Baudhuin Henri, Puttemans Janik, Hanssens Heleen, Vanwolleghem Philippe, Hernot Sophie, Raes Geert, Xavier Catarina, Lahoutte Tony, Debie Pieterjan
Department of Medical Imaging (MIMA), Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium.
Unit of Cellular and Molecular Immunology (CMIM), Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium.
Pharmaceuticals (Basel). 2021 Apr 25;14(5):407. doi: 10.3390/ph14050407.
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 Ga (NOTA) or In (DTPA) to assess the impact of the conjugation degree on pharmacokinetics. AEX could successfully be applied to separate fractions of (chelator)-anti-HER2 and (chelator)-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)-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)-anti-MMR, which showed a significantly higher liver uptake for (DTPA)-anti-MMR compared to (DTPA)-anti-MMR and (DTPA)-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.
单域抗体（sdAbs）已被证明是分子成像的重要探针。为了制备此类探针，一种策略是用螯合剂对赖氨酸的反应性胺侧链进行功能化，从而得到具有不同共轭程度的化合物混合物。在本研究中，我们采用阴离子交换色谱（AEX）分离不同的化合物或组分，这些化合物或组分经过进一步表征和评估，以研究共轭程度对药代动力学性质和功能的影响。抗HER2和抗MMR单域抗体用NOTA或DTPA螯合剂进行功能化。阴离子交换色谱使用0.02 mol/L Tris pH 7.5作为第一种溶剂，0.25 M或0.4 M NaCl（分别针对NOTA螯合剂或DTPA螯合剂）作为第二种溶剂进行梯度洗脱。通过质谱（MS）、表面等离子体共振（SPR）和等电聚焦凝胶电泳（IEF）对各组分进行表征，同时在用Ga（NOTA）或In（DTPA）进行放射性标记后进行体内研究，以评估共轭程度对药代动力学的影响。AEX能够成功地用于分离（螯合剂）-抗HER2和（螯合剂）-抗MMR单域抗体构建体的组分。MS证实了分离过程中获得的不同峰的身份。SPR测量表明（螯合剂）-抗单域抗体的亲和力略有下降，而IEF显示等电点（pI）随共轭螯合剂数量的增加而相应降低。有趣的是，对于两种单域抗体，DTPA螯合剂导致的亲和力和pI降低都比NOTA螯合剂更强。体内数据显示，除了（DTPA）-抗MMR外，任何构建体的器官摄取均无显著差异，（DTPA）-抗MMR的肝脏摄取显著高于（NOTA）-抗MMR和（DTPA）-抗MMR。总体而言，由于基于单域抗体的示踪剂具有典型的肾脏清除特性，所有构建体均观察到较高的肾脏摄取。同一构建体的不同组分之间肾脏摄取存在显著差异，表明较高的共轭程度可改善肾脏清除。AEX能够分离具有不同共轭程度的单域抗体，并为进一步表征各个组分提供了机会。螯合剂与单域抗体的共轭可改变示踪剂的某些性质，如pI；然而，对总体生物分布谱和肿瘤靶向的影响最小。
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