Build-up functionalization of anti-EGFR \xd7 anti-CD3 bispecific diabodies by integrating high-affinity mutants and functional molecular formats

Ryutaro Asano,Mai Okada,Katsuhiro Hosokawa,Kyoko Arai,Shota Konno,Hiromi Ogata,Masayoshi Onitsuka,Mitsuo Umetsu,Shintaro Taki,Takeshi Nakanishi,Izumi Kumagai,Takeshi Omasa,Ippei Shimomura

doi:10.1038/s41598-020-61840-3

Abstract

Designing non-natural antibody formats is a practical method for developing highly functional next-generation antibody drugs, particularly for improving the therapeutic efficacy of cancer treatments. One approach is constructing bispecific antibodies (bsAbs). We previously reported a functional humanized bispecific diabody (bsDb) that targeted epidermal growth factor receptor and CD3 (hEx3-Db). We enhanced its cytotoxicity by constructing an Fc fusion protein and rearranging order of the V domain. In this study, we created an additional functional bsAb, by integrating the molecular formats of bsAb and high-affinity mutants previously isolated by phage display in the form of Fv. Introducing the high-affinity mutations into bsDbs successfully increased their affinities and enhanced their cytotoxicity in vitro and in vivo. However, there were some limitations to affinity maturation of bsDb by integrating high-affinity Fv mutants, particularly in Fc-fused bsDb with intrinsic high affinity, because of their bivalency. The tetramers fractionated from the bsDb mutant exhibited the highest in vitro growth inhibition among the small bsAbs and was comparable to the in vivo anti-tumor effects of Fc-fused bsDbs. This molecule shows cost-efficient bacterial production and high therapeutic potential.

Highlights

Designing non-natural antibody formats is a practical method for developing highly functional nextgeneration antibody drugs, for improving the therapeutic efficacy of cancer treatments
Increased affinity was observed in LH-HY52W; additional improvements were not observed with the other hEx3-LH mutants, higher affinity mutants of h528 Fv (2HH11, 2HH11-2L1, and 2HH11-2L6) were used in their construction
BsAbs were the first non-natural antibody formats approved by the Food and Drug Administration (FDA); to date only one has been approved for the treatment of cancer

Summary

Introduction

Designing non-natural antibody formats is a practical method for developing highly functional nextgeneration antibody drugs, for improving the therapeutic efficacy of cancer treatments. We created an additional functional bsAb, by integrating the molecular formats of bsAb and high-affinity mutants previously isolated by phage display in the form of Fv. Introducing the high-affinity mutations into bsDbs successfully increased their affinities and enhanced their cytotoxicity in vitro and in vivo. To create additional functional bsAbs, we integrated the high-affinity h528 VH and VL mutants into hEx3-LH and Fc-fused hEx3-Dbs. The affinity of hEx3-LH was successfully improved by introducing mutations, which enhanced the cytotoxicity of the bsAbs in vitro and in vivo. A more sensitive in vivo model is needed to evaluate these molecules, we successfully developed a highly potent small bsDb by integrating a high-affinity mutant; the hEx3-LH mutant tetramer showed comparable in vivo therapeutic effects to Fc-fused hEx3-Dbs

Methods

Results

Conclusion