Alemtuzumab

Monocytes were cultured at a density of 0.5 to 1.0 × 10⁶/mL in RPMI-1640 (Gibco, Karlsruhe, Germany) supplemented with 2% human AB serum (PAN Biotech, Aidenbach, Germany), l-glutamine (2 mmol/L), penicillin (50 U/mL), and streptomycin (50 mg/mL; all from Life Technologies, Karlsruhe, Germany). For differentiation into DC, 0.5 to 1.0 × 10⁶ monocytes/mL were cultured for six days in medium as described above, supplemented with 10% fetal calf serum (PAN Biotech), IL-4 (144 U/mL), and granulocyte macrophage colony-stimulating factor (GM-CSF, 225 U/mL; both from PeproTech, Hamburg, Germany). Monocytes and DC were incubated for 4 h, 24 h, and 48 h, respectively, with or without LPS (from Salmonella abortus equi S-form, Enzo Life Sciences, Lörrach, Germany), ATG (Fresenius, Bad Homburg, Germany) (now named Grafalon^®, distributed by Neovii Biotech, Gräfelfing, Germany) (100 µg/mL), IgG isotype control (polyclonal, rabbit, Molecular Innovations, Novi, MI, USA) (100 µg/mL) or Alemtuzumab (monoclonal, human, anti-CD52, Genzyme, Cambridge, MA, USA) (100 µg/mL).

J774A.1 macrophages were cultivated in 96-well plates at 1 × 10⁴ cells per well. After 4 h of incubation at 37 °C, 1.5 × 10⁵ hMB “double-hit” lymphoma or CD19⁺ B-cells derived from CLL patients per well were co-cultured with respective macrophages. Subsequently, this co-culture was treated for 17 h with tyrosine kinase inhibitors and monoclonal antibodies in combination or as mono treatment. To stimulate the ADCP of human cells the human specific anti-CD52 monoclonal antibody alemtuzumab (Genzyme, Cambridge, MA, USA; 10 µg/mL), anti-CD20 obinutuzumab (GA101, Roche, Basel, Switzerland; 1 µg/mL), and anti-CD20 rituximab (Roche, Basel, Switzerland; 20 µg/mL) antibodies were used. Primary human CLL cells were stained with CD19 (fluorescein isothiocyanate (FITC), #363008, BioLegend, San Diego, CA, USA) fluorescent antibody for 15 min at 4 °C before measurement by flow cytometry. Each condition was performed with five replicates. For determination of ADCP GFP⁺, target cells were analyzed using a MACSQuant VYB flow cytometer (Miltenyi Biotec, Berg. Gladbach, Germany). The percentage of ADCP was calculated as follows:

The human CML cell line K562 was obtained from the Japanese Collection of Research Bioresource Cell Bank. The human AML cell line MOLM-13 and MOLM-14 were purchased from DSMZ (German Collection of Microorganisms and Cell Cultures). Primary human AML samples from newly diagnosed patients were obtained from the sample archive at the Aichi Medical University Hospital. All samples were obtained after written informed consent, and the use of biological samples for research was approved by the ethics committee of the Aichi Medical University Hospital (Approval Number 2020-156), in accordance with the Declaration of Helsinki. The cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) at 37 °C in a 5% CO₂ humidified atmosphere. Afuresertib, gilteritinib, and quizartinib were purchased from Selleck Chemicals (Houston, TX, USA). Sorafenib was obtained from ChemScene (Monmouth Junction, NJ, USA). Pimozide was obtained from Sigma-Aldrich (St. Louis, MO, USA). Alemtuzumab, a recombinant humanized monoclonal antibody against human CD52, was purchased from Sanofi K.K. (Tokyo, Japan).