title: Bi-specific antibodies with high antigen-binding affinity identified by flow cytometry.
abstract: Using conventional approaches, the antigen-binding affinity of a novel format of bi-specific antibody (BsAb) cannot be determined until purified BsAb is obtained. Here, we show that new lipoprotein A (NlpA)-based bacteria display technology, combined with flow cytometry (FCM), can be used to detect antigen-binding affinity of BsAbs, in the absence of expression and purification work. Two formats of BsAb, scFv2-CH/CL and Diabody-CH/CL, specific for human interleukin 1β (hIL-1β) and human interleukin 17A (hIL-17A), were constructed and displayed in Escherichia coli using NlpA-based bacteria display technology. Conversion of these cells to spheroplasts, and their incubation with fluorescently conjugated antigens resulted in the selective labeling of spheroplasts expressing BsAb; enabling their antigen-binding affinity to be analyzed with FCM. The association and dissociation of BsAbs for binding to hIL-1β and hIL-17A were analyzed using FCM-based assays. The results showed that antigen-binding affinity of Diabody-CH/CL was significantly higher than that of scFv2-CH/CL. To confirm these results of FCM-based assays, BsAbs were expressed, purified and subjected to relative affinity measurements, in vitro and in vivo bioactivity analysis. The results showed that Diabody-CH/CL had greater relative affinities for both antigens, resulting in better blocking bioactivities on cellular level and effects on alleviating joint inflammation, and cartilage destruction and bone damage in collagen induced arthritis (CIA) mice model. These results indicate that BsAbs with good antigen-binding affinity can be identified by FCM-based assays without expression and purification work, and the indentified BsAb can serve as a lead compound for further drug development.
authors.affiliation: Heilongjiang River Fishery Research Institute CAFS, Harbin 150070, China.
authors.forename: Liming
authors.initials: L
authors.lastname: Xu
authors.affiliation: Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, CAAS, Harbin 150001, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Biopharmaceutical Laboratories, Northeast Agricultural University, Harbin 150030, China.
authors.forename: Qiuying
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Heilongjiang River Fishery Research Institute CAFS, Harbin 150070, China.
authors.forename: Jingzhuang
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Heilongjiang River Fishery Research Institute CAFS, Harbin 150070, China.
authors.forename: Miao
authors.initials: M
authors.lastname: Liu
authors.affiliation: Biopharmaceutical Laboratories, Northeast Agricultural University, Harbin 150030, China.
authors.forename: Mo
authors.initials: M
authors.lastname: Guo
authors.affiliation: Harbin Pharmaceutical Groups Bioengineering Co., Harbin 150025, China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: College of Biological Science and Technology, HeiLongJiang BaYi Agricultural University, DaQing 163319, China.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Cao
authors.affiliation: Biopharmaceutical Laboratories, Northeast Agricultural University, Harbin 150030, China.
authors.forename: Qingcui
authors.initials: Q
authors.lastname: Li
authors.affiliation: Biopharmaceutical Laboratories, Northeast Agricultural University, Harbin 150030, China.
authors.forename: Guiping
authors.initials: G
authors.lastname: Ren
authors.affiliation: Biopharmaceutical Laboratories, Northeast Agricultural University, Harbin 150030, China. Electronic address: deshanli@163.com.
authors.forename: Deshan
authors.initials: D
authors.lastname: Li
mesh_terms: D041721:3T3-L1 Cells; D000818:Animals; D018033:Antibodies, Bispecific / Q000097:blood / Q000276:immunology* / Q000493:pharmacokinetics / Q000627:therapeutic use; D000941:Antigens / Q000276:immunology*; D001169:Arthritis, Experimental / Q000097:blood / Q000188:drug therapy / Q000276:immunology* / Q000473:pathology; D001842:Bone and Bones / Q000473:pathology; D002356:Cartilage / Q000473:pathology; D005434:Flow Cytometry; D033023:Foot Joints / Q000473:pathology; D006367:HeLa Cells; D006801:Humans; D020381:Interleukin-17 / Q000276:immunology*; D053583:Interleukin-1beta / Q000276:immunology*; D008297:Male; D051379:Mice; D008811:Mice, Inbred DBA
chemical_list: D018033:Antibodies, Bispecific; D000941:Antigens; C508717:IL17A protein, human; D020381:Interleukin-17; D053583:Interleukin-1beta
keywords: Bacteria display technology; Bi-specific antibody; Collagen induced arthritis; Flow cytometry; Rheumatoid arthritis