Site-specific conjugation of monodispersed DOTA-PEGn to a thiolated diabody reveals the effect of increasing peg size on kidney clearance and tumor uptake with improved 64-copper PET imaging.
Site-specific conjugation of monodispersed DOTA-PEGn to a thiolated diabody reveals the effect of increasing peg size on kidney clearance and tumor uptake with improved 64-copper PET imaging.
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DOI：
10.1021/bc100464e
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发表时间：
2011-04-20
影响因子：
4.7
通讯作者：
Shively, John E.
中科院分区：
文献类型：
--
作者：
Li, Lin;Crow, Desiree;Turatti, Fabio;Bading, James R.;Anderson, Anne-Line;Poku, Erasmus;Yazaki, Paul J.;Carmichael, Jenny;Leong, David;Wheatcroft, Michael P.;Raubitschek, Andrew A.;Hudson, Peter J.;Colcher, David;Shively, John E.
用放射性标记的工程抗体对肿瘤进行最佳PET成像需要将血液清除率和肿瘤摄取与工程抗体的半衰期相匹配，以及其他参数。尽管双抗体具有用于快速血液清除（t12 = 30-60 min）的有利分子大小（50 kDa）并且是二价的，从而增加肿瘤摄取，但它们表现出大量肾摄取作为其主要清除途径，这在它们用PET同位素64 Cu（t12 = 12 hr）标记时尤其明显。为了克服这个缺点，可以将双抗体缀合至PEG，这是一种增加双抗体的表观分子大小并减少肾摄取而不会不利地影响肿瘤摄取或肿瘤与血液比率的修饰。我们在此表明，增加分子大小（n= 12、24和48）的单分散PEGn的位点特异性附着可以均匀地增加PEG-双抗体缀合物的表观分子大小，降低肾摄取并增加肿瘤摄取，后者是由于缀合物在血液中的停留时间增加。由于单分散的PEG与螯合剂DOTA预缀合，缀合物能够结合放射性金属，例如分别可用于SPECT和PET成像的111 In和64 Cu。为了允许DOTA-PEG与双抗体缀合，DOTA-PEG掺入与乙烯基砜部分缀合的末端半胱氨酸。为了控制缀合化学，我们设计了表面硫醇化的双抗体，其每个单体并入两个半胱氨酸（每个双抗体四个）。从细菌发酵中表达和纯化硫醇化的双抗体，并且仅需要在缀合至DOTA-PEGn-Cys-VS之前还原。当用111 In和37.9%ID/g放射性标记时，在24小时，D 0 TA-PEG 48-Cys-VS（具有连接至引入的硫醇的DOTA-PEG 48-Cys-VS的双抗体）给出高达80%ID/g的肿瘤摄取，肿瘤与血液的比率（T/B）为8。在动物模型中，当在PET成像中用64 Cu放射性标记时，44 h时的肿瘤摄取（T/B= 8）为1.5g。肿瘤摄取从用表面赖氨酸残基上聚乙二醇化的双抗体观察到的24小时的50%ID/g显著改善。重要的是，与亲本未缀合的双抗体相比，位点特异性Cys缀合的双抗体对其抗原（TAG-72）的免疫反应性没有损失。我们提出，巯基化的双抗体缀合DOTA化的单分散PEG在临床环境中具有上级SPECT和PET成像的潜力。
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 (t1/2= 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 64Cu (t1/2= 12 hr). 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 pre-conjugated to the chelator DOTA, the conjugates were able to bind radiometals such as 111In and 64Cu 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 24h when radiolabeled with 111In and 37.9% ID/g of tumor uptake (T/B= 8) at 44h when radiolabeled with 64Cu in PET imaging in an animal model. Tumor uptake was significantly improved from the 50% ID/g at 24 hours 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.
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DOI：
10.2967/jnumed.109.074153
发表时间：
2010-07
期刊：
Journal of nuclear medicine : official publication, Society of Nuclear Medicine
影响因子：
--
作者：
Li L;Turatti F;Crow D;Bading JR;Anderson AL;Poku E;Yazaki PJ;Williams LE;Tamvakis D;Sanders P;Leong D;Raubitschek A;Hudson PJ;Colcher D;Shively JE
通讯作者：
Shively JE
DOI：
10.1073/pnas.94.17.9261
发表时间：
1997-08-19
影响因子：
11.1
作者：
Cai, XH;Garen, A
通讯作者：
Garen, A
影响因子：
11.5
作者：
Robinson, Matthew K.;Shaller, Calvin;Adams, Gregory P.
通讯作者：
Adams, Gregory P.
影响因子：
3.1
作者：
Wittel, UA;Jain, M;Batra, SK
通讯作者：
Batra, SK
DOI：
10.1007/s00259-004-1664-0
发表时间：
2005-03-01
影响因子：
9.1
作者：
Chauhan, SC;Jain, M;Batra, SK
通讯作者：
Batra, SK