Journal: Nature metabolism

Article Title: CD38 ecto-enzyme in immune cells is induced during aging regulating NAD + and NMN levels

doi: 10.1038/s42255-020-00298-z

Figure Lengend Snippet: Analysis of WAT ( a , b , f ) and liver ( c , d , e , g ) of young and old mice. ( a,c ) Representative immunofluorescence image for CD38 (red) and CD45 (green) in WAT and liver of young (3–4 month-old) and old (28–32 month-old) C57BL/6 mice (4 mice per group), compared to isotype control or CD38 Knockout (KO). ( b,d ) CD38 + , CD38 + CD45 − , and CD38 + CD45 + cells in young (4 month-old) and old (32 month-old) mice were determined by flow cytometry in WAT (CD38 + , CD38 + CD45 + n=9 mice per group; CD38 + CD45 − n=5 mice for young, 6 mice for old) and liver (young n=4 mice per group; old n=5 mice per group), changes are relative to young mice. ( e ) In the left panel, flow cytometry-derived color-coded cell clusters of CD38 + immune cell types in liver of young (4 month-old) and old (32 month-old) mice analyzed by Uniform Manifold Approximation and Projection (UMAP). In the right panel, heat maps show CD38 protein expression (lowest in blue and highest in red) in the respective immune clusters (n=4 for young and n=5 for old). ( f ) Graph shows CD45 + F4/80 + CD38 + cell population in WAT of young (4 month-old, n=5 mice) and old mice (32 month-old, n=6 mice). Fold changes are relative to young mice. ( g ) CD38 + CD45 + subsets increase with age in liver tissue (Classical monocytes, monocyte-derived macrophages, CD8 + T cells, and granulocytes n=6 mice; CD4 + T cells n=4 mice). Change is relative to young mice. ( h ) CD38 activity in mouse immune cells isolated from spleen (n=3 biologically independent samples). ( i-k ) Macrophages isolated from 3 month-old and 18 month-old mice were stimulated with different concentrations of LPS for 20 hours. ( i ) Cd38 mRNA expression measured by qRT-PCR analysis (n=4 biologically independent samples). ( j ) CD38 activity measured in protein lysates (RFU-relative fluorescent units) (n=5, except for 18 mo 50 ng/mL LPS where n=4 biologically independent samples). ( k ) Representative immunoblotting of three biologically independent samples from macrophages treated with different concentrations of LPS. Data are mean ± SEM, analyzed by unpaired two-sided t-test, except for CD38 + CD45 + in , where samples were analyzed by unpaired on-sided t-test.

Article Snippet: In experiments where 293T cells were incubated with human recombinant CD38 (hrCD38, R&D Systems) in the cell culture medium, the first step involved incubation of the hrCD38 (100 ng/mL) with 5 μg/mL isatuximab for 30 minutes at 37°C in cell culture media containing 1% FBS.

Techniques: Immunofluorescence, Control, Knock-Out, Flow Cytometry, Derivative Assay, Expressing, Activity Assay, Isolation, Quantitative RT-PCR, Western Blot

Journal: Nature metabolism

Article Title: CD38 ecto-enzyme in immune cells is induced during aging regulating NAD + and NMN levels

doi: 10.1038/s42255-020-00298-z

Figure Lengend Snippet: ( a ) Graphs show no change in CD38 + CD31 + cell population in WAT and liver tissues between young (4 month-old) and old (32 month-old) mice (n=3 for liver and n=9 mice for WAT). ( b ) Gating strategy used to show the CD45 + CD38 + population in WAT of young (4 month-old) and old (22 month-old) mice. ( c ) Histograms of CD38 + population in young (4 month-old) and old (22 month-old) WAT showing the shift in CD38 expression. The graph shows the relative area under the curve (AUC) for young and old (n=5 mice per group). ( d ) Histograms showing the CD38 expression in different immune cells in young (4 month-old) and old (32 month-old) liver. Table shows cell counts for each subset of immune cells, the data are representative of n=5 mice per group. ( e ) Gating strategy for CD45 + immune subsets in CD38 + liver cells in young (4 month-old) and old mice (32 month-old). Data are mean ± SEM, analyzed by unpaired two-sided t -test.

Article Snippet: In experiments where 293T cells were incubated with human recombinant CD38 (hrCD38, R&D Systems) in the cell culture medium, the first step involved incubation of the hrCD38 (100 ng/mL) with 5 μg/mL isatuximab for 30 minutes at 37°C in cell culture media containing 1% FBS.

Techniques: Expressing

Journal: Nature metabolism

Article Title: CD38 ecto-enzyme in immune cells is induced during aging regulating NAD + and NMN levels

doi: 10.1038/s42255-020-00298-z

Figure Lengend Snippet: ( a-g ) Sub-lethally irradiated 3 month-old CD38 KO mice were subjected to bone marrow transplant (BMT) with 1×10 6 bone marrow cells (BMC) per animal from either WT (WT>KO) or CD38 KO donors (KO>KO). Twelve weeks after transplantation, μice were subcutaneously injected with LPS (300 μg/kg) or vehicle daily for 5 days and harvested at day 5 (n=4 mice per group). Figure shows analyses in WAT. ( a ) Schematic of experiment. ( b ) Relative Cd38 mRNA expression measured by qRT-PCR. Expression is relative to WT>KO group. ND denotes non-detectable. ( c ) CD38 activity. ( d ) Representative immunoblotting showing CD38 levels in 3 mice from each treatment group. ( e ) mRNA expression of Cd45 and F4/80 measured by qRT-PCR. Expression is relative to CD38 KO (KO>KO). ( f ) Immunofluorescent staining of CD38 (red) and CD45 (green), representative of 4 mice per group. The graph shows quantification of CD38 and CD45 positive cells from the images (n=3 fields obtained from a representative mouse from each treatment group). ( g ) NAD + and NMN levels. Data are mean ± SEM, analyzed by unpaired two-sided t-test.

Article Snippet: In experiments where 293T cells were incubated with human recombinant CD38 (hrCD38, R&D Systems) in the cell culture medium, the first step involved incubation of the hrCD38 (100 ng/mL) with 5 μg/mL isatuximab for 30 minutes at 37°C in cell culture media containing 1% FBS.

Techniques: Irradiation, Transplantation Assay, Injection, Expressing, Quantitative RT-PCR, Activity Assay, Western Blot, Staining

Journal: Nature metabolism

Article Title: CD38 ecto-enzyme in immune cells is induced during aging regulating NAD + and NMN levels

doi: 10.1038/s42255-020-00298-z

Figure Lengend Snippet: ( a-b ) 12 month-old mice received daily subcutaneous injection of vehicle (Ctrl) or LPS (300 μg/kg) for 5 days (n=9 mice per group). ( a ) mRNA expression of Cd38 , F4/80 , and Cd45 in subcutaneous WAT (Subq WAT) measured by qRT-PCR analysis and expressed relative to Ctrl. ( b ) Immunofluorescent staining for CD38 (red) and CD45 (green) in Subq WAT, showing accumulation of CD38 + CD45 + cells with LPS treatment. Images are representative of 6 mice per group. ( c-h ) Sub-lethally irradiated CD38 KO mice were subjected to bone marrow transplant (BMT) with 1×10 6 bone marrow cells (BMC) per animal from either WT (WT>KO) or CD38 KO donors (KO>KO). Twelve weeks after transplantation, mice were subcutaneously injected with LPS (300 μg/kg) or vehicle daily for 5 days and harvested at day 5. ( c , d ) CD38 activity and NAD + levels in spleen and jejunum (n=4 mice per group). ( e ) CD38 expression by immunoblot (n=3 mice per group). ( f ) Number of CD38 + cells by flow cytometry was measured in spleen and jejunum with and without LPS treatment (n=5 mice per group). ( g , h ) CD38 activity and NAD + levels in liver and pancreas (n=4 mice per group). ( i ) CD38 activity was measured in macrophages isolated from WT or CD38 KO mice and treated or not with LPS (100 ng/mL) with and without the CD38 inhibitor 78c (0.5 μM) for 24 hours. CD38 activity is relative to Ctrl WT (n=3 biologically independent samples). ( j ) Macrophages isolated from WT or CD38 KO mice were treated or not with LPS (100 ng/mL) for 24 hours and mRNA expression of Cd38 and other markers of macrophage activation were measured. Expression was measured by qRT-PCR analysis and expressed relative to Ctrl WT (n=4 biologically independent samples). Data are mean ± SEM, analyzed by unpaired two-sided t -test, NS=non-significant.

Article Snippet: In experiments where 293T cells were incubated with human recombinant CD38 (hrCD38, R&D Systems) in the cell culture medium, the first step involved incubation of the hrCD38 (100 ng/mL) with 5 μg/mL isatuximab for 30 minutes at 37°C in cell culture media containing 1% FBS.

