Journal: mAbs

Article Title: Effect of a tail piece cysteine deletion on biochemical and functional properties of an epidermal growth factor receptor-directed IgA2 m(1) antibody

doi: 10.4161/mabs.26396

Figure Lengend Snippet: Figure 1. Biochemical characterization of 225-IgA2-d471 and 225-IgA2-wt antibody. ( A ) Antibody content in supernatants was analyzed by ELISA, and specific production rates were calculated. ( B ) Affinity-purified antibodies were separated by size exclusion to isolate monomeric antibodies. ( C1 ) Purity of antibodies was analyzed by denaturing SDS-PAGE stained with silver nitrate. ( C2 ) Formation of non-native aggregates was analyzed using native-PAGE stained with Coomassie. Proteins were transferred onto PVDF membranes and probed using polyclonal antibodies against κ- ( C3 ) or α-chain (C4). Lanes: (1) control IgA2, (2) 225-IgA2-wt, 3) 225-IgA2-d471. ( D ) 225-IgA2-wt and –d471 producing cells were transfected with a pIRESpuro3 plasmid encoding human J-chain to allow the formation of stable dimeric IgA. Proteins were transferred onto PVDF membranes and stained using polyclonal antibodies against human α-chain. Lanes: (1) control IgA2, (2) purified monomeric 225-IgA2-wt, (3) purified monomeric 225-IgA2-d471, (4) 225-IgA2-wt containing SUP from CHO cells transfected with human J chain, (5) 225-IgA2-d471 containing SUP from CHO cells transfected with human J chain, (6) purified dimeric 225-IgA2-wt. ( E ) The composition of freshly prepared (E1), two (E2) and four (E3) year old preparations of 225-IgA2-wt (lane 1) and 225-IgA2-d471 (lane 2) was analyzed by analytical size exclusion chromatography on a Superdex200 10 × 300 column. (E4) Relative areas under the curve (rAUC) of monomeric (F M , elution volumes 10‒14 ml) and polymeric (F P , elution volumes 6.5–10 ml) IgA were calculated. ( F ) Thermal stability was analyzed by incubating antibodies at denaturing temperatures and measuring maintenance of functionality in chromium release assays using A431 as targets and freshly isolated human PMN as effector cells. Results are presented as “mean ± SEM” of “relative specific lysis [%]” of three independent experiments.

Article Snippet: Purified human myeloma IgA2 m(1) antibody was used as control IgA2 (Meridian Life Science).

Techniques: Enzyme-linked Immunosorbent Assay, Affinity Purification, SDS Page, Staining, Clear Native PAGE, Control, Transfection, Plasmid Preparation, Purification, Size-exclusion Chromatography, Isolation, Lysis

Journal: mAbs

Article Title: Effect of a tail piece cysteine deletion on biochemical and functional properties of an epidermal growth factor receptor-directed IgA2 m(1) antibody

doi: 10.4161/mabs.26396

Figure Lengend Snippet: Figure 2. Fab-mediated effector functions were not impaired by the d471 mutation. ( A ) Binding of 225-IgA2-wt and –d471 to EGFR-expressing A431 cells was analyzed by indirect immunofluorescence analyses using anti-human kappa light chain directed secondary antibody. ( B ) Blocking of EGF binding was detected by FACS analyses treating A431 cells simultaneously with FITC-labeled EGF and excess of specific or nonspecific antibodies. ( C ) DiFi colon carcinoma cells were incubated with IgA2 antibodies for 72 h before viability was measured by MTS assay. Results are shown as “mean ± SEM” of “EGFR binding [RFI]” ( A ), “inhibition of EGF binding [%]” ( B ) and “cell viability [%]” ( C ) of five different experiments. Significant differences (p ≤ 0.001) between EGFR-specific and non-specific (control IgA2) antibodies are indicated by *, between mutant and wild type IgA2 by #.

Article Snippet: Purified human myeloma IgA2 m(1) antibody was used as control IgA2 (Meridian Life Science).

