X‐ray structures of fragments from binding and nonbinding versions of a humanized anti‐CD18 antibody: Structural indications of the key role of V_H residues 59 to 65

Abstract

X-ray crystal structures of fragments from two different humanized anti-CD18 antibodies are reported. The Fv fragment of the nonbinding version has been refined in space group C2 with a = 64.2 A, b = 61.3 A, c = 51.8 A, and beta = 99 degrees to an R-value of 18.0% at 1.9 A, and the Fab fragment of the tight-binding version has been refined in space group P3 with a = 101. A and c = 45.5 A to an R-value of 17.8% at 3.0 A resolution. The very large difference in their binding affinity (> 1000-fold) is attributed to large and local structural differences in the C-terminal part of CDR-H2, and from this we conclude there is direct contact between this region and antigen when they combine. X-ray structures of antibody-antigen complexes available in the literature have yet to show this part of CDR-H2 in contact with antigen, despite its hypervariable sequence. Implications of this result for antibody humanization are discussed.

Antibody Humanization Using Monovalent Phage Display

“…Given this consideration, we feel that the general phage method described will usually suffice for the selection of tight binding humanized antibodies. Finally, the success of this humanization, in combination with previous results (13)(14)(15)25), again illustrates the feasibility of using a single human framework as a generic scaffold for humanized antibodies.Acknowledgments-We thank Marcus Ballinger for assistance with the oligonucleotide preassembly, James Bourell for mass spectrometric analyses, Allan Padua for amino acid analyses, and the oligonucleotide synthesis group. …”

“…Unfortunately, simple grafting of CDR sequences often yields humanized antibodies that bind antigen much more weakly than the parent murine mAb (4, 10 -16), and decreases in affinity of up to several hundredfold have been reported (13)(14)(15). To restore high affinity, the antibody must be further engineered to fine tune the structure of the antigen-binding loops.…”

“…For this reason, humanization of non-human antibodies is commonly referred to as CDR grafting. The low content of nonhuman sequence in humanized antibodies (ϳ5%) has proven effective in both reducing the immunogenicity and prolonging the serum half-life in humans (7,9).Unfortunately, simple grafting of CDR sequences often yields humanized antibodies that bind antigen much more weakly than the parent murine mAb (4, 10 -16), and decreases in affinity of up to several hundredfold have been reported (13)(14)(15). To restore high affinity, the antibody must be further engineered to fine tune the structure of the antigen-binding loops.…”

“…However, the method used in that work differs from that presented here in several key features. For reasons enunciated earlier, we used the same human V L I-V H III framework that has been used in previous antibody humanizations (13)(14)(15)25), whereas Rosok et al selected a novel human framework based on homology to the sequence of the parent murine mAb. Furthermore, we humanized the entire framework region (except for CDRs), then randomized only those framework residues that have been empirically found to be important to antigen binding.…”

“…This method has been successfully used to humanize a number of murine antibodies (13)(14)(15)25) using a framework derived from consensus sequences of the most abundant human subclasses, namely V L subgroup I (V L I) and V H subgroup III (V H III) (26). Use of the most common human V L and V H frameworks minimizes any potential immunogenicity of the humanized antibody and also eliminates possible idiosyncracies associated with any one particular framework.…”