TY - JOUR
T1 - Association behavior and control of the quality of cancer therapeutic bispecific diabodies expressed in Escherichia coli
AU - Nakazawa, Hikaru
AU - Onodera-Sugano, Tomoko
AU - Sugiyama, Aruto
AU - Tanaka, Yoshikazu
AU - Hattori, Takamitsu
AU - Niide, Teppei
AU - Ogata, Hiromi
AU - Asano, Ryutaro
AU - Kumagai, Izumi
AU - Umetsu, Mitsuo
N1 - Funding Information:
Funding: This work was supported by Scientific Research Grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan [H.N., grant numbers 16K18296 , 18K04842 ; M.U., 16H04570 , 16K14483 , and I.K., 24000011 ); and by Precursory Research for Embryonic Science and Technology from the Japan Science and Technology Agency (M.U.). The X-ray diffraction experiments were performed at the Photon Factory (proposals 18G050) and SPring-8 (proposals 2015A1124, 2015B2114, 2015B2117, and 2015B6524).
Funding Information:
Funding: This work was supported by Scientific Research Grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan [H.N. grant numbers 16K18296, 18K04842; M.U. 16H04570, 16K14483, and I.K. 24000011); and by Precursory Research for Embryonic Science and Technology from the Japan Science and Technology Agency (M.U.). The X-ray diffraction experiments were performed at the Photon Factory (proposals 18G050) and SPring-8 (proposals 2015A1124, 2015B2114, 2015B2117, and 2015B6524).
Publisher Copyright:
© 2020 Elsevier B.V.
PY - 2020/8/15
Y1 - 2020/8/15
N2 - Diabody 31, a bispecific therapeutic antibody, is a heterodimer comprised of two types of chimeric single-chain variable fragments (scFv), that has been identified as a clone with high cytotoxicity against cancer cells. Diabody 31 is inactive as a homodimer and monomer, yet active as a heterodimer in solution. Herein, we examined the association between two kinds of diabodies, LH- and HL type, based on the behavior of four kinds of their constituent chimera scFvs, by size analysis. More than half of the LH diabody fraction, including two types of chimeric scFV that were equally expressed, and nearly all HL diabody fraction components, including one chimeric HL-L scFv, were in inactive form and changed components dynamically as time passed. The association of these diabodies corresponded to that of the scFv chimera, indicating that analysis of these chimeras could predict diabody quality. Furthermore, addition of the deficient HL-R chimera scFv, comprised of an unbalanced chimeric scFv ratio, to the HL diabody fraction increased cancer cell cytotoxicity, with maximal effects obtained upon repeating the process five times. These findings provide insights into the efficient construction of a functional diabody by noting the characteristics of associated diabodies and the molar ratio of expressed chimera scFvs.
AB - Diabody 31, a bispecific therapeutic antibody, is a heterodimer comprised of two types of chimeric single-chain variable fragments (scFv), that has been identified as a clone with high cytotoxicity against cancer cells. Diabody 31 is inactive as a homodimer and monomer, yet active as a heterodimer in solution. Herein, we examined the association between two kinds of diabodies, LH- and HL type, based on the behavior of four kinds of their constituent chimera scFvs, by size analysis. More than half of the LH diabody fraction, including two types of chimeric scFV that were equally expressed, and nearly all HL diabody fraction components, including one chimeric HL-L scFv, were in inactive form and changed components dynamically as time passed. The association of these diabodies corresponded to that of the scFv chimera, indicating that analysis of these chimeras could predict diabody quality. Furthermore, addition of the deficient HL-R chimera scFv, comprised of an unbalanced chimeric scFv ratio, to the HL diabody fraction increased cancer cell cytotoxicity, with maximal effects obtained upon repeating the process five times. These findings provide insights into the efficient construction of a functional diabody by noting the characteristics of associated diabodies and the molar ratio of expressed chimera scFvs.
KW - Antibody
KW - Association
KW - Diabody
KW - Protein engineering
KW - Quality control
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U2 - 10.1016/j.bej.2020.107636
DO - 10.1016/j.bej.2020.107636
M3 - Article
AN - SCOPUS:85085143694
VL - 160
JO - Biochemical Engineering Journal
JF - Biochemical Engineering Journal
SN - 1369-703X
M1 - 107636
ER -
