ABSTRACT
BACKGROUND: Targeted therapy of human cancers is an attractive approach and has been investigated with limited success. We have developed novel cytotoxic agents for targeted therapy of human cancers based on the extracellular cytotoxicity domain of CD178 (FasL) and the specificity offered by single chain antibodies (scFv) against dominant human tumor Ag TAG-72 (cc49scFv) and TAL6 (L6scFv). RESULTS: The cc49scFv-FasLext is highly effective in in vitro killing of human TAG-72+ Jurkat-Ras tumor cells with a 30,000 fold greater cytotoxicity as compared to soluble FasL (sFasL). On the other hand, L6scFv-FasLext only increased cytotoxicity 500-fold as compared with sFasL against TAL6+ HeLa cells in in vitro assays. The high specificity and strong cytotoxicity of cc49scFv-FasLext made it feasible to cure IP-implanted Jurkat-Ras tumors in SCID mice. CONCLUSION: Our study demonstrated that scFv-FasLext with a strong cytotoxicity against sensitive human tumor targets may be useful as effective chemotherapeutic agents.
Subject(s)
Fas Ligand Protein/genetics , Neoplasms/drug therapy , Single-Chain Antibodies/genetics , Animals , Antineoplastic Agents/immunology , Antineoplastic Agents/pharmacology , Fas Ligand Protein/pharmacology , HeLa Cells , Humans , Immunoglobulin Fragments/immunology , Jurkat Cells , Mice , Mice, SCID , Neoplasms/pathology , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/therapeutic use , Single-Chain Antibodies/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
The CD25(+)Foxp3(+) regulatory T-cells (Treg) that had lost CD25 and Foxp3 in vivo (ex-Treg) exist but are difficult to study. We generated antigen (Ag)-specific Treg hybridomas from iTreg clones (iTreg-hyb) using iTreg of DO11.10.Foxp3-GFP mice and presented evidence that they behave like ex-Treg. The iTreg-hyb displayed little CD25 and Foxp3-GFP but strong expression could be induced with OVA(323-339) in the presence of Ag-presenting cells, rIL-2 and rTGF-ß1. They displayed all of the iTreg-associated markers examined except CTLA-4, the latter was also absent in the ex-Treg. They lacked the Helios transcription factor, suggesting they were derived from iTreg. Similar to ex-Treg, the iTreg-hyb produced high level of IL-2 and Foxp3 under specific activation conditions. Two unusual properties were observed. First, the ability to induce Foxp3-GFP upon activation is progressively lost in culture over a period of 2-4 weeks. Second, Rag2(-/-) spleen cells alone selectively induced Foxp3-GFP expression albeit 30 times less efficient than Ag-specific activation. We identified cell-free supernatant, IL-6, IL-9, and IL-27 as Foxp3-inducing factors. Our study has significant implications to the stability, plasticity and fate of Treg. The usefulness and limitation of iTreg-hyb as a novel tool to study Foxp3 regulation and the fate of specific Treg subsets are discussed.
