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Bacterial production and structure-functional validation of a recombinant antigen-binding fragment (Fab) of an anti-cancer therapeutic antibody targeting epidermal growth factor receptor.
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- Author(s): Kim JH;Kim JH; Sim DW; Sim DW; Park D; Park D; Jung TG; Jung TG; Jung TG; Lee S; Lee S; Oh T; Oh T; Ha JR; Ha JR; Seok SH; Seok SH; Seo MD; Seo MD; Seo MD; Kang HC; Kang HC; Kim YP; Kim YP; Won HS; Won HS
- Source:
Applied microbiology and biotechnology [Appl Microbiol Biotechnol] 2016 Dec; Vol. 100 (24), pp. 10521-10529. Date of Electronic Publication: 2016 Jul 28.- Publication Type:
Journal Article- Language:
English - Source:
- Additional Information
- Source: Publisher: Springer International Country of Publication: Germany NLM ID: 8406612 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1432-0614 (Electronic) Linking ISSN: 01757598 NLM ISO Abbreviation: Appl Microbiol Biotechnol Subsets: MEDLINE
- Publication Information: Original Publication: Berlin ; New York : Springer International, c1984-
- Subject Terms: Structure-Activity Relationship*; Antineoplastic Agents/*metabolism ; Cetuximab/*metabolism ; ErbB Receptors/*antagonists & inhibitors ; Escherichia coli/*metabolism ; Immunoglobulin Fab Fragments/*metabolism ; Recombinant Proteins/*metabolism; Antineoplastic Agents/chemistry ; Cell Line, Tumor ; Cetuximab/chemistry ; Cetuximab/genetics ; Crystallography, X-Ray ; Escherichia coli/genetics ; Humans ; Immunoglobulin Fab Fragments/chemistry ; Immunoglobulin Fab Fragments/genetics ; Mass Spectrometry ; Protein Binding ; Protein Conformation ; Protein Engineering ; Recombinant Proteins/chemistry ; Recombinant Proteins/genetics
- Abstract: Fragment engineering of monoclonal antibodies (mAbs) has emerged as an excellent paradigm to develop highly efficient therapeutic and/or diagnostic agents. Engineered mAb fragments can be economically produced in bacterial systems using recombinant DNA technologies. In this work, we established recombinant production in Escherichia coli for monovalent antigen-binding fragment (Fab) adopted from a clinically used anticancer mAB drug cetuximab targeting epidermal growth factor receptor (EGFR). Recombinant DNA constructs were designed to express both polypeptide chains comprising Fab in a single vector and to secrete them to bacterial periplasmic space for efficient folding. Particularly, a C-terminal engineering to confer an interchain disulfide bond appeared to be able to enhance its heterodimeric integrity and EGFR-binding activity. Conformational relevance of the purified final product was validated by mass spectrometry and crystal structure at 1.9 Å resolution. Finally, our recombinant cetuximab-Fab was found to have strong binding affinity to EGFR overexpressed in human squamous carcinoma model (A431) cells. Its binding ability was comparable to that of cetuximab. Its EGFR-binding affinity was estimated at approximately 0.7 nM of Kd in vitro, which was quite stronger than the binding affinity of natural ligand EGF. Hence, the results validate that our construction could serve as an efficient platform to produce a recombinant cetuximab-Fab with a retained antigen-binding functionality.
- Contributed Indexing: Keywords: Antibody fragment engineering; Bacterial production; Cetuximab; EGFR; Recombinant Fab; Therapeutic antibody
- Accession Number: 0 (Antineoplastic Agents)
0 (Immunoglobulin Fab Fragments)
0 (Recombinant Proteins)
EC 2.7.10.1 (ErbB Receptors)
PQX0D8J21J (Cetuximab) - Publication Date: Date Created: 20160730 Date Completed: 20170124 Latest Revision: 20181202
- Publication Date: 20221213
- Accession Number: 10.1007/s00253-016-7717-z
- Accession Number: 27470143
- Source:
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