The authors thank Prof. Ralph Weissleder, Dr. Pat Zanzonico, and Dr. NagaVaraKishore Pillarsetty for helpful conversations and the NIH for funding (BMZ: 1K99CA178205-01A1)

伦理声明：以上所描述的体内动物实验是根据经批准的协议，并在纪念Sloan Kettering癌症中心的机构动物护理和使用委员会（IACUC）的道德准则进行。 1.合成TZ-BN-NOTA的<ol><li>在一个小的反应容器中，在600微升的NaHCO 3缓冲液（0.1M，pH值8.1）溶解7毫克的NH 2 -Bn-NOTA（1.25×10 -2毫摩尔）。检查溶液的pH值。如果需要的话，用0.1M的钠2小等份CO 3调节溶液的pH至8.1。 </li><li>在NH 2 -Bn-NOTA解决方案添加0.5毫克TZ-NHS（1.25×10 -3毫摩尔）在1.7 ml离心管。 注：TZ-NHS既可以称出干燥或无水的DMF或DMSO（&lt;50微升）的储备溶液。 </li><li>允许将得到的反应溶液中进行30分钟，在RT下的反应适度搅拌。 </li><li> 30分钟后，使用纯化逆相C 18 HPLC色谱以除去未反应的NH 2 -Bn-NOTA的产物。所述NH 2 -Bn-NOTA可以在254纳米的波长进行监控，而TZ-NHS和TZ-BN-NOTA最好在525纳米的波长监测。 注：保留时间明显高度依赖于每个实验室（泵，柱，管， 等等 ）的高效液相色谱法的设备的设置。然而，到目前的例子中，如果为0的梯度：100的MeCN / H 2 O（均含有0.1％TFA）至100：0的MeCN / H 2 O在25分钟内和分析4.6×250毫米C18 柱使用，TZ-BN-NOTA，TZ-NHS，和NH 2的保留时间-Bn-NOTA将大约15分钟，16.5分钟和10分钟，分别。该产品可以与其它反应组分中任一单次运行或使用半制备或制备-C 18 HPLC柱多个运行进行纯化。1 H-NMR，肛门ytical HPLC和ESI-MS是可用于验证完成TZ-BN-NOTA前体的纯度的所有方法。11 </li><li>冻结使用液氮收集HPLC洗脱液。 </li><li>包装在不透明的铝箔冷冻收集管。 </li><li>放置在冷冻干燥容器澳冰冻收集管/ N去除HPLC流动相。 </li><li>存储所述纯化的产物（一个明亮的粉红色固体）在黑暗中在-80℃。 注意：这是在程序中可接受的停止点。已完成的TZ-BN-NOTA的前体可稳定至少1年在这些条件下。 </li></ol> 2.准备huA33-TCO免疫偶联物<ol><li>在1.7 ml离心管，制备1mg / ml的TCO-NHS的（2.7毫摩尔）的无水DMF溶液。 </li><li>在1.7 ml离心管，制备2mg / ml的huA33的（13.3μM）溶液在1ml磷酸盐缓冲盐水，pH 7.4中（0.01M PO 4 3-， </sub&gt; 0.154摩尔NaCl）。 </li><li>使用小的等分试样（&lt;5微升）为0.1M的Na 2 CO 3中 ，调整的抗体溶液的pH至8.8-9.0。请使用pH试纸或pH计用微电极监测pH值，并注意不要让pH值升高pH值高于9.0。 </li><li>一旦抗体溶液是在正确的pH值，添加了对应于活化酯8摩尔当量的TCO-NHS溶液的体积。例如，添加7.9微升1mg / ml的TCO-NHS溶液（1.07×10 -7摩尔的TCO-NHS），以1毫升2毫克/毫升huA33抗体溶液（1.33×10 -8摩尔huA33）。不通过在溶液体积超过5％的DMF。 </li><li>轻轻颠倒的离心管几次混合溶液。 </li><li>包装在不透明的铝箔的离心管中。 </li><li>让溶液孵育1小时，在室温温和搅拌。 </li><li> 1小时在室温之后，使用预填充的一次性SIZ纯化所得免疫Ë排除脱盐柱。首先，如所描述的供应商到储存过程中除去存在于列中的任何防腐剂冲洗尺寸排阻柱。然后，将反应混合物添加到尺寸排阻柱，冲洗用1.5ml 0.9％的无菌盐水的柱，并随后收集用2-毫升0.9％的无菌盐水作为洗脱液的产物。 注：此步骤将产生完成huA33-TCO为2 ml的溶液。 </li><li>测定所得huA33-TCO上的紫外可见分光光度计的浓度。 </li><li>如果浓度较高是理想的，集中使用离心过滤器单元具有50000分子量截止的huA33-TCO溶液。 