How much do you know about the Bioactivity analysis of bispecific antibody？

Bispecific antibody (BsAb) is a type of antibody molecule that can simultaneously bind to two different antigens or two different epitopes on the same antigen. Bispecific antibodies can exert the synergistic effect of two monoclonal antibodies, and may be more advantageous than mixed use of two monoclonal antibodies.

According to Bispecific Antibody Development Programs Guidance for Industry (FDA, 2019), quality attributes may affect pharmacology and should be studied.

Case Study

>>> Case 1 antigen-antibody affinity

ACROBiosystems developed a 1:1 heterodimer CD3E&CD3D recombinant protein to support the potential applications. We verified the purity and bioactivity of the CD3E&CD3D protein using the bispecific antibody (AMG420) through MALS, SPR and ELISA.

• Purity verified by MALS

Fig.1 Human CD3E&CD3D Heterodimer Protein, His Tag&Tag Free (Cat. No. CDD-H52W1) on SDS-PAGE under reducing (R) and non-reducing（NR）condition. The purity of the protein was more than 85% and around 35-43 kDa verified by SEC-MALS.

• Bioactivity verified by SPR

Fig.2 Bispecific T-cell Engager (CD3 X BCMA) immobilized on CM5 Chip can bind Human CD3E&CD3D Heterodimer Protein, His Tag&Tag Free (Cat. No. CDD-H52W1) with an affinity constant of 31.8 nM as determined in a SPR assay (Biacore T200).

• Bioactivity verified by ELISA

Fig 3. Immobilized Human CD3E&CD3D Heterodimer Protein, His Tag&Tag Free (Cat. No. CDD-H52W1) at 2 μg/mL, add increasing concentrations of Bispecific T cell Engager (CD3 X BCMA) and then add Biotinylated BCMA Fc,Avitag (Cat. No. BC7-H82F0) at 0.2 μg/mL. Detection was performed using HRP-conjugated streptavidin with sensitivity of 4 ng/mL.

>>> Case 2 FcRn-antibody affinity

IgG like BsAb maintains the same bioactivity and PK/PD characteristics as the traditional IgG monoclonal antibody. We verified the affinity between IgG like BsAb and Fc gamma receptors using Biacore.

Fig 4. Bispecific antibody can bind to Fc receptors

>>> Case 3 Intact assay

The preclinical and clinical pharmacology studies for a BsAb, such as the PK assessment, are usually complex due to the interference of anti-drug antibodies and free target proteins in biological samples.

Fig 5. PK assessment methods

Based on the intact assay method, we established an ELISA method for free CTLA-4 x OX40 BsAb measurement using MALS verified OX40 protein and biotin-labeled CTLA-4 protein.

Fig 6. Immobilized Human OX40 Protein, His Tag (MALS verified) (Cat. No. OX0-H5224) at 2 μg/mL, add increasing concentrations of CTLA-4 x OX40 bispecific antibody in 50% Human serum and then add Biotinylated Human CTLA-4, Fc,Avitag (Cat. No. CT4-H82F3) at 0.2 μg/mL. Detection was performed using HRP-conjugated streptavidin with sensitivity of 4 ng/mL (Intact assay).

Click to learn more targets for bispecific antibodies:

CD3              CTLA-4               CD47

CD20                       CD19                         PD-L1