Review



c3c  (Lee Biosolutions)


Bioz Verified Symbol Lee Biosolutions is a verified supplier
Bioz Manufacturer Symbol Lee Biosolutions manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 93
      Buy from Supplier

    Structured Review

    Lee Biosolutions c3c
    a Cp40-C3b structure solved at 2.0-Å resolution showing a single molecule of Cp40 (stick representation) binding to the β-chain of C3b (green, cartoon representation; the α-chain is shown in cyan). b Cp40 binding site at the MG core of C3b highlighted in red. c Superimposition of the current Cp40-C3b structure (green/cyan) with the previous <t>Cp01-C3c</t> structure (grey). The dotted box shows an enlarged view of the compstatin binding region at the interface of the MG4 and MG5 domains of <t>C3b/C3c.</t> The two compstatin analogs are shown as sticks. d – f Structure of Cp40 in its target-bound conformation. d Cp40 is bound as cyclic 14-amino-acid peptide (green stick representation) engaging in intermolecular contacts with the MG4 (cyan) and MG5 (purple) domains of C3b. e Backbone trace (main chain as cartoon, side chain as sticks) revealing a twisted, O-shaped bound conformation of the disulfide-bridged peptide f Superimposition of Cp01 (grey; from C3c-Cp01 complex) and Cp40 (green; from C3b-Cp40 complex) showing an overall structural similarity with deviations primarily found in side chains not engaging in tight intermolecular interactions. CTC C-terminal complement domain, CUB complement C1r/C1s, Uegf Bmp1 domain, MG macroglobulin domain, TED thioester-containing domain.
    C3c, supplied by Lee Biosolutions, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+complement+plasma/Complement+C3c+(C3c)%2C+Human+Plasma/pmc09488889-347-21-28
    Average 93 stars, based on 1 article reviews
    c3c - by Bioz Stars, 2026-10
    93/100 stars

    Images

    1) Product Images from "Insight into mode-of-action and structural determinants of the compstatin family of clinical complement inhibitors"

    Article Title: Insight into mode-of-action and structural determinants of the compstatin family of clinical complement inhibitors

    Journal: Nature Communications

    doi: 10.1038/s41467-022-33003-7

    a Cp40-C3b structure solved at 2.0-Å resolution showing a single molecule of Cp40 (stick representation) binding to the β-chain of C3b (green, cartoon representation; the α-chain is shown in cyan). b Cp40 binding site at the MG core of C3b highlighted in red. c Superimposition of the current Cp40-C3b structure (green/cyan) with the previous Cp01-C3c structure (grey). The dotted box shows an enlarged view of the compstatin binding region at the interface of the MG4 and MG5 domains of C3b/C3c. The two compstatin analogs are shown as sticks. d – f Structure of Cp40 in its target-bound conformation. d Cp40 is bound as cyclic 14-amino-acid peptide (green stick representation) engaging in intermolecular contacts with the MG4 (cyan) and MG5 (purple) domains of C3b. e Backbone trace (main chain as cartoon, side chain as sticks) revealing a twisted, O-shaped bound conformation of the disulfide-bridged peptide f Superimposition of Cp01 (grey; from C3c-Cp01 complex) and Cp40 (green; from C3b-Cp40 complex) showing an overall structural similarity with deviations primarily found in side chains not engaging in tight intermolecular interactions. CTC C-terminal complement domain, CUB complement C1r/C1s, Uegf Bmp1 domain, MG macroglobulin domain, TED thioester-containing domain.
    Figure Legend Snippet: a Cp40-C3b structure solved at 2.0-Å resolution showing a single molecule of Cp40 (stick representation) binding to the β-chain of C3b (green, cartoon representation; the α-chain is shown in cyan). b Cp40 binding site at the MG core of C3b highlighted in red. c Superimposition of the current Cp40-C3b structure (green/cyan) with the previous Cp01-C3c structure (grey). The dotted box shows an enlarged view of the compstatin binding region at the interface of the MG4 and MG5 domains of C3b/C3c. The two compstatin analogs are shown as sticks. d – f Structure of Cp40 in its target-bound conformation. d Cp40 is bound as cyclic 14-amino-acid peptide (green stick representation) engaging in intermolecular contacts with the MG4 (cyan) and MG5 (purple) domains of C3b. e Backbone trace (main chain as cartoon, side chain as sticks) revealing a twisted, O-shaped bound conformation of the disulfide-bridged peptide f Superimposition of Cp01 (grey; from C3c-Cp01 complex) and Cp40 (green; from C3b-Cp40 complex) showing an overall structural similarity with deviations primarily found in side chains not engaging in tight intermolecular interactions. CTC C-terminal complement domain, CUB complement C1r/C1s, Uegf Bmp1 domain, MG macroglobulin domain, TED thioester-containing domain.

