Monovalent TNF receptor 1-selective antibody with improved affinity and neutralizing activity |
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Authors: | Fabian Richter Kirstin A. Zettlitz Oliver Seifert Andreas Herrmann Peter Scheurich Klaus Pfizenmaier |
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Affiliation: | 1. Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany;2. Stuttgart Research Center Systems Biology, University of Stuttgart, Stuttgart, Germany;3. Baliopharm, Basel, Switzerland |
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Abstract: | Selective inhibition of tumor necrosis factor (TNF) signaling through the proinflammatory axis of TNF-receptor 1 (TNFR1) while leaving pro-survival and regeneration-promoting signals via TNFR2 unaffected is a promising strategy to circumvent limitations of complete inhibition of TNF action by the approved anti-TNF drugs. A previously developed humanized antagonistic TNFR1-specific antibody, ATROSAB, showed potent inhibition of TNFR1-mediated cellular responses. Because the parental mouse antibody H398 possesses even stronger inhibitory potential, we scrutinized the specific binding parameters of the two molecules and revealed a faster dissociation of ATROSAB compared to H398. Applying affinity maturation and re-engineering of humanized variable domains, we generated a monovalent Fab derivative (13.7) of ATROSAB that exhibited increased binding to TNFR1 and superior inhibition of TNF-mediated TNFR1 activation, while lacking any agonistic activity even in the presence of cross-linking antibodies. In order to improve its pharmacokinetic properties, several Fab13.7-derived molecules were generated, including a PEGylated Fab, a mouse serum albumin fusion protein, a half-IgG with a dimerization-deficient Fc, and a newly designed Fv-Fc format, employing the knobs-into-holes technology. Among these derivatives, the Fv13.7-Fc displayed the best combination of improved pharmacokinetic properties and antagonistic activity, thus representing a promising candidate for further clinical development. |
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Keywords: | tumor necrosis factor tumor necrosis factor receptor 1 neutralizing antibody monovalent antibody inflammatory diseases affinity maturation antibody humanization |
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