glycoengineered anti cd20 mabs  (Sino Biological)

Journal: mAbs

Article Title: Effects of terminal galactose residues in mannose α1-6 arm of Fc-glycan on the effector functions of therapeutic monoclonal antibodies

doi: 10.1080/19420862.2019.1608143

Figure Lengend Snippet: FcγR activation properties of glycoengineered anti-CD20 mAbs. FcγRIIa (a) and FcγRIIIa (b) activation properties of glycoengineered mAbs. Jurkat/FcγRIIa/NFAT-Luc or Jurkat/FcγRIIIa/NFAT-Luc reporter cells were incubated with serially diluted anti-CD20 mAbs in the presence of Raji cells. FcγR activation was evaluated by assessing the luminescence intensity.

Article Snippet: The binding of glycoengineered anti-CD20 mAbs to recombinant human FcγRs with C-terminal polyhistidine tag (Cat. No. 10256-H08H, 10374-H08H1, 10259-H08H, 10389-H08H1, and 11046-H08H, Sino Biological) was assessed as described previously.

Techniques: Activation Assay, Incubation

Journal: mAbs

Article Title: Effects of terminal galactose residues in mannose α1-6 arm of Fc-glycan on the effector functions of therapeutic monoclonal antibodies

doi: 10.1080/19420862.2019.1608143

Figure Lengend Snippet: CDC and C1q-binding activities of glycoengineered anti-CD20 mAbs. (a, b) CDC activities of glycoengineered anti-CD20 mAbs. Raji cells were incubated with 16% human serum and were serially diluted with glycoengineered anti-CD20 mAbs. (a) Percentages of cell lysis plotted against mAb concentrations. (b) CDC activity of 1 µg/ml of G1aF mAb and G1bF mAb mixtures in different ratios. (c) C1q binding of glycoengineered anti-CD20 mAbs. Raji cells were opsonized with anti-CD20 mAbs and incubated with human serum. The cells were stained with FITC-conjugated anti-C1q antibody and the C1q-binding level was analyzed by flow cytometer. Data are presented as mean ± SD ( n = 3).

Article Snippet: The binding of glycoengineered anti-CD20 mAbs to recombinant human FcγRs with C-terminal polyhistidine tag (Cat. No. 10256-H08H, 10374-H08H1, 10259-H08H, 10389-H08H1, and 11046-H08H, Sino Biological) was assessed as described previously.

Techniques: Binding Assay, Incubation, Lysis, Activity Assay, Staining, Flow Cytometry

Journal: mAbs

Article Title: Effects of terminal galactose residues in mannose α1-6 arm of Fc-glycan on the effector functions of therapeutic monoclonal antibodies

doi: 10.1080/19420862.2019.1608143

Figure Lengend Snippet: Comparison of structural stabilities of the CH2 domain of glycoengineered anti-CD20 mAbs using HDX/MS . Deuterium uptake plots of peptides Phe245–Leu255 (FLFPPKPKDTL) (a), Leu246–Leu255 (LFPPKPKDTL) (b), and Leu246–Met256 (LFPPKPKDTLM) (c) in the H-chain. (d) Physical representations of the crystal structures (PDB 1HZH) of peptides at Phe245–Met256 (magenta ribbons).

Article Snippet: The binding of glycoengineered anti-CD20 mAbs to recombinant human FcγRs with C-terminal polyhistidine tag (Cat. No. 10256-H08H, 10374-H08H1, 10259-H08H, 10389-H08H1, and 11046-H08H, Sino Biological) was assessed as described previously.

Techniques:

Journal: mAbs

Article Title: Effects of terminal galactose residues in mannose α1-6 arm of Fc-glycan on the effector functions of therapeutic monoclonal antibodies

doi: 10.1080/19420862.2019.1608143

Figure Lengend Snippet: Glycan profiles of mAbs with homogeneous glycans. Glycan profiles of commercially available anti-CD20 mAb (a) and glycoengineered G0F mAb (b), G1aF mAb (c), G1bF mAb (d), and G2F mAb (e). The glycan profiles were obtained using HPLC analysis of 2-AB-labeled glycans.

