| Abstract: |
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 IgGI 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. |
| Keywords: |
immunohistochemistry; signal transduction; controlled study; protein expression; unclassified drug; human cell; sequence deletion; nonhuman; animal cell; mouse; animals; mice; animal tissue; apoptosis; tumor necrosis factor receptor 1; tumor necrosis factor receptor 2; tumor markers, biological; culture medium; cell line; animal experiment; animal model; membrane proteins; antineoplastic activity; drug effect; xenograft model antitumor assays; cell line, tumor; mice, inbred balb c; hybrid protein; antigens, neoplasm; recombinant fusion proteins; serine endopeptidases; tumor necrosis factor-alpha; immunoglobulin g; recombinant tumor necrosis factor; endothelium, vascular; antigen recognition; cytotoxicity, immunologic; neutrophils; dimerization; protein structure, tertiary; tumor necrosis factor receptor; hydrogen peroxide; neoplasm transplantation; immunoglobulin g1; macrophage; oxidation-reduction; tumor necrosis factor; protein antibody; thromboplastin; solubility; binding sites, antibody; dose time effect relation; dimer; target organ; immunoglobulin fc fragment; guanosine phosphate; recombinant dna technology; humans; human; male; female; priority journal; article; fibroblast activating protein antibody
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