Techniques: Injection, Expressing, Quantitative RT-PCR, Staining, Irradiation, Transplantation Assay, Activity Assay, Western Blot, Flow Cytometry, Isolation, Activation Assay

Journal: Nature metabolism

Article Title: CD38 ecto-enzyme in immune cells is induced during aging regulating NAD + and NMN levels

doi: 10.1038/s42255-020-00298-z

Figure Lengend Snippet: ( a-b ) Immunofluorescent staining of WAT from young (2 month-old) and old (28 month-old) C57BL/6 mice. ( a ) Images of WAT stained with CD38 (red), lamin B1 (green), and ORF1 (yellow), representative of 3 mice per group. Insets show an enlarged portion of old tissue, with and without ORF1 signal, to highlight that CD38 + cells are present near ORF1 + lamin B1 − cells. ( b ) Images of WAT stained with CD38 (red), CD45 (green), and ORF1 (yellow), representative of 6 mice per group. Insets show the image of old WAT with removal of green color channel CD45 signal (left) and with removal of red color channel CD38 signal (right) to show accumulation of CD38 + CD45 + cells near ORF1 + cells. ( c-e ) 12 month-old INK-ATTAC mice were treated with vehicle or AP20187 for 16 months. At 28 months these mice were euthanized and compared with 12 month-old INK-ATTAC mice. Analyses were performed in WAT. ( c ) Schematic of experiment. ( d ) p16 and Cd38 mRNA levels detected by qRT-PCR analysis (12 mo n=5 mice; 28 mo n=6 mice; 28 mo+AP n=12 mice) ( e ) NAD + levels (12 mo n=5 mice; 28 mo n=6 mice; 28 mo+AP n=14 mice). ( f - g ) 18 month-old WT C57BL/6 mice were injected IP with DiI-labelled (yellow) senescent (SEN) or non-senescent (NS) mouse pre-adipocytes (mPA) from WT mice. ( f ) Schematic of experiment. ( g ) Immunofluorescent staining of CD38 (red) and CD45 (green) in WAT (representative of n=4 mice per group). Accumulation of CD38 + CD45 + cells was increased when senescent cells were injected. ( h-j ) 12 month-old CD38 KO mice were injected IP with vehicle, senescent (SEN), or non-senescent (NS) mPA from WT mice. 48 hours later, mice were injected with luciferase labeled PBMCs. Control mice received PBMCs only. ( h ) Schematic of experiment. ( i ) Representative images showing increase in luciferase + PBMCs in mice injected with SEN mPA. ( j ) Relative Cd38 mRNA levels measured by qRT-PCR in WAT (Ctrl and SEN mPA n=7 mice; NS mPA n=8 mice). Data are mean ± SD ( d,e ) and SEM ( j ), analyzed by unpaired two-sided t-test.

Article Snippet: In experiments where 293T cells were incubated with human recombinant CD38 (hrCD38, R&D Systems) in the cell culture medium, the first step involved incubation of the hrCD38 (100 ng/mL) with 5 μg/mL isatuximab for 30 minutes at 37°C in cell culture media containing 1% FBS.

Techniques: Staining, Quantitative RT-PCR, Injection, Luciferase, Labeling, Control

Journal: Nature metabolism

Article Title: CD38 ecto-enzyme in immune cells is induced during aging regulating NAD + and NMN levels

doi: 10.1038/s42255-020-00298-z

Figure Lengend Snippet: ( a ) WT macrophages were incubated with LPS (100 ng/mL) in the presence or absence of 100 nM AP20187 for 20 hours. CD38 activity was measured in cell lysates (n=4 biologically independent samples). ( b ) WT macrophages were treated with LPS (100 ng/mL) for 20 hours and cell lysates were prepared. Lysates were incubated with or without AP20187 for 15 min before CD38 activity was measured (n=3 biologically independent samples). ( c - g ) 12 month-old INK-ATTAC mice were treated with vehicle or with AP20187 for 16 months (28 month-old groups) and were compared with 12 month-old animals. ( c ) Representative images of immunofluorescent staining for CD38 (red), ORF1 (yellow), and CD45 (green) in WAT. Insets show the image of 28 month-old WAT with removal of green color channel CD45 signal (left) and with removal of red color channel CD38 signal (right) to show accumulation of CD38 + CD45 + cells near ORF1 + cells. Graph shows quantification of CD45 + immune clusters in WAT, based on 20 5x fields per sample (12 mo, n=5; 28 mo, n=4; 28 mo+AP, n=3 mice). ( d ) IL6 levels in WAT detected by ELISA (12 mo n=5; 28 mo n=7; 28 mo+AP n=6 mice). ( e ) NMN levels measured in WAT (12 mo n=5; 28 mo n=6; 28 mo+AP n=13 mice). ( f ) Relative mRNA levels of p16 (12 mo n=6; 28 mo n=7; 28 mo+AP n=5 mice) and Cd38 (12 mo n=10; 28 mo n=9; 28 mo+AP n=6 mice) detected by qRT-PCR analysis in liver. Levels are relative to 12 month-old mice. ( g ) NAD + levels in liver (12 mo n=6; 28 mo n=7; 28 mo+AP n=15 mice). ( h ) Relative mRNA levels of inflammatory and senescence-related genes in X-ray irradiated WT mouse pre-adipocytes determined by qRT-PCR. Levels are relative to control non-senescent cells (Ctrl) (Ctrl n=5; X-ray n=6 biologically independent samples). ( i ) Quantitative analysis of cytokines/chemokines in conditioned media harvested from irradiated (CM-SEN) or non-senescent (CM-NS) mouse pre-adipocytes. Heat maps reflect analytes (pg/mL) measured in 3–25-plex Luminex assays. Heat map shows average of 5 biologically independent samples. Data are mean ± SEM, except letters e-g that are mean ± SD, analyzed by unpaired two-sided t -test.

Article Snippet: In experiments where 293T cells were incubated with human recombinant CD38 (hrCD38, R&D Systems) in the cell culture medium, the first step involved incubation of the hrCD38 (100 ng/mL) with 5 μg/mL isatuximab for 30 minutes at 37°C in cell culture media containing 1% FBS.

Techniques: Incubation, Activity Assay, Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Irradiation, Control, Luminex

Journal: Nature metabolism

Article Title: CD38 ecto-enzyme in immune cells is induced during aging regulating NAD + and NMN levels

doi: 10.1038/s42255-020-00298-z

Figure Lengend Snippet: ( a - b ) WT macrophages were incubated with conditioned media from senescent (CM-SEN) and non-senescent (CM-NS) mouse pre-adipocytes for 20 hours. CD38 expression and activity are relative to CM-NS. ( a ) Cd38 mRNA expression in the macrophages was measured by qRT-PCR analysis. (n=5 biologically independent samples). ( b ) Relative CD38 activity in macrophage lysates (n=5 biologically independent samples). ( c ) WT macrophages were treated with LPS (100 ng/mL) with and without 3TC (10 μM) for 20 hours and CD38 activity was measured in cell lysates (n=3 biologically independent samples). ( d ) WT macrophages were incubated with conditioned media from senescent (CM-SEN) and non-senescent (CM-NS) mouse pre-adipocytes for 20 hours. Conditioned media was pre-incubated for 2 hours with or without IL6 or TNF-α antibody (5 μg/mL) before addition to the macrophages. CD38 activity was measured in cell lysates, and expressed relative to CM-NS (CM-NS and CM-SEN n=8; CM-NS+IL6 Ab and CM-SEN+IL6 n=6; CM-NS+TNF Ab and CM-SEN+TNF n=4 biologically independent samples). ( e ) HUVECs were treated with conditioned media from senescent (CM-SEN) and non-senescent (CM-NS) mouse embryonic fibroblasts for 20 hours. CM was pre-incubated for 2 hours with or without TNF-α antibody (5 μg/mL) before addition to the HUVECs. Cd38 mRNA expression was measured by qRT-PCR analysis (CM-NS n=12; CM-NS+TNF Ab n=4; CM-SEN n=10; CM-SEN+TNF Ab n=4 biologically independent samples). Data are mean ± SEM, analyzed by unpaired two-sided t -test.

Article Snippet: In experiments where 293T cells were incubated with human recombinant CD38 (hrCD38, R&D Systems) in the cell culture medium, the first step involved incubation of the hrCD38 (100 ng/mL) with 5 μg/mL isatuximab for 30 minutes at 37°C in cell culture media containing 1% FBS.

Techniques: Incubation, Expressing, Activity Assay, Quantitative RT-PCR

Journal: Nature metabolism

Article Title: CD38 ecto-enzyme in immune cells is induced during aging regulating NAD + and NMN levels

doi: 10.1038/s42255-020-00298-z

Figure Lengend Snippet: ( a - b )12 month-old WT mice received daily subcutaneous injection of conditioned media from senescent (CM-SEN) or non-senescent (CM-NS) mouse pre-adipocytes for 5 days, and were harvested at day 5. ( a ) CD38 activity (n=7 mice per group) and NAD + levels (CM-NS n=7 mice; CM-SEN n=6 mice) were measured in subcutaneous WAT (Subq WAT). ( b ) Relative Cd38 (n=11 mice per group), Cd45, and F4/80 (CM-NS n=14 mice; CM-SEN n=12 mice) mRNA expression measured by qRT-PCR in Subq WAT. Levels were relative to CM-NS. ( c-f ) 26 month-old mice were treated with vehicle (Ctrl) or 3TC for 2 weeks. ( c ) Schematic of experiment. ( d ) Relative mRNA levels for p16 and SASP components measured by qRT-PCR in WAT (n=8 mice per group for all conditions except Pai1 3TC where n=7 mice). ( e ) Relative mRNA levels for F4/80 (n=8 mice per group), Cd38, Parp1, and Sirt1 (n=5 mice per group) measured by qRT-PCR in WAT. ( f ) Relative NAD + levels in WAT (n=5 mice per group). Levels were relative to vehicle-treated mice. ( g ) Immunofluorescent staining of CD38 (red), lamin B1 (green), and IL6 (yellow) in WAT of young (2 month-old) and old (28 month-old) C57BL/6 mice, representative of 4 young and 6 old mice. Insets show enlarged area of old tissue with and without IL6 staining, to highlight that CD38 can be found near IL6 + lamin B1 − cells. Data are mean ± SEM, analyzed by unpaired two-sided t -test. Two statistical outliers were identified in the Cd45 mRNA expression of the CM-SEN-treated animals (with values higher than 16) using the appropriate test in GraphPad Prism 6. These samples were excluded from all the PCR analyses for this experiment. Data were also analyzed by non-parametric Mann-Whitney using the full data set (including the identified statistical outliers) and the p value for Cd45 was 0.002.

Article Snippet: In experiments where 293T cells were incubated with human recombinant CD38 (hrCD38, R&D Systems) in the cell culture medium, the first step involved incubation of the hrCD38 (100 ng/mL) with 5 μg/mL isatuximab for 30 minutes at 37°C in cell culture media containing 1% FBS.

