antibody specific production rates [pg/d/cell] growth rates [%/d] culture flask miniperm CL1000 culture flask miniperm CL1000

Transcription

1 Table 1: Growth rates of transfectomas and production, concentrations and yields of monomeric (2-IgA) and dimeric 2-IgA (2-dIgA) under different culture conditions. antibody specific production rates [pg/d/cell] growth rates [%/d] culture flask miniperm L culture flask miniperm L 2-IgA.8 (±.3).34 (±.17) 1.6 (±.4) 47.5 (± 26.9) (± 73.2) 91.4 (± 216.7) 2-dIgA.9 (±.4).32 (±.11) 2.6 (±.6) 44.1 (± 22.3) (± 35.2) (± 234.2) antibody antibody concentrations in supernatants Antibody yields [mg/week/flask] [µg/ml] culture flask miniperm L Miniperm L 2-IgA 4. (± 1.1) 44.8 (± 16.5) 2.8 (± 73.3) 1.7 (±.6) 6.2 (± 1.5) 2-dIgA 4.2 (± 1.3) 34.5 (± 11.4) (± 139.5) 2.1 (±.4) 7.9 (± 1.4)

2 A 1 kappa-purified antibody Fraction 1 Fraction 2 Fraction 3 2 His-Tag-purified polymeric IgA Fraction 1 Fraction E1 E purified antibodies dimeric IgA monomeric IgA -kappafractions His-Tagfractions 1 2 F purified antibodies 1 2

3 A dimeric 2-IgA monomeric 2-IgA control Side scatter (log) number of cells Side scatter (log) number of cells

4 A control alu3 alu3 2-IgG 2-mIgA 2-dIgA ontrol SIgA1 miga1 t antihuman migg1 EGFR * S pigr * IgG 4 3 RFI dimeric 2-IgA monomeric 2-IgA control IgA [nm] 2 IgA hours

5 Supplemental Legends Supplemental Table 1: Growth rates of transfectomas and productions, concentrations and yields of monomeric (2-IgA) and dimeric 2-IgA (2-dIgA) under different culture conditions. Supplemental Figure 1: Purification of J-chain-containing dimeric 2-IgA. Serum-free supernatants from non-adherent HO-K1 cells transfected with vectors for human J-chain, IgA1 heavy and α-light chains were run over an XK16 affinity chromatography column containing apture Select Fab anti-human κ sepharose beads. The eluted samples were analyzed by gel filtration on a Superdex2 26x6 column (A). The indicated fractions 1-3 were separately collected, and analyzed by Western blot using anti-human α-chain specific peroxidase labelled antibody for staining (). Fraction 1 - containing monomeric, dimeric and multimeric IgA - was then run on an anti-his- (IMA) column to selectively purify His-tagged, J-chain-containing antibody. The resulting eluate was again analyzed by gel filtation (). Western blot analyses of the collected two fractions () demonstrated that fraction 2 contained predominantly dimeric IgA with an estimated molecular weight of approximately 32. Finally, purified dimeric IgA was again analyzed by gel filtration - demonstrating a homogenous peak at 135 ml (E1). In (E2), purified recombinant monomeric 2-IgA1 is shown for comparison. Western blots of purified dimeric and monomeric IgA (lanes 1 and 2, respectively) demonstrated the expected size of the respective isoforms (F). Supplemental Figure 2. inding of recombinant dimeric 2-IgA to EGFR and FcαRI (89). Representative density plots (A and ) and histograms ( and ) demonstrate similar binding of saturating concentrations of monomeric (dark gray curves) and dimeric (black curves) 2-IgA to EGFR expressing A431 (A and ) and FcαRI-transfected HK-21 cells ( and ). inding of antibodies was detected using a fluoresceine labeled anti-human kappa antibody. Irrelevant IgA and IgG antibodies served as negative control in (A, ) and (,, light gray curves), respectively. ata shown are representative for at least three independent experiments. Supplemental Figure 3. Transcytosis of dimeric IgA by alu3 cells. (A) Transcytosis of monomeric and dimeric 2-IgA through a confluent monolayer of human alu3 cells was analyzed by Western blot experiments using secretory component (S), IgG (IgG) or IgA (IgA) specific antibodies for detection. 2-IgG, added to the lateral compartment, served as leakage control and did not enter the compartment. As demonstrated, monomeric 2-IgA (miga) and control IgA (ciga) were retained in the lateral compartment, while dimeric 2- IgA (diga) was transcytosed from the lateral to the compartment ( ) within 24 h. This transport was unidirectional, since diga was not transported from the to the lateral compartment ( ). After transcytosis, dimeric 2-IgA stained positive for secretory component, and demonstrated a higher molecular weight compared to dimeric 2-IgA - demonstrating in vitro generation of secretory 2-IgA. () Expression of EGFR and pigr by alu3 cells was analyzed by indirect immunofluorescence using m4 and SPM217 antibodies to detect EGFR and pigr, respectively. A fluoresceine labeled goat anti mouse IgG antibody was used as secondary antibody. alu3 cells simultaneously express pigr and EGFR. () Measuring transcytosis efficiency by an IgA-specific ELISA demonstrated that dimeric 2-IgA was transported in a time-dependent manner.