The construction of antibody fragments has the potential to reduce the high cost of therapeutic antibody production, but the structures of these fragments, with monovalency and the lack of an Fc region, can lead to reduced function. Multimerization is one strategy for recovering function that also yields better tumor-to-blood ratios than IgGs or monomeric antibody fragments because of rapid tumor uptake and clearance. Here, we constructed single-chain variable fragment (scFv) multimers by modifying the linker length and domain order of humanized anti-(epidermal growth factor receptor) IgG 528 (h528) and tested their ability to inhibit tumor growth. h528 scFv multimers, expressed using a bacterial expression system, were successfully fractionated and inhibited cancer growth in a multimerization-dependent manner, whereas the h528 scFv monomer showed no inhibition. h528 scFv trimers with variable heavy-light domain order and no linkers showed the highest in vitro and in vivo antitumor effects, which were comparable with those of the approved anti-(epidermal growth factor receptor) therapeutic IgG Cetuximab and Panitumumab. The trimers were also structurally stable in vitro and in vivo, which may be attributable to a strong interaction between the variable heavy and variable light domains of h528 Fv. Thus, h528 scFv multimers, especially trimers, are attractive as the next generation of anti-(epidermal growth factor receptor) therapeutic IgG and offer the possibility of low-cost production.

The development of single‐chain variable fragments (scFvs) as therapeutic agents has the potential to reduce the high cost of antibody production, but the development process often impairs scFv functions such as binding affinity and pharmacokinetics. Multimerization is one strategy for recovering or enhancing these lost functions. Previously, we constructed several antiepidermal growth factor receptor ( EGFR ) scFv multimers by modifying linker length and domain order. Antitumor effects comparable with those of the currently approved anti‐ EGFR therapeutic antibodies were observed for scFv trimers. In the present study, we fractionated an anti‐ EGFR scFv tetramer from the intracellular soluble fraction of an Escherichia coli transformant. Compared with the trimer, the tetramer showed higher affinity, greater cancer cell growth inhibition, and prolonged blood retention time. Furthermore, the tetramer did not dissociate into the trimer or other smaller species during long‐term storage (up to 33 weeks). Thus, our developed scFv tetramer is an attractive candidate next‐generation anti‐ EGFR therapeutic antibody that can be produced via a low‐cost bacterial expression system.