Techniques: Injection, Activity Assay, Expressing, Quantitative RT-PCR, Staining, MANN-WHITNEY

Journal: Nature metabolism

Article Title: CD38 ecto-enzyme in immune cells is induced during aging regulating NAD + and NMN levels

doi: 10.1038/s42255-020-00298-z

Figure Lengend Snippet: ( a-f ) 12 month-old WT and transgenic catalytically inactive CD38 (CI) mice were treated with vehicle (Ctrl) or a single dose of doxorubicin (Doxo, 15 mg/kg), and WAT was harvested 10 days later for analyses (CI n=4 mice; WT, WT+Doxo, CI+Doxo n=5 mice per group). ( a ) Schematic of experiment. ( b ) p21, II6, Il1b , and F4/80 mRNA expression measured by qRT-PCR. ( c ) Cd38 mRNA expression measured by qRT-PCR. ( d ) Representative immunoblotting of CD38 protein levels and graph showing quantification of CD38 levels. The quantification is a ratio of CD38/tubulin levels (n=3 mice per group). ( e ) CD38 activity. ( f ) NAD + levels. Data are mean ± SEM, analyzed by unpaired two-sided t-test.

Article Snippet: In experiments where 293T cells were incubated with human recombinant CD38 (hrCD38, R&D Systems) in the cell culture medium, the first step involved incubation of the hrCD38 (100 ng/mL) with 5 μg/mL isatuximab for 30 minutes at 37°C in cell culture media containing 1% FBS.

Techniques: Transgenic Assay, Expressing, Quantitative RT-PCR, Western Blot, Activity Assay

Journal: Nature metabolism

Article Title: CD38 ecto-enzyme in immune cells is induced during aging regulating NAD + and NMN levels

doi: 10.1038/s42255-020-00298-z

Figure Lengend Snippet: ( a-c ) 293T cells were transfected with vector (V), CD38 WT, CD38 catalytically-inactive (CI), or CD38 Δ49 expression plasmid. ( a ) Representative immunoblotting (of four biologically independent samples) showing CD38 expression in whole cell lysates of 293T cells transfected with CD38 plasmids. ( b ) Immunofluorescence of fixed 293T cells transfected with CD38 plasmids (n=3 biologically independent samples). Cells were labeled with CD38 antibody and a membrane dye, followed by Hoechst staining. Yellow arrows indicate membrane CD38, white arrows show intracellular CD38. ( c ) NAD + levels in transfected 293T cells (V, WT, Δ49 n=6; CI n=4 biologically independent samples). ( d - e ) 293T cells were transfected with vector, CD38 WT, or CD38 Δ49 and 20 hours later were incubated with isatuximab (isa, 5 μg/mL) or 78c (0.5 μM). When NMN was added, it was 4 hours after drugs. NAD + levels were measured 20 hours after drug treatments (n=5 biologically independent samples). Levels are relative to vector-transfected cells. ( f ) NAD + levels in AML12 cells co-cultured with 293T cells expressing CD38 plasmids. 293T cells were treated with and without 200 μM NMN 20 hours after transfection. AML 12 cells were collected 20 hours after addition of NMN (n=5 biologically independent samples). Levels are relative to vector-transfected cells. Data are mean ± SEM, analyzed by unpaired two-sided t -test, NS=non-significant.

Article Snippet: In experiments where 293T cells were incubated with human recombinant CD38 (hrCD38, R&D Systems) in the cell culture medium, the first step involved incubation of the hrCD38 (100 ng/mL) with 5 μg/mL isatuximab for 30 minutes at 37°C in cell culture media containing 1% FBS.

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Immunofluorescence, Labeling, Membrane, Staining, Incubation, Cell Culture

Journal: Nature metabolism

Article Title: CD38 ecto-enzyme in immune cells is induced during aging regulating NAD + and NMN levels

doi: 10.1038/s42255-020-00298-z

Figure Lengend Snippet: ( a ) 293T cells were transfected with vector (V), CD38 WT (WT), CD38 CI (CI) or CD38 Δ49 (Δ49). Representative immunoblotting of 4 independent experiments showing CD38 expression in cytosol of 293T cells transfected with CD38 plasmids. ( b ) Immunofluorescence of fixed 293T cells transfected with CD38 plasmids. Images are representative of 3 independent experiments. Cells were labeled with CD38 antibody and a membrane dye, followed by Hoechst staining. Images show separate CD38 and membrane staining. ( c - d ) Lysates of transfected 293T cells were incubated with varied concentrations of the human CD38 antibody isatuximab (isa) or 0.5 μM 78c for 15 min before CD38 activity was measured. Activity is relative to control (no antibody). ( c ) Lysates of 293T transfected with CD38 WT plasmid (n=4 biologically independent samples). ( d ) Lysates of 293T transfected with CD38 Δ49 plasmid (n=3 biologically independent samples). ( e ) Human recombinant CD38 (hCD38) (100 ng/mL) was incubated with and without isatuximab (5 μg/mL) for 2 hours and then added to 293T cells together with NMN (300 μM). Control samples had no CD38 and no NMN. 293T were incubated with NMN and hCD38 for 20 hours and NAD + levels were measured in the 293T cells. Values show the difference from untreated control (n=3 except for NMN+hCD38+isa where n=4 biologically independent samples). ( f ) Scheme representing the coculture model. ( g ) NAD + levels in AML 12 cells co-cultured with 293T cells expressing vector or CD38 WT. 293T cells were treated with and without 200 μM NMN 20 hours after transfection in the presence or absence of 5 μg/ml isatuximab. AML 12 cells were collected 20 hours after incubation with 293T cells (n=3 except for V+NMN where n=4 biologically independent samples). Levels are relative to vector-transfected cells. Data are mean ± SEM, analyzed by unpaired two-sided t -test.

Article Snippet: In experiments where 293T cells were incubated with human recombinant CD38 (hrCD38, R&D Systems) in the cell culture medium, the first step involved incubation of the hrCD38 (100 ng/mL) with 5 μg/mL isatuximab for 30 minutes at 37°C in cell culture media containing 1% FBS.

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Immunofluorescence, Labeling, Membrane, Staining, Incubation, Activity Assay, Control, Recombinant, Cell Culture

Journal: Nature metabolism

Article Title: CD38 ecto-enzyme in immune cells is induced during aging regulating NAD + and NMN levels

doi: 10.1038/s42255-020-00298-z

Figure Lengend Snippet: ( a-b ) NAD + levels in BMDM from 3 month-old WT ( a ) and CD38 KO ( b ) mice. Macrophages were treated with vehicle (Ctrl) or LPS (100 ng/mL), with or without 78c (0.5 μM) (n=7; with exception of WT LPS+78c where n=6 biologically independent samples). ( c-k ) BMDM isolated from 18 month-old WT mice. ( c ) NAD + levels in macrophages treated with LPS (100 ng/mL), Ab68 (5 μg/mL), 78c (0.5 μM), and olaparib (olap, 5 μM) (Ctrl, LPS n=11; LPS+78c n=4; LPS+78c, LPS+olap, LPS+78c+olap n=9 biologically independent samples). ( d ) Macrophages were treated with LPS (100 ng/mL), Ab68 (5 μg/mL), 78c (0.5 μM), and olaparib (olap, 5 μM). 3 hours later, 300 μM NMN was added and cells were collected 20 hours later for NAD + measurements (n=7 except for NMN+LPS+olap and NMN+LPS+olap+78c where n=6 biologically independent samples). ( e ) Representative immunoblotting of three biologically independent samples of M0 or M1 (LPS-treated) macrophages and AML12 lysates showing CD38 and Slc12a8. ( f ) NAD + levels in AML 12 cells cocultured with macrophages. Macrophages were treated with LPS (100 ng/mL), Ab68 (10 μg/mL), and NMN (200 μM) before incubation with AML 12 cells (n=7 biologically independent samples except for LPS+Ab68 where n=5). ( g ) Extracellular NMN in culture media after treatment of macrophages with 100 μM NMN, LPS (100 ng/mL), and Ab68 (5 μg/mL). LPS was given 9 hours before NMN, and Ab68 was given 3 hours before NMN. Cells were incubated with NMN for 12 hours before media was collected (n=5 biologically independent samples). ( h ) NAD + levels in AML 12 cells cocultured with macrophages. Macrophages were treated with LPS (100 ng/mL), Ab68 (10 μg/mL), and NR (200 μM) before incubation with AML 12 cells (n=5 biologically independent samples). ( i ) NAD + levels in macrophages treated with CM from non-senescent (CM-NS) or senescent mouse pre-adipocytes (CM-SEN or CM-SEN + 78c (0.5 μM)) (n=6 biologically independent samples). ( j-k ) NAD + (n=8 biologically independent samples except for CM SEN+NMN+Ab68 where n=7) and NMN levels (n=4 biologically independent samples) in AML 12 cells cocultured with macrophages. Macrophages were treated with CM-NS or CM-SEN from mouse preadipocytes, Ab68 (10 μg/mL), and NMN (200 μM) before incubation with AML 12 cells. All levels are relative to their respective control (Ctrl or CM-NS). Data are mean ± SEM, analyzed by unpaired two-sided t-test.

Article Snippet: In experiments where 293T cells were incubated with human recombinant CD38 (hrCD38, R&D Systems) in the cell culture medium, the first step involved incubation of the hrCD38 (100 ng/mL) with 5 μg/mL isatuximab for 30 minutes at 37°C in cell culture media containing 1% FBS.

