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"Superhumanized" Antibodies: Reduction of Immunogenic Potential by Complementarity-Determining Region Grafting with Human Germline Sequences: Application to an Anti-CD28¹

Philip Tan^*, David A. Mitchell^2,, Timothy N. Buss^*, Margaret A. Holmes^*, Claudio Anasetti and Jefferson Foote^3,*,

Divisions of ^* Human Biology and Clinical Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and Department of Immunology, University of Washington, Seattle, WA 98195

Humanized Abs are created by combining, at the genetic level, the complementarity-determining regions of a murine mAb with the framework sequences of a human Ab variable domain. This leads to a functional Ab with reduced immunogenic side effects in human therapy. In this study, we report a new approach to humanizing murine mAbs that may reduce immunogenicity even further. This method is applied to humanize the murine anti-human CD28 Ab, 9.3. The canonical structures of the hypervariable loops of murine 9.3 were matched to human genomic V gene sequences whose hypervariable loops had identical or similar canonical structures. Framework sequences for those human V genes were then used, unmodified, with the 9.3 complementarity-determining regions to construct a humanized version of 9.3. The humanized 9.3 and a chimeric 9.3 control were expressed in Escherichia coli as Fab. The humanized Fab showed a moderate loss in avidity in a direct binding ELISA with immobilized CD28-Ig fusion protein (CD28-Ig). Humanized 9.3 blocked ligation of CD28-Ig to cells expressing the CD28 receptor CD80. Lastly, the humanized 9.3 showed biological activity as an immunosuppressant by inhibiting a MLR.

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