Luminescence imaging of the subcutaneous injected cells (exposure times 1 s, 10 s, and 60 s) was performed using a dedicated small animal multimodal imaging system (values 0
Luminescence imaging of the subcutaneous injected cells (exposure times 1 s, 10 s, and 60 s) was performed using a dedicated small animal multimodal imaging system (values 0.05 were considered significant. SUPPLEMENTARY MATERIALS FIGURE Click here to view.(1.5M, pdf) Acknowledgments This study was supported by grants provided to MPB including by the german government (BMBF, TurbiCAR) and the DKTK. cells and thereby lead to their destruction. So far, we constructed TMs with a short half-life. The fast turnover of such a TM allows to rapidly interrupt the treatment in case severe side effects occur. After elimination of most of the tumor cells, however, longer lasting TMs which have not to be applied via continous infusion would be more convenient for the patient. Here we describe and characterize a TM for retargeting UniCAR T cells to CD19 positive tumor cells. Moreover, we show that the TM can efficiently be produced from producer cells housed in a sponge-like biomimetic cryogel and, thereby, serving as an TM factory for an extended retargeting of UniCAR T cells to CD19 positive leukemic cells. synthesized TM(A) Schematic view of the UniCAR system. For retargeting of UniCAR T cells to CD19 positive tumor cells a TM against the CD19 antigen (anti-CD19 TM) had to be constructed. In its presence, UniCAR T cells will be cross-linked to CD19 positive tumor cells which will finally lead to lysis of the latter. In the absence of the TM, UniCAR T cells will automatically be switched off. (B) For both and TMB synthesis the reading frame encoding the anti-CD19 TM had to be transduced into a producer cell line. To this, murine 3T3 cells were selected. For synthesis the transduced cells were housed in starPEG-heparin cryogels. (C) For proof of concept, it had to be analyzed whether or not the amount of anti-CD19 TM that can be released form producer cells housed in the cryogel is sufficient for retargeting of UniCAR T cells to CD19 positive tumor cells and production of the therapeutic molecule. RESULTS The aims of the presented manuscript are schematically summarized in Figure ?Figure1:1: We wanted to (i) develop and functionally characterize a TM for redirection of UniCAR T cells to CD19 positive tumor cells (Figure ?(Figure1A)1A) and (ii) challenge the idea to manufacture the TM from the producer cell line housed in a starPEG-heparin cryogel (Figure ?(Figure1B)1B) for retargeting of UniCAR T cells in experimental mice (Figure ?(Figure1C).1C). For this purpose, we had to (i) clone the TM, (ii) establish a cell collection permanently expressing the TM, (iii) isolate the TM from your supernatant, (iv) characterize the TM biochemically, (v) display its features 0.05, ** 0.01, *** 0.001; ns, not significant). Killing of CD19 positive tumor cells by retargeted UniCAR T cells happens inside a TM-dependent- and target-specific manner For functional analysis, we used a FACS-based killing assay  [observe also MATERIALS AND METHODS]. A total of 1 1 TMB 104 TMB Nalm-6 cells were labeled with eFluor670? and incubated with T cells engrafted with the UniCAR signaling construct (Number ?(Number3B,3B, UniCAR CD28/) at an e:t percentage of 1 1:1. T cells expressing either the vector control encoding EGFP marker protein (Number ?(Number3B,3B, vector control) or the UniCAR stop construct lacking the intracellular signaling website (Number ?(Number3B,3B, UniCAR stop) served as bad controls. The number of surviving tumor cells was identified via circulation cytometry after coculturing genetically altered T cells with CD19 positive tumor cells for 24h and CD207 48h as indicated in the presence or absence of 0.1 nM to 5 nM of anti-CD19 TM. As demonstrated in Number ?Number3B,3B, only T cells equipped with a signaling UniCAR construct efficiently eliminate target cells. CD19 bad cells were not attacked by UniCAR T cells either in the presence or absence of the anti-CD19 TM (data not demonstrated). Related data were acquired for other CD19 positive tumor cells e.g. Raji and Daudi cells (data not demonstrated). In order to estimate the EC50 value of the anti-CD19 TM, titration.