Restoration of membrane TNF-like activity by cell surface targeting and matrix metalloproteinase-mediated processing of a TNF prodrug.
Tumor necrosis factor (TNF) prodrugs are fusion proteins comprised of an N-terminal single-chain antibody variable fragment (scFv) targeting a TNF effector and a C-terminal TNF receptor (TNFR)1-derived inhibitor module. Introduction of matrix metalloproteinase (MMP)-2 recognition motifs between TNF and the TNFR1 fragment allowed activation by recombinant MMP-2 and MMP-expressing HT1080 cells. Processing by endogeneous MMPs required specific membrane binding of the TNF prodrug via the targeting scFv, ensuring strictly antigen-dependent activation. Interestingly, TNF bioactivity of the processed prodrug was approximately 1000-fold higher upon scFv-mediated targeting, and signaled juxtatropic cell death also to antigen-negative cells. Microscopical analyses of TNFR2 clustering and TNF receptor-associated factor 2 recruitment at contact sites to adjacent cells revealed the formation of stable TNFR complexes by target-bound, processed prodrug, resembling the increased signal capacity of natural, membrane-expressed TNF. MMP-2-sensitive TNF prodrugs represent novel cytokine-based reagents for targeted cancer therapy, which should be exploitable for MMP-overexpressing tumors.
Gerspach
J
J
1Institute of Cell Biology and Immunology, University of Stuttgart, Germany.
Müller
D
D
Münkel
S
S
Selchow
O
O
Nemeth
J
J
Noack
M
M
Petrul
H
H
Menrad
A
A
Wajant
H
H
Pfizenmaier
K
K
D014408:Biomarkers, Tumor / Q000032:analysis; D015153:Blotting, Western; D016923:Cell Death; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects*; D005434:Flow Cytometry; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D011355:Prodrugs / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
D014408:Biomarkers, Tumor; D011355:Prodrugs; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D010447:Peptide Hydrolases; D020778:Matrix Metalloproteinase 2