Construction of bispecific antibodies by specific pairing between the heavy chain and the light chain using removable SpyCatcher/SnoopCatcher units

Jyunna Yoshida; Yuki Kato; Ai Isogawa; Yoshikazu Tanaka; Izumi Kumagai; Ryutaro Asano; Takeshi Nakanishi; Koki Makabe

doi:10.1186/s13036-024-00454-z

Construction of bispecific antibodies by specific pairing between the heavy chain and the light chain using removable SpyCatcher/SnoopCatcher units

J Biol Eng. 2024 Oct 14;18(1):57. doi: 10.1186/s13036-024-00454-z.

Authors

Jyunna Yoshida¹, Yuki Kato², Ai Isogawa³, Yoshikazu Tanaka⁴, Izumi Kumagai², Ryutaro Asano², Takeshi Nakanishi³, Koki Makabe⁵

Affiliations

¹ Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jyonan, Yonezawa, Yamagata, 992-8510, Japan.
² Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan.
³ Department of Chemistry and Bioengineering, Division of Science and Engineering for Materials, Chemistry and Biology, Graduate School of Engineering, Osaka Metropolitan University, Sugimoto 3-3-138, Sumiyoshi-ku, Osaka, 558-8585, Japan.
⁴ Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, 980-8577, Japan.
⁵ Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jyonan, Yonezawa, Yamagata, 992-8510, Japan. makabe@yz.yamagata-u.ac.jp.

Abstract

During the production of bispecific antibodies (bsAbs), nonspecific pairing results in low yields of target bsAb molecules, an issue known as the "mispairing problem." Several antibody engineering techniques have been developed to overcome mispairing issues. Here, we introduce "bsAb by external pairing and excision" (BAPE), a novel chain pairing method that induces specific chain pairing by fusing external SpyCatcher/Tag and SnoopCatcher/Tag units. These tags are then removed via protease cleavage. In this study, we applied this method to force the correct pairings of heavy and light chains while the heavy-chain pairing was achieved by the Knobs-into-Holes mutation. We then confirmed the formation of interchain bridges with covalent isopeptide bonds. Both anti-CD3/anti-Her2 and anti-CD3/anti-EGFR bsAbs displayed satisfactory target binding activities and in vitro cell-killing activity with activated T-cells.

Keywords: Bispecific antibody; Cancer; Chain pairing; SnoopCatcher; SpyCatcher.

Abstract

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