Abstract
Agonistic antibodies, which bind specifically to death receptor 5 (DR5), can trigger apoptosis in tumor cells through the extrinsic pathway. In this present study, we describe the use of a phage display to isolate a novel fully human agonistic single chain fragment variable (scFv) antibody, which targets DR5. After five rounds of panning a large (1.2 × 108 clones) phage display library on DR5, a total of over 4000 scFv clones were screened by the phage ELISA. After screening for agonism in a cell-viability assay in vitro, a novel DR5-specific scFv antibody TR2-3 was isolated, which inhibited COLO205 and MDA-MB-231 tumor cell growth without any cross-linking agents. The activity of TR2-3 in inducing apoptosis in cancer cells was evaluated by using an Annexin V-PE apoptosis detection kit in combination with flow cytometry and the Hoechst 33342 and propidium iodide double staining analysis. In addition, the activation of caspase-dependent apoptosis was evaluated by Western blot assays. The results indicated that TR2-3 induced robust apoptosis of the COLO205 and MDA-MB-231 cells in a dose-dependent and time-dependent manner, while it remarkably upregulated the cleavage of caspase-3 and caspase-8. Furthermore, TR2-3 suppressed the tumor growth significantly in the xenograft model. Taken together, these data suggest that TR2-3 exhibited potent antitumor activity both in vitro and in vivo. This work provides a novel human antibody, which might be a promising candidate for cancer therapy by targeting DR5.
Highlights
Apoptosis is a process of programmed cell death event, which eliminates harmful cells from the body in order to maintain tissue integrity [1]
We selected fully human anti-death receptor 5 (DR5) antibodies from the phage display library
DR5 as a critical component of initiating the extrinsic apoptotic pathway was mainly expressed in cancer cells and rarely in normal cells
Summary
Apoptosis is a process of programmed cell death event, which eliminates harmful cells from the body in order to maintain tissue integrity [1]. The second is the extrinsic pathway, which is initiated by death receptors belonging to the tumor necrosis factor receptor (TNF) receptor superfamily [2,3,4]. The TNF-related apoptosis-inducing ligand (TRAIL) is a type-II membrane protein, which initiates apoptosis in a wide variety of human cancer cell lines, but not in most normal human cells [5]. AAfftteerr beeiinngg puurriififieeddbbyy mmeettaall aafffifinniittyycchhrroommaattooggrraapphhyyaanndd ssiizzee--eexxcclluussiioonn chrroommaattooggrraapphhyy,,tthheessDDRR55pprrootteeiinnwwaass cchhaarraacctteerriizzeedd bbyy SSDDSS––PPAAGGEEaannddCCoooommaassssiieeBBlluueessttaaiinniinngg..TThhee rreessuullttsssshhoowweeddtthhaatt tthhee ssDDRR55 pprrootteeiinn wwaass hhiigghhllyy ppuurree ((>>90%) with an apparreenntt mmoolleeccuullaarr wweeiigghhtt ooffaabboouutt1166kkDDaa((FFiigguurree11aa)). Construction of Single Chain Fragment Variable (scFv) Phage Display Library. A total 110 independent transformations generated a scFv phage display library with a repertoire size of 1.2 × 108
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