Characterization of scFv-Ig constructs generated from the anti-CD20 mAb 1F5 using linker peptides of varying lengths

The heavy (V-H) and light (V-L) chain variable regions of the murine anti-h uman CD20 mAb 1F5 were cloned, and four single-chain Ab (scFv) molecules we re constructed using linker peptides of variable lengths to join the V-H an d 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 hu man IgG1 (hinge plus CH2 plus CH3) to facilitate purification using staphyl ococcal protein A. The aggregation and CD20 binding properties of these fou r 1F5 scFv-Ig derivatives produced were investigated. Both size-exclusion H PLC 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 si milar to 55 kDa, The CD20 binding properties of the four 1F5 scFv-Ig were s tudied by ELISA and how cytometry, The 1F5 scFv-Ig with the 5-aa linker (GS 1) demonstrated significantly superior binding to CD20-expressing target ce lls, compared with the other scFv-Ig constructs. Scatchard analysis of the radiolabeled monovalent GS1 scFv-Ig revealed a binding avidity of 1.35 x 1( 8) M-1 compared with an avidity of 7.56 x 10(8) M-1 for the native bivalent 1F5 Ab, These findings suggest that the GS1 scFv-Ig with a short linker pe ptide of similar to 5 aa is the best of the engineered constructs for futur e studies.