Techniques: Isolation, Western Blot, Incubation, Control

Journal: Nature metabolism

Article Title: CD38 ecto-enzyme in immune cells is induced during aging regulating NAD + and NMN levels

doi: 10.1038/s42255-020-00298-z

Figure Lengend Snippet: ( a ) Relative mRNA expression of Nampt and Cd157 after treatment of BMDMs of WT and CD38 KO mice with and without 100 ng/mL LPS for 24 hours. Expression, assessed by qRT-PCR, is relative to Ctrl WT (n=4 biologically independent samples). ( b-g ) Characterization of the mouse anti-CD38 antibodies used in this study. ( b ) Schematic representation of a human heavy chain-only antibody (UniAbs). ( c ) Workflow to discover CD38-specific UniAbs. Antibodies were derived from transgenic rats, called UniRats that produce heavy-chain antibodies with fully human V H domains (UniAbs) in response to an antigen challenge. ( d ) Development of UniRat transgenic animals. ( e ) Protein and cell-based screens to discover anti-CD38 UniAbs. ( f ) Graph showing the binding of FITC-labeled Ab68 to freshly isolated mouse spleen cells (n=3 biologically independent samples). ( g ) Effect of Ab68 on the rate of CD38 hydrolase activity at different concentrations of substrate (left, n=8; except for no e-NAD where n=4 biologically independent samples and respective Lineweaver-Burk plots (right, n=6 biologically independent samples). ( h ) Table shows the binding affinities (KD) of anti-mouse CD38 antibodies Ab68 and Ab69 and a mouse control antibody OKT3. NB=No Binding. ( i ) Apoptosis of CHO cells stably transfected with mouse CD38 after incubation with antibodies Ab68, Ab69, and NIMR-5 for 24 hours (n=2 samples per concentration). ( j ) Cell viability of WT macrophages treated with LPS (100 ng/mL), with or without Ab68 or Ab69 (5 μg/mL) for 24 hours (n=4 except for LPS+Ab69 where n=3 biologically independent samples). ( k ) Graph shows the internalization of Ab68 compared to control antibody NIMR-5 after 45 minutes and 1.5 hours. ( l ) NAD + levels in AML 12 treated with 200 μM NMN in the presence or absence of LPS (100 ng/mL), Ab68 (5 μg/mL), and 78c (0.5 μM). LPS was given for 18 hours, then Ab68 was added, and 3 hours later NMN was added. Cells were collected 20 hours after NMN was added. NAD + levels were relative to control (Ctrl) (n=4 biologically independent samples). ( m ) NAD + levels in AML 12 cells cocultured with macrophages from CD38 KO mice. LPS was given for 18 hours to the macrophages, then NMN or NR (200 μM) were added for 3 hours. Next, macrophages were incubated with AMLs, and AML cells were collected 20 hours later. NAD + levels were relative to control (Ctrl) (n=4 biologically independent samples). ( n ) NAD + levels in AML 12 cocultured with macrophages. Macrophages were treated with 200 μM NA in the presence or absence of LPS (100 ng/mL), and Ab68 (5 μg/mL). LPS was given for 18 hours, then Ab68 was added, and 3 hours later nicotinic acid (NA) was added to the macrophages. Three hours after addition of NA, macrophages were incubated with AML cells. AML cells were collected 20 hours later and NAD + levels were calculated relative to control (Ctrl) (n=5 biologically independent samples). ( o ) Macrophages were incubated with conditioned media from senescent (CM-SEN) and non-senescent (CM-NS) mouse pre-adipocytes, and with LPS and without (Ctrl) for 20 hours. Cd38 mRNA expression (CM-NS and CM-SEN n=5; Ctrl and LPS n=4 biologically independent samples), CD38 activity (n=5 biologically independent samples), and NAD + levels (n=6 biologically independent samples) were measured in the macrophages. Samples from cells incubated with CM-SEN, were calculated relative to cells incubated with CM-NS. Samples from cells treated with LPS, were calculated relative to vehicle-treated cells (Ctrl). Data are mean ± SEM, analyzed by unpaired two-sided t -test, NS= non-significant.

Article Snippet: In experiments where 293T cells were incubated with human recombinant CD38 (hrCD38, R&D Systems) in the cell culture medium, the first step involved incubation of the hrCD38 (100 ng/mL) with 5 μg/mL isatuximab for 30 minutes at 37°C in cell culture media containing 1% FBS.

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Transgenic Assay, Binding Assay, Labeling, Isolation, Activity Assay, Control, Stable Transfection, Transfection, Incubation, Concentration Assay

Journal: Nature metabolism

Article Title: CD38 ecto-enzyme in immune cells is induced during aging regulating NAD + and NMN levels

doi: 10.1038/s42255-020-00298-z

Figure Lengend Snippet: ( a-c ) 4 month-old WT and CD38 KO mice were treated with vehicle (Ctrl), Ab68 (5 mg/kg), or Ab69 (5 mg/kg) by intraperitoneal (i.p.) injection on day 1 and day 5, and euthanized on day 8. ( a ) Relative CD38 activity in WAT. Levels are relative to control WT (Ctrl) (WT Ctrl n=6; WT Ab68, CD38 KO n=5; WT Ab69 n=3 mice). ( b ) NAD + levels in WAT (WT n=4 mice; CD38 KO n=5 mice). ( c ) Heat map showing levels of metabolites in WT mice treated with Ab68 and Ab69 relative to Ctrl (n=4 mice per group). ( d ) 4 month-old WT mice were treated withFK866 (FK) (25 mg/kg) and/or Ab68 (5 mg/kg) (n=5 mice per group). NMN and NAD + levels were measured in WAT. Levels are relative to Ctrl. ( e ) 22 month-old WT mice were injected i.p. with a single dose of vehicle (Ctrl) or Ab68 (5 mg/kg), euthanized at different time points, and NMN and NAD + levels measured in WAT. Graphs show the time course of the relative increase of NMN or NAD + in vehicle and Ab68-treated over levels at time 0 (n=4 mice per group). ( f ) 18 month-old mice were injected i.p. with a single dose of vehicle (Ctrl) or Ab68 (5 mg/kg) and mice were euthanized at different time points. NMN levels were measured in serum (Ctrl, 0, 24, 72 hours, and Ab68 72 hours n=4 mice per group; Ab68 24 hours n=5 mice; Ctrl 6 hours n=8 mice; Ab68 6 hours n=9 mice). ( g ) 18 month-old WT mice were injected i.p. with vehicle (Ctrl, NMN groups), 5 mg/kg Ab68 (Ab68, Ab68+NMN groups), with and without 500 mg/kg NMN. Serum NMN levels were determined (n=5 mice per group). ( h ) Absolute NAD + levels in WAT of 3 month-old (young) and 18 month-old (old) mice were determined 3 days after treatment with a single injection i.p. injection of vehicle (Ctrl) or 5 mg/kg Ab68 (n=5 mice per group). ( i ) 3 month old and 18 month-old mice were treated with Ab68 and NMN as described in panel ( g ) and WAT levels of NAD + were measured. NAD + levels after each treatment are expressed relative to its age-matched control (vehicle treatment) (n=5 mice per group). Data are mean ± SEM, analyzed by unpaired two-sided t -test, except for (e-f) where data are analyzed by two-way ANOVA.

Article Snippet: In experiments where 293T cells were incubated with human recombinant CD38 (hrCD38, R&D Systems) in the cell culture medium, the first step involved incubation of the hrCD38 (100 ng/mL) with 5 μg/mL isatuximab for 30 minutes at 37°C in cell culture media containing 1% FBS.

Techniques: Injection, Activity Assay, Control

Journal: Nature metabolism

Article Title: CD38 ecto-enzyme in immune cells is induced during aging regulating NAD + and NMN levels

doi: 10.1038/s42255-020-00298-z

Figure Lengend Snippet: ( a - b ) 4 month-old WT and CD38 KO mice were treated with vehicle (Ctrl), Ab68 (5 mg/kg), or Ab69 (5 mg/kg) on day 1 and day 5, and euthanized on day 8. ( a ) CD38 activity in several tissues (liver n=5 except for Ctrl CD38 KO where n=4 mice; skeletal muscle n=3 except for Ab68 where n=2 mice; jejunum and spleen n=5 mice per group). ( b ) NAD + levels in several tissues (liver and spleen n=5 mice except for Ctrl CD38 KO where n=4; skeletal muscle and spleen n=5 mice per group). ( c ) 4 month-old WT mice were treated with a single injection of vehicle (Ctrl) or Ab68 (5 mg/kg). After 3 days, tissues were harvested and gene expression was measured in WAT by qRT-PCR. Values are relative to Ctrl (Ctrl n=5 mice; Ab68 n=4 mice). ( d ) 4 month-old WT mice were treated with vehicle (Ctrl) or Ab68 (5 mg/kg) on day 1 and day 5, and euthanized on day 8. NMN and nicotinamide (NAM) levels were measured in liver and skeletal muscle. Levels are relative to Ctrl (n=5 mice per group). ( e ) Relative NMN levels in WAT, liver and skeletal muscle of 4 month-old WT and CD38 KO mice. Levels are relative to WT mice (n=5 mice per group except for liver CD38 KO where n=3 mice and skeletal muscle CD38 KO where n=4 mice). ( f ) 22 month-old WT mice were injected with a single dose of vehicle (Ctrl) or Ab68 (5 mg/kg). Mice were euthanized at different time points, and NMN and NAD + levels were measured in liver. Graphs show the time course of the relative increase of NMN or NAD + in vehicle and Ab68-treated over levels at time 0 (n=4 mice per group). ( g ) Graph shows the difference between NMNase activity of CD38 in the blood of WT and CD38 KO mice (n=3 mice per group). ( h ) Human recombinant CD38 enzyme was incubated with NMN at different pHs, and levels of nicotinamide were measured by an enzymatic coupled reaction (n=3 biologically independent samples). ( i ) Relative levels of NAD + and NR in the serum of mice injected with a single injection of vehicle (Ctrl) or Ab68 (5 mg/kg). Mice were euthanized 3 days later and NAD + and NR levels in the serum were measured (n=6 mice per group). (j) Relative mRNA expression of inflammatory and SASP markers in WAT of 3 month-old (young) and 18 month-old mice (old) treated or not with a single injection of vehicle (Ctrl) or 5 mg/kg Ab68. WAT was collected 3 days after injection of Ab68 (n=5 except for Ctrl old where n=4). Data are mean ± SEM, analyzed by unpaired two-sided t -test, except for (f-g) where data are analyzed by two-way ANOVA.

Article Snippet: In experiments where 293T cells were incubated with human recombinant CD38 (hrCD38, R&D Systems) in the cell culture medium, the first step involved incubation of the hrCD38 (100 ng/mL) with 5 μg/mL isatuximab for 30 minutes at 37°C in cell culture media containing 1% FBS.

