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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.

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.

We previously reported the construction and activity of a humanized, bispecific diabody (hEx3) that recruited T cells towards an epidermal growth factor receptor (EGFR) positive tumor. Herein, we describe the construction of a second functional, fully humanized, anti-EGFR bispecific diabody that recruits another subset of lymphocyte effectors, the natural killer cells, to EGFR-expressing tumor cells. After we confirmed that an anti-EGFR × anti-CD16 bispecific diabody (Ex16) consisting of a previously humanized anti-EGFR variable fragment (Fv) and a mouse anti-CD16 Fv had growth inhibitory activity, we designed a humanized anti-CD16 Fv to construct the fully humanized Ex16 (hEx16). However, the humanized form had lower activity for inhibition of cancer growth. To restore its growth inhibitory activity, we introduced mutations into the Vernier zone, which is located near the complementarity-determining regions and is involved in their binding activity. We efficiently prepared 15 different hEx16 mutants by expressing each chimeric single-chain component for hEx16 separately. We then used our in vitro refolding system to select the most functional mutant, which had a growth inhibitory effect comparable with that of the commercially available chimeric anti-EGFR antibody, cetuximab. Our refolding system could aid in the efficient optimization of other proteins with heterodimeric structure.

Antibodies and their fragments are attractive binding proteins because their high binding strength is generated by several hypervariable loop regions, and because high-quality libraries can be prepared from the vast gene clusters expressed by mammalian lymphocytes. Recent explorations of new genome sequences and protein structures have revealed various small, nonantibody scaffold proteins. Accurate structural descriptions of protein-protein interactions based on X-ray and NMR analyses allow us to generate binding proteins by using grafting and library techniques. Here, we review approaches for generating binding proteins from small scaffold proteins on the basis of tertiary structural information. Identification of binding sites from visualized tertiary structures supports the transfer of function by peptide grafting. The local library approach is advantageous as a go-between technique for grafted foreign peptide sequences and small scaffold proteins. The identification of binding sites also supports the construction of efficient libraries with a low probability of denatured variants, and, in combination with the design for library diversity, opens the way to increasing library density and randomized sequence lengths without decreasing density. Detailed tertiary structural analyses of protein-protein complexes allow accurate description of epitope locations to enable the design of and screening for multispecific, high-affinity proteins recognizing multiple epitopes in target molecules.

We previously reported the use of a humanized bi-specific diabody that targets epidermal growth factor receptor and CD3 (hEx3-Db) for cancer immunotherapy. Bacterial expression can be used to express small recombinant antibodies on a large scale; however, their overexpression often results in the formation of insoluble aggregates, and in most cases artificial affinity peptide tags need to be fused to the antibodies for purification by affinity chromatography. Here, we propose a novel method for preparing refined, functional, tag-free bi-specific diabodies from IgG-like bi-specific antibodies (BsAbs) in a mammalian expression system. We created an IgG-like BsAb in which bi-specific diabodies were fused to the human Fc region via a designed human rhinovirus 3C (HRV3C) protease recognition site. The BsAb was purified by protein A affinity chromatography, and the refined tag-free hEx3-Db was efficiently produced from the Fc fusion format by protease digestion. The tag-free hEx3-Db from the Fc fusion format showed a greater inhibition of cancer growth than affinity-tagged hEx3-Db prepared directly from Chinese hamster ovary cells. We also applied our novel method to another small recombinant antibody fragment, hEx3 single-chain diabody (hEx3-scDb), and demonstrated the versatility and advantages of our proposed method compared with papain digestion of hEx3-scDb. This approach may be used for industrial-scale production of functional tag-free small therapeutic antibodies.

Recombinant proteins are often expressed in the form of insoluble inclusion bodies in bacteria. To facilitate refolding of recombinant proteins obtained from inclusion bodies, 0.1 to 1 M arginine is customarily included in solvents used for refolding the proteins by dialysis or dilution. In addition, arginine at higher concentrations, e.g., 0.5-2 M, can be used to extract active, folded proteins from insoluble pellets obtained after lysing Escherichia coli cells. Moreover, arginine increases the yield of proteins secreted to the periplasm, enhances elution of antibodies from Protein-A columns, and stabilizes proteins during storage. All these arginine effects are apparently due to suppression of protein aggregation. Little is known, however, about the mechanism. Various effects of solvent additives on proteins have been attributed to their preferential interaction with the protein, effects on surface tension, or effects on amino acid solubility. The suppression of protein aggregation by arginine cannot be readily explained by either surface tension effects or preferential interactions. In this review we show that interactions between the guanidinium group of arginine and tryptophan side chains may be responsible for suppression of protein aggregation by arginine.