title: Antibody engineering of a cytotoxic monoclonal antibody 84 against human embryonic stem cells: Investigating the effects of multivalency on cytotoxicity.
abstract: Antibody fragments have shown targeted specificity to their antigens, but only modest tissue retention times in vivo and in vitro. Multimerization has been used as a protein engineering tool to increase the number of binding units and thereby enhance the efficacy and retention time of antibody fragments. In this work, we explored the effects of valency using a series of self-assembling polypeptides based on the GCN4 leucine zipper multimerization domain fused to a single-chain variable fragment via an antibody upper hinge sequence. Four engineered antibody fragments with a valency from one to four antigen-binding units of a cytotoxic monoclonal antibody 84 against human embryonic stem cells (hESC) were constructed. We hypothesized that higher cytotoxicity would be observed for fragments with increased valency. Flow cytometry analysis revealed that the trimeric and tetrameric engineered antibody fragments resulted in the highest degree of cytotoxicity to the undifferentiated hESC, while the engineered antibody fragments were observed to have improved tissue penetration into cell clusters. Thus, a trade off was made for the trimeric versus tetrameric fragment due to improved tissue penetration. These results have direct implications for antibody-mediated removal of undifferentiated hESC during regenerative medicine and cell therapy.
authors.affiliation: Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, 138668, Singapore; Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, 117576, Singapore.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Klement
authors.affiliation: Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, 138668, Singapore; NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 28 Medical Drive, #05-01, 117456, Singapore.
authors.forename: Jiyun
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, 138668, Singapore.
authors.forename: Chengcheng
authors.initials: C
authors.lastname: Liu
authors.affiliation: Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, 138668, Singapore.
authors.forename: Heng-Liang
authors.initials: HL
authors.lastname: Tan
authors.affiliation: Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, 138668, Singapore.
authors.forename: Victor Vai Tak
authors.initials: VV
authors.lastname: Wong
authors.affiliation: Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, 138668, Singapore; Department of Biomedical Engineering, National University of Singapore, 9 Engineering Drive 1, 117575, Singapore.
authors.forename: Andre Boon-Hwa
authors.initials: AB
authors.lastname: Choo
authors.affiliation: Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, 138668, Singapore; Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, 117576, Singapore; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 28 Medical Drive, 117456, Singapore. Electronic address: cheld@nus.edu.sg.
authors.forename: Dong-Yup
authors.initials: DY
authors.lastname: Lee
authors.affiliation: Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, 138668, Singapore. Electronic address: dave_ow@bti.a-star.edu.sg.
authors.forename: Dave Siak-Wei
authors.initials: DS
authors.lastname: Ow
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D000915:Antibody Affinity; D000941:Antigens / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003601:Cytotoxicity Tests, Immunologic; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D000066449:Human Embryonic Stem Cells / Q000187:drug effects* / Q000276:immunology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies
keywords: Antibody fragments; Avidity; Multimerization; Self-assembling polypeptides; hESC therapy; scFv