Article Snippet: The binding of glycoengineered anti-CD20 mAbs to recombinant human FcγRs with C-terminal polyhistidine tag (Cat. No. 10256-H08H, 10374-H08H1, 10259-H08H, 10389-H08H1, and 11046-H08H, Sino Biological) was assessed as described previously.

Techniques: High Performance Liquid Chromatography, Labeling

Journal: mAbs

Article Title: Effects of terminal galactose residues in mannose α1-6 arm of Fc-glycan on the effector functions of therapeutic monoclonal antibodies

doi: 10.1080/19420862.2019.1608143

Figure Lengend Snippet: Binding of glycoengineered anti-CD20 mAbs to human FcγRs. SPR analysis was used to measure the binding of anti-CD20 mAbs to human FcγRI, FcγRIIa, and FcγRIIIa. Binding sensorgrams corrected by both the surface blank and buffer injection control are represented.

Article Snippet: The binding of glycoengineered anti-CD20 mAbs to recombinant human FcγRs with C-terminal polyhistidine tag (Cat. No. 10256-H08H, 10374-H08H1, 10259-H08H, 10389-H08H1, and 11046-H08H, Sino Biological) was assessed as described previously.

Techniques: Binding Assay, SPR Assay, Injection

Journal: mAbs

Article Title: Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

doi: 10.1080/19420862.2015.1078054

Figure Lengend Snippet: Expression of anti-CD20 mAb in transgenic silkworms. ( A ) The protein lysates extracted from MSGs of H line or L line transgenic silkworms were separated by SDS-PAGE followed by staining with CBB or by western blotting with an anti-Human IgG(H + L) antibody. The protein lysates extracted form MSGs of transgenic silkworms that harbored only the Ser1-GAL4 construct were used as negative controls. The numbers above the gels or western blots indicate the line number of each transgenic strain. ( B ) The protein lysates extracted form MSGs or cocoons of the H + L line transgenic silkworms were separated by SDS-PAGE under reducing or non-reducing conditions. Gels were stained with CBB or subjected to protein gel blotting using an anti-Human IgG(H + L) antibody. The lysates obtained from the transgenic silkworms harboring only the Ser1-GAL4 construct were used as a negative control.

Article Snippet: Serially diluted anti-CD20 mAbs were injected into the flow cells and association and dissociation were monitored.

Techniques: Expressing, Transgenic Assay, SDS Page, Staining, Western Blot, Construct, Negative Control

Journal: mAbs

Article Title: Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

doi: 10.1080/19420862.2015.1078054

Figure Lengend Snippet: FcγRIIIa activation and antibody-dependent cell-mediated cytotoxicity (ADCC). ( A ) CD20 binding-dependent activation of FcγRIIIa was measured by using Daudi (target) and Jurkat/FcγRIIIa/NFAT-Luc (effector) cells. Fold increases in luciferase activity were plotted against the mAbs concentration (n = 3, bars indicate SEM). ( B ) The levels of ADCC against Daudi cells were measured by using human PBMCs as effector cells. The percentages of cell lysis were plotted against the mAbs concentration (n = 3, bars indicate SEM).

Article Snippet: Serially diluted anti-CD20 mAbs were injected into the flow cells and association and dissociation were monitored.

Techniques: Activation Assay, Binding Assay, Luciferase, Activity Assay, Concentration Assay, Lysis

Journal: mAbs

Article Title: Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

doi: 10.1080/19420862.2015.1078054

Figure Lengend Snippet: CD20-binding properties. ( A ) Daudi cells were incubated with anti-CD20 mAbs and their binding properties were assessed by flow cytometric analysis using Alexa488-conjugated F(ab')2 anti-human IgG Fc. The mean fluorescence intensities were plotted against the mAbs concentration (n = 3, bars indicate SEM). ( B ) Daudi cells were incubated with DyLight 488-labeled MabThera and serially diluted unlabeled mAbs. Percentages of DyLight 488-labeled MabThera binding were calculated by the mean fluorescence intensities and plotted against the unlabeled mAbs concentration (n = 3, bars indicate SEM).

Article Snippet: Serially diluted anti-CD20 mAbs were injected into the flow cells and association and dissociation were monitored.

