Decorating sdAbs with Chelators: Effect of Conjugation on Biodistribution and Functionality.

Area of Science:

• Bioconjugation Chemistry
• Molecular Imaging Probes
• Antibody Engineering

Background:

• Single domain antibodies (sdAbs) are valuable for molecular imaging.
• Functionalizing sdAbs with chelators for radiolabeling creates mixtures with varying conjugation degrees.
• Separating these mixtures is crucial for understanding structure-activity relationships.

Purpose of the Study:

• To implement anion exchange chromatography (AEX) for separating sdAb-chelator conjugates with different conjugation degrees.
• To characterize these separated fractions and evaluate the impact of conjugation degree on pharmacokinetic properties and functionality.
• To assess the utility of AEX in optimizing sdAb-based imaging probes.

Main Methods:

• Functionalization of anti-HER2 and anti-MMR sdAbs with NOTA or DTPA chelators.
• Separation of functionalized sdAbs using anion exchange chromatography (AEX).
• Characterization of fractions via mass spectrometry (MS), surface plasmon resonance (SPR), and isoelectric focusing (IEF).
• In vivo evaluation after radiolabeling with ⁶⁸Ga or ¹¹¹In to assess pharmacokinetics.

Main Results:

• AEX successfully separated sdAb constructs with varying degrees of chelator conjugation.
• MS confirmed fraction identities; SPR indicated a slight affinity loss with higher conjugation (especially DTPA).
• IEF showed a decrease in isoelectric point (pI) correlated with increased chelator numbers.
• In vivo studies revealed minimal impact on general biodistribution, except for higher liver uptake in mono-DTPA-anti-MMR; kidney uptake increased with higher conjugation, indicating improved renal clearance.

Conclusions:

• AEX is effective for separating sdAbs based on chelator conjugation degree.
• Chelator conjugation can alter sdAb properties (e.g., pI, affinity) but has minimal impact on overall biodistribution and tumor targeting.
• Higher chelator conjugation can enhance kidney clearance of sdAb-based tracers.