Abstract
Proviral integration site of Moloney virus-2 (PIM2) is overexpressed in multiple human cancer cells and high level is related to poor prognosis; thus, PIM2 kinase is a rational target of anti-cancer therapeutics. Several chemical inhibitors targeting PIMs/PIM2 or their downstream signaling molecules have been developed for treatment of different cancers. However, their off-target toxicity is common in clinical trials, so they could not be advanced to official approval for clinical application. Here, we produced human single-chain antibody fragments (HuscFvs) to PIM2 by using phage display library, which was constructed in a way that a portion of phages in the library carried HuscFvs against human own proteins on their surface with the respective antibody genes in the phage genome. Bacterial derived-recombinant PIM2 (rPIM2) was used as an antigenic bait to fish out the rPIM2-bound phages from the library. Three E. coli clones transfected with the HuscFv genes derived from the rPIM2-bound phages expressed HuscFvs that bound also to native PIM2 from cancer cells. The HuscFvs presumptively interact with the PIM2 at the ATP binding pocket and kinase active loop. They were as effective as small chemical drug inhibitor (AZD1208, which is an ATP competitive inhibitor of all PIM isoforms for ex vivo use) in inhibiting PIM kinase activity. The HuscFvs should be engineered into a cell-penetrating format and tested further towards clinical application as a novel and safe pan-anti-cancer therapeutics.
Highlights
The proviral integration site of Moloney murine leukemia virus proteins are kinases of the serine/threonine kinase family
The DNA was cloned into pLATE52 vector and the recombinant pLATE52-pim2 plasmid was put into NiCo21 (DE3) E. coli
Development of anti-PIMs/Proviral integration site of Moloney virus-2 (PIM2) agents that are biocompatible to humans for long-term treatment of cancers through repeated administration is rational. We offer another ramification of the PIM kinase inhibition, i.e., fully human single-chain antibodies directed to the residues critical for the PIMs/PIM2 kinase activity
Summary
The proviral integration site of Moloney murine leukemia virus proteins (acronymPIMs) are kinases of the serine/threonine kinase family. A single pim transcript gives rise to three PIM2 variants of molecular masses 34, 37 and 40 kDa due to in-frame alternative translation initiation sites; the three variants share an identical catalytic/kinase domain (residues 32–286) but differ at their N-termini [4]. The intracellularly expressed PIM2 is constitutively active regardless of cytokines or mitogenic signals [5]. PIM2 mediates survival signaling through phosphorylation of several pro-apoptotic proteins resulting in arrest of cell death. PIM2 phosphorylates BAD (Bcl-2 associated agonist of cell death) and reverses the pro-apoptotic property of BAD, preventing cell death [7]. PIM2 phosphorylates c-Myc to increase c-Myc stability and transcriptional activity [8].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