Techniques: Activity Assay, Injection, Gene Expression, Quantitative RT-PCR, Recombinant, Incubation, Expressing

Journal: Nature metabolism

Article Title: CD38 ecto-enzyme in immune cells is induced during aging regulating NAD + and NMN levels

doi: 10.1038/s42255-020-00298-z

Figure Lengend Snippet: In young mice, levels of CD38 + inflammatory cells and senescent cells in tissues are lower than older mice. During aging there is an increase in senescent cells that, at least in part, through their senescence associated secretory phenotype (SASP), promotes accumulation of CD38 + immune cells. The ecto-enzymatic activity of CD38 in immune cells degrades NMN extracellularly, preventing the NMN-induced NAD + boosting in other cells in the tissue.

Article Snippet: In experiments where 293T cells were incubated with human recombinant CD38 (hrCD38, R&D Systems) in the cell culture medium, the first step involved incubation of the hrCD38 (100 ng/mL) with 5 μg/mL isatuximab for 30 minutes at 37°C in cell culture media containing 1% FBS.

Techniques: Activity Assay

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: a , Cartoon and structural model of ISB 2001 BEAT. On the cartoon, immunoglobulin domains are shown as rectangles. VH domains of the anti-CD38, anti-BCMA and anti-CD3ε binders are depicted in blue, orange and magenta, respectively. All binders make use of a cLC depicted in gray. Fc-silencing mutations are depicted by the orange dots. The BEAT interface shown in the CH3 domains is depicted by the green and black dots. Chain A encompasses an engineered human IgG1 CH2 domain with an engineered human IgG3 CH3 domain. Chain B has engineered human IgG1 CH2 and CH3 domains. CHx, constant domain x; TCR Cα or TCR Cβ, BEAT interface proprietary mutations based on the T cell receptor constant domain α or β, respectively. ISB 2001 BEAT was generated by homology modeling. b , Human T cell activation of anti-CD3ε produced as human IgG1 LALA and control isotype by incubating with a dose–response of the cLC Fab bound to the plate. Graph shows mean ± s.d. ( n = 6 independent T cell donors from two independent experiments). c , SPR sensorgrams from a single replicate show blockade of BCMA/APRIL interaction (top sensorgram) and blockade of BCMA/BAFF interaction (bottom sensorgram) upon binding of anti-BCMA-E6 Fab to recombinant human BCMA protein. Curves are colored by anti-BCMA-E6 Fab concentration in BCMA/anti-BCMA premix solution. Data provided are from a single experiment (no repeats). d , Competition binding assay by Octet BLI. Curves represent injection of anti-CD38-B3 Fab/daratumumab Fab premix (dashed line) or daratumumab at twofold concentration of saturating solution (solid line) over CD38 immobilized surface saturated with daratumumab Fab from a single replicate in one experiment (no repeats). e , Binding sensorgrams of respective representative measurements from three independent replicates show the binding of ISB 2001 to human CD3εδ, human BCMA and human CD38 by SPR. Colored curves represent single concentration injections with serial dilutions of 1:3. Black curves represent 1:1 kinetic fits (BCMA and CD38). For binding to CD3εδ, the K d was inferred from a steady-state affinity model.

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: Generated, Activation Assay, Produced, Control, Binding Assay, Recombinant, Concentration Assay, Injection

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: (a) Curves representing the binding of ISB 2001 to CD38 neg T cells compared to teclistamab, EM801 and alnuctamab (n = 6 T cell donors for ISB 2001 and EM801 and n = 5 for teclistamab and alnuctamab performed in 2 independent experiments). Symbols represent the mean ± SD. (b-c) Quantification of the maximum binding (RFI) (b) and K D (nM ) (c) to T cells (n = 6 T cell donors for ISB 2001 and n = 5 T cell donors for teclistamab and alnuctamab). Average of maximal binding or Log 10 (K D ) was compared using repeated measure ANOVA and Dunnett’s multiple comparisons to ISB 2001. Statistical differences are shown in the graphs when significant (P < 0.05). (d) Size-Exclusion chromatography profiles of ISB 2001. ISB 2001 eluted mainly as a monodisperse sample (main peak=99.7%) with a retention time of 7.35 min. (e) Non-reducing capillary Gel Electrophoresis (cGE (NR)). ISB 2001 accounts for 88.1% of the total species detected. Traces of disulfide bond variants, with one (4%) or two (3.6%) reduced interchain disulfide bonds was observed. The other species detected at smaller molecular weights (ranging from 0.5 to 2% of the total of the species detected), likely arise from artefactual degradation of the molecule in the analysis sample buffer. (f) cGE profile in reducing conditions (R) showing three main peaks corresponding to the common light chain (cLC) (37.6%), BEAT chain A (BTA) (26.0%), and BEAT chain B (BTB) (35.0%) at respectively 15.2, 19.0 and 20.8 min, and accounting for 98.6% of the total species detected. (g) Thermal stability by Differential Scanning Calorimetry (DSC) shows a first transition at 70.0 °C, corresponding to the melting of ISB 2001 CH2 and CH3 domains, and a second transition at 83.1 °C corresponding to the cooperative unfolding of the different Fabs in ISB 2001. A280=Absorbance at 280 nm; AU=Absorbance Units; Tm=melting temperature; Cp=specific heat capacity .

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: Binding Assay, Size-exclusion Chromatography, Nucleic Acid Electrophoresis, Differential Scanning Calorimetry

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: a – c , Cytotoxicity of KMS-12-BM cells at different concentrations of CD3 ×DU × CD38 and CD3 × CD38 × DU ( a ), CD3 × BCMA × DU and CD3 × DU × BCMA ( b ) and ISB 2001 and CD3 × CD38 × BCMA ( c ). RDL assays were performed at a 5:1 effector to target ratio for 48 h with purified T cells. Graphs show four-parameter logistic curve fitting with symbols representing mean ± s.d. ( n = 6 independent T cell donors from two independent experiments). d – f , T cell activation in a HD-PBMC at different concentrations of CD3 × DU × CD38 and CD3 × CD38 × DU ( d ), CD3 × BCMA × DU and CD3 × DU × BCMA ( e ) and ISB 2001 and CD3 × CD38 × BCMA ( f ). Graphs show four-parameter logistic curve fitting with symbols representing mean ± s.d. ( n = 6 independent T cell donors from two independent experiments). g , Cytotoxicity of the KMS-12-BM cell line at different concentrations of ISB 2001, CD3 × BCMA × DU, CD3 × DU × CD38 and the combination of CD3 × BCMA × DU and CD3 × DU × CD38. Graphs show four-parameter logistic curve fitting with symbols representing mean ± s.d. ( n = 6 independent T cell donors from three independent experiments). h – j , EC 50 values for cytotoxicity on KMS-12-BM ( h ), NCI-H929 ( i ) and MOLP-8 ( j ). log 10 (EC 50 ) ( n = 6 independent T cell donors from three independent experiments; EC 50 values for CD3 × DU × CD38 were not quantifiable except for n = 2 on MOLP-8) were compared using repeated measure ANOVA followed by Tukey’s multiple comparison in h – j . k , Representative confocal image (from three independent experiments) of ISB 2001 (white) and the synapse between a T cell (green) and a KMS-12-BM cell (blue), acquired with a Zeiss LSM 800 inverted confocal microscope, magnification ×40. l , Quantification of T cell and KMS-12-BM tumor cell interaction over time using Incucyte live imaging for ISB 2001, CD3 × BCMA × DU and CD3 × DU × CD38. Quantification of T cell and tumor interaction using Incucyte. Graph shows mean of n = 6 (ISB 2001) or 5 (CD3 × BCMA × DU and CD3 × DU × CD38) technical replicates from two independent experiments. Statistical differences from post-hoc comparison are shown in the graphs as exact P value when statistically significant ( P < 0.05). NQ, not quantifiable. WT, wild-type.

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: Purification, Activation Assay, Comparison, Microscopy, Imaging

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: (a-c) Quantification of cytokines and cytolytic factors (granzyme B, IFN-γ, IL-2, IL-6, IL-10, perforin and TNF-α) in a high-density PBMC assay comparing the secretion induced by ( a ) CD3 × DU ×CD38 and CD3 × CD38 × DU, ( b ) CD3 × BCMA ×DU and CD3 × DU × BCMA and ( c ) ISB 2001 and CD3 × CD38 × BCMA. Graphs show the maximum secretion in pg/mL. LLOD for each cytokine are the following: granzyme B = 43.4 pg/ml, IFN-γ = 13.7 pg/ml, IL-2 = 39.5 pg/ml, IL-6 = 3.4 pg/ml, IL-10 = 3 pg/ml, perforin = 9.3 pg/ml and TNF-α = 2.9 pg/ml. Each dot corresponds to a different PBMC donor (n = 6 individual PBMC donors from 3 independent experiment). Samples were compared for each cytokines using multiple paired T-Test (two-sided, using false discovery with the two-stage step-up method) and statistical differences are shown in the graphs when statistically significant (P < 0.05). LLOD=Lower limit of detection ( d-e ) BCMA and CD38 human antibody binding capacity (BioCytex) on three MM cell lines (KMS-12-BM, NCI-H929 and MOLP-8) and on MM tumor cells from five patients. Each dot represents one patient sample, bars the mean and error bars the SD. The staining on the cell lines were done in parallel to each patient sample. Dotted line corresponds to the mean of all patients tested ( f ) Representative histograms (from 5 repeats) of sABC staining for BCMA (top, orange) and CD38 (bottom, blue) on KMS-12-BM, NCI-H929 and MOLP-8 MM cell lines compared to the isotype antibody (gray). Numbers show the mean of the experiments for specific antigen binding capacity with mouse anti-BCMA and anti-CD38 antibodies followed by anti-mouse FITC-labeled secondary antibody (QIFIKIT). See Supplementary Figure for gating strategy.

