Multimerization of anti‐(epidermal growth factor receptor) IgG fragments induces an antitumor effect: the case for humanized 528 scFv multimers

Abstract

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.

Anti‐EGFR scFv tetramer (tetrabody) with a stable monodisperse structure, strong anticancer effect, and a long in vivo half‐life

“…Here, we evaluated the inhibitory effect of HLG0 tetramer on the growth of tumor cells, and compared it with those of an h528 scFvs monomer, dimer, and trimer, and also with those of panitumumab and cetuximab using the MTS assay. Although HLG1 dimer and HLG0 trimer both inhibited cancer growth more than the HLG3 monomer, which was consistent with previous results [17], HLG0 tetramer had the greatest overall inhibitory effect (Fig. 4A).…”

“…Gel‐filtration chromatography after immobilized metal ion affinity chromatography purification showed that HLG1 prepared from ICS fraction predominantly formed dimers, as did the HLG1 prepared previously from secretion fraction (bacterial supernatant plus periplasmic fraction) (Fig. A) . The final yield of HLG1 dimer prepared from ICS fraction was 0.70 mg·L ‐1 culture, which was fourfold of that obtained from secretion fraction.…”

“…Next, we applied our ICS preparation method to the production of HLG0 trimer and tetramer because we previously confirmed that HLG0 prepared from secretion fraction predominantly formed trimers and that h528 scFv multimers inhibited cancer growth in a multimerization‐dependent manner . Two equivalent peaks, which were attributed to the trimer and tetramer species, were observed on the gel‐filtration chromatogram (Fig.…”

“…In contrast, anti‐EGFR scFv multimers, including the tetramers presented in this study, exert their functions in a precise, multimerization‐dependent manner, suggesting that all of the possible binding sites of the anti‐EGFR scFv multimers are active and that EGFR is an ideal target for specific scFv multimer‐based therapeutics. Note, however, that differences in the domain order of anti‐EGFR scFv multimers did affect their function , and therefore different results are likely to be observed with other anti‐EGFR antibody clones.…”

Novel EGFR-specific immunotoxins based on panitumumab and cetuximab show in vitro and ex vivo activity against different tumor entities

“…Single-chain fragment variable (scFvs) antibodies comprise the immunoglobulin variable domains of the parent mAb joined by a flexible polypeptide linker, and these smaller molecules (~30 kDa) achieve better tumor penetration (Asano et al 2013;Kampmeier et al 2010). Fusion proteins based on scFvs and truncated toxins derived from plants or bacteria result in recombinant immunotoxins (ITs), these are promising tools for targeted cancer treatment (Pastan et al 2007).…”