ISOLATION OF HIGH-AFFINITY MONOMERIC HUMAN ANTI-C-ERBB-2 SINGLE-CHAINFV USING AFFINITY-DRIVEN SELECTION

The use of antibodies to target tumor antigens has had limited success , partially due to the large size of IgG molecules, difficulties in co nstructing smaller single chain Fv (scFv) antibody fragments, and immu nogenicity of murine antibodies. These limitations can be overcome by selecting human scFv directly from non-immune or semi-synthetic phage antibody libraries; however, the affinities are typically too low for therapeutic application. For hapten antigens, higher-affinity scFv can be isolated from phage antibody libraries where the V-H and V-L genes of a binding scFv are replaced with repertoires of V genes (chain shu ffling). The applicability of this approach to protein binding scFv is unknown. For this work, chain shuffling was used to increase the affi nity of a non-immune human scFv, which binds the glycoprotein tumor an tigen c-erbB-2 with an affinity of 1.6 x 10(-8) M. The affinity of the parental scFv was increased sixfold (K-d = 2.5 x 10(-9) M) by light-c hain shuffling and fivefold (K-d = 3.1 x 10(-9)M) by heavy-chain shuff ling, values comparable to those for antibodies against the same antig en produced by hybridomas. When selections were performed on antigen i mmobilized on polystyrene, spontaneously dimerizing scFv were isolated , the best of which had only a slightly lower K-d than wild type (K-d = 1.1 x 10(-8) M). These scFv dimerize on phage and are preferentially selected as a result of increased avidity Compared to scFv which form ed only monomer, dimerizing scFv had mutations located at the V-H-V-L interface, suggesting that V-H-V-L complementarity determines the exte nt of dimerization. Higher-affinity monomeric scFv were only obtained by selecting in solution using limiting concentrations of biotinylated antigen, followed by screening mutant scFv from bacterial periplasm b y k(off) in a BIAcore. Using the proper selection and screening condit ions, protein binding human scFv with affinities comparable to murine hybridomas can be produced without immunization. (C) 1996 Academic Pre ss Limited