Probing the binding mechanism and affinity of tanezumab, a recombinant humanized anti-NGF monoclonal antibody, using a repertoire of biosensors.
We describe the use of four complementary biosensors (Biacore 3000, Octet QK, ProteOn XPR36, and KinExA 3000) in characterizing the kinetics of human nerve growth factor (NGF) binding to a humanized NGF-neutralizing monoclonal antibody (tanezumab, formerly known as RN624). Tanezumab is a clinical candidate as a therapy for chronic pain. Our measurements were consistent with the NGF/tanezumab binding affinity being tighter than 10 pM due to the formation of an extremely stable complex that had an estimated half-life exceeding 100 h, which was beyond the resolution of any of our methods. The system was particularly challenging to study because NGF is an obligate homodimer, and we describe various assay orientations and immobilization methods that were used to minimize avidity in our experiments while keeping NGF in as native a state as possible. We also explored the interactions of NGF with its natural receptors, TrkA and P75, and how tanezumab blocks them. The Biacore blocking assay that we designed was used to quantify the potency of tanezumab and is more precise and reproducible than the currently available cell-based functional assays.
Abdiche
Yasmina Noubia
YN
Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080, USA. Yasmina.abdiche@pfizer.com
Malashock
Dan Stephen
DS
Pons
Jaume
J
D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001667:Binding, Competitive; D015374:Biosensing Techniques / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D020932:Nerve Growth Factor / Q000276:immunology / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011485:Protein Binding; D020917:Receptor, trkA / Q000378:metabolism; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism
D000911:Antibodies, Monoclonal; C484065:NGFR protein, human; D009419:Nerve Tissue Proteins; D017475:Receptors, Nerve Growth Factor; D020932:Nerve Growth Factor; D020917:Receptor, trkA