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: PBMC Assay, Binding Assay, Staining, Labeling

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: ( a-c ) Binding to MM cell lines of ISB 2001 (pink), CD3 × BCMA ×DU (orange) and CD3 ×DUxCD38 (blue). Graphs show 4-parameters logistic curve fitting using variable slope with symbols representing the mean ± SD of 3 experiments (top) and maximum binding and K D for the three molecules are shown (bottom), each dot represents one independent experiment. Log 10 (K D ) and Max binding of 3 experiments were compared using repeated measure ANOVA model and Tukey HSD comparison. Statistical differences are shown on the graph when statistically significant (P < 0.05) ( d ) Curves showing the binding of ISB 2001 to NCI-H929 cell line expressing CD38 and BCMA (WT, full line), not expressing CD38 (CD38 KO, dashed line) or not expressing BCMA (BCMA KO, dotted line). Graph shows 4-parameters logistic curve fitting using variable slope with symbols representing mean ± SD (n = 3 independent experiments). ( e ) EC 50 values for cytotoxicity of ISB 2001, CD3xDUxCD38 and CD3 × BCMAxDU on MM cell lines expressing different sABC levels of BCMA and CD38. Log 10 (EC 50 ) (n = 6 individual T cell donors from 2 experiments; before exclusion based on acceptance criteria) were compared using repeated measure ANOVA or mixed-effect model (for RPMI-8226 only) followed by Tukey’s multiple comparisons test. (f ) Representative images captured at 20X of Incucyte data (from 2 independent experiments) from one donor after 18 h of incubation with ISB 2001 (left), CD3 × BCMAxDU (middle) and CD3xDUxCD38 (right), at 2000 pM. Green show T cells, red KMS-12-BM and yellow the interaction between effector and target cells. (g-i) Quantification of T cell and KMS-12-BM tumor cell interaction over time using Incucyte live imaging for ISB 2001, CD3 × BCMAxDU and CD3xDUxCD38 at (g) 200 pM, (h) 20 pM and (i) 2 pM. Graphs show mean (n = 6 technical replicates performed on 2 independent experiments using 2 different T cell donors).

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: Binding Assay, Comparison, Expressing, Incubation, Imaging

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: a – d , Cytotoxicity of the KMS-12-BM at different concentrations of ISB 2001, teclistamab, alnuctamab and EM801 ( a ) and EC 50 values for cytotoxicity of KMS-12-BM ( b ), NCI-H929 ( c ) and MOLP-8 cell lines ( d ). Graph in a shows four-parameter logistic curve fitting with symbols representing mean ± s.d. of six independent donors. log 10 (EC 50 ) ( n = 6 independent T cell donors from three independent experiments) were compared using repeated measure ANOVA model and Dunnett’s comparison in b – d . e , f , CD8 + T cell activation ( e ) and proliferation ( f ) after treatment in an RDL assay against KMS-12-BM. log 10 (EC 50 ) ( n = 6 independent T cell donors from two independent experiments) were compared using repeated measure ANOVA model and Dunnett’s comparison. g , h , ISB 2001, teclistamab, alnuctamab and EM801 killing curves ( g ) and EC 50 values for cytotoxicity ( h ) of NCI-H929 WT, CD38 −/− and BCMA −/− cell lines. log 10 (EC 50 ) ( n = 4 independent T cell donors (before acceptance criteria exclusion) from two independent experiments) were compared using repeated measure ANOVA model and Dunnett’s comparison to ISB 2001. Graphs in g show four-parameter logistic curve fitting with symbols representing mean ± s.d. ( n = 4 independent T cell donors). i , EC 50 of cytotoxicity, IL-6, IFNγ, TNF and IL-10 release in an RDL assay against KMS-12-BM. Boxplots show 25th to 75th percentile and whiskers minimum and maximum values of n = 6 donors from two independent experiments. j , k , Cytotoxicity in presence (+) or absence (−) of soluble factors after treatment with ISB 2001, teclistamab, alnuctamab and EM801 ( j ) or ISB 2001 and CD3 × BCMA × BCMA molecule ( k ) in an RDL assay. log 10 (EC 50 ) ( n = 6 independent PBMC donors from two experiments) were compared using two-way ANOVA and Tukey’s multiple comparisons in k (only differences between ISB 2001 and TCEs with soluble factors are shown). RDL assays were performed at a 5:1 effector to target ratio with purified T cells or PBMCs with six donors for 48 or 72 h. Statistical differences from post-hoc comparison are shown in the graphs as exact P value when statistically significant ( P < 0.05). NQ, not quantifiable.

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: Comparison, Activation Assay, Purification

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: a , T cell activation and cytolytic molecules secretion in HD-PBMC assay treated with ISB 2001, teclistamab, alnuctamab and CD3 × CD38 control. Percentage of CD25 + (left), granzyme B (middle) and perforin (right) of CD8 + and CD4 + T cells are shown in a four-parameter logistic curve fitting with symbols representing mean ± s.d. ( n = 6 independent PBMC donors from two independent experiments for CD25 + ; n = 3 independent PBMC donors from two independent experiments for granzyme B and perforin). Dashed line represents the EC 90 of ISB 2001 killing potency in RDL assay on KMS-12-BM. b , PBMC counts in HD-PBMC assay treated with 1 nM of ISB 2001, teclistamab, alnuctamab and CD3 × CD38 control. Total counts of CD8 + , CD4 + and CD20 + (left) and total counts of CD14 + and CD56 + from live cells (right). Cell counts were compared using RM ANOVA (CD4 + and CD20 + ) or Friedman test (CD8 + and CD56 + ), when donor pairing was effective or Kruskal–Wallis (CD14 + ) test when it was not. Multiple comparisons were performed using Tukey (following RM ANOVA) or Dunn’s multiple comparisons (following Friedman or Kruskal–Wallis). Bars represent the mean and error bars the s.d. ( n = 6 independent PBMC donors from two independent experiments). c , d , Quantification of CD38-expressing cells (progenitors, B cell committed progenitors and mature T cells) in the bone marrow of HIS-NXG mice 3 days after treatment with 1.5 mg kg −1 ISB 2001, teclistamab, CD3 × CD38 control or vehicle control ( n = 5 bone marrow samples from independent mice from one experiment). Gating of cell populations used for the plots in d from one vehicle mouse ( c ). Red numbers located in the top left corner of the last four dot plots refer to the gated populations shown in the first dot plot. Plots summarizing the total count and percentage of CD38 + cells of the indicated populations in the bone marrow ( d ). Mice reconstituted with different CD34 + donor cells are represented with different symbols; bars represent the mean and error bars the s.d. ( n = 5 bone-marrow samples from independent mice from one experiment). Samples were compared using Friedman test or one-way ANOVA (only for CD4 + T cells) followed by a Dunn’s multiple comparisons. Differences are shown in the graphs as exact P value when statistically significant ( P < 0.05).

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: Activation Assay, PBMC Assay, Control, Expressing

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: a , Competition binding assay by Octet BLI shows the absence of competition between ISB 2001 and daratumumab, as suggested by additive binding signal upon sequential exposure of a daratumumab-saturated CD38 sensor surface to ISB 2001 (dotted line). Saturation of the CD38 sensor surface was verified by dipping the daratumumab-saturated CD38 sensor surface into a solution of daratumumab at twofold concentration of saturation solution (solid line). Representative plot shows binding to the sensor tip as a wavelength shift (response) versus time ( n = 2 independent measurements). b , Surface representation of CD38 illustrating the hypothetical epitope bin of ISB 2001 (dashed line), as determined from epitope binning assays including daratumumab and isatuximab. The epitopes of daratumumab (PDB 7DHA ) and isatuximab (PDB 4CMH ) are colored blue and orange, respectively. c , Cytotoxicity of the KMS-12-BM cell line at different concentrations of ISB 2001 and teclistamab in the presence or absence of 100 nM daratumumab in a MMoAK assay. Dotted lines show the no-treatment and daratumumab at 100 nM-only conditions. Four-parameter logistic curve ( c ) fitting from a representative donor ( n = 9 individual PBMC donors and n = 8 individual donors for ISB 2001 + daratumumab, from n = 3 independent experiments). d , e , Cytotoxicity of ISB 2001 and teclistamab at 10 pM ( d ) or 100 pM ( e ) daratumumab (at 100 nM) and a combination of ISB 2001 or teclistamab (at 10 and 100 pM, respectively) plus daratumumab (at 100 nM) in a MMoAK assay. Each dot represents one individual donor ( n = 9 or n = 8 for ISB 2001 + daratumumab) and bars show the mean ± s.d. from four independent experiments. Means were compared using a mixed-effects model followed by a Tukey’s multiple comparison and statistical differences are shown as exact P value when statistically significant ( P < 0.05).

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: Binding Assay, Concentration Assay, Comparison

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: a , b , Representative curves of cytotoxicity of KMS-12-BM cell line at different concentrations of ISB 2001 and teclistamab by T cells isolated from HD- or PS-PBMCs ( n = 5 and n = 2, respectively) or BMA ( n = 5 and n = 2 donors, respectively) ( a ) and cytotoxicity at 10 pM ( b ). Percentage of cytotoxicity of n = 3 (PS-BMMC donors) or n = 5 (HD- and PS-PBMC and HD-BMMC donors, before acceptance criteria application) from n = 5 independent experiments were compared using REML followed by Šidák’s multiple comparison for each population of T cells. c , Experimental setup schema to assess ISB 2001 and teclistamab cytotoxic capacity of CD138 + tumor cells in BMA and T cell activation. d , Representative dot plots of CD138 + cell killing (top) and CD69 + of CD8 + T cells (bottom). e , f , Cytotoxicity of CD138 + tumor cells ( e ) or T cell activation (CD69 + ) ( f ) on samples from patients with MM treated with ISB 2001 or teclistamab at 0.01 ( n = 10 PS for cytotoxicity and n = 9 PS for T cell activation), 0.1 ( n = 6 PS for cytotoxicity and T cell activation) and 1 nM ( n = 10 PS for cytotoxicity and n = 9 PS for T cell activation). CD138 + cell killing and T cell activation were compared using REML followed by Šidák’s multiple comparison for each concentration. g , Cytotoxicity of CD138 + tumor cells on samples from newly diagnosed (left, n = 4 PS) or patients with r/r MM (right, n = 6 PS), treated with ISB 2001 or teclistamab at 0.1 nM. Graph shows dots for individual samples. Previous treatments are stated in the graph as § for anti-CD38-treated or # for anti-BCMA-treated PS. Percentage of CD138 + cell killing was compared using a Holm–Šidák’s multiple two-sided paired t -test. h , Cytotoxicity curve of CD138 + MM cells by ISB 2001, teclistamab and isotype controls at 20 h in a sample from PCL ( n = 1 PS). Graph shows four-parameter logistic curve fitting and symbols represent the mean of replicates ( n = 2 replicates for ISB 2001 and teclistamab and n = 4 replicates for isotype controls). PCL, plasma cell leukemia. Statistical differences are shown in graphs as exact P value when statistically significant ( P < 0.05).