Techniques: Mutagenesis, Binding Assay, Expressing, Immunofluorescence, Blocking Assay, Labeling, Incubation, MTS Assay, Inhibition, Control

Journal: mAbs

Article Title: Effect of a tail piece cysteine deletion on biochemical and functional properties of an epidermal growth factor receptor-directed IgA2 m(1) antibody

doi: 10.4161/mabs.26396

Figure Lengend Snippet: Figure 3. FcαRI-binding and recruitment of myeloid effector cells. In ( A ), binding of 225-IgA2-wt and –d471 to BHK-21 cells co-transfected with human FcαRI and FcRγ was measured by indirect immunofluorescence using FITC-labeled anti-human kappa light chain directed secondary antibody. Efficiency of IgA2 antibodies in mediating ADCC against A431 targets cells was analyzed in chromium release assays using whole blood and increasing antibody concentrations ( B ). Next, increasing volumes of whole blood ( C ) and increasing ratios of isolated granulocytes ( D ), monocytes ( E ) and monocyte-derived macrophages ( F ) were used as effectors cells. Results are shown as “mean ± SEM” of “FcαRI binding [RFI %]” ( A ) or “specific lysis [%]” ( B–F ) of at least five independent experiments. Significant differences between EGFR-specific and control antibodies (control IgG1 in [ A ], control IgA2 in [ B‒F ]) are indicated by * ( P ≤ 0.001).

Article Snippet: Purified human myeloma IgA2 m(1) antibody was used as control IgA2 (Meridian Life Science).

Techniques: Binding Assay, Transfection, Immunofluorescence, Labeling, Isolation, Derivative Assay, Lysis, Control

Journal: mAbs

Article Title: Effect of a tail piece cysteine deletion on biochemical and functional properties of an epidermal growth factor receptor-directed IgA2 m(1) antibody

doi: 10.4161/mabs.26396

Figure Lengend Snippet: Figure 4. Glycosylation of wild type and d471-mutated IgA2 antibodies. Single clones producing 225-IgA2-wt and –d471 were cultured in tissue culture flasks (TCF), and glycosylation of affinity-purified antibodies was analyzed using lectin blots. Different sugar moieties were detected using biotinylated lectins specific for terminal galactosylation ( A1 ), α(1,6)-linked fucosylation ( A2 ), α-linked mannosylation ( A3 ), α(1,3)-linked mannosylation ( A4 ), N -acetylglucosamine ( A5 ), terminal sialic acid ( A6 ), and human α-chain as loading control ( A7 ). Lanes: 1. 225-IgA2-wt (CL1000 produced), 2. 225-IgA2-wt clone 1, 3. 225-IgA2-wt clone 2, 4. 225-IgA2-wt clone 3, 5. 225-IgA2-d471 (CL1000 produced), 6. 225-IgA2-d471 clone 1, 7. 225-IgA2-wt clone 2, 8. 225-IgA2-wt clone 3, 9. control IgA2. ( A8 ) To evaluate whether that glycosylation of wild type and mutant IgA2 antibodies was different, lectin blots were analyzed densitometrically using ImageJ software. Results are presented as “pixel density (dots per inch [dpi])” and significant differences between wild type and mutant IgA2 glycosylation are indicated by * ( P ≤ 0.001).

Article Snippet: Purified human myeloma IgA2 m(1) antibody was used as control IgA2 (Meridian Life Science).

Techniques: Glycoproteomics, Clone Assay, Cell Culture, Affinity Purification, Control, Produced, Mutagenesis, Software

Journal: mAbs

Article Title: Effect of a tail piece cysteine deletion on biochemical and functional properties of an epidermal growth factor receptor-directed IgA2 m(1) antibody

doi: 10.4161/mabs.26396

Figure Lengend Snippet: Figure 5. Recruitment of myeloid effector cells by differently glycosylated IgA2 antibodies. The capacity of differently glycosylated wild type and d471-mutated IgA2 antibodies to mediate ADCC of human A431 tumor cells was analyzed in chromium release assays using isolated granulocytes or monocytes as effector cells. Results are shown as “mean ± SEM” of “specific lysis [%]” of at least three independent experiments.

Article Snippet: Purified human myeloma IgA2 m(1) antibody was used as control IgA2 (Meridian Life Science).

Techniques: Isolation, Lysis