Synthesis of an Anti-CD7 Recombinant Immunotoxin Based on PE24 in CHO and E. coli Cell-Free Systems

Simple item page
dc.contributor.authorKrebs, Simon K.
dc.contributor.authorStech, Marlitt
dc.contributor.authorJorde, Felix
dc.contributor.authorRakotoarinoro, Nathanaël
dc.contributor.authorRamm, Franziska
dc.contributor.authorMarinoff, Sophie
dc.contributor.authorBahrke, Sven
dc.contributor.authorDanielczyk, Antje
dc.contributor.authorWüstenhagen, Doreen A.
dc.contributor.authorKubick, Stefan
dc.date.accessioned2023-01-19T14:48:48Z
dc.date.available2023-01-19T14:48:48Z
dc.date.issued2022-11-08
dc.date.updated2022-12-07T13:00:02Z
dc.description.abstractRecombinant 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.
dc.description.sponsorshipBMBF, 031B0078A, Basistechnologien: Zellfreie Systeme für die Funktionsanalyse transmembranärer Proteine (SysMem2016), Teilprojekt A
dc.description.sponsorshipBMBF, 031B0831C, Maßgeschneiderte Inhaltsstoffe 2 - Verbundvorhaben: "CEFOX - Teilprojekt C"
dc.identifier.eissn1422-0067
dc.identifier.issn1661-6596
dc.identifier.urihttps://depositonce.tu-berlin.de/handle/11303/18022
dc.identifier.urihttps://doi.org/10.14279/depositonce-16814
dc.language.isoen
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subject.ddc540 Chemie und zugeordnete Wissenschaftende
dc.subject.othercell-free protein synthesis
dc.subject.otherCFPS
dc.subject.othercell-free expression
dc.subject.otherCFE
dc.subject.otherRIT
dc.subject.otherPseudomonas Exotoxin A
dc.subject.otherGroEL/GroES
dc.subject.otherDnaK/DnaJ/GrpE
dc.subject.othertrigger factor
dc.titleSynthesis of an Anti-CD7 Recombinant Immunotoxin Based on PE24 in CHO and E. coli Cell-Free Systems
dc.typeArticle
dc.type.versionpublishedVersion
dcterms.bibliographicCitation.articlenumber13697
dcterms.bibliographicCitation.doi10.3390/ijms232213697
dcterms.bibliographicCitation.issue22
dcterms.bibliographicCitation.journaltitleInternational Journal of Molecular Sciences
dcterms.bibliographicCitation.originalpublishernameMDPI
dcterms.bibliographicCitation.originalpublisherplaceBasel
dcterms.bibliographicCitation.volume23
dcterms.rightsHolder.referenceCreative-Commons-Lizenz
tub.accessrights.dnbfree
tub.affiliationFak. 3 Prozesswissenschaften::Inst. Biotechnologie::N/A (Not Applicable)
tub.publisher.universityorinstitutionTechnische Universität Berlin

Files

Original bundle
Now showing 1 - 2 of 2
Thumbnail Image
Name:
ijms-23-13697.pdf
Size:
7.51 MB
Format:
Adobe Portable Document Format
Download
No Thumbnail Available
Name:
ijms-23-13697-s001.zip
Size:
2.65 MB
Format:
ZIP archive format.
Download
License bundle
Now showing 1 - 1 of 1
No Thumbnail Available
Name:
license.txt
Size:
4.86 KB
Format:
Item-specific license agreed upon to submission
Description:
Download

Collections

Publications