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Characterization of scFv-Ig Constructs Generated from the Anti-CD20 mAb 1F5 Using Linker Peptides of Varying Lengths¹

Daming Shan^2,*, Oliver W. Press^*,,, Theta T. Tsu^3,§, Martha S. Hayden^3,§ and Jeffrey A. Ledbetter^3,§

Departments of ^* Biological Structure and Medicine, University of Washington, Seattle, WA 98195; Fred Hutchinson Cancer Research Center, Seattle, WA 98104; and ^§ Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA 98121

The heavy (V_H) and light (V_L) chain variable regions of the murine anti-human CD20 mAb 1F5 were cloned, and four single-chain Ab (scFv) molecules were constructed using linker peptides of variable lengths to join the V_H and V_L domains. Three constructs were engineered using linker peptides of 15, 10, and 5 aa residues consisting of (GGGGS)₃, (GGGGS)₂, and (GGGGS)₁ sequences, respectively, whereas the fourth was prepared by joining the V_H and V_L domains directly. Each construct was fused to a derivative of human IgG1 (hinge plus CH2 plus CH3) to facilitate purification using staphylococcal protein A. The aggregation and CD20 binding properties of these four 1F5 scFv-Ig derivatives produced were investigated. Both size-exclusion HPLC column analysis and Western blots of proteins subjected to nonreducing SDS-PAGE suggested that all four 1F5 scFv-Ig were monomeric with m.w. of 55 kDa. The CD20 binding properties of the four 1F5 scFv-Ig were studied by ELISA and flow cytometry. The 1F5 scFv-Ig with the 5-aa linker (GS1) demonstrated significantly superior binding to CD20-expressing target cells, compared with the other scFv-Ig constructs. Scatchard analysis of the radiolabeled monovalent GS1 scFv-Ig revealed a binding avidity of 1.35 x 10⁸ M^-1 compared with an avidity of 7.56 x 10⁸ M^-1 for the native bivalent 1F5 Ab. These findings suggest that the GS1 scFv-Ig with a short linker peptide of 5 aa is the best of the engineered constructs for future studies.

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