Krebs, Simon K.Stech, MarlittJorde, FelixRakotoarinoro, NathanaëlRamm, FranziskaMarinoff, SophieBahrke, SvenDanielczyk, AntjeWüstenhagen, Doreen A.Kubick, Stefan2023-01-192023-01-192022-11-081661-6596https://depositonce.tu-berlin.de/handle/11303/18022https://doi.org/10.14279/depositonce-16814Recombinant immunotoxins (RITs) are an effective class of agents for targeted therapy in cancer treatment. In this article, we demonstrate the straight-forward production and testing of an anti-CD7 RIT based on PE24 in a prokaryotic and a eukaryotic cell-free system. The prokaryotic cell-free system was derived from Escherichia coli BL21 StarTM (DE3) cells transformed with a plasmid encoding the chaperones groEL/groES. The eukaryotic cell-free system was prepared from Chinese hamster ovary (CHO) cells that leave intact endoplasmic reticulum-derived microsomes in the cell-free reaction mix from which the RIT was extracted. The investigated RIT was built by fusing an anti-CD7 single-chain variable fragment (scFv) with the toxin domain PE24, a shortened variant of Pseudomonas Exotoxin A. The RIT was produced in both cell-free systems and tested for antigen binding against CD7 and cell killing on CD7-positive Jurkat, HSB-2, and ALL-SIL cells. CD7-positive cells were effectively killed by the anti-CD7 scFv-PE24 RIT with an IC50 value of 15 pM to 40 pM for CHO and 42 pM to 156 pM for E. coli cell-free-produced RIT. CD7-negative Raji cells were unaffected by the RIT. Toxin and antibody domain alone did not show cytotoxic effects on either CD7-positive or CD7-negative cells. To our knowledge, this report describes the production of an active RIT in E. coli and CHO cell-free systems for the first time. We provide the proof-of-concept that cell-free protein synthesis allows for on-demand testing of antibody–toxin conjugate activity in a time-efficient workflow without cell lysis or purification required.en540 Chemie und zugeordnete Wissenschaftencell-free protein synthesisCFPScell-free expressionCFERITPseudomonas Exotoxin AGroEL/GroESDnaK/DnaJ/GrpEtrigger factorArticle2022-12-071422-0067