Tumor-Specific Detection of an Optically Targeted Antibody Combined with a Quencher-Conjugated Neutravidin "Quencher-Chaser": A Dual "Quench and Chase" Strategy to Improve Target to Nontarget Ratios for Molecular Imaging of Cancer
Tumor-Specific Detection of an Optically Targeted Antibody Combined with a Quencher-Conjugated Neutravidin "Quencher-Chaser": A Dual "Quench and Chase" Strategy to Improve Target to Nontarget Ratios for Molecular Imaging of Cancer
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DOI：
10.1021/bc8003765
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发表时间：
2009-01-01
影响因子：
4.7
通讯作者：
Kobayashi, Hisataka
中科院分区：
文献类型：
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作者：
Ogawa, Mikako;Kosaka, Nobuyuki;Kobayashi, Hisataka
在体单抗分子肿瘤成像不仅在癌症检测方面具有巨大的潜力，而且在癌症定性方面也具有很大的潜力。然而，静脉注射抗体在血液中的滞留时间较长，导致靶区肿瘤与背景比(TBR)较低。亲和素已被用作清除未结合的循环中的生物素化抗体并降低背景信号的“追踪器”。在这里，我们利用荧光共振能量转移(FRET)猝灭抗体和“亲和素追逐”相结合的方法来增加总胆红素受体。将人源化抗人表皮生长因子受体2型(HER2)的单抗曲妥珠与近红外(NIR)荧光团Alexa680偶联，合成了反式Alexa680生物素。接下来，FRET猝灭剂QSY-21与亲和素、中性亲和素(NAV)或链霉亲和素(SAV)结合，从而产生Av-QSY21、NAV-QSY21或Sav-QSY21作为“追赶者”。通过将Tra-Alexa680-生物素与Av-QSY21、NAV-QSY21或Sav-QSY21结合，体外荧光被猝灭。采用靶向(3T3/HER2+)和非靶向(Balb3T3/ZsGreen)荷瘤小鼠，采用“猝灭和追赶”双重策略，评价注射猝灭结合的亲和素衍生物是否能改善靶区TBR。所有基于QSY21亲和素的结合物引起的“FRET猝灭”效应都有所减弱，但并未完全消除血池中的背景信号。NAV-QSY21的加入增加了靶的总胆红素受体，主要是由于“追逐”效应，即未结合的结合抗体优先清除到肝脏。未结合的NAV-QSY21的清除相对缓慢，导致背景信号进一步减少，从血管空间泄漏出来，并与肿瘤血管外空间的未结合抗体结合，导致非靶向肿瘤与背景的比率降低，但由于靶向结合的抗体内化而不能与NAV-QSY21结合，导致靶区TBR增加。综上所述，提出的“猝灭-追逐”系统结合了两种策略，即荧光猝灭和亲和素追逐，以改善靶区TBR和降低非靶区TBR，这将导致肿瘤敏感性和特异性的提高。
In vivo molecular cancer imaging with monoclonal antibodies has great potential not only for cancer detection, but also for cancer characterization. However, the prolonged retention of intravenously injected antibody in the blood causes low target tumor-to-background ratio (TBR). Avidin has been used as a "chase" to clear the unbound, circulating biotinylated antibody and decrease the background signal. Here, we utilize a combined approach of a fluorescence resonance energy transfer (FRET) quenched antibody with an "avidin chase" to increase TBR. Trastuzumab, a humanized monoclonal antibody against human epidermal growth factor receptor type 2 (HER2), was biotinylated and conjugated with the near-infrared (NIR) fluorophore Alexa680 to synthesize Tra-Alexa680-biotin. Next, the FRET quencher, QSY-21, was conjugated to avidin, neutravidin (nAv), or streptavidin (sAv), thus creating Av-QSY21, nAv-QSY21, or sAv-QSY21 as "chasers". The fluorescence was quenched in vitro by binding Tra-Alexa680-biotin to Av-QSY21, nAv-QSY21, or sAv-QSY21. To evaluate if the injection of quencher-conjugated avidin derivatives can improve target TBR by using a dual "quench and chase" strategy, both target (3T3/HER2+) and nontarget (Balb3T3/ZsGreen) tumor-bearing mice were employed. The "FRET quench" effect induced by all the QSY21 avidin-based conjugates reduced but did not totally eliminate background signal from the blood pool. The addition of nAv-QSY21 administration increased target TBR mainly because of the "chase" effect where unbound conjugated antibody was preferentially cleared to the liver. The relatively slow clearance of unbound nAv-QSY21 leads to further reductions in background signal by leaking out of the vascular space and binding to unbound antibodies in the extravascular space of tumors, resulting in decreased nontarget tumor-to-background ratios but increased target TBR due to the "FRET quench" effect, because target-bound antibodies were internalized and could not bind to nAv-QSY21. In conclusion, the proposed "quench-and-chase" system combines two strategies, fluorescent quenching and avidin chasing, to improve target TBR and reduce nontarget TBR, which should result in both improved tumor sensitivity and improved specificity.