注意：需要注意的是，虽然huA33和其他各种公知的抗体（ 例如，贝伐单抗，曲妥单抗，西妥昔单抗，和J591）非常宽容被浓缩，凝聚，沉淀可以在其他情况下，会发生在浓度是很重要的。研究人员尝试此procedURE用新的抗体应该相信文献或自身抗体的知识问题方面是否集中的抗体。 </li><li>存储完成huA33-TCO免疫在4°C黑暗环境中。 注意：这是在程序中可接受的停止点。已完成的单抗的TCO偶联物应为这些储存条件下至少3个月是稳定的。 </li></ol> TZ-BN-NOTA 3. 64铜放射性同位素标记注：该协议的这一步涉及的处理和操纵的放射性。在执行这些步骤 - 或进行任何其他工作与放射性 - 研究人员应该与他们的家机构的辐射安全部咨询。采取一切可能的措施，以尽量减少暴露于电离辐射。 <ol><li>在1.7 ml离心管，制备0.5毫克/毫升（723μM）TZ-BN-NOTA的溶液。 </li><li>在一个1.7毫升的MicroCentrifuge管中，加入10微升的TZ-BN-NOTA溶液（5微克）的400微升的0.2M的NH 4 OAC pH值为5.5的缓冲。 </li><li>在适当的放射化学音符维持兴趣，测量和使用剂量校准之前和在下面的协议（3.4-3.8）的随后的步骤后记录样品中放射性的量。这将有助于准确测定放射化学产量。 </li><li>加入2000微居里64铜（74活度）将TZ-BN-NOTA的解决方案。 注意：通常情况下，[64铜]的CuCl 2中的0.1N的HCl的小体积（&lt;30微升）被提供，并且因此只有少量（&lt;10微升）此原料液都需要的放射性标记反应。如果需要更大的[64铜] 氯化铜库存量，放射性标记反应是宽容提高整体反应体积。然而，放射性标记反应溶液的pH值应仔细监测，以确保它不低于pH值4.0。 </li><li>使该溶液温育在室温下10分钟，温和搅拌。 </li><li>之后孵育10分钟，用纯化逆相C 18 HPLC层析产物。保留时间明显高度依赖于每个实验室（泵，柱，管， 等等 ）的高效液相色谱法的设备的设置。然而，到目前的例子中，如果使用梯度5:95的MeCN / H 2 O（均含有0.1％TFA）至95：5的MeCN / H 2 O在15分钟时，64的Cu-Tz-的保留时间BN-NOTA应约为9.8分钟，同时游离的未络合铜64会与溶剂前沿洗脱在约2-4分钟。 </li><li>使用旋转蒸发器，取出的HPLC洗脱剂。 </li><li>再溶解64的Cu-TZ-BN-NOTA产物在0.9％无菌盐水。 注意：由于64 Cu的12.7小时物理半衰期，这不是在程序中一个可接受的停止点。 -NOT的Cu-TZ-Bn的执行器64的合成一个紧接注射放射性的，并遵循立即步骤4.5步骤3.7。 </li></ol> 4. 体内预靶向PET成像注意：由于协议中的第3节，该协议的这一步涉及到处理和操作放射性。在执行这些步骤，研究人员应该与他们的家机构的辐射安全部咨询。采取一切可能的措施，以尽量减少暴露于电离辐射。 <ol><li>在一个雌性无胸腺裸小鼠皮下植入1×10 6 SW1222结肠癌细胞，并允许这些（接种后9-12天），以长成100-150毫米3异种移植物。11 </li><li>稀从协议第2节的huA33-TCO溶液至0.5毫克/毫升的浓度在0.9％无菌盐水的等分试样。 </li><li>注入200微升huA33-TCO溶液（100微克）至尾静脉的异种移植物承载鼠标。 </li><li>允许24小时为huA33-TCO在小鼠的肿瘤积累。 </li><li>稀释64的Cu-TZ-BN-NOTA放射性为1.5毫居里/ ml，在0.9％无菌盐水中的浓度。 </li><li>注入200微升64的Cu-TZ-BN-NOTA放射性溶液（300微居里; 11.1活度; 1.6纳摩尔64的Cu-TZ-BN-NOTA，假设6.7活度/纳摩尔的特定活性）至尾静脉的异种移植物的小鼠。 </li><li>在所需的成像时间点（ 例如，2个，6个，12个，或24小时后喷射），麻醉的小鼠用2％异氟烷：氧混合气。 </li><li>将鼠标放在小动物PET扫描仪的床。氧混合气：使用1％异氟烷在扫描期间保持麻醉。在此之前放置在扫描仪床上的动物，用脚趾捏法验证麻醉和适用兽医药膏鼠​​标，以防止在麻醉过程中干燥的眼睛。 </li><li>通过静态获得为鼠标在PET数据扫描用最少的使用350-700千电子伏的能量窗和6纳秒重合定时窗口20000000同步事件。完成收购的图像后，不要离开鼠标无人看管，不把它关在笼子里与其他老鼠，直到它恢复意识。 </li></ol>

