Figure 1. Design and biophysical analysis of the humanization of tanezumab by LSM. (A) Schematic diagram depicting the development of tanezumab by cloning the mouse 911 hybridoma-derived antibody (1), identifying the CDRs and constructing a human framework template (2), humanizing and adjusting the template (3), employing library scanning mutagenesis (LSM) on the CDR3s (4) and merging mutations into the final antibody (5). The template affinity for NGF was KD 25 nM. Following CDR 1 and 2 optimizations, the intermediate affinity for NGF was 1 nM. LSM analysis was then combined on the intermediate for a final set of library screening to arrive at tanezumab (KD < 12 pM). (B) LSM histogram for each complete mutagenesis at each position in L3 except P95 and all residues in H3. Mutants were better off-rate than wild-type (w.t., red), same as w.t. (beige), worse than w.t. (light blue) or no binding (dark blue). The rectangular box around L3 K92 highlights a 100% permissive position. Asterisks (*) identify positions targeting for combination in the last round of LSM. (C) Tanezumab binding competition for NGF receptors, p75NTR (green) and TrkA (pink) by percent capture of NGF to biosensor chip coated with either receptor. (D) 911 binding competition for NGF receptors, p75NTR (green) and TrkA (pink) by percent capture of NGF to biosensor chip coated with either receptor. (E) Percent neuron survival as a function of increasing antibody concentration of either tanezumab (purple) or 911 (orange).