Construction and humanization of a functional bispecific EGFR × CD16 diabody using a refolding system

Abstract

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.

Heterodimeric Bispecific Single Chain Variable Fragments (scFv) Killer Engagers (BiKEs) Enhance NK-cell Activity Against CD133+ Colorectal Cancer Cells

“…Tetravalent diabodies as well as duovalent triplebodies show high anti-cancer reactivity [21-23]. Evidence grows revealing beneficial properties in treatment with scFv including lower immunogenicy, rapid blood clearance and improved tumor penetration compared to larger molecules with high efficacy in cancer therapy when targeting immune effectors like T-cells or NK-cells [21, 22]. …”

NK cells and cancer: you can teach innate cells new tricks

“…Given the potency of ADCC, many of these bispecific proteins were designed in order to provide stronger and more stable binding to CD16 than the Fc region of conventional antibodies, given their relatively low affinity for CD16. Various approaches, such as using CD16-targeting Fv domains with higher affinity 175 and tetravalent proteins with two binding sites for CD16 for added stability 176 , have proved successful in redirecting NK cell lysis towards tumour cells. Different tumour types have been targeted by linking these anti-CD16 Fv domains to Fv domains against CD19 (REF.…”

“…Different tumour types have been targeted by linking these anti-CD16 Fv domains to Fv domains against CD19 (REF. 177) and HLA class II for B cell malignancies 178 , CD30 for Hodgkin's lymphoma 176,179 , epidermal growth factor receptor (EGFR), which is overexpressed in several epithelial cancer types 175 , HER2 (REFS 180,181) for breast cancer, CD33 for AML 182,183 and EPCAM 184 for carcinomas. The in vitro and in vivo efficacy of these optimized proteins in eliciting specific antitumour activity is encouraging and should be further developed in clinical settings.…”

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

“…We previously reported the humanization of anti-EGFR IgG 528, which inhibits tumor growth by blocking the phosphorylation of a tyrosine kinase, and also reported the construction of recombinant antibodies based on humanized 528 (h528) Fv [13][14][15][16]. Here, we prepared scFv multimers using two types of h528 scFvs each with a different domain order, because the domain order of VH and VL in scFv may influence multimerization [8,9].…”