<ol>
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The authors have nothing to disclose.

这个预靶向PET成像策略的主要优点在于，它能够描绘肿瘤靶 - 背景图像的对比度，在通过直接标记的抗体所产生的辐射剂量的一小部分的。例如，在此处描述的结肠直肠癌的成像系统，从急性生物分布实验数据进行了用来执行剂量计算为64 Cu基预靶向策略连同直接标记64的Cu-NOTA-huA33和89锆DFO-huA33。相比，更临床相关的89 Zr的标记的抗体，这些计算清楚地说明了预靶?...

在过去的三十年来，正电子发射断层扫描（PET）已经成为在癌症的诊断和管理的一个不可缺少的临床工具。抗体长期以来被认为是有前途的载体用于递送的发射正电子的放射性同位素，以由于其精致的亲和力和特异性的癌症生物标志物的肿瘤。1,2-然而，相对缓慢的体内药物动力学的抗体的要求使用放射性同位素具有多天的物理半衰期。此组合可以产生高辐射剂量的患者的非靶器官，一个重要的并发症，具​​有特别的临床意义，因为放射性免疫静脉内，因此注射​​ - 不像偏身CT扫描 - 结果在吸收剂量在体内的每一个部分，不论审问组织。 为了绕过这个问题，显著的努力一直致力于开发板pment那解耦放射性同位素和靶向部分，从而利用抗体的有利特性，同时避开它们的内在药动学局限性PET成像策略。这些策略-最经常被称为预定位或多步目标 - 通常使用四个步骤：（1）能够结合两个抗原和放射性配体的抗体的施用; （2）在靶组织和从血液中其间隙抗体的积累; （3）小分子放射的管理;和（4） 在体内结扎的放射性配体的抗体，然后用过量的放射性配体的快速清除。3-8。在一些情况下，一个额外的清除剂是步骤2和3之间，以加速的任何抗体的排泄注入即尚未结合肿瘤并保留在血液中。5 从广义上讲，总重量预定位策略的O型是最常见的文献。虽然两人都在临床前模型证明是成功的，他们也拥有了阻碍其临床应用主要限制。第一个策略依赖于链霉亲和标记的抗体和生物素修饰的放射性之间的高亲和性;然而，链霉亲和修饰的抗体的免疫原性已被证明是一个令人担心的问题，相对于平移。5,6,9,10第二种策略，与此相反，采用已基因工程改造以结合两个癌症的双特异性抗体生物标志物抗原和小分子放射性标记的半抗原。3,11-14虽然这后一种途径是肯定的创作，其广泛的适用性是由复杂性，费用，以及缺乏系统的模块化的限制。 最近，我们开发并发布一个预靶向PET成像方法的基础上的逆电子需求狄尔斯 - 阿尔德（我EDDA） 反 -cyclooctene（TCO）和四嗪（Tz的之间的环加成反应;。） 图1 11虽然反应本身就已经知道了几十年，IEDDA化学经历了复兴近年来的点击化学生物耦合技术，就说明了拉尔夫·惠斯勒，约瑟夫·福克斯，和彼得·孔蒂等。12-15 IEDDA环已经在广泛的设置得到应用，包括肽，抗体，和纳米粒子的荧光成像小组的迷人工作以及核成像。既放射性卤素和放射性金属16-26的结扎是高产，清洁，快速（K 1&gt; 30000 M -1秒-1），有选择性的，以及-危重-生物正交27虽然许多类型点击化学的-包括铜催化叠氮炔环加成，应变促进叠氮炔环加成，和施陶丁格LIGations -是生物正交为好，它是快速反应动力学和bioorthogonality，使IEDDA化学所以非常适合在整个生物体的预定位应用的独特组合28,29沿着这条思路，值得注意的是，从最近的报告是非常重要的我们实验室是不是第一个IEDDA化学适用于预靶向：预靶向成像IEDDA的第一份报告源于ROSSIN的工作， 等人 ，配备了SPECT方法采用了111标记嗪30。 如我们上面讨论的，预靶向方法有四个相当简单的步骤（ 图2）。在协议中，在另一方面，一预靶向策略的结肠直肠癌的PET成像，其使用64的Cu-NOTA标记四嗪放射性和huA33抗体的TCO改性共轭进行说明。但是，这种方法的最终模块化是其GR之一eatest资产，如反式 -cyclooctene部分可以被附加到任何非内化抗体，和四嗪可以连接到各种各样的放射性记者的。

