Targeted Bioactivity of Membrane-Anchored TNF by an Antibody-Derived TNF Fusion Protein

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 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.

Single-Chain TNF, a TNF Derivative with Enhanced Stability and Antitumoral Activity

“…As targeting component, typically an Ab derivative against a tumor-associated Ag is used. Several studies have successfully detailed the creation of these rTNF proteins, strongly supporting the potential and viability of this approach (12)(13)(14). The main disadvantages and limitations for these TNF-fusion protein approaches are the obligation to create TNF trimers for activity, and a potential agonistic Ab response due to oligomerization.…”

Serological identification of breast cancer-related antigens from aSaccharomyces cerevisiae surface display library

“…In addition, the identification of cell membrane associated antigens opens the possibility to generate new antibody-derived targeting devices for the treatment of breast cancer. 51 …”

Chimeric form of tumor necrosis factor-α has enhanced surface expression and antitumor activity

“…22 Some investigators have devised strategies to mitigate the problems associated with the systemic release of soluble TNF through techniques such as hypothermic isolated limb perfusion of cells transduced in situ, 23,24 physically linking soluble TNF to the tumor-cell membrane, 25 using TNF in the form of a fusion molecule, 26,27 or by driving expression of TNF in transduced cells with weak, tissue-specific or radiation-inducible promoters. [27][28][29][30] In addition, others have used vectors encoding a truncated TNF that lacks the TACE cleavage site. [7][8][9][10] However, such strategies still have the potential to release soluble TNF into the systemic circulation.…”