Abstract
Single domain antibodies (sdAbs) have proven to be valuable probes for molecular imaging. In order to produce such probes, one strategy is the functionalization of the reactive amine side chain of lysines with a chelator, resulting in a mixture of compounds with a different degree of conjugation. In this study, we implemented anion exchange chromatography (AEX) to separate the different compounds or fractions that were further characterized and evaluated to study the impact of the conjugation degree on pharmacokinetic properties and functionality. Anti-HER2 and anti-MMR sdAbs were functionalized with NOTA or DTPA chelator. Anion exchange chromatography was performed using 0.02 mol/L Tris pH 7.5 as the first solvent and 0.25 M or 0.4 M NaCl (in case of NOTA chelator or DTPA chelator, respectively) as the second solvent applied as a gradient. The fractions were characterized via mass spectrometry (MS), surface plasmon resonance (SPR), and isoelectric focusing gel electrophoresis (IEF), while in vivo studies were performed after radiolabeling with either 68Ga (NOTA) or 111In (DTPA) to assess the impact of the conjugation degree on pharmacokinetics. AEX could successfully be applied to separate fractions of (chelator)n-anti-HER2 and (chelator)n-anti-MMR sdAb constructs. MS confirmed the identity of different peaks obtained in the separation process. SPR measurement suggests a small loss of affinity for (chelator)3-anti-sdAb, while IEF revealed a correlated decrease in isoelectric point (pI) with the number of conjugated chelators. Interestingly, both the reduction in affinity and in pI was stronger with the DTPA chelator than with NOTA for both sdAbs. In vivo data showed no significant differences in organ uptake for any construct, except for (DTPA)n-anti-MMR, which showed a significantly higher liver uptake for (DTPA)1-anti-MMR compared to (DTPA)2-anti-MMR and (DTPA)3-anti-MMR. For all constructs in general, high kidney uptake was observed, due to the typical renal clearance of sdAb-based tracers. The kidney uptake showed significant differences between fractions of a same construct and indicates that a higher conjugation degree improves kidney clearance. AEX allows the separation of sdAbs with a different degree of conjugation and provides the opportunity to further characterize individual fractions. The conjugation of a chelator to sdAbs can alter some properties of the tracers, such as pI; however, the impact on the general biodistribution profile and tumor targeting was minimal.
Highlights
Nuclear molecular imaging can be used for cancer diagnosis, staging, and characterization
Clusters of peaks could be determined, which contained either1-sdAb,2-sdAb, or3sdAb, and within one cluster, we identified amidated and deamidated forms, which have a different pI and already count for at least two peaks per cluster
Recombinant sdAbproteins were produced without terminal tags by the Vlaams Instituut voor Biotechnologie (VIB) Protein Service Facility (VIB, Gent) in Pichia pastoris and were formulated in phosphate-buffered saline 1× (PBS) (0.01 M phosphate buffer/0.14 M NaCl) pH 7.4 during the final batch purification. p-SCN-Bn-NOTA and p-SCN-Bn-CHX-A”-DTPA were purchased from Macrocyclics (Macrocyclics, Inc., Plano, TX, USA). 68 Ga was obtained from a 68 Ge/68 Ga Galli EoTM generator (IRE Elit, Fleurus, Belgium), indium chloride
Summary
Nuclear molecular imaging can be used for cancer diagnosis, staging, and characterization. Due to the ease of their generation and their wide availability, monoclonal antibodies (mAbs) were the basis for the design of several target-specific tracers [1]. Due to their long blood retention—caused by their large size and the presence of an intact. Fc-region, the optimal imaging timepoint for mAb-based tracers is typically several days after administration [2]. This requires the use of long-lived radionuclides and leads to complex logistics and non-negligible radiation doses to healthy tissues in patients [3]. Antibody fragments [4], scaffold proteins [5], and peptides [6] have all been shown to be capable of this
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