csl362 (anti-human cd123)  (CSL Limited)

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