Targeted bioactivity of membrane-anchored TNF by an antibody-derived TNF fusion protein.
We describe the generation and characterization of a fusion protein consisting of a humanized anti-fibroblast-activating protein (anti-FAP) Ab and human TNF replacing the IgG1 CH2/CH3 Fc domain. The construct was generated by recombinant DNA technology and preserved its IgG1-derived dimeric structure with the TNF molecule linked as a dimer. Expression in CHO cells was optimized in serum-free medium under GMP conditions to achieve production levels up to 15 mg/liter. Recognition of the FAP Ag by the construct was as good as that by the parental anti-FAP Ab. TNF signaling was induce able via both TNF receptor types. When acting in solution, the Ab-linked TNF dimer exhibited a 10- to 20-fold lower activity compared with recombinant trimeric TNF. However, after binding to FAP-expressing cells, immobilized anti-FAP-TNF dimer was equivalent to membrane-anchored TNF with regard to bioactivity. Amplification of TNF-related pathways by mimicking the membrane-integrated TNF signaling was detectable in various systems, such as apoptosis induction or tissue factor production. The difference in TNF receptor type 1 and 2 signaling by the anti-FAP-TNF construct correlated well with its Ag-bound or -soluble status. Translating the approach into a xenograft animal model (BALB/c nu/nu mice), we demonstrated low toxicity with measurable antitumor efficacy for the TNF fusion protein after i.v. application. Immunohistochemical analysis of tumor sections showed restricted TNF-mediated macrophage recruitment to the targeted tissue in a time- and dose-dependent manner. These data warrant transfer of the anti-FAP-TNF immunocytokine into clinical trials for the treatment of FAP-positive tumors.
Medical Department I, Universität des Saarlandes, Homburg/Saar, Germany.
Stefan
S
Bauer
Nicole
N
Adrian
Barbara
B
Williamson
Con
C
Panousis
Natalie
N
Fadle
Joanna
J
Smerd
Ilknur
I
Fettah
Andrew M
AM
Scott
Michael
M
Pfreundschuh
Christoph
C
Renner
D000818:Animals; D000951:Antigens, Neoplasm / Q000276:immunology* / Q000378:metabolism; D001666:Binding Sites, Antibody / Q000235:genetics; D014408:Biomarkers, Tumor / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D003602:Cytotoxicity, Immunologic / Q000235:genetics; D019281:Dimerization; D010450:Endopeptidases; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D018093:Gelatinases; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007074:Immunoglobulin G / Q000008:administration & dosage / Q000235:genetics* / Q000378:metabolism* / Q000633:toxicity; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009368:Neoplasm Transplantation; D009504:Neutrophils / Q000378:metabolism; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D017384:Sequence Deletion; D012697:Serine Endopeptidases / Q000276:immunology* / Q000378:metabolism; D012995:Solubility; D013925:Thromboplastin / Q000096:biosynthesis; D014409:Tumor Necrosis Factor-alpha / Q000008:administration & dosage / Q000235:genetics* / Q000378:metabolism* / Q000633:toxicity; D023041:Xenograft Model Antitumor Assays; D000099167:Fibroblast Activation Protein Alpha
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D010450:Endopeptidases; D018093:Gelatinases; D006861:Hydrogen Peroxide; D007074:Immunoglobulin G; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D013925:Thromboplastin; D014409:Tumor Necrosis Factor-alpha; D000099167:Fibroblast Activation Protein Alpha