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Production of bispecific antibodies in "knobs-into-holes" using a cell-free expression system.

ABSTRACT: Bispecific antibodies have emerged in recent years as a promising field of research for therapies in oncology, inflammable diseases, and infectious diseases. Their capability of dual target recognition allows for novel therapeutic hypothesis to be tested, where traditional mono-specific antibodies would lack the needed mode of target engagement. Among extremely diverse architectures of bispecific antibodies, knobs-into-holes (KIHs) technology, which involves engineering CH3 domains to create either a "knob" or a "hole" in each heavy chain to promote heterodimerization, has been widely applied. Here, we describe the use of a cell-free expression system (Xpress CF) to produce KIH bispecific antibodies in multiple scaffolds, including 2-armed heterodimeric scFv-KIH and one-armed asymmetric BiTE-KIH with tandem scFv. Efficient KIH production can be achieved by manipulating the plasmid ratio between knob and hole, and further improved by addition of prefabricated knob or hole. These studies demonstrate the versatility of Xpress CF in KIH production and provide valuable insights into KIH construct design for better assembly and expression titer.

SUBMITTER: Xu Y

PROVIDER: S-EPMC4623329 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Production of bispecific antibodies in "knobs-into-holes" using a cell-free expression system.

Xu Yiren Y Lee John J Tran Cuong C Heibeck Tyler H TH Wang Willie D WD Yang Junhao J Stafford Ryan L RL Steiner Alexander R AR Sato Aaron K AK Hallam Trevor J TJ Yin Gang G

mAbs 20150101 1

Bispecific antibodies have emerged in recent years as a promising field of research for therapies in oncology, inflammable diseases, and infectious diseases. Their capability of dual target recognition allows for novel therapeutic hypothesis to be tested, where traditional mono-specific antibodies would lack the needed mode of target engagement. Among extremely diverse architectures of bispecific antibodies, knobs-into-holes (KIHs) technology, which involves engineering CH3 domains to create eit ...[more]

PMID: 25427258

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Project description:Shigella spp. invade the colonic epithelium and cause bacillary dysentery in humans. Individuals living in areas that lack access to clean water and sanitation are the most affected. Even though infection can be treated with antibiotics, Shigella antimicrobial drug resistance complicates clinical management. Despite decades of effort, there are no licensed vaccines to prevent shigellosis. The highly conserved invasion plasmid antigens (Ipa), which are components of the Shigella type III secretion system, participate in bacterial epithelial cell invasion and have been pursued as vaccine targets. However, expression and purification of these proteins in conventional cell-based systems have been challenging due to solubility issues and extremely low recovery yields. These difficulties have impeded manufacturing and clinical advancement. In this study, we describe a new method to express Ipa proteins using the Xpress+TM cell-free protein synthesis (CFPS) platform. Both IpaB and the C-terminal domain of IpaH1.4 (IpaH-CTD) were efficiently produced with this technology at yields > 200 mg/L. Furthermore, the expression was linearly scaled in a bioreactor under controlled conditions, and proteins were successfully purified using multimode column chromatography to > 95% purity as determined by SDS-PAGE. Biophysical characterization of the cell-free synthetized IpaB and IpaH-CTD using SEC-MALS analysis showed well-defined oligomeric states of the proteins in solution. Functional analysis revealed similar immunoreactivity as compared to antigens purified from E. coli. These results demonstrate the efficiency of CFPS for Shigella protein production; the practicality and scalability of this method will facilitate production of antigens for Shigella vaccine development and immunological analysis. KEY POINTS : • First report of Shigella IpaB and IpaH produced at high purity and yield using CFPS • CFPS-IpaB and IpaH perform similarly to E. coli-produced proteins in immunoassays • CFPS-IpaB and IpaH react with Shigella-specific human antibodies and are immunogenic in mice.

Dataset Information

Production of bispecific antibodies in "knobs-into-holes" using a cell-free expression system.

Publications

Production of bispecific antibodies in "knobs-into-holes" using a cell-free expression system.

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