Abs.

Efgartigimod used in nonclinical experiments was transiently produced in CHO cells and purified using MabSelect SuRe resin (Evitria). Efgartigimod for human use was produced by Lonza Biologics using a CHOK1SV GS-KO cell line (Lonza Group Ltd.) stably transfected with an expression vector encoding efgartigimod. Purification was performed in a 3-step process including protein A column chromatography, anion exchange chromatography, and hydrophobic interaction chromatography. Production yield and glycosylation profile were in line with what is typically observed for full-length IgG Abs expressed using this system.

FR70 Ab is a rat anti-mouse CD70 mAb produced by hybridoma technology (45). Following sequencing of its variable regions using standard procedures, mammalian expression vectors were made encoding FR70-hIgG1. Subsequently, FR70-hIgG1 was produced transiently in HEK293-E cells and purified using protein A beads at U-Protein Express. IVIg (Octagam 10%) was bought from Octapharma. An anti-FcRn Ab (DX-2507) was previously described (18), and sequences for the heavy and light chains were retrieved from patent application WO2012/167039. cDNA sequences for the heavy and light chains of the Ab were cloned into mammalian expression vectors. Subsequently, the anti-FcRn Ab was produced and purified as described for FR70-hIgG1. The humanized anti-HEL Ab (hIgG1) was expressed and purified from culture supernatants of transfected NS0 cells using HEL-sepharose and previously described methods (46).

For in vitro use, Abs or efgartigimod was labeled with Alexa Fluor 647 N-hydroxysuccinimide (NHS) ester, tris(triethylammonium salt) (Invitrogen, Thermo Fisher Scientific), using methods recommended by the manufacturer, except that the molar ratio of dye:Ab/Fc fragment was 2:1. Degrees of labeling for each Ab/Fc fragment were as follows: efgartigimod, 2.2 dye:Fc; hIgG1, 1.7 dye:Ab; anti-FcRn, and 1.45 dye:Ab. Labeled Ab/Fc fragments were analyzed to ensure that there were no detectable aggregates using HPLC (Superdex 200 or Yarra 3 μm SEC 3000).

Anti–CD4–Pacific Blue (BD Biosciences — Pharmingen, clone RPA-T4), anti–CD45-Krome Orange (Beckman Coulter, clone J.33), anti-CD8 FITC (Dako, clone DK25), anti–CD56-PE (BD, clone NCAM16.2), anti–CD16-PE (BD, clone Leu 11c), anti–CD19-PC7 (Beckman Coulter, clone J3.119), and anti–CD3-APC (BD, clone SK7) were used for flow cytometric analysis of the human lymphocyte subsets. The dermal-derived microvasculature cell line HMEC-1.CDC was provided by F. Candal (CDC, Atlanta, Georgia, USA).