    Techniques Used: Binding Assay

    a Contact residues on the target protein engaging in H-bonds (yellow), lipophilic contacts (blue) or both (purple) are highlighted on the surface of C3b. Contacts only present in Cp01 but not Cp40 are show in pastel shades. The structures of Cp01 and Cp40 are shown in grey and green ball-and-stick representation, respectively; residues not engaging in target binding are shown as lines. c Intramolecular contact networks in Cp01 (left) and Cp40 (right) showing H-bonds in yellow and hydrophobic contacts in grey. Analog-specific H-bonds and hydrophobic contacts are marked yellow and cyan arrows, respectively. b , d Differences in binding free energies per residue between Cp01-C3c and Cp40-C3b based on three independent MD simulation runs with distinct initiation parameters. (Supplementary Tables – ). Closeup view of the interaction profiles of e D-Tyr1, f (1Me)Trp5, g Sar9, h mIle14, i Asp7, and j Arg12. C3b is shown in stick representation with grey semitransparent surface and Cp40 in green ball-and-stick representation. H-bonds and hydrophobic contacts are marked as yellow and brown dotted lines, respectively.
    Figure Legend Snippet: a Contact residues on the target protein engaging in H-bonds (yellow), lipophilic contacts (blue) or both (purple) are highlighted on the surface of C3b. Contacts only present in Cp01 but not Cp40 are show in pastel shades. The structures of Cp01 and Cp40 are shown in grey and green ball-and-stick representation, respectively; residues not engaging in target binding are shown as lines. c Intramolecular contact networks in Cp01 (left) and Cp40 (right) showing H-bonds in yellow and hydrophobic contacts in grey. Analog-specific H-bonds and hydrophobic contacts are marked yellow and cyan arrows, respectively. b , d Differences in binding free energies per residue between Cp01-C3c and Cp40-C3b based on three independent MD simulation runs with distinct initiation parameters. (Supplementary Tables – ). Closeup view of the interaction profiles of e D-Tyr1, f (1Me)Trp5, g Sar9, h mIle14, i Asp7, and j Arg12. C3b is shown in stick representation with grey semitransparent surface and Cp40 in green ball-and-stick representation. H-bonds and hydrophobic contacts are marked as yellow and brown dotted lines, respectively.

