title: Cytotoxic enhancement of a bispecific diabody by format conversion to tandem single-chain variable fragment (taFv): the case of the hEx3 diabody.
abstract: Diabodies (Dbs) and tandem single-chain variable fragments (taFv) are the most widely used recombinant formats for constructing small bispecific antibodies. However, only a few studies have compared these formats, and none have discussed their binding kinetics and cross-linking ability. We previously reported the usefulness for cancer immunotherapy of a humanized bispecific Db (hEx3-Db) and its single-chain format (hEx3-scDb) that target epidermal growth factor receptor and CD3. Here, we converted hEx3-Db into a taFv format to investigate how format affects the function of a small bispecific antibody; our investigation included a cytotoxicity assay, surface plasmon resonance spectroscopy, thermodynamic analysis, and flow cytometry. The prepared taFv (hEx3-taFv) showed an enhanced cytotoxicity, which may be attributable to a structural superiority to the diabody format in cross-linking target cells but not to differences in the binding affinities of the formats. Comparable cross-linking ability for soluble antigens was observed among hEx3-Db, hEx3-scDb, and hEx3-taFv with surface plasmon resonance spectroscopy. Furthermore, drastic increases in cytotoxicity were found in the dimeric form of hEx3-taFv, especially when the two hEx3-taFv were covalently linked. Our results show that converting the format of small bispecific antibodies can improve their function. In particular, for small bispecific antibodies that target tumor and immune cells, a functional orientation that avoids steric hindrance in cross-linking two target cells may be important in enhancing the growth inhibition effect.
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai 980-8579, Japan.
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Asano
authors.forename: Keiko
authors.initials: K
authors.lastname: Ikoma
authors.forename: Ippei
authors.initials: I
authors.lastname: Shimomura
authors.forename: Shintaro
authors.initials: S
authors.lastname: Taki
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nakanishi
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Umetsu
authors.forename: Izumi
authors.initials: I
authors.lastname: Kumagai
mesh_terms: D018033:Antibodies, Bispecific / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D017252:CD3 Complex / Q000276:immunology*; D003603:Cytotoxins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D066246:ErbB Receptors / Q000276:immunology*; D006801:Humans; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D018033:Antibodies, Bispecific; D000970:Antineoplastic Agents; D017252:CD3 Complex; D003603:Cytotoxins; D057127:Single-Chain Antibodies; C512478:EGFR protein, human; D066246:ErbB Receptors