title: 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.
authors.affiliation: a Sutro Biopharma, Inc. ; South San Francisco , CA USA.
authors.forename: Yiren
authors.initials: Y
authors.lastname: Xu
authors.forename: John
authors.initials: J
authors.lastname: Lee
authors.forename: Cuong
authors.initials: C
authors.lastname: Tran
authors.forename: Tyler H
authors.initials: TH
authors.lastname: Heibeck
authors.forename: Willie D
authors.initials: WD
authors.lastname: Wang
authors.forename: Junhao
authors.initials: J
authors.lastname: Yang
authors.forename: Ryan L
authors.initials: RL
authors.lastname: Stafford
authors.forename: Alexander R
authors.initials: AR
authors.lastname: Steiner
authors.forename: Aaron K
authors.initials: AK
authors.lastname: Sato
authors.forename: Trevor J
authors.initials: TJ
authors.lastname: Hallam
authors.forename: Gang
authors.initials: G
authors.lastname: Yin
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000235:genetics; D016466:CHO Cells; D002474:Cell-Free System / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D018033:Antibodies, Bispecific; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies
keywords: BiTE, bispecific T-cell engager; BiTE-KIH; CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay; EpCAM, epithelial cell adhesion molecule; FACS, fluorescence-activated cell sorting; Fab, antigen-binding fragment; Fc, fragment crystallizable; FcR, Fc receptor; HC, immunoglobulin heavy chain; HER2, human epidermal growth factor receptor 2; IgG, immunoglobulin G; KIH, knob-into-hole; LC, immunoglobulin light chain; LC-MS, liquid chromatography-mass spectrometry; PK, pharmacokinetics; bispecific antibody; cell-free protein expression; knob-into-hole; prefabrication; scFv, single-chain fragment variable; scFv-KIH