    Techniques Used: Binding Assay, Residue

    a Compstatin-based drugs may interact with circulating (i.e., C3, C3c) or surface-tethered targets (i.e., C3b, C3bBb, iC3b). Depending on their valency, avidity may occur. b ITC analysis of solution interactions between C3c and compstatin analogs Cp01, Cp05*, Cp05-PEG-Cp05, and Cp40 shows 1:1 binding for monovalent analogs ( N = 1) whereas the data of the bivalent Cp05-PEG-Cp05 fits a 2:1 ratio ( N = 0.5). The displayed curve is a representative of two independent experiments per compound, obtained by peak-to-peak integration of the processed thermogram, as a function of At/Mt (titrant-to-titrate molar ratio). The integrated signal (black) is fitted to a 1:1 binding model (red curve) for determination of affinity (K D ) and molecular ratio ( N ). Error bars represent the uncertainty associated to the integral calculation of each peak in the binding isotherm (as determined by AFFINImeter). Cp05* contains the linker necessary for the conjugation to PEG-40kDa; its binding profile is comparable to that of Cp05 (Supplementary Fig. ). c SPR analysis between immobilized C3b and mono- and bivalent Cp05 analogs reveal a strong enhancement of surface binding for Cp05-PEG-Cp05 over monovalent Cp05. d Isoaffinity kinetic plot for surface interactions of compstatin analogs with immobilized C3b, demonstrating that the development steps from Cp01 to Cp40 were enabled by improving dissociation rates with little impact on association rates. In contrast, the improved stability of the C3b complex with Cp05-PEG-Cp05 is accompanied by a marked drop in the association rate. PEG polyethylene glycol, RU resonance units.
    Figure Legend Snippet: a Compstatin-based drugs may interact with circulating (i.e., C3, C3c) or surface-tethered targets (i.e., C3b, C3bBb, iC3b). Depending on their valency, avidity may occur. b ITC analysis of solution interactions between C3c and compstatin analogs Cp01, Cp05*, Cp05-PEG-Cp05, and Cp40 shows 1:1 binding for monovalent analogs ( N = 1) whereas the data of the bivalent Cp05-PEG-Cp05 fits a 2:1 ratio ( N = 0.5). The displayed curve is a representative of two independent experiments per compound, obtained by peak-to-peak integration of the processed thermogram, as a function of At/Mt (titrant-to-titrate molar ratio). The integrated signal (black) is fitted to a 1:1 binding model (red curve) for determination of affinity (K D ) and molecular ratio ( N ). Error bars represent the uncertainty associated to the integral calculation of each peak in the binding isotherm (as determined by AFFINImeter). Cp05* contains the linker necessary for the conjugation to PEG-40kDa; its binding profile is comparable to that of Cp05 (Supplementary Fig. ). c SPR analysis between immobilized C3b and mono- and bivalent Cp05 analogs reveal a strong enhancement of surface binding for Cp05-PEG-Cp05 over monovalent Cp05. d Isoaffinity kinetic plot for surface interactions of compstatin analogs with immobilized C3b, demonstrating that the development steps from Cp01 to Cp40 were enabled by improving dissociation rates with little impact on association rates. In contrast, the improved stability of the C3b complex with Cp05-PEG-Cp05 is accompanied by a marked drop in the association rate. PEG polyethylene glycol, RU resonance units.

    Techniques Used: Binding Assay, Conjugation Assay

    Related Articles

    Binding Assay:

    Article Title: Insight into mode-of-action and structural determinants of the compstatin family of clinical complement inhibitors
    Article Snippet: .. Human C3, C3b, FH, FB, and FD used in binding and functional studies were purchased from Complement Technology (Tyler, TX, USA); C3c for ITC experiments was obtained from Lee Biosolutions (Maryland Heights, MO, USA). ..

    Functional Assay:

    Article Title: Insight into mode-of-action and structural determinants of the compstatin family of clinical complement inhibitors
    Article Snippet: .. Human C3, C3b, FH, FB, and FD used in binding and functional studies were purchased from Complement Technology (Tyler, TX, USA); C3c for ITC experiments was obtained from Lee Biosolutions (Maryland Heights, MO, USA). ..



