Journal: mAbs

Article Title: Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

doi: 10.1080/19420862.2015.1046665

Figure Lengend Snippet: CEX fractionation profiles of hybridoma-derived batches of a CD20 mAb ( A ) and a CD38 mAb ( B ). Fractions containing K0, K1 and K2 isoforms were collected and pooled. Fractions are highlighted in gray. The fractions were incubated with (+) or without (−) carboxypeptidase B (CPB) to demonstrate the presence of C-terminal lysines and analyzed the samples on SDS-PAGE ( C, D ) and IEF gel electrophoresis ( E, F ). The changes in charge on IEF gel suggest the absence of C-terminal lysines in K0, and the presence of one lysine in K1 and 2 lysines in K2. Unfr. indicates the original unfractionated material.

Article Snippet: Hybridomas producing human mAbs against human CD38 (HuMab 005) and CD20 (2F2) were developed at Genmab and have been described previously.

Techniques: Fractionation, Derivative Assay, Incubation, SDS Page, Nucleic Acid Electrophoresis

Journal: mAbs

Article Title: Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

doi: 10.1080/19420862.2015.1046665

Figure Lengend Snippet: Dose-response curves and EC50 values of complement-mediated lysis (CDC) induced by the CD20 mAb of Daudi cells ( A, B ; n = 3) and of Raji cells ( C, D ; n = 2) and induced by the CD38 mAb of Daudi cells ( E, F ; n = 3). For both mAbs the unfractionated (purple symbols and bars) and K2 isoform (blue symbols and bars) with and without CPB treatment were analyzed. NHS (20% vol/vol) was used as complement source and cells were incubated at 37°C for 45 min. Lysis was assessed by flow cytometry using a PI exclusion assay and the level of CDC is expressed as percentage of total cells. The data represents mean ± SEM and statistical significance was assessed by one-way ANOVA followed by a Tukey post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Hybridomas producing human mAbs against human CD38 (HuMab 005) and CD20 (2F2) were developed at Genmab and have been described previously.

Techniques: Lysis, Incubation, Flow Cytometry, Exclusion Assay

Journal: mAbs

Article Title: Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

doi: 10.1080/19420862.2015.1046665

Figure Lengend Snippet: Overlay of capillary isoelectric focusing profiles of CD38 C-terminal mutants ( A ). The mutant abbreviations K0, K2, K4, K6 and E2 ( Table S1 ) and pI markers 7.65 and 10.10 are indicated and detection occurred at 280 nm. Dose-response curves of CDC mediated cell lysis of the C-terminal mutants (n = 3) ( B ). NHS (20% vol/vol) was used as complement source and cells were incubated at 37°C for 45 min. Lysis was assessed by flow cytometry using a PI exclusion assay and the level of CDC is expressed as percentage of total cells. CD38 mAb wt produced in HEK-293F was used as positive control.

Article Snippet: Hybridomas producing human mAbs against human CD38 (HuMab 005) and CD20 (2F2) were developed at Genmab and have been described previously.

Techniques: Mutagenesis, Lysis, Incubation, Flow Cytometry, Exclusion Assay, Produced, Positive Control