Journal
JOURNAL OF IMMUNOLOGY
Volume 165, Issue 12, Pages 7050-7057Publisher
AMER ASSOC IMMUNOLOGISTS
DOI: 10.4049/jimmunol.165.12.7050
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Due to their multispecificity and versatility, bispecific Abs (BsAbs) are promising therapeutic tools in tomorrow's medicine. Especially intermediate-sized BsAbs that combine body retention with tissue penetration are valuable for therapy but necessitate expression systems that favor heterodimerization of the binding sites for large-scale application. To identify heterodimerization domains to which single-chain variable fragments (scFv) can be fused, we compared the efficiency of heterodimerization of CL and CH1 constant domains with complete L and Ed chains in mammalian cells. We found that the isolated CL:CH1 domain interaction was inefficient for secretion of heterodimers. However, when the complete L and Ed chains, were used, secretion of L:Fd heterodimers was highly successful, Because these Fab chains contribute a binding moiety, C-terminal fusion of a scFv molecule to the L and/or Fd chains generated BsAbs or trispecific Abs (TsAbs) of intermediate size (75-100 kDa), These disulfide-stabilized bispecific Fab-scFv (bibody) and trispecific Fab-(scFv)(2) (tribody) heterodimers represent up to 90% of all secreted Ab fragments in the mammalian expression system and possess fully functional binding moieties, Furthermore, both molecules recruit and activate T cells in a tumor cell-dependent way, whereby the trispecific derivative can exert this activity to two different tumor cells. Thus we propose the use of the disulfide-stabilized L:Fd heterodimer as an efficient platform for production of intermediate-sized BsAbs and TsAbs in mammalian expression systems.
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