    Similar Products

    human complement sera  (Innovative Research Inc)
    93
    Innovative Research Inc human complement sera
    Human Complement Sera, supplied by Innovative Research Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+complement+plasma/Pooled+Human+Complement+Plasma/pm40314805-41-1-14
    Average 93 stars, based on 1 article reviews
    human complement sera - by Bioz Stars, 2026-10
    93/100 stars
    		  Buy from Supplier
    iplasnac2ml  (Assaypro)
    91
    Assaypro iplasnac2ml
    Iplasnac2ml, supplied by Assaypro, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+complement+plasma/Human+Complement+Factor+H+Deficient+Plasma/pm37191558-245-60-64
    Average 91 stars, based on 1 article reviews
    iplasnac2ml - by Bioz Stars, 2026-10
    91/100 stars
    		  Buy from Supplier
    human male ab plasma as a source of human complement  (Millipore)
    90
    Millipore human male ab plasma as a source of human complement
    Anti-KLRG1 monoclonal antibodies exert no apoptosis but induce ADCP, ADCC, and <t>complement-mediated</t> cytotoxicity (CDC). A, SMZ1-WT and SMZ1-KLRG1 cells were incubated with the indicated antibodies (mAb208, 13F12F2, or its isotype control mIgG2aκ) at increasing concentrations from 1 to 100 μg/mL or 0.2 or 1 μmol/L staurosporine (positive control) for 24 hours, and the percentage of apoptotic cells was quantified by flow cytometry. Three independent experiments were conducted. Shown are the mean values of technical triplicates with error bars representing SEM from one representative experiment. B–C, CFSE-labeled SMZ1-WT and SMZ1-KLRG1 cells were incubated with human monocyte-derived macrophages (hMDM) from three healthy donors and bone-marrow–derived murine macrophages (mBMDM) from different lots of mice in the presence of indicated antibodies for 2 hours. Anti-CD47 mAb, MIAP410-mediated phagocytosis of Jurkat cells relative to its isotype control, mIgG1κ, was used as a positive control. The percentage of CFSE + macrophages (of human CD14 + or murine CD11b + cells) was determined by flow cytometry and plotted. D–E, CFSE-labeled SMZ1-WT and SMZ1-KLRG1 cells were incubated with human-derived PBMCs from three healthy donors in the presence of indicated antibodies at increasing concentrations for 4 hours. The percentage of propidium iodide–positive (% PI pos; of CFSE + ) cells was determined by flow cytometry and plotted. Rituximab and Raji cells were used as a positive control. F–G, ADCC reporter assay measuring luminescence with murine FcγRIIIA-expressing Jurkat cells was performed in triplicate in three independent experiments at an effector:target ratio of 25:1 with indicated antibodies. Fold induction is relative to isotype control (mIgG2aκ for mAb208 and 13F12F2). Human anti-CD20 mAb and Raji cells were used as positive control along with its isotype control, hIgG1. Representative results from one experiment are shown. H–K, CDC assay with 30% murine and human complement was performed in triplicate in three independent experiments, and % PI pos cells were reported. Rituximab and Raji cells were used as positive controls. Shown are the mean values of technical triplicates, with error bars representing SEM from one representative experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-sided Welch t test.
    Human Male Ab Plasma As A Source Of Human Complement, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+complement+plasma/human+plasma+complement+factor+h++cfh/pmc11145167-34-8-13
    Average 90 stars, based on 1 article reviews
    human male ab plasma as a source of human complement - by Bioz Stars, 2026-10
    90/100 stars
    		  Buy from Supplier
    human plasma complement factor h (cfh  (Millipore)
    90
    Millipore human plasma complement factor h (cfh
