NSG CDX mouse model

Animal experiments were performed with 8–12 week old male and female NSG mice according to the German animal protection law (Landesdirektion Sachsen, TVV 61/2017). On day zero, 1 × 10⁵ MOLM-13 AML cells in 100 µl PBS were administered IV via the tail vein into NSG mice. Three days later, 5 × 10⁶ UniCAR-T (82% CAR⁺) were applied IV via the tail vein in 100 µl PBS. Right away, the first treatment cycle of TM therapy started with a total of two TM therapy cycles, each lasting 10 days with a short break of five days in-between. Both TM123-41BBL and TM123 were administered by IP injections of 1 μg/g_BW twice a day. Mice were sacrificed either at a pre-defined endpoint after the 1^st TM therapy cycle (n = 3), or if stop criteria according to the German Animal Welfare Act were met (n = 10), including paralysis of the hind extremities, weight loss >20%, weakness or morbid state. Sacrificed mice were analyzed for human cells in peripheral blood, bone marrow, spleen, or metastatic tumor samples. For organ sample preparation, RO obtained blood samples and single-cell suspensions from spleen, metastases, as well as bone marrow obtained from femur and tibia of both hind legs were prepared. Erythrocytes were removed by lysis and cells were stained with indicated mAbs. Human T cell chimerism was defined as the percentage of CD3-positive cells over the entire populations of both human tumor cells and murine CD45 leukocytes together. For MOLM-13, chimerism was defined as the percentage of target cells over the populations of CD3-positive cells and murine CD45 leukocytes. Here, MOLM-13 tumor cells were characterized via expression of CD33, human CD45 and CD4. Stained organ samples were measured on the MACSQuant® Analyzer 10 and analyzed using FlowJo™v10.