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Figure S1. ADCC assay with human PBMC. (A) Specific killing of A1207 cells by human PBMC in a 4 h 51 Cr-release assay. (B) Specific killing of A1207 cells with 1 μg/mL EGFR antibodies by monocytes isolated from PBMC fractions and tested in a 4 h 51 Cr-release assay. Source data is available for this figure in the Supporting Information.

Figure S2. Cytofluorimetric analysis of G-CSF-primed mouse whole blood. (A) Mice were injected subcutaneously with 20 μg of G-CSF and bled via the retro-orbital plexus four days later. Blood was analysed by cytofluorimetry and the number of effector cells was analysed relative to known amount of beads. (B) Expression of mouse FcγR and human FcαRI on different populations in the blood was analysed by staining with specific antibodies. Unstimulated and PEG-G-CSF-stimulated wild-type and FcαRI transgenic mice are compared. Source data is available for this figure in the Supporting Information.

Figure S3. N-glycan sequencing profiles of antibodies N-glycoprofiling of IgA1 EGFR (A) and IgA2 EGFR (B) antibodies: the reduced and alkylated antibodies were immobilized in a polyacrylamide gel block before releasing the N-glycans by PNGaseF. The glycans were fluorescently labelled with 2-AB and analysed on an ACQUITYUPLC-BEH-Glycan column. A combination of the retention times standardized to glucose units (GU) and exoglycosidase digestions were used to determine the sequence of the glycans. Source data is available for this figure in the Supporting Information.

Figure S4. Efficacy of depletion of specific cell types (A) PMN depletion efficacy. WT Balb/c mice were injected with tumor cells and two times with 200 μg isotype control or with Gr-1 depleting antibody (Ly6G/C-specific). The effector cells from the peritoneal lavage were identified by FACS staining. (B) Macrophage depletion efficacy. WT Balb/c mice were injected with 200 μL chlodronate liposomes or PBS before the experiment to deplete macrophages. Mice received 50 μg IgA2 EGFR and the effector cells from the peritoneal lavage were identified by FACS staining. Source data is available for this figure in the Supporting Information.

Table S1. Half-life of IgG1 and IgA1 EGFR. Source data is available for this figure in the Supporting Information.

Table S2. A summary of the types of N-glycans on IgA1 EGFR and IgA2 EGFR. Source data is available for this figure in the Supporting Information.

emmm201201929.reviewer_comments.pdfPDF document479KReview Process File

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