    Anti-KLRG1 monoclonal antibodies exert no apoptosis but induce ADCP, ADCC, and <t>complement-mediated</t> cytotoxicity (CDC). A, SMZ1-WT and SMZ1-KLRG1 cells were incubated with the indicated antibodies (mAb208, 13F12F2, or its isotype control mIgG2aκ) at increasing concentrations from 1 to 100 μg/mL or 0.2 or 1 μmol/L staurosporine (positive control) for 24 hours, and the percentage of apoptotic cells was quantified by flow cytometry. Three independent experiments were conducted. Shown are the mean values of technical triplicates with error bars representing SEM from one representative experiment. B–C, CFSE-labeled SMZ1-WT and SMZ1-KLRG1 cells were incubated with human monocyte-derived macrophages (hMDM) from three healthy donors and bone-marrow–derived murine macrophages (mBMDM) from different lots of mice in the presence of indicated antibodies for 2 hours. Anti-CD47 mAb, MIAP410-mediated phagocytosis of Jurkat cells relative to its isotype control, mIgG1κ, was used as a positive control. The percentage of CFSE + macrophages (of human CD14 + or murine CD11b + cells) was determined by flow cytometry and plotted. D–E, CFSE-labeled SMZ1-WT and SMZ1-KLRG1 cells were incubated with human-derived PBMCs from three healthy donors in the presence of indicated antibodies at increasing concentrations for 4 hours. The percentage of propidium iodide–positive (% PI pos; of CFSE + ) cells was determined by flow cytometry and plotted. Rituximab and Raji cells were used as a positive control. F–G, ADCC reporter assay measuring luminescence with murine FcγRIIIA-expressing Jurkat cells was performed in triplicate in three independent experiments at an effector:target ratio of 25:1 with indicated antibodies. Fold induction is relative to isotype control (mIgG2aκ for mAb208 and 13F12F2). Human anti-CD20 mAb and Raji cells were used as positive control along with its isotype control, hIgG1. Representative results from one experiment are shown. H–K, CDC assay with 30% murine and human complement was performed in triplicate in three independent experiments, and % PI pos cells were reported. Rituximab and Raji cells were used as positive controls. Shown are the mean values of technical triplicates, with error bars representing SEM from one representative experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-sided Welch t test.
    Human Plasma Complement Factor H (Cfh, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+complement+plasma/human+plasma+complement+factor+h++cfh/pmc10451625-76-18-25
    Average 90 stars, based on 1 article reviews
    human plasma complement factor h (cfh - by Bioz Stars, 2026-10
    90/100 stars
    		  Buy from Supplier
    plasma  (Assaypro)
    91
    Assaypro plasma
    Anti-KLRG1 monoclonal antibodies exert no apoptosis but induce ADCP, ADCC, and <t>complement-mediated</t> cytotoxicity (CDC). A, SMZ1-WT and SMZ1-KLRG1 cells were incubated with the indicated antibodies (mAb208, 13F12F2, or its isotype control mIgG2aκ) at increasing concentrations from 1 to 100 μg/mL or 0.2 or 1 μmol/L staurosporine (positive control) for 24 hours, and the percentage of apoptotic cells was quantified by flow cytometry. Three independent experiments were conducted. Shown are the mean values of technical triplicates with error bars representing SEM from one representative experiment. B–C, CFSE-labeled SMZ1-WT and SMZ1-KLRG1 cells were incubated with human monocyte-derived macrophages (hMDM) from three healthy donors and bone-marrow–derived murine macrophages (mBMDM) from different lots of mice in the presence of indicated antibodies for 2 hours. Anti-CD47 mAb, MIAP410-mediated phagocytosis of Jurkat cells relative to its isotype control, mIgG1κ, was used as a positive control. The percentage of CFSE + macrophages (of human CD14 + or murine CD11b + cells) was determined by flow cytometry and plotted. D–E, CFSE-labeled SMZ1-WT and SMZ1-KLRG1 cells were incubated with human-derived PBMCs from three healthy donors in the presence of indicated antibodies at increasing concentrations for 4 hours. The percentage of propidium iodide–positive (% PI pos; of CFSE + ) cells was determined by flow