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: Isolation, Comparison, Activation Assay, Concentration Assay, Clinical Proteomics

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: Tumor cell killing of bone marrow aspirates (BMA) of ISB 2001 (pink), CD3 × BCMA ×DU (orange) and CD3 ×DU ×CD38 (blue). Graphs show three Multiple Myeloma patients performed in 3 independent experiments. Each dot represents one dose for each molecule.

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques:

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: a , Workflow for developing QSP model. b , Simplified binding schematic with accompanying equations below, where [ D ] is ISB 2001 concentration; [ T 1], [ T 2] and [ T 3] are the concentrations of free CD3, BCMA and CD38; [ D : T 1] is a concentration of dimer complex of ISB 2001-CD3; [ D : T 1: T 2] is a concentration of trimer complex of ISB 2001–CD3–BCMA; [ D : T 1: T 2: T 3] is a concentration of tetramer complex of ISB 2001–CD3–BCMA–CD38; k on T 1, k on T 2 and k on T 3 are the association rate constants for CD3, BCMA and CD38, respectively; and k off T 1, k off T 2 and k off T 3 are the dissociation rate constants for CD3, BCMA and CD38, respectively. c , d , Goodness-of-fit plots for ISB 2001 and teclistamab dose–response data in vitro and in vivo post-calibration of killing models. RSE for in vitro (CD8, CD4 and tumor) ISB 2001 (30%, 29% and 12%) and teclistamab (20%, 33% and 10%). RSE in vivo ISB 2001 4.7% teclistamab 9.9%. Symbols represent individual experimental data points (tumor, CD4 + T and CD8 + T cell counts for n = 1 representative donor out of 6 ( c ) and tumor volume for n = 8 mice per group ( d )). Lines show model simulations. e , nACT plots showing maximum nACT for a range of doses. f , Repeated dose nACT levels reach the EC 50 and EC 90 thresholds (ISB 2001) and EC 50 threshold for teclistamab. g , MPAD predictions for ISB 2001 and teclistamab. All RSE values are calculated at the EC 50 of the effect modeled . Graphs in e – g show model predictions of nACT levels in simulated patients.

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: Binding Assay, Concentration Assay, In Vitro, In Vivo

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: ( a ) Full Schematic of binding model (in vitro). Green circles show species with CD38 binding not found with teclistamab. ( b ) Goodness of fit plot for in vitro parameterization. RSE CD8-30%, CD4-29%, Tumor-12% Residual distribution plots for tumor, CD4 and CD8 T cells ( c ) Goodness of fit plot for in vivo parameterization. RSE KMS-116%, NCI-35% ( d ) TG32 SCID single-dose PK. (5 mg/kg).

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: Binding Assay, In Vitro, In Vivo

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: In vitro killing model with target engagement on left and cell dynamics on right. Species not produced by teclistamab due to lack of a CD38 binder are circled in green and trimers labeled Tri 1, Tri 2, Tri 3 and tetramer Tet 1. Active species (Tri 2, Tri 3, Tet 1) are shown with a light gray background.

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: In Vitro, Drug discovery, Produced, Labeling

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: (a) In vivo mouse PK/PD model with PK model (left) and tumor compartment cell dynamics upon trimer formation (right). (b) Human PBPK model with specific bone marrow compartment (illustrated in blue box). (c) Human QSP model with binding dynamics in compartments. Species in green circles are not produced by teclistamab due to lack of CD38 binder.

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: In Vivo, Binding Assay, Produced

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: Generation of CRISPR-engineered CD38 KO /CD38-CAR human primary NK cells using Cas9/RNP and AAV. (A) Schemata of steps for CRISPR/RNP knockout of the CD38 gene and directed insertion of a CD38-CAR encoding DNA delivered by AAV6 vector with homology arms for CD38 targeting site. (B) CD38 (PE) and CAR (APC) expression levels measured by flow cytometry for binding of CD38 antigen, 7 days after stimulation. Constructs contain a 41BB signaling domain, a CD8α transmembrane domain/hinge, a CD3ζ stimulatory domain, and reversed orderings of light and heavy chain orientations. (C) Relative percentage and intensity of CD38-CAR expression (n = 10; mean ± standard deviation [SD]). (D) Fold expansion of WT and CD38-CAR NK cells over 12 days after activation with irradiated, modified mbIL21-K562 cells and IL-2 show no significant change from WT human NK cells (n = 10; mean ± SD). P values were calculated using a 2-way analysis of variance (ANOVA); ∗ P = .0332; ∗∗ P = .0021; ∗∗∗ P = .0002; ∗∗∗∗ P < .0001. (E) Cytotoxicity observed for V3 and V4 CD38 KO /CD38-CAR NK cells against high CD38-expressing MM (H929), BL (Raji), and AML (MV-11) (n = 5; mean ± SD). P values were calculated using a 2-way ANOVA; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P = .001; ∗∗∗∗ P < .0001.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: CRISPR, Knock-Out, Plasmid Preparation, Expressing, Flow Cytometry, Binding Assay, Construct, Standard Deviation, Activation Assay, Irradiation, Modification

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: CD38-CAR NK cells exhibit enhanced cytotoxic function and cytokine secretion. CD38-CAR NK cells were tested against CD38-expressing AML, MM, and T-cell malignancies collected from patients at baseline. (A) NK and CD38-CAR NK killing of AML-1 (n = 3; mean ± SD). (B) NK and CD38-CAR NK killing of samples from patients with MM (MM-1 and MM-2; n = 1; mean ± SD). (C) NK and CD38-CAR NK killing of T-cell malignancies (hepatosplenic T-cell lymphoma and T-PLL [T-PLL-1 and T-PLL-2]; n = 1; mean ± SD). All cytotoxicity P values were calculated using a 2-way ANOVA; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P = .001; ∗∗∗∗ P < .0001. (D) Bio-Plex Pro Human Cytokine assay was performed on the supernatant of WT and CD38-CAR NK cells cocultured with CD38 + malignancies (n = 9; mean ± SD). P values were calculated using a paired Student t test; ∗ P = .05; ∗∗ P = .01; ∗∗∗ P = .001. GM-CSF, IFN-γ, MCP-1, MIP-1α, TNF-α.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: Expressing, Cytokine Assay

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: ATRA upregulates CD38 expression on tumor cells and can enhance antitumor activity. (A) CD38 cell surface expression as measured by flow cytometry across the hematologic malignancies MM, AML, BL, and T-ALL after treatment with 10 nM of ATRA for 48 hours. (B) Mean fluorescence intensity (MFI) of CD38 expression on cell lines with and without ATRA treatment. (C) Cytotoxicity assays performed by coculturing WT and CD38-CAR NK cells against AML, MM, BL, and T-cell malignancies with and without 48-hour, 10-nM ATRA pretreatments. MM1S (n = 4), H929 (n = 4), AML-10 (n = 4), MV4-11 (n = 4), Raji (n = 4), Daudi (n = 4), and primary cells from patients with T-ALL (n = 3; mean ± SD). P values were calculated using a 2-way ANOVA; ∗ P < .05; ∗∗ P <.01; ∗∗∗ P = .001; ∗∗∗∗ P < .0001.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: Expressing, Activity Assay, Flow Cytometry, Fluorescence

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: Mass cytometry analysis shows combination ATRA and CD38-CAR NK-cell treatment decreases live AML population. Mass cytometry analysis was performed 24 hours after the coculture of a primary AML cell line (AML-1) with WT or CD38-CAR NK cells with and without 48-hour, 10-nM ATRA pretreatment. Eight distinct conditions were analyzed: WT NK cells alone (A), CD38-CAR NK cells alone (B), AML cells alone (C), AML plus WT NK cells (D), AML plus CD38-CAR NK cells (E), AML cells with ATRA (F), AML plus ATRA plus WT NK cells (G), and AML plus ATRA plus CD38-CAR NK cells (H). (I) Heat map of surface marker appearing on the live AML cells after treatment analyzed by mass cytometry. Original values are ln(x)-transformed. Rows are centered; unit variance scaling is applied to rows. Both rows and columns are clustered using correlation distance and average linkage using ClustVis.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: Mass Cytometry, Marker, Transformation Assay

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: CD38 KO /CD38-CAR T cells generated by Cas9/RNP and AAV6 show antitumor activity. (A) CD38 (PE) and CAR (APC) expression levels measured by flow cytometry. (B) Relative percentage of CD38-CAR expression (n = 5; mean ± SD). (C) Ten-day fold expansion of generated CD38-CAR T cells with matched donors (n = 4; mean ± SD). P values were calculated using a 2-way ANOVA; ∗ P = .0332; ∗∗ P = .0021; ∗∗∗ P = .0002; ∗∗∗∗ P < .0001. (D) Cytotoxicity of CD38 KO /CD38-CAR T cells toward H929, Raji, and MV4-11 cell lines (n = 4; mean ± SD). P values were calculated using a 2-way ANOVA; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P = .001; ∗∗∗∗ P < .0001.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: Generated, Activity Assay, Expressing, Flow Cytometry

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: AAVS1KO/CD38-CAR NK cells avoid fratricide. (A) CD38 (PE) and CAR (APC) expression levels measured by flow cytometry for the CD38-CAR inserted into different loci. All CAR NK cells were generated with matched donors. (B) Fold expansion of WT and CD38-CAR NK cells over 12 days (n = 6; mean ± SD). P values were calculated using a 2-way ANOVA; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P = .001; ∗∗∗∗ P < .0001. (C) Relative percentage of CD38-CAR expression (n = 6; mean ± SD). (D) Reverse-transcription qPCR was performed using CD38 primer probes to detect transcription of the CD38 gene in the WT and AAVS1KO/CD38-CAR NK cells. (E) CD38 expression measured by flow cytometry using a polyclonal anti-CD38 antibody.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: Expressing, Flow Cytometry, Generated, Reverse Transcription

Journal: Blood Neoplasia

Article Title: CD38-CAR human NK cells in combination with ATRA enhance cytotoxicity against CD38-expressing hematologic malignancies ∗

doi: 10.1016/j.bneo.2024.100032

Figure Lengend Snippet: AAVS1KO/CD38-CAR NK cells display enhanced cytotoxicity and metabolism. CD38-CAR NK cells were tested against CD38-expressing AML, MM, and BL. (A) CD38-CAR NK-cell killing of CD38-expressing cell lines (n = 3; mean ± SD). P values were calculated using a 2-way ANOVA; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P = .001; ∗∗∗∗ P < .0001. (B) Oxygen consumption rate (OCR) for CD38-CAR NK cells. (C) Glycolytic capacity, measured by the extracellular acidification rate (ECAR), observed in CD38-CAR NK cells. (D) Spare respiratory capacity, a measure of the cell's ability to produce adenosine triphosphate (ATP) in response to stress, measured in CAR NK cells. (E) CAR NK-cell maximal respiration rates compared with WT.

