What pharmacokinetic (PK) assays would you recommend for a bispecific antibody with one arm targeting a non-soluble antigen and the other arm targeting a soluble antigen with high concentration?

Pharmacokinetic Assays for Bispecific Antibodies with Non-Soluble and High-Concentration Soluble Targets

For a bispecific antibody with one arm targeting a non-soluble antigen and the other arm targeting a soluble antigen with high concentration, a total drug concentration assay using a sandwich ELISA with anti-idiotype antibody capture and target-based detection is recommended.

Understanding the Bispecific Antibody Challenge

Bispecific antibodies present unique pharmacokinetic challenges compared to standard monoclonal antibodies:

• They bind two different epitopes or antigens simultaneously, creating complex binding dynamics 1
• When one target is soluble at high concentration and the other is membrane-bound (non-soluble), traditional free drug assays become problematic 2
• Target-mediated drug disposition (TMDD) significantly impacts pharmacokinetics, especially with high-concentration soluble targets 3

Recommended Primary PK Assay Approach

Total Drug Concentration Assay

• Implement a sandwich ELISA format that measures total bispecific antibody regardless of target binding status 2
• Use a non-blocking anti-idiotype antibody to one arm of the bispecific for capture 2
• Use an antibody to the target bound to the other arm for detection 2
• This approach enables detection of total bispecific antibody even in the presence of high levels of both targets 2

Validation Parameters

• Ensure the assay demonstrates acceptable precision, accuracy, dilutional linearity, and reproducibility 2
• Confirm specificity by testing with individual targets alone and in combination 2
• Validate the assay's ability to detect drug-target complexes in various binding states 2

Complementary PK Assay Approaches

SPR-Based Assays

• Implement surface plasmon resonance (SPR) assays to assess binding activity to both targets simultaneously 4
• Use an alternative SPR-based assay that allows individual assessment of both targets in solution 4
• Compare data between assays to calculate simultaneous binding based on individual readouts 4

Mass Spectrometry Confirmation

• Consider LC-MS/MS as a complementary method to confirm total drug concentrations 2
• This approach provides orthogonal validation of the total drug concentration measurements 2

Considerations for Target-Mediated Drug Disposition

Impact of Soluble Target

• High concentrations of soluble target can significantly affect drug clearance through TMDD 3
• For antibodies binding to soluble antigens (e.g., cytokines), TMDD can dramatically alter PK profiles 3
• Mechanistic PK models incorporating TMDD are necessary when doses are low and TMDD cannot be ignored 3

Non-Soluble Target Considerations

• Binding to membrane-bound targets can lead to internalization and degradation 5
• Only about 0.1-1% of dosed bispecific antibody typically reaches tumor tissue with membrane-bound targets 5
• Tissue distribution is influenced by organ volumes, blood flow rates, and lymphatic circulation 5

Pitfalls to Avoid

• Avoid relying solely on free drug concentration assays, as they may be misleading with high-concentration soluble targets 2
• Do not use target proteins as capture reagents if they might interfere with drug binding 2
• Be cautious about changing antigen activity upon immobilization in SPR-based assays 4
• Don't overlook the impact of target expression levels in healthy tissues, which can significantly affect PK 5

Translational Considerations

• Use PK data to develop mechanistic models incorporating TMDD for accurate dose prediction 3
• Consider that dual-positive tumor cell targeting specificity is likely higher at lower drug concentrations 5
• Be aware that higher doses may shift exposure balance but increase target engagement in healthy tissues 5
• Use physiologically based PK modeling to guide bispecific antibody development from target evaluation to dose selection 5