Techniques: Binding Assay, Incubation, Fluorescence, Concentration Assay, Labeling

Journal: mAbs

Article Title: Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

doi: 10.1080/19420862.2015.1078054

Figure Lengend Snippet: Complement-dependent cytotoxicity (CDC). Daudi cells were cultured in the presence of human serum (16%) and anti-CD20 mAbs (1, 3 or 10 µg/ml). The percentages of 7-AAD positive-dead cells were calculated by flow cytometric analysis and represented as the mean + SEM (n = 3). **, p

Article Snippet: Serially diluted anti-CD20 mAbs were injected into the flow cells and association and dissociation were monitored.

Techniques: Cell Culture

Journal: mAbs

Article Title: Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

doi: 10.1080/19420862.2015.1078054

Figure Lengend Snippet: Structures of the plasmids used to generate transgenic silkworms. Each plasmid has right and left arms of piggyBac and the 3 × P3-fluorescent gene cassette for a screening marker (EYFP, AmCyan, or DsRed2). Plasmids pBac[UAS_anti-CD20 mAb HC/3 × P3-EYFP] and pBac[UAS_anti-CD20 mAb LC/3 × P3-AmCyan] encode the anti-CD20 mAb H chain gene and the anti-CD20 mAb L chain gene, respectively, under the control of a UAS promoter, and contain an BmNPV- derived hr5 enhancer and an A3-Blasticidin cassette. The anti-CD20 H and L genes were fused to the signal peptide sequence of the sericin 1 gene encoded by exon 1 and 18 bp of exon 2. The plasmid pBac[Ser1-GAL4/3 × P3-DsRed] encodes the GAL4 gene under the control of the sericin1 promoter.

Article Snippet: Serially diluted anti-CD20 mAbs were injected into the flow cells and association and dissociation were monitored.

Techniques: Transgenic Assay, Plasmid Preparation, Marker, Derivative Assay, Sequencing

Journal: mAbs

Article Title: Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

doi: 10.1080/19420862.2015.1078054

Figure Lengend Snippet: Purification of anti-CD20 mAbs from the transgenic silkworms. ( A ) Anti-CD20 mAbs were purified from the lysates of MSGs or cocoons derived from transgenic silkworms by affinity chromatography using a Protein G column. The lysates (original), flow-through fractions from the Protein G column, and eluted fractions were analyzed by non-reducing SDS-PAGE. ( B ) MabThera and anti-CD20 mAbs derived from transgenic silkworms showed the same migration pattern on non-reducing SDS-PAGE.

Article Snippet: Serially diluted anti-CD20 mAbs were injected into the flow cells and association and dissociation were monitored.

Techniques: Purification, Transgenic Assay, Derivative Assay, Affinity Chromatography, SDS Page, Migration

Journal: mAbs

Article Title: Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

doi: 10.1080/19420862.2015.1078054

Figure Lengend Snippet: Fc glycosylation analysis of anti-CD20 mAbs. ( A ) Comparison of mass spectra acquired at the elution point of the glycopeptides of EEQYNSTYR, corresponding to 293–301 amino acids (EU numbering) in the heavy chain of anti-CD20 mAbs. ( B ) Percentage distributions of glycoforms were calculated by the relative peak area average values. Symbols: blue square, N-acetylglucosamine; green circle, mannose; yellow circle, galactose; red triangle, fucose.

Article Snippet: Serially diluted anti-CD20 mAbs were injected into the flow cells and association and dissociation were monitored.

Techniques:

Journal: mAbs

Article Title: Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

doi: 10.1080/19420862.2015.1078054

Figure Lengend Snippet: Binding affinity to human Fc receptors. Binding affinities of anti-CD20 mAbs to FcγRI, FcγRIIIa and FcRn were measured by an SPR analysis. ( A ) Binding sensorgrams corrected for both the surface blank and the buffer injection control are represented. Dashed lines indicate the fitting curves generated by 1:1 binding model for FcγRs and bivalent model for FcRn, respectively. ( B ) Dissociation constant (K D ) values are represented as mean + SD (n = 3). *, p

Article Snippet: Serially diluted anti-CD20 mAbs were injected into the flow cells and association and dissociation were monitored.

Techniques: Binding Assay, SPR Assay, Injection, Generated