TY - JOUR
T1 - In vitro selection of bispecific diabody fragments using covalent bicistronic DNA display
AU - Nakayama, Masanao
AU - Komiya, Shoko
AU - Fujiwara, Kei
AU - Horisawa, Kenichi
AU - Doi, Nobuhide
N1 - Funding Information:
We thank Takeshi Sumida and Hiroshi Yanagawa for early contributions to this project. This research was supported in part by a Grant-in-Aid for Scientific Research ( 19360377 , 22360351 , 25289298 , 15K12545 , 15H01543 and 16H05291 ) from the JSPS or MEXT of Japan, by the Mochida Memorial Foundation for Medical and Pharmaceutical Research , and by the Strategic Research Foundation Grant-aided Project for Private Universities ( S1411003 ) from the MEXT of Japan.
Publisher Copyright:
© 2016 Elsevier Inc.
PY - 2016/9/16
Y1 - 2016/9/16
N2 - Bispecific antibodies with two different antigen-binding sites have been widely used for a variety of medical applications. The activity and stability of antibody fragments can be improved by in vitro evolution. Although the affinity and stability of small bispecific antibody fragments such as diabodies can be further optimized by in vitro display technologies, cell-free display of bispecific antibody fragments has not been reported. In this study, we applied a covalent bicistronic DNA display for the in vitro selection of heterodimeric diabodies. First, we confirmed the antigen-binding activities of a diabody synthesized by an in vitro transcription and translation system. However, when we performed DNA-display selection of a model diabody library in a proof-of-principle experiment, no enrichment of the diabody gene was observed, likely due to a low yield of the diabody heterodimer. To overcome this issue, we introduced cysteine residues at the VH-VL interface of the diabody heterodimer. Using the disulfide-stabilized diabodies, we successfully enriched the diabody gene from a model library. Our results indicate that the covalent bicistronic DNA display technique could be useful for improving the stability and affinity of bispecific diabody fragments.
AB - Bispecific antibodies with two different antigen-binding sites have been widely used for a variety of medical applications. The activity and stability of antibody fragments can be improved by in vitro evolution. Although the affinity and stability of small bispecific antibody fragments such as diabodies can be further optimized by in vitro display technologies, cell-free display of bispecific antibody fragments has not been reported. In this study, we applied a covalent bicistronic DNA display for the in vitro selection of heterodimeric diabodies. First, we confirmed the antigen-binding activities of a diabody synthesized by an in vitro transcription and translation system. However, when we performed DNA-display selection of a model diabody library in a proof-of-principle experiment, no enrichment of the diabody gene was observed, likely due to a low yield of the diabody heterodimer. To overcome this issue, we introduced cysteine residues at the VH-VL interface of the diabody heterodimer. Using the disulfide-stabilized diabodies, we successfully enriched the diabody gene from a model library. Our results indicate that the covalent bicistronic DNA display technique could be useful for improving the stability and affinity of bispecific diabody fragments.
KW - Bispecific antibody
KW - Cell-free protein synthesis
KW - Display technology
KW - Disulfide stabilization
KW - PURE system
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U2 - 10.1016/j.bbrc.2016.07.113
DO - 10.1016/j.bbrc.2016.07.113
M3 - Article
C2 - 27473655
AN - SCOPUS:84995546063
SN - 0006-291X
VL - 478
SP - 606
EP - 611
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 2
ER -