cytometry and plotted. Rituximab and Raji cells were used as a positive control. F–G, ADCC reporter assay measuring luminescence with murine FcγRIIIA-expressing Jurkat cells was performed in triplicate in three independent experiments at an effector:target ratio of 25:1 with indicated antibodies. Fold induction is relative to isotype control (mIgG2aκ for mAb208 and 13F12F2). Human anti-CD20 mAb and Raji cells were used as positive control along with its isotype control, hIgG1. Representative results from one experiment are shown. H–K, CDC assay with 30% murine and human complement was performed in triplicate in three independent experiments, and % PI pos cells were reported. Rituximab and Raji cells were used as positive controls. Shown are the mean values of technical triplicates, with error bars representing SEM from one representative experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-sided Welch t test.
    Plasma, supplied by Assaypro, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+complement+plasma/Human+Complement+Factor+H+Deficient+Plasma/pm37191558-245-63-64
    Average 91 stars, based on 1 article reviews
    plasma - by Bioz Stars, 2026-10
    91/100 stars
    		  Buy from Supplier
    fluid phase plasma complement bb  (Quidel)
    96
    Quidel fluid phase plasma complement bb
    Anti-KLRG1 monoclonal antibodies exert no apoptosis but induce ADCP, ADCC, and <t>complement-mediated</t> cytotoxicity (CDC). A, SMZ1-WT and SMZ1-KLRG1 cells were incubated with the indicated antibodies (mAb208, 13F12F2, or its isotype control mIgG2aκ) at increasing concentrations from 1 to 100 μg/mL or 0.2 or 1 μmol/L staurosporine (positive control) for 24 hours, and the percentage of apoptotic cells was quantified by flow cytometry. Three independent experiments were conducted. Shown are the mean values of technical triplicates with error bars representing SEM from one representative experiment. B–C, CFSE-labeled SMZ1-WT and SMZ1-KLRG1 cells were incubated with human monocyte-derived macrophages (hMDM) from three healthy donors and bone-marrow–derived murine macrophages (mBMDM) from different lots of mice in the presence of indicated antibodies for 2 hours. Anti-CD47 mAb, MIAP410-mediated phagocytosis of Jurkat cells relative to its isotype control, mIgG1κ, was used as a positive control. The percentage of CFSE + macrophages (of human CD14 + or murine CD11b + cells) was determined by flow cytometry and plotted. D–E, CFSE-labeled SMZ1-WT and SMZ1-KLRG1 cells were incubated with human-derived PBMCs from three healthy donors in the presence of indicated antibodies at increasing concentrations for 4 hours. The percentage of propidium iodide–positive (% PI pos; of CFSE + ) cells was determined by flow cytometry and plotted. Rituximab and Raji cells were used as a positive control. F–G, ADCC reporter assay measuring luminescence with murine FcγRIIIA-expressing Jurkat cells was performed in triplicate in three independent experiments at an effector:target ratio of 25:1 with indicated antibodies. Fold induction is relative to isotype control (mIgG2aκ for mAb208 and 13F12F2). Human anti-CD20 mAb and Raji cells were used as positive control along with its isotype control, hIgG1. Representative results from one experiment are shown. H–K, CDC assay with 30% murine and human complement was performed in triplicate in three independent experiments, and % PI pos cells were reported. Rituximab and Raji cells were used as positive controls. Shown are the mean values of technical triplicates, with error bars representing SEM from one representative experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-sided Welch t test.
    Fluid Phase Plasma Complement Bb, supplied by Quidel, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+complement+plasma/pm37093125-119-0-18
    Average 96 stars, based on 1 article reviews
    fluid phase plasma complement bb - by Bioz Stars, 2026-10
    96/100 stars
    		  Buy from Supplier