<table border="0" cellpadding="0" cellspacing="0" width="565">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrazine NHS Ester</td>
			<td>Sigma-Aldrich</td>
			<td>764701</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trans-cyclooctene NHS Ester</td>
			<td>Sigma-Aldrich</td>
			<td>764523</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p-NH2-Bn-NOTA</td>
			<td>Macrocyclics</td>
			<td>B-601</td>
			<td>Store at -80 &#176;C</td>
		</tr>
	</tbody>
</table>

harnessing the bioorthogonal inverse electron demand diels-alder cycloaddition for pretargeted pet imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64Cu or 89Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes.

最初的三个步骤在实验- TZ-BN-NOTA，TCO对huA33缀合的合成，和TZ-BN-NOTA的放射性标记构造（ 图3和4） -是高度可靠的。在上述过程中的情况下，合成在高产率和高纯度的TZ-BN-NOTA结构。所述huA33抗体与4.2±0.6的TCO /单抗进行了修改，并TZ-BN-NOTA是放射性标记的64铜，得到纯化的放射性配体在&gt; 99％放射化学纯度，&gt; 85％的衰变校正收率和比活性〜 6.7活度/纳摩尔（ 图...

Watch this Scientific Journal Video about Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging at JoVE.com

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

利用生物正交反电子需求的Diels-Alder环的预靶向PET成像

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for ...

harnessing-bioorthogonal-inverse-electron-demand-diels-alder

Research

JoVE Journal

Bioengineering

8.7K 次观看.  Memorial Sloan Kettering Cancer Center. The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand. 