Dako qifikit

HER2 binding capacity of tumor cell lines was quantified by DAKO QIFIKIT (DAKO Cytomation), according to the manufacturer's protocol using mAb ER-23 (Santa Cruz) as primary antibody. HER2 quantity was expressed as specific antibody-binding capacity units after subtraction of isotype control (mouse IgG2b) background. For binding experiments, HER2bsFab and trastuzumab were biotinylated in vitro using Ez-link micro NMHS-PEO4-biotinylation kit (Perbio science). The impact of biotinylation on antigen binding was checked by comparing binding activities of biotinylated antibodies and their unlabeled counterparts by flow cytometry. Binding and apparent affinities were determined by flow cytometry as previously described [59 (link)] using 2×10⁵ Jurkat-huFcγRIIIA, SK-BR-3, stably transfected CHO cells expressing FLAG-tagged human or mouse FcγRs and biotinylated antibodies (0.05 nM to 2000 nM). Stability assays in human serum were performed as previously described [30 (link)] for 21 days with biotinylated antibodies. Competition assays with endogenous IgGs were performed by incubating Jurkat-huFcγRIIIA cells in the presence of human serum (20 or 100%) and sub-saturating concentration of biotinylated HER2bsFab (50 nM) or trastuzumab (200 nM). Bound antibodies were detected by flow cytometry using PE-labeled streptavidin [30 (link)].

Antibodies were purchased from BioLegend (anti-human IL-10R(CD210), 308806; anti-human PD-1, 329912; anti-human PD-L1-APC, 329708), Miltenyi Biotech (anti-His-PE, 130-092-691), KPL (goat anti-mouse IgG H + L; 01-10-06) and R&D Systems (anti-human CD3ε, MAB100; anti-human TGF-β1,2,3, MAB1835; anti-IDO Alexa488, IC6030G; anti-His-HRP, sc-8036 HRP). Human IFN-γ (DY285), IL-2 (DY202), IL-10 (DY217B) and TGF-β (DY240) DuoSet^® ELISA kits were purchased from R&D Systems. A431, Colo205, HCT-116, LS174T and NCI-H460 were cultured in RPMI 1640 (Life Technologies, 11875), 10% FBS (PAN Biotech, P30-3309). Lovo and A549 were cultured in RPMI 1640, and 5% FBS and SKBR3 were cultured in DMEM (Life Technologies, 41965), 10% FBS. Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coat of healthy donors (Klinikum Stuttgart, Germany) by Ficoll density gradient centrifugation (Lymphocyte Separation Medium 1077, Promocell, C-44010) and cultivated in RPMI 1640, 10% FBS. DAKO QIFIKIT (K007811-8) was purchased from Agilent Technologies.

Experiments were performed on a MACSQuant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany) or BD LSR II (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed via FlowJo v10 software (FlowJo, Ashland, OR, USA). Antibodies used for analysis of cells were as follows: anti-CD4 (SK3), anti-CD8 (RPA-T8), anti-CD10 (HI10a), anti-CD14 (M5E2), anti-CD19 (HIB-19), anti-CD25 (BC96), anti-CD28 (CD28.2), anti-CD33 (WM53), anti-CD34 (8G12), anti-CD38 (HIT2), anti-CD45RO (UCHL1), anti-CD45RA (HI100), anti-CD66b (G10F5), anti-CD95 (DX2), anti-CD107a (H4A3), anti-CD123 (9F5), and anti-CD197 (150503) (all purchased from BD Biosciences, Franklin Lakes, NJ, USA); anti-human leukocyte antigen (HLA)-DR (L243) and anti-CD3 (UCHT1) (both purchased from Thermo Fisher Scientific, Waltham, MA, USA); anti-CD45 (HI30), anti-CD123 (6H6), and anti-HuLin (OKT3/M5E2/3G8/HIB19/2H7/HCD56) (all purchased from BioLegend, San Diego, CA, USA); anti-CD49f (REA518) (Miltenyi Biotec, Bergisch Gladbach, Germany); and anti-La (5B9) and anti-La (7B6)³⁰ (link)^,61 (link) (in-house) or GaM-Alexa Fluor 647 (Dianova, Hamburg, Germany). Antigen quantification was performed using Dako QIFIKIT (Agilent Technologies, Santa Clara, CA, USA).