Structure-Activity Profiles of Ab-Derived TNF Fusion Proteins

Abstract

TNF application in humans is limited by severe side effects, including life-threatening symptoms of shock. Therefore, TNF can be successfully applied as a tumor therapeutic reagent only under conditions that prevent its systemic action. To overcome this limitation, genetic fusion of TNF to tumor-selective Abs is a favored strategy to increase site-specific cytokine targeting. Because wild-type TNF displays its bioactivity as noncovalently linked homotrimer, the challenge is to define structural requirements for a TNF-based immunokine format with optimized structure-activity profile. We compared toxicity and efficacy of a dimerized CH2/CH3 truncated IgG1-TNF fusion protein and a single-chain variable fragment-coupled TNF monomer recognizing fibroblast-activating protein. The former construct preserves its dimeric structure stabilized by the natural disulfide bond IgG1 hinge region, while the latter trimerizes under native conditions. Analysis of complex formation of wild-type TNF and of both fusion proteins with TNFR type 1 (TNF-R1) using surface plasmon resonance correlated well with in vitro and in vivo toxicity data. There is strong evidence that TNF subunits in a trimeric state display similar toxicity profiles despite genetic fusion to single-chain variable fragment domains. However, LD50 of either immunodeficient BALB/c nu/nu or immunocompetent BALB/c mice was significantly decreased following administration of TNF in the formation of IgG1-derived dimeric fusion protein. Reduction of unspecific peripheral complexation of TNF-R1 resulted in higher anticancer potency by immunotargeting of fibroblast-activating protein-expressing xenografts. The broader therapeutic window of the IgG1-derived TNF fusion protein favors the dimeric TNF-immunokine format for systemic TNF-based tumor immunotherapy.

Targeted therapy of renal cell carcinoma: Synergistic activity of cG250‐TNF and IFNg

“…The cG250-based TNF fusion protein was designed in analogy to a previously generated IgG-TNF construct allowing for efficient systemic TNF administration. 13,33 Intensity of TNF-R1 mediated signaling in vitro using the cytotoxicity assay system was comparable with the well-known range of the formerly characterized dimeric TNF-construct. 13 Overall, cG250-TNF displays bioactivity in tumoricidal effector systems in vitro like induction of respiratory burst and triggering of endothelial cell injury.…”

“…13 Abandoning the natural homotrimeric symmetry of TNF resulted in significantly reduced toxicity as seen both in immunodeficient and immunocompetent mouse strains. 33 The dimeric IgG-TNF molecules displayed significantly stronger antitumor activity in-vivo than wild type TNF or trimeric TNF-antibody conjugates. Dose escalation increased the therapeutic index of IgG-TNF and repeated administration additionally delayed tumor growth with tolerable side effects.…”

“…Initial studies revealed that the toxicity profile of cG250-TNF were analogous to our previously defined LD 50 of dimeric-TNF-fusion proteins such as anti-FAP-TNF for BALB/c nu/nu mice. 33 Tumorbearing mice treated with 30 lg of rhTNF all died within 24 hr and could not be challenged with a second injection. Mice treated with 100 lg of cG250-TNF showed some signs of stress but all completed treatment without any lethal side effects.…”

Radioimmunotherapy of Fibroblast Activation Protein Positive Tumors by Rapidly Internalizing Antibodies

“…For example, an antibody-maytansinoid conjugate (26) and antibody-TNF fusion proteins (27,28) have shown promise at the preclinical stage and are pending clinical development.…”

Antitumor activity of TNF-αafter intratumoral injection using anin situthermosensitive hydrogel

“…Thus, TNF-a is described as a double-edged sword 8 and the clinical application has been a challenge. Nevertheless, to date tremendous efforts have been made to improve the antitumor value and reduce the occurrence of systemic toxicity, including the following strategies: (a) development of regional administration techniques such as isolated limb perfusion (ILP) and isolated limb infusion (ILI), which were introduced to exert TNF-a's advantage in the treatment of metastatic melanoma and soft tissue sarcoma confined to a limb because of safety and excellent response rates 9 , (b) increase of antitumor potency by conjugation to macromolecules such as gelatin 10 , polyvinyl pyrrolidone (PVP) 11 and polyethylene glycol (PEG) [12][13][14][15] , and creat various TNF mutants 16,17 , (c) construction of targeting fusion proteins based on DNA recombination technology [18][19][20][21][22][23][24][25][26][27][28][29][30] and (d) design of targeting nanoparticle delivery system [31][32][33][34] .…”