Humanization by Resurfacing

Abstract

Different techniques have been developed to reduce the antigenicity of murine antibodies while preserving their affinity. Joining the variable domains of a murine antibody to the constant domains of a human antibody to construct a chimeric antibody was the first strategy to reduce antigenicity (Morrison et al. 1984; Boulianne et al. 1984). Although some successfully designed chimeric antibodies have been reported (Maloney 1999; Wagner et al. 2003; LoBuglio et al. 1989), the majority of them still induced pathologically relevant immune responses against the variable domains (Khazaeli et al. 1991). Since it has been shown that foreign framework regions (FRs) can evoke immune responses (Bruggemann et al. 1987), the next step in the development of humanized antibodies was the “grafting” of the complementarity determining regions (CDRs) on a human acceptor antibody (Jones et al. 1986; Verhoeyen et al. 1988). This procedure usually leads to a significant reduction or complete loss of binding affinity, because certain framework residues are important for maintaining the conformation of the CDRs (Foote and Winter 1992; Chothia et al. 1989) or are even directly involved in antigen binding (Mian et al. 1991). This problem could be solved by reintroducing murine residues into the human framework at positions that are deemed to be critical for CDR loop conformation (Co et al. 1991; Queen et al. 1989; Riechmann et al. 1988). Although Foote and Winter (1992) defined a “Vernier” zone of framework residues that are important for CDR loop conformation, the necessary mutations should nevertheless be determined experimentally for every antibody.