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Competition assay for bispecific antibody therapeutics
Writer: Alex Jang
Reference: Antiviral Research (2023, V. 212, 105576)
Since the first monoclonal antibody therapeutics obtained US FDA approval in 1986, antibody therapeutics technology has been developed in very diverse forms. Next-generation antibodies with improved efficacy compared to traditional antibodies are emerging one after another, and bispecific antibodies are one of them. Lee et al. (Kookmin University, Korea) published K202.B, a successful bispecific antibody drug candidate targeting multiple strains of SARS-CoV-2, in Antiviral Research (2023, V. 212, 105576). This short introduction was written about an experiment (Competition assay) to check whether the developed bispecific antibody K202.B, which is part of the thesis, recognizes different regions of RBD (Receptor binding domain, target protein).
Lee et al. discovered K102.1 and K102.2 that do not compete with each other and have good binding with RBD using bacteriophage-based biopanning technology and ELISA analysis technique. As a strategy to combine these two types of scFv, two types of IgG4-based bispecific antibodies were developed. As shown in Figure A, K202.A is placed farthest from K101.1 and K102.2 scFvs, and K202.B is placed side by side. The kinetics evaluation of these two types of bispecific antibodies was carried out using the iMSPR-mini device and the COOH-Au chip for various variants of RBD, and it was confirmed that K202.B has better binding ability than K202.A.
Lee et al. conducted an SPR-based competition assay to confirm that K202.B recognizes different regions of RBD. After wild-type RBD was covalently immobilized on the COOH-Au chip, K102.1 (512nM) or K102.2 (512nM) was sufficiently bound, and then K202.B (128nM) was reacted. As shown in Figure B, you can see that K202.B binds well. This can be explained that even though K102.1 binds to RBD, K202.B binds because there is an additional K102.2 epitope. For the same reason, even though K102.2 binds to the RBD, K202.B can bind to it because it has an additional K102.1 epitope.
For further verification, Lee et al. conducted an experiment in reverse this time. K202.B (512nM) was first bound to the RBD-fixed sensor chip, and then K102.1 (256nM) or K102.2 (256nM) was reacted. As shown in Figure C, K202.B is combined first, and then both K102.1 and K102.2 are not combined. This can be interpreted as K202.B blocking the region where K102.1 and K102.2 are to be combined.
If you use iMSPR, you can check the competition very quickly and conveniently without labeling. This is an experimental technique very similar to epitope binning, which must be analyzed when developing antibody drugs. This sandwich-based competition assay method can also be applied to immunogenicity testing.
*This introductory material has been edited from the original thesis image for better understanding.
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