    Image Search Results


    Anti-KLRG1 monoclonal antibodies exert no apoptosis but induce ADCP, ADCC, and complement-mediated cytotoxicity (CDC). A, SMZ1-WT and SMZ1-KLRG1 cells were incubated with the indicated antibodies (mAb208, 13F12F2, or its isotype control mIgG2aκ) at increasing concentrations from 1 to 100 μg/mL or 0.2 or 1 μmol/L staurosporine (positive control) for 24 hours, and the percentage of apoptotic cells was quantified by flow cytometry. Three independent experiments were conducted. Shown are the mean values of technical triplicates with error bars representing SEM from one representative experiment. B–C, CFSE-labeled SMZ1-WT and SMZ1-KLRG1 cells were incubated with human monocyte-derived macrophages (hMDM) from three healthy donors and bone-marrow–derived murine macrophages (mBMDM) from different lots of mice in the presence of indicated antibodies for 2 hours. Anti-CD47 mAb, MIAP410-mediated phagocytosis of Jurkat cells relative to its isotype control, mIgG1κ, was used as a positive control. The percentage of CFSE + macrophages (of human CD14 + or murine CD11b + cells) was determined by flow cytometry and plotted. D–E, CFSE-labeled SMZ1-WT and SMZ1-KLRG1 cells were incubated with human-derived PBMCs from three healthy donors in the presence of indicated antibodies at increasing concentrations for 4 hours. The percentage of propidium iodide–positive (% PI pos; of CFSE + ) cells was determined by flow cytometry and plotted. Rituximab and Raji cells were used as a positive control. F–G, ADCC reporter assay measuring luminescence with murine FcγRIIIA-expressing Jurkat cells was performed in triplicate in three independent experiments at an effector:target ratio of 25:1 with indicated antibodies. Fold induction is relative to isotype control (mIgG2aκ for mAb208 and 13F12F2). Human anti-CD20 mAb and Raji cells were used as positive control along with its isotype control, hIgG1. Representative results from one experiment are shown. H–K, CDC assay with 30% murine and human complement was performed in triplicate in three independent experiments, and % PI pos cells were reported. Rituximab and Raji cells were used as positive controls. Shown are the mean values of technical triplicates, with error bars representing SEM from one representative experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-sided Welch t test.

    Journal: Clinical Cancer Research

    Article Title: KLRG1 Cell Depletion as a Novel Therapeutic Strategy in Patients with Mature T-Cell Lymphoma Subtypes

    doi: 10.1158/1078-0432.CCR-23-3504

    Figure Lengend Snippet: Anti-KLRG1 monoclonal antibodies exert no apoptosis but induce ADCP, ADCC, and complement-mediated cytotoxicity (CDC). A, SMZ1-WT and SMZ1-KLRG1 cells were incubated with the indicated antibodies (mAb208, 13F12F2, or its isotype control mIgG2aκ) at increasing concentrations from 1 to 100 μg/mL or 0.2 or 1 μmol/L staurosporine (positive control) for 24 hours, and the percentage of apoptotic cells was quantified by flow cytometry. Three independent experiments were conducted. Shown are the mean values of technical triplicates with error bars representing SEM from one representative experiment. B–C, CFSE-labeled SMZ1-WT and SMZ1-KLRG1 cells were incubated with human monocyte-derived macrophages (hMDM) from three healthy donors and bone-marrow–derived murine macrophages (mBMDM) from different lots of mice in the presence of indicated antibodies for 2 hours. Anti-CD47 mAb, MIAP410-mediated phagocytosis of Jurkat cells relative to its isotype control, mIgG1κ, was used as a positive control. The percentage of CFSE + macrophages (of human CD14 + or murine CD11b + cells) was determined by flow cytometry and plotted. D–E, CFSE-labeled SMZ1-WT and SMZ1-KLRG1 cells were incubated with human-derived PBMCs from three healthy donors in the presence of indicated antibodies at increasing concentrations for 4 hours. The percentage of propidium iodide–positive (% PI pos; of CFSE + ) cells was determined by flow cytometry and plotted. Rituximab and Raji cells were used as a positive control. F–G, ADCC reporter assay measuring luminescence with murine FcγRIIIA-expressing Jurkat cells was performed in triplicate in three independent experiments at an effector:target ratio of 25:1 with indicated antibodies. Fold induction is relative to isotype control (mIgG2aκ for mAb208 and 13F12F2). Human anti-CD20 mAb and Raji cells were used as positive control along with its isotype control, hIgG1. Representative results from one experiment are shown. H–K, CDC assay with 30% murine and human complement was performed in triplicate in three independent experiments, and % PI pos cells were reported. Rituximab and Raji cells were used as positive controls. Shown are the mean values of technical triplicates, with error bars representing SEM from one representative experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-sided Welch t test.

    Article Snippet: Human male AB plasma as a source of human complement was obtained from Sigma-Aldrich.

    Techniques: Flow Cytometry Complement-Mediated Cytotoxicity Assay, Incubation, Positive Control, Flow Cytometry, Labeling, Derivative Assay, Reporter Assay, Expressing, CDC Assay