Fab antibody fragment-functionalized liposomes for specific targeting of antigen-positive cells.

Liposomes functionalized with monoclonal antibodies or their antigen-binding fragments have attracted much attention as specific drug delivery devices for treatment of various diseases including cancer. The conjugation of antibodies to liposomes is usually achieved by covalent coupling using cross-linkers in a reaction that might adversely affect the characteristics of the final product. Here we present an alternative strategy for liposome functionalization: we created a recombinant Fab antibody fragment genetically fused on its C-terminus to the hydrophobic peptide derived from pulmonary surfactant protein D, which became inserted into the liposomal bilayer during liposomal preparation and anchored the Fab onto the liposome surface. The Fab-conjugated liposomes specifically recognized antigen-positive cells and efficiently delivered their cargo, the Alexa Fluor 647 dye, into target cells in vitro and in vivo. In conclusion, our approach offers the potential for straightforward development of nanomedicines functionalized with an antibody of choice without the need of harmful cross-linkers.

Soluble M6P/IGF2R released by TACE controls angiogenesis via blocking plasminogen activation.

RATIONALE: The urokinase plasminogen activator (uPA) system is among the most crucial pericellular proteolytic systems associated with the processes of angiogenesis. We previously identified an important regulator of the uPA system in the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R). OBJECTIVE: Here, we wanted to clarify whether and how did the soluble form of M6P/IGF2R (sM6P/IGF2R) contribute to modulation of the uPA system. METHODS AND RESULTS: By using specific inhibitors and RNA interference, we show that the tumor necrosis factor α convertase (TACE, ADAM-17) mediates the release of the ectodomain of M6P/IGF2R from human endothelial cells. We demonstrate further that sM6P/IGF2R binds plasminogen (Plg) and thereby prevents Plg from binding to the cell surface and uPA, ultimately inhibiting in this manner Plg activation. Furthermore, peptide 18-36 derived from the Plg-binding site of M6P/IGF2R mimics sM6P/IGF2R in the inhibition of Plg activation and blocks cancer cell invasion in vitro, endothelial cell invasion in vivo, and tumor growth in vivo. CONCLUSIONS: The interaction of sM6P/IGF2R with Plg may be an important regulatory mechanism to inhibit migration of cells using the uPA/uPAR system.

Large-scale production and characterization of novel CD4+ cytotoxic T cells with broad tumor specificity for immunotherapy.

Immune-cell-based approaches using cytotoxic and dendritic cells are under constant scrutiny to design novel therapies for the treatment of tumors. These strategies are hampered by the lack of efficient and economical large-scale production methods for effector cells. Here we describe the propagation of large amounts of a unique population of CD4(+) cytotoxic T cells, which we termed tumor killer T cells (TKTC), because of their potent and broad antitumor cell activity. With this cultivation strategy, TKTCs from peripheral blood mononuclear cells are generated within a short period of time using a pulse with a stimulating cell line followed by continuous growth in serum-free medium supplemented with a mixture of interleukin-2 and cyclosporin A. Expression and functional profiling did not allow a classification of TKTCs to any thus far defined subtype of T cells. Cytotoxic assays showed that TKTCs kill a panel of tumor targets of diverse tissue origin while leaving normal cells unaffected. Blocking experiments revealed that TKTC killing was, to a significant extent, mediated by tumor necrosis factor-related apoptosis-inducing ligand and was independent of MHC restriction. These results suggest that TKTCs have a high potential as a novel tool in the adoptive immunotherapy of cancer.