Granted by their dual target recognition capability, bispecific antibodies have allowed testing novel mechanisms for therapies in oncology, inflammable diseases and infectious diseases, which cannot be achieved by traditional mono-specific antibodies. Among extremely diverse architectures of bispecific antibody, Knobs-into-holes (KIHs) are in particular for promoting heavy chain heterodimerization by introducing either a “knob” or a “hole” in each C<sub>H</sub>3 domain. Because of their high resemblance to natural human IgGs, KIHs offers advantageous pharmacokinetic properties and lower risk of immunogenicity. We report use of a cell-free expression system (Xpress CF) to produce KIH bispecific antibodies in multiple scaffolds. Efficient KIH production can be achieved by expeditious plasmid ratio optimization between knob and hole, and further improved by adding prefabricated knob or hole. Xpress CF produced KIHs exhibit potent cell killing and prolonged PK properties. These studies demonstrate the versatility of Xpress CF in KIH production, and provide valuable insights into KIH construct design for better assembly and titer.

Production of bispecific antibodies in “Knobs-into-Holes” using a cell-free expression system

249th National Meeting (2015)

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