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Bioactivity analysis of bispecific antibody
Release time: 2020-10-21 Source: ACROBiosystems Read: 6147

Traditional monoclonal antibodies bind mono specifically to certain cells or  proteins and exert biological activities. However, it is important to  block multiple signaling pathways to avoid compensatory effects,  especially for cancer and autoimmune treatments. Viruses are usually susceptible to mutations and easy to develop resistance.

So,  it is common to use multi-targeting therapies to prevent the emergence  of resistance. Therefore, monoclonal antibodies have limited efficacy  under these circumstances. Advances in technology led to the development  of bispecific antibodies (BsAb). Bispecific antibodies can bring out a  synergy effect, making them one of the most popular drug research areas.  Currently, there are hundreds of BsAb in clinical trials, and the  number will continue to grow in the next few years.

There  are many formats of BsAb including IgG-like and non-IgG-like,  symmetric, and asymmetric. For these new molecules, the characterization  and pharmacokinetic (PK)/PD assessment are vital. Quality attributes  such as antigen specificity; affinity and on- and off-rates; avidity  (for bispecific antibodies that target two molecules on the same cell);  potency; process-related impurities such as aggregates, fragments, and  homodimers; stability; and half-life may affect pharmacology and should  be studied.[2]
 As bispecific antibodies may present as a  mixture of biologically active and inactive forms, it is important to  identify the bispecific antibody form(s) that is most pharmacologically  relevant to PK/PD assessment and to develop validated assays that  measure the appropriate form(s) accordingly. Due to the synergetic  effect BsAb brings, the dosage is relatively low. Therefore, it requires  a more sensitive assay for analysis.

Case study
Antigen-antibody affinity

Case 1 antigen-antibody affinity

It  is critical to choose the building blocks of BsAb with the best binding  affinity, stability, and functionality based on the binding affinities  and kinetics, especially when dual-targeting the effector cell and the  target cell. Targeting CD3 may over-activate T cells or other immune  cells, resulting in excessive cytokines release and leading to biosafety  issues. Therefore, it is critical to reduce the binding of the Fc  fragment of CD3-targeting antibodies and Fc gamma receptors, thereby  reducing the side effects.
 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  SPR, ELISA, and SDS-PAGE.

MALS verification:


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 is more than 85% and around 35-43 kDa verified by SEC-MALS.

SPR verification:


Bispecific T-cell Engager (CD3 X BCMA, AMG420) 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).


ELISA Assay verification:


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.



PK assessment

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.

Intact Assay:

For  the intact BsAb molecule, all their functional domains are not blocked  by ADA or free target proteins. The intact assay is developed to  evaluate the intact BsAb molecule. Recombinant protein targets or  competitive anti-idiotypic antibodies can be utilized for this kind of  assay development. The intact assay has advantages such as high  specificity, small matrix effect, and high sensitivity.

Free A/B Assay:

Free  A/B assay can be developed to study one of the functional domains. This  method can be used to evaluate the exposure and activity of the  specific functional domain as well as evaluate the interference of ADA  and free target proteins. We can use the specific target protein and  detection antibody for assay development. Due to the matrix effect and  steric hindrance in clinical PK study, the sensitivity is relatively  low.

Total Assay:

The total assay will not be  interfered by ADA and free target proteins. The method can be developed  based on the structural domain of the BsAb molecule using  non-competitive ADA and detection antibodies, which is helpful for  toxicity evaluation.


Case 3

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.


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).

Methodology validation:


When developing a PK method, we have to  consider all the advantages and disadvantages of different detection  methods. Based on the requirement of sensitivity, we make the most  appropriate choices and establish a satisfying method through  optimizations.

Based on the Biacore T200/8K platform and ForteBio Octet platform, ACROBiosystems provides SPR /BLI analytical services to our valued customers. The SPR/BLI analytical services include antibody screening, antibody pairing/group, kinetic analysis, and interaction measurement.

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