title: Improvement in soluble expression levels of a diabody by exchanging expression vectors.
abstract: Accumulating evidence suggests that bispecific antibody fragments (BsAbs) seem set to join monoclonal antibodies as powerful therapeutic and diagnostic agents, particularly for targeting cancer. One type of recombinant BsAbs are diabodies, which are constructed from heterogeneous single-chain antibodies and can increase antigen-specific cytotoxicity in T cells by cross-linking tumor antigens with T cell associated antigens. Some diabodies, however, cannot be solubly expressed in sufficient quantities, which limits their use in clinical therapeutics. Previously we constructed an anti-CD20 x CD3 diabody, which effectively directs the lytic potential of cytolytic T cells toward CD20(+) malignant B cells and shows marked antitumor efficacy in vivo. Here, to increase the amount of soluble product for clinical trials, we used an alternative expression vector under the T7 promoter but retained the stII signal sequences to ensure that the expressed protein is secreted to the periplasm of Escherichia coli. We achieved a periplasmic, soluble product by optimizing the conditions for induction with a yield of 8-9mg/L after affinity chromatography purification. This is nearly a five times greater yield than obtained with the previous vector. The diabodies generated from this modified vector retain dual binding specificity for both CD20-positive and CD3-positive cell lines. Taken together, these results suggest that changing expression vectors may be an alternative strategy to accomplish high-level expression of active BsAb proteins from E. coli.
authors.affiliation: State Key Laboratory of Experimental Hematology, Department of Pharmacy, Institute of Hematology & Hospital of Blood Diseases, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, PR China.
authors.forename: Fang
authors.initials: F
authors.lastname: Liu
authors.forename: Zhong
authors.initials: Z
authors.lastname: Chen
authors.forename: Wenguo
authors.initials: W
authors.lastname: Jiang
authors.forename: Chunzheng
authors.initials: C
authors.lastname: Yang
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Xiong
authors.forename: Zhenping
authors.initials: Z
authors.lastname: Zhu
mesh_terms: D018033:Antibodies, Bispecific / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D018951:Antigens, CD20 / Q000276:immunology / Q000378:metabolism; D001666:Binding Sites, Antibody; D017252:CD3 Complex / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D016393:Lymphoma, B-Cell / Q000276:immunology; D012995:Solubility
chemical_list: D018033:Antibodies, Bispecific; D018951:Antigens, CD20; D017252:CD3 Complex