Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

Yin, Gang; Garces, Eudean; Yang, Jun; Zhang, Juan; Tran, Cuong; Steiner, Alexander; Roos, C. M.; Bajad, Sunil; Hudak, Susan; Penta, Kalyani; Zawada, James; Pollitt, S; Murray, Christopher J L

doi:10.4161/mabs.4.2.19202

Cited by 127 publications

(159 citation statements)

References 49 publications

(63 reference statements)

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“…46,47 Without the limitation of cell viability imposed by conventional expression systems, Xpress CF is capable of protein expression at g/L scale within hours, and has been demonstrated to retain high productivity in transition from micro-to manufacturing scale. Its open and flexible nature can tolerate multifarious manipulations in extract, DNA template, and chemical and protein additives, 46,48,49 which makes it amenable for high-throughput screening by automation. 50 Over the years, its utilization has expanded to include production of IgGs and antibody derivatives, 46 antibody-drug conjugates, 49 vaccines, 51 membrane proteins, 52 metalloproteins, 53 and viral proteins.…”

Section: Introductionmentioning

confidence: 99%

“…Its open and flexible nature can tolerate multifarious manipulations in extract, DNA template, and chemical and protein additives, 46,48,49 which makes it amenable for high-throughput screening by automation. 50 Over the years, its utilization has expanded to include production of IgGs and antibody derivatives, 46 antibody-drug conjugates, 49 vaccines, 51 membrane proteins, 52 metalloproteins, 53 and viral proteins. 54 We report bispecific antibody expression, in particular KIH, using Xpress CF in this study.…”

Section: Introductionmentioning

confidence: 99%

“…54 We report bispecific antibody expression, in particular KIH, using Xpress CF in this study. The high manipulability of the system 46,50,55 enables expeditious optimization of knob:hole plasmid ratio to achieve most efficient KIH assembly, as well as an "add-back" approach with prefabricated knob or hole for higher titer. Multiple KIH scaffolds have been investigated, including 2-armed heterodimeric scFv-KIH and one-armed asymmetric BiTE-KIH with tandem scFv (Fig.…”

Section: Introductionmentioning

confidence: 99%

“…1), providing insights to guide rational KIH design for better assembly and titer. Furthermore, Xpress CF has been demonstrated to scale in a linear fashion from small scale expression to pilot scale production, 46,47 making it capable of KIH production at a large scale.…”

Section: Introductionmentioning

confidence: 99%

“…46 Thus, we adapted a similar concept for KIH production, suspecting prefabricated knob or hole polypeptide might serve as a chaperon to facilitate the folding of its partner and promote KIH assembly. Using 5-10-Fc(K)CdiL2K-Fc(H) (scFv-KIH r ) as an example, indeed more 5-10-Fc(K) expressed in the presence of its partner diL2K-Fc(H) protein, and vice versa.…”

Section: Comparison Of Kih Scaffoldsmentioning

confidence: 99%

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Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

Lee

Tran

et al. 2015

mAbs

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(2015) Production of bispecific antibodies in "knobs-into-holes" using a cell-free expression system , mAbs, 7:1, 231-242, DOI: 10.4161/19420862.2015.989013 To link to this article: https://doi.org/10. 4161/19420862.2015 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 C H 3 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.

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