Department of Immunology, Beckman Research Institute of City of Hope , Duarte, California 91010, United States.
Bioconjug Chem. 2011 Apr 20;22(4):709-16. doi: 10.1021/bc100464e. Epub 2011 Mar 24.
Optimal PET imaging of tumors with radiolabeled engineered antibodies requires, among other parameters, matching blood clearance and tumor uptake with the half-life of the engineered antibody. Although diabodies have favorable molecular sizes (50 kDa) for rapid blood clearance (t(1/2) = 30-60 min) and are bivalent, thereby increasing tumor uptake, they exhibit substantial kidney uptake as their major route of clearance, which is especially evident when they are labeled with the PET isotope (64)Cu (t(1/2) = 12 h). To overcome this drawback, diabodies may be conjugated to PEG, a modification that increases the apparent molecular size of the diabody and reduces kidney uptake without adversely affecting tumor uptake or the tumor to blood ratio. We show here that site-specific attachment of monodispersed PEGn of increasing molecular size (n = 12, 24, and 48) can uniformly increase the apparent molecular size of the PEG-diabody conjugate, decrease kidney uptake, and increase tumor uptake, the latter due to the increased residence time of the conjugate in the blood. Since the monodispersed PEGs were preconjugated to the chelator DOTA, the conjugates were able to bind radiometals such as (111)In and (64)Cu that can be used for SPECT and PET imaging, respectively. To allow conjugation of the DOTA-PEG to the diabody, the DOTA-PEG incorporated a terminal cysteine conjugated to a vinyl sulfone moiety. In order to control the conjugation chemistry, we have engineered a surface thiolated diabody that incorporates two cysteines per monomer (four per diabody). The thiolated diabody was expressed and purified from bacterial fermentation and only needs to be reduced prior to conjugation to the DOTA-PEGn-Cys-VS. This novel imaging agent (a diabody with DOTA-PEG48-Cys-VS attached to introduced thiols) gave up to 80%ID/g of tumor uptake with a tumor to blood ratio (T/B) of 8 at 24 h when radiolabeled with (111)In and 37.9% ID/g of tumor uptake (T/B = 8) at 44 h when radiolabeled with (64)Cu in PET imaging in an animal model. Tumor uptake was significantly improved from the 50% ID/g at 24 h observed with diabodies that were pegylated on surface lysine residues. Importantly, there was no loss of immunoreactivity of the site-specific Cys-conjugated diabody to its antigen (TAG-72) compared to the parent, unconjugated diabody. We propose that thiolated diabodies conjugated to DOTAylated monodisperse PEGs have the potential for superior SPECT and PET imaging in a clinical setting.
使用放射性标记的工程抗体进行肿瘤的最佳 PET 成像需要匹配血液清除率和肿瘤摄取与工程抗体半衰期等参数。尽管二价体具有有利的分子大小（50 kDa），可实现快速血液清除（t(1/2) = 30-60 分钟），并且是二价的，从而增加肿瘤摄取，但它们表现出大量的肾脏摄取作为其主要清除途径，当用 PET 同位素（64）Cu（t(1/2) = 12 h）标记时，这种情况尤其明显。为了克服这一缺点，二价体可以与 PEG 偶联，这种修饰可以增加二价体的表观分子大小，并减少肾脏摄取，而不会对肿瘤摄取或肿瘤与血液的比值产生不利影响。我们在这里表明，通过特定位置连接大小逐渐增加的单分散性 PEGn（n = 12、24 和 48）可以均匀增加 PEG-二价体的表观分子大小，减少肾脏摄取并增加肿瘤摄取，后者是由于共轭物在血液中的停留时间增加所致。由于单分散性 PEG 预先与螯合剂 DOTA 偶联，因此这些共轭物能够结合放射性金属，例如分别用于 SPECT 和 PET 成像的（111）In 和（64）Cu。为了允许 DOTA-PEG 与二价体偶联，DOTA-PEG 包含一个末端半胱氨酸，与乙烯砜部分连接。为了控制偶联化学，我们设计了一种表面巯基化的二价体，每个单体包含两个半胱氨酸（每个二价体四个）。巯基化的二价体通过细菌发酵表达和纯化，仅在与 DOTA-PEGn-Cys-VS 偶联之前需要还原。这种新型成像剂（带有连接到引入的硫醇的 DOTA-PEG48-Cys-VS 的二价体）在动物模型中用（111）In 标记时，肿瘤摄取高达 80%ID/g，肿瘤与血液的比值（T/B）为 8，而用（64）Cu 标记时，肿瘤摄取为 37.9%ID/g（T/B = 8）在 PET 成像 44 小时时。与在表面赖氨酸残基上聚乙二醇化的二价体相比，肿瘤摄取从 24 小时观察到的 50%ID/g 显著提高。重要的是，与未偶联的二价体相比，该位点特异性 Cys 偶联的二价体对其抗原（TAG-72）的免疫反应性没有丧失。我们提出，与 DOTA 化的单分散性 PEG 偶联的巯基化二价体具有在临床环境中进行 SPECT 和 PET 成像的潜力。
