JNJ 56022473] was kindly provided by Janssen Research and Development, LLC.

Techniques: Release Assay, Control, Binding Assay, Negative Control, Flow Cytometry

Journal: Blood Cancer Journal

Article Title: Humanized anti-CD123 antibody facilitates NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) of Hodgkin lymphoma targets via ARF6/PLD-1

doi: 10.1038/s41408-018-0168-2

Figure Lengend Snippet: Results of one representative donor, tested in triplicate. a CSL362-mediated ADCC validation using primary interleukin-15 (10 ng/mL) activated NK cells against CD123-positive HL cells (KM-H2 and L-428) in a 4-h chromium release assay; 5:1 effector-to-target ratio. Estimation of the half-maximal stimulatory effective concentration (EC 50 ) of CSL362 (1 µg/mL–1 pg/mL) in combination with haNK cells or activated primary NK cells against KM-H2 ( b ) and L-428 cells ( c )

Article Snippet: The fully humanized IgG1 anti-CD123 mAb [CSL362 or JNJ 56022473] was kindly provided by Janssen Research and Development, LLC.

Techniques: Biomarker Discovery, Release Assay, Concentration Assay

Journal: Blood Cancer Journal

Article Title: Humanized anti-CD123 antibody facilitates NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) of Hodgkin lymphoma targets via ARF6/PLD-1

doi: 10.1038/s41408-018-0168-2

Figure Lengend Snippet: a haNK cells and target cells were co-cultured at 5:1 effector-to-target (E:T) ratio for 4 h with and without CSL362 (1 µg/mL). Using flow cytometry and gating for CD56+ cells, the dot plot expression of the degranulation marker CD107a vs standard side scatter is shown. b Cumulative expression of CD107a compared to the CD123-negative control cell line K562. The presence of interferon-γ ( c ) and tumor necrosis factor-α ( d ) was estimated by comparing the relative variation of the median fluorescence intensity (RMFI) of haNK cells in co-culture with L-428 cells (5:1 E:T ratio) in the presence or absence of CSL362 (1 µg/mL), to that of untreated haNK cells alone

Article Snippet: The fully humanized IgG1 anti-CD123 mAb [CSL362 or JNJ 56022473] was kindly provided by Janssen Research and Development, LLC.

Techniques: Cell Culture, Flow Cytometry, Expressing, Marker, Negative Control, Fluorescence, Co-Culture Assay

Journal: Blood Cancer Journal

Article Title: Humanized anti-CD123 antibody facilitates NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) of Hodgkin lymphoma targets via ARF6/PLD-1

doi: 10.1038/s41408-018-0168-2

Figure Lengend Snippet: HaNK cells were cultured for 16 h with CD123-positive Hodgkin lymphoma a KM-H2 and b L-428 cells at 5:1 effector-to-target ratio, in the presence or absence of CSL362 (1 µg/mL). Later, cells were stained for PE anti-human CD16 and sorted using anti-PE magnetic microbeads. The expression of ARF6 , c-JUN , PLD-1 , and RAC-1 RNA was analyzed using real-time reverse-transcriptase PCR and expressed relative to haNK cells under baseline culture conditions. c , d show a similar experiment using primary NK cells activated with IL-15 (10 ng/mL) as effector cells

Article Snippet: The fully humanized IgG1 anti-CD123 mAb [CSL362 or JNJ 56022473] was kindly provided by Janssen Research and Development, LLC.

Techniques: Cell Culture, Staining, Expressing, Reverse Transcription