Engineered Protein G for Creating Multivalent, Bispecific Recombinant Antibody Affinity Reagents

SUMMARY

• Antibody fragments such as Fabs have demonstrated their utility as research tools, diagnostics, and in therapeutic applications. However, the monovalent nature of Fabs and lack of avidity is particularly limiting for binding antigens at low concentration. 
• The inventors engineered protein G to have higher affinity and specificity for Fabs as compared to native protein G and protein A. The engineered protein G variant also binds Fabs in a pH dependent fashion.
• The product is an engineered variant of protein G (eProtein G) that can be covalently tethered together and complexed with Fabs to create multivalent recombinant antibody reagents to desired targets. eProtein G enhances valency as compared to current methods such as in vitro Fab fusions.
• eProtein G was determined to have 100-fold higher affinity to Fabs over native Protein G and a 50-fold lower affinity to the Fc region of full-length antibodies. In a validation test using Fabs that recognize yeast protein Asf1, the Fabs tethered with eProtein G demonstrated a 10-fold increase in avidity to the target antigen. (All measured using ELISA).

FIGURE

ADVANTAGES

ADVANTAGES

• Increased specificity to Fab region
• Increased pH dependence for binding (1,000-fold change in affinity)
• Improve avidity of Fab reagents to target antigen
• Easily mix-and-match docking of Fabs to generate bispecific complexes.

APPLICATIONS

• Fab purification
• Multivalent affinity reagent generation (diagnostics, therapeutics)

PUBLICATIONS

• Bailey, LJ; et al. Applications for an engineered Protein-G variant with a pH controllable affinity to antibody fragments. J Immunol Methods. 2014 Dec 15; 415: 24-30. 

• US: 15/519,576
• Counterpart in the EU