All surviving cells, i.e., 293T CFSElow, 7-AAD unfavorable cells and A375M CFSEhigh, 7-AAD unfavorable cells, were set to 100%. work is usually descriptive rather than investigative in nature, we expect RPS6KA6 that providing this clinically applicable protocol to generate sufficient numbers of mRNA-transfected CAR-T cells will help in moving the field of adoptive cell therapy of cancer forward. Keywords: CAR-T cell, melanoma, CSPG4, clinical scale production, full GMP compliance, clinical study, consistency runs 1. Introduction Autologous T cells, reprogrammed to target malignant cells via the expression of a chimeric antigen receptor (CAR-T cells) represent a promising tool in the adoptive cellular therapy of cancer. Impressive clinical regressions of leukemias or lymphomas have been achieved using CD19-specific CAR-T cells in several clinical trials. This culminated in the approval by the FDA and EMA of Kymriah? (Tisagenlecleucel), a one-time treatment for B-cell acute lymphoblastic leukemia (ALL) that has shown an 83% remission rate after three months in clinical trials with patients that do not respond to standard treatments, and Yescarta? (Axicabtagen-Ciloleucel), which induced remissions in 72% of the patients with aggressive B-cell non-Hodgkin lymphoma [1]. However, most clinical trials focus on the Palmatine chloride elimination of these so-called liquid tumors; the development of CAR-T cells against solid tumors lags behind (reviewed in [2,3,4,5]). This is due to the lack of real tumor-specific antigens that can be targeted by CAR-T cells, causing potential on-target/off-tumor toxicity due to the accidental killing of non-malignant bystander cells co-expressing Palmatine chloride the target antigen [6]. The expression of the target antigen on healthy tissue usually bears the risk of severe side effects due to tissue toxicity. This is probably the reason that very few CAR-T cells against different antigens expressed on melanoma (e.g., VEGFR2, CD70, GD2, c-Met) were tested in clinical trials (“type”:”clinical-trial”,”attrs”:”text”:”NCT03060356″,”term_id”:”NCT03060356″NCT03060356, “type”:”clinical-trial”,”attrs”:”text”:”NCT01218867″,”term_id”:”NCT01218867″NCT01218867, “type”:”clinical-trial”,”attrs”:”text”:”NCT02107963″,”term_id”:”NCT02107963″NCT02107963, “type”:”clinical-trial”,”attrs”:”text”:”NCT02830724″,”term_id”:”NCT02830724″NCT02830724). Chondroitin sulfate proteoglycan 4 (CSPG4), also known as melanoma-associated- chondroitin-sulfate-proteoglycan (MCSP), high molecular weight-melanoma-associated antigen (HMW-MAA), or neuron-glial antigen 2 (NG2) is usually a 450 kDa large, heavily glycosylated proteoglycan [7,8]. CSPG4 is usually expressed on almost all melanoma cells [9], but also on uveal melanoma [10,11], and on other tumors like sarcomas, astrocytomas, gliomas, neuroblastomas [12,13,14,15], leukemias [16,17,18,19,20], and triple unfavorable breast malignancy [21]. In many of those malignancies, CSPG4-expression is associated with poor prognosis and aggressive growth [22]. On non-pathologic tissue, CSPG4 is usually expressed on precursors of hair-follicle and epidermis cells, as well as on endothelial cells and on activated pericytes, however, not on mature vasculature [23,24]. Moreover, CSPG4 is expressed on chondrocytes of the articular cartilage [25], on easy muscle cells [26], on brain pericytes [27], and on cells of the neuromuscular synapse of human postnatal skeleton muscles [28]. The antigen is also expressed on fetal melanocytes, but not on healthy melanocytes of adults [29]. The expression of CSPG4 on healthy tissues is, however, clearly weaker than on tumor cells [8,30,31]. Nevertheless, CSPG4 is usually a primary tumor target antigen [30], since it plays a role in the metastasizing of melanoma [32], and is expressed on activated pericytes during angiogenesis in tumors and hypoxia [33,34,35], the latter making targeting of tumor vasculature possible. CSPG4-specific monoclonal antibodies [36], radio-immunoconjugates [37], or immunotoxins [38,39,40] were already applied in animal models and melanoma patients, with partially promising results [41]. Other strategies to specifically eliminate CSPG4-positive targets include fusion proteins linking a CSPG4 binding domain name to soluble TRAIL (TNF-related apoptosis-inducing ligand) agonists to initiate cell death upon CSPG4 binding through the extrinsic apoptosis pathway [42]. T cells, virally transduced with a CSPG4-specific CAR, exerted potent cytotoxicity in response to various CSPG4-expressing tumors, such as melanoma, breast malignancy, mesothelioma, glioblastoma and osteosarcoma [43,44,45,46,47,48,49] in animal models or in vitro. Additionally, intracranial application of CSPG4-CAR T cells in a murine model Palmatine chloride of glioblastoma imposed efficient tumor control [50]. To circumvent concerns about potential on-target/off-tumor toxicities, we have previously exhibited that transient transfection of T cells with CSPG4-CARs using mRNA electroporation might be an effective and safe tool in cancer immunotherapy [51,52,53]. Using RNA-transfected CAR-T cells offers the advantage that this receptor expression is usually temporally restricted, rendering potential off-target and on-target/off-tumor toxicity transient as well. For safety reasons, an initial use of repetitive injections of RNA-transfected CSPG4-CAR-T cells may be beneficial to probe for toxicity. In the case of no serious side-effects, a switch to permanently transfected CSPG4-CAR-T cells may be conceivable. No CSPG4-specific CAR-T cells have been used in humans so far. Therefore, it was.