Fcil 7

Once per week, 20 µg of the NHS-IL12 fusion protein together with 20 µg FcIL-7 (both from Merck, Germany) was administered to engrafted humanized NSG mice via puncture of the tail vein after the tumor had grown to ≥150 mm³. As stated above, one additional cohort of mice received a mixture of 1.5 µg IL-2 plus 15 µg anti-IL-2 mAb MAB602 per week. Recombinant human IL-2 (PROLEUKIN, Aldesleukin, Chiron) and MAB602 (anti-hIL-2 mABCD122, clone 5355, R&D Systems) were co-incubated for 15 min at room temperature before injection.

HuCD34⁺ stem cells were derived from a surplus of granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood stem cells from parental donors that had been T-cell depleted by CD34⁺ selection (CliniMACS, Miltenyi, Germany). Cells were suspended at a 1:2 ratio in a solution of DMSO/5% HSA (20%/80%) and cryopreserved with a Sylab Icecube device and a controlled freezing rate. After thawing, cells were stained with trypan blue and counted in a Neubauer cell count chamber. Informed consent regarding the scientific use of surplus cells was obtained from all donors in accordance with the Declaration of Helsinki. Purity of the CD34⁺ population was further increased to >99.99% by a second round of CD3⁺ depletion after thawing (LS MACS, Miltenyi). Stem cell donors were all HLA-mismatched to the RMS A204 cell line. huCD34⁺ cells (1 × 10⁶ cells in 100 µl prewarmed PBS) were injected in the tail vein of sublethally irradiated (250 cGy) NSG mice. Engraftment was supported by weekly applications of 20 µg FcIL-7 (Merck, Germany). In each of the NHS-IL12 treatment groups, 4 animals received long-term NHS-IL12 cytokine treatment with FcIL-7 or IL-2MAB602 for a maximum of 15 weeks (100 days).