Humanization of a murine monoclonal antibody by simultaneous optimisation of framework and CDR residues

Optimal protein function often depends on co-operative interactions between amino acid residues distant in the protein primary sequence yet spatially near one another following protein folding. For example, antibody affinity is influenced by interactions of framework residues with complementarity-de termining region (CDR) residues. However, despite the abundance of antibody structural information and computational tools the humanization of rodent antibodies for clinical use often results in a significant loss of affinity . To date, antibody engineering efforts have focused either on optimizing C DR residues involved in antigen binding or on optimizing antibody framework residues that serve critical roles in preserving the conformation of CDRs. In the present study a new approach which permits the rapid identification of co-operatively interacting framework and CDR residues was used to simul taneously humanize and optimize a murine antibody directed against CD40. Sp ecifically, a combinatorial library that examined eight potentially importa nt framework positions concomitantly with focused CDR libraries consisting of variants containing random single amino acid mutations in the third CDR of the heavy and light chains was expressed. Multiple anti-CD40 Fab variant s containing as few as one murine framework residue and displaying up to ap proximate to 500-fold higher affinity than the initial chimeric Fab were id entified. The higher affinity humanized variants demonstrated a cooperative interaction between light chain framework residue Y49 and heavy chain CDR3 residue R/K101 (coupling energy, Delta G(I) = 0.9 kcal/mol). Screening of combinatorial framework-CDR libraries permits identification of monoclonal antibodies (mAb) with structures optimized for function, including instance s in which the antigen induces conformational changes in the mAb. Moreover, the enhanced humanized variants contain fewer murine framework residues an d could not be identified by sequential in vitro humanization and affinity muturation strategies. This approach to identifying co-operatively interact ing residues is not restricted to antibody-antigen interactions and consequ ently, may be used broadly to gain insight into protein structure-function relationships, including proteins that serve as catalysts. (C) 1999 Academi c Press.