Article Snippet: Cells were stained in phosphate-buffered saline containing 2% FBS using anti-CD38/PE (Miltenyi Biotec; clone REA572) and CD38 Fc chimera protein (R&D Systems; rhCD38), followed by secondary staining with Alexa Fluor 647 AffiniPure goat anti-human immunoglobulin G (Jackson ImmunoResearch) and Tonbo Ghost Dye 450 (Tonbo Biosciences, San Diego, CA).

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: CD38 mediates nicotinamide mononucleotide base exchange to yield nicotinic acid mononucleotide

doi: 10.1016/j.jbc.2025.108248

Figure Lengend Snippet: CD38 mediates NMN base exchange with nicotinic acid . A , experimental scheme to identify NMN base exchange. Unlabeled NMN (M + 0) and 13 C 6 -labeled–nicotinic acid (M + 6) are coincubated, and the formation of M + 6–labeled nicotinic acid mononucleotide (NaMN) is indicative of base exchange of the unlabeled nicotinamide group from NMN for an M + 6–labeled nicotinic acid. B , targeted mass spectrometry for these metabolites in the presence or absence of recombinant CD38 protein, with or without cotreatment with the small molecule CD38 inhibitor 78c , showing CD38-dependent formation of M + 6–labeled NaMN. This base exchange is preferential for nicotinic acid over nicotinamide, as ( C ) coincubation of unlabeled NMN with both 13 C 6 -nicotinic acid (M + 6) and D 4 -nicotinamide (M + 4) leads to the greater formation of M + 6 NaMN over M + 4 NMN. D , technical control to show the absence of labelled NaMN in the absence of 13 C 6 -nicotinic acid (M + 6). E , scheme to test whether CD38-mediated base exchange is selective to nicotinic acid. Double-labeled NMN containing D 2 -ribose and D 4 -nicotinamide, for an overall mass shift of M + 6 was coincubated with nicotinic acid or its structural orthologs picolinic acid or isonicotinic acid, which are identical in molecular weight. CD38-mediated base exchange would result in exchange of the D 4 -nicotinamide label for an unlabeled group, resulting in the formation of a product with a molecular weight identical to NaMN plus an M + 2 mass shift, due to retention of the ribose label. Base exchange was not observed for ( F ) picolinic acid, but was observed for ( G ) nicotinic acid and ( H ) isonicotinic acid. I , chromatograms showing the detection of M + 2 ribose-labeled NaMN in each group. Data points represent technical replicates within a single experiment, and experiments were performed three times. Error bars are SDs. NMN, nicotinamide mononucleotide.

Article Snippet: CD38 base-exchange activity was assessed in vitro using recombinant human CD38 (UniProt P28907, Val54–Ile300) corresponding to its extracellular domain, with a C-terminal 6xHis tag produced in NS0-derived mouse myeloma cell line from R&D Systems (catalog number RDS2404-AC), with activity of >2500 pmol/min/μg, as measured by its ability to convert nicotinamide guanine dinucleotide to cyclic GDP ribose.

Techniques: Labeling, Mass Spectrometry, Recombinant, Control, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: CD38 mediates nicotinamide mononucleotide base exchange to yield nicotinic acid mononucleotide

doi: 10.1016/j.jbc.2025.108248

Figure Lengend Snippet: CD38 base exchange is selective to NMN, and not NaMN, NR, or NaR . CD38-mediated base exchange was confirmed through three different labelling combinations. A , 5,5-D 2 -ribose labelled NMN (M + 2) was coincubated with 13 C 6 -nicotinic acid, leading to the formation of D 2 -ribose, 13 C 6 -nicotinic acid labeled NaMN (M + 8). B , 5,5-D 2 -ribose, D 4 -nicotinamide–labeled NMN (M + 6) was coincubated with unlabeled nicotinic acid, leading to the formation of D 2 -ribose–labeled NaMN (M + 2). C , unlabeled NMN was coincubated with 13 C 6 -nicotinic acid, leading to the formation of 13 C 6 -nicotinic acid–labeled NaMN (M + 6). D , to test whether CD38 could mediate base exchange on NaMN rather than NMN, unlabeled NaMN was coincubated with D 4 -labeled nicotinamide (M + 4), however M + 4–labeled NMN was not identified. E , to test whether there is direct base exchange between NMN and NaMN, D 4 -nicotinic acid–labeled NaMN (M + 4) was coincubated with D 2 -ribose–labeled NMN (M + 2), however the hypothetical base exchange products were not detected. F and G , to test whether nicotinamide riboside (NR) or nicotinic acid riboside (NaR) could act as a substrate for base exchange, ( F ) unlabeled NR was coincubated with 13 C 6 -nicotinic acid (M + 6), and ( G ) unlabeled NaR was coincubated with D 4 -nicotinamide (M + 4), with no base-exchange products observed in either case. Experiments were performed three separate times; data are shown from a single experiment that included all reaction combinations in parallel, using one technical observation per reaction condition. NaMN, nicotinic acid mononucleotide; NMN, nicotinamide mononucleotide.

Article Snippet: CD38 base-exchange activity was assessed in vitro using recombinant human CD38 (UniProt P28907, Val54–Ile300) corresponding to its extracellular domain, with a C-terminal 6xHis tag produced in NS0-derived mouse myeloma cell line from R&D Systems (catalog number RDS2404-AC), with activity of >2500 pmol/min/μg, as measured by its ability to convert nicotinamide guanine dinucleotide to cyclic GDP ribose.

Techniques: Labeling

Journal: The Journal of Biological Chemistry

Article Title: CD38 mediates nicotinamide mononucleotide base exchange to yield nicotinic acid mononucleotide

doi: 10.1016/j.jbc.2025.108248

Figure Lengend Snippet: CD38 inhibition blocks NMN-induced increases in NaMN and NaAD in vivo . Mice received a single oral dose of the amidated NAD + precursor NMN (500 mg/kg) in the presence or absence of the small molecule CD38 inhibitor 78c (“CD38i”) (10 mg/kg). Two hours later, animals were euthanized and liver, muscle (quadriceps), and kidneys were collected for metabolomics to measure levels of the Preiss–Handler/ de novo pathway deamidated intermediates ( A ) nicotinic acid mononucleotide (NaMN) and ( B ) nicotinic acid adenine dinucleotide (NaAD). Each point represents data from a separate animal, data analyzed by two-way ANOVA followed by Tukey’s test for post hoc comparisons, n = 5/group, error bars are SD. NMN, nicotinamide mononucleotide.

Article Snippet: CD38 base-exchange activity was assessed in vitro using recombinant human CD38 (UniProt P28907, Val54–Ile300) corresponding to its extracellular domain, with a C-terminal 6xHis tag produced in NS0-derived mouse myeloma cell line from R&D Systems (catalog number RDS2404-AC), with activity of >2500 pmol/min/μg, as measured by its ability to convert nicotinamide guanine dinucleotide to cyclic GDP ribose.

Techniques: Inhibition, In Vivo

Journal: The Journal of Biological Chemistry

Article Title: CD38 mediates nicotinamide mononucleotide base exchange to yield nicotinic acid mononucleotide

doi: 10.1016/j.jbc.2025.108248

Figure Lengend Snippet: Mammalian NAD + biosynthesis and proposed role for CD38 . A , NAD + can be synthesized via the salvage pathway, which recycles the amidated precursor nicotinamide via an amidated intermediate, nicotinamide mononucleotide (NMN). In the Preiss–Handler pathway, nicotinic acid is incorporated into NAD + via the deamidated intermediates nicotinic acid mononucleotide (NaMN) and nicotinic acid adenine dinucleotide (NaAD). Treatment with the amidated precursor NMN can increase levels of the deamidated intermediates NaMN and NaAD. B , the enzyme CD38 is well-studied for its NAD + glycohydrolase activity, cleaving NAD + into free nicotinamide and ADP ribose (ADPR). C , CD38 also has base-exchange activity toward NADP + , yielding the Ca 2+ signaling intermediate NaADP + . D , this investigation identified NMN and nicotinic acid as new base-exchange substrates for CD38, yielding NaMN and explaining the crossover between the amidated salvage/recycling pathways and the deamidated Preiss–Handler/ de novo pathways of NAD + biosynthesis.

Article Snippet: CD38 base-exchange activity was assessed in vitro using recombinant human CD38 (UniProt P28907, Val54–Ile300) corresponding to its extracellular domain, with a C-terminal 6xHis tag produced in NS0-derived mouse myeloma cell line from R&D Systems (catalog number RDS2404-AC), with activity of >2500 pmol/min/μg, as measured by its ability to convert nicotinamide guanine dinucleotide to cyclic GDP ribose.

Techniques: Synthesized, Activity Assay