Name: preparation and evaluation of 99mtc-labeled tridentate chelates for pre-targeting using bioorthogonal chemistry
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Description: Watch this Scientific Journal Video about Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry at JoVE.com

This work supported by research grant funding from the Natural Sciences and Engineering Research Council (NSERC) of Canada, the Ontario Institute for Cancer Research (OICR, #P.SI.015.8), and the Canadian Cancer Society (CCS, #703857). The authors acknowledge the contributions of Dr. Denis Snider who provided assistance in preparing the manuscript.

动物实验是由动物研究伦理委员会麦克马斯特大学按照加拿大议会关于动物饲养（廉政公署）的指导方针批准。 1. 99m锝TZ-齿配体的放射性标记注意:以下步骤需要使用放射性化合物。工作只应在有执照的实验室进行与遵守安全和处置法规。微波反应应该专门为化学合成设计的微波炉进行。 <ol><li> 合成[99m锝（CO）3（H 2 O）3] + 19,20 <ol><li>在微波小瓶中，合并8毫克K 2 [BH 3 CO 2]，15毫克的 Na 2 CO 3，20毫克的 Na 2 B 4 O 7·10H 2 O和25毫克KOCO [CH（OH）]2 COONa·4H 2 O吹扫小瓶10分钟用氩气。 </li><li>在0.9％的盐水（〜1100活度，〜30毫居里）到小瓶-添加4mL的标99m TCO 4。 </li><li>热在微波反应在110℃下10秒后搅拌3.5分钟，以确保试剂的彻底混合。 </li><li>使用〜400微升的1M HCl调节溶液的pH至3.5-4。验证使用pH试纸。 </li></ol></li><li> 四嗪的放射性标记配体1-5 <ol><li>在250μL甲醇溶解21毫克2每配体（化合物1-5）。 </li><li>添加250微升[99m锝（CO）3（H 2 O）3] +（〜74活度，〜2毫居里）向每个溶液中。 </li><li>加热在60℃下用20分钟微波反应混合物中。 注:此步骤是所有5个四嗪相同。 </li><li>对化合物2-5，使溶剂蒸发并重新dissol已经将所得产物在1毫升1:1的体积/体积的DCM:TFA。 </li><li>在60℃下加热溶解反应产物（2-5）在6分钟（2-4）或10分钟（5）一台微波炉。 </li><li>在冷却至室温后，蒸发用蒸发器（36℃，8毫巴，3分钟，6000转）的溶剂，并溶解在1干燥化合物:1的ACN:H 2 O或1:1的MeOH:H 2 O，之前HPLC纯化。 </li><li>净化99m锝标记的化合物（1-5），包括使用HPLC（C18反相）分离未标记的四嗪配体的标记产物。通常情况下，使用30:70的ACN梯度洗脱:H 2 O（均含有0.1％TFA），以40:60的ACN:H 2 O在20分钟内（18分钟）和C 18的分析4.6×100毫米柱。同时使用UV（254纳米）和伽马探测。 <ol><li>每次取标记产品的一个小样本及其HPLC保留时间比较，一个共同的注射ected，无放射性，重新标记的标准（0.125毫克20％甲醇- H 2 O）。 [重新标记的标准是在UV HPLC迹识别，并且将在同一时间，在γ-HPLC跟踪该99m锝标记的化合物洗脱。此共注射示出了在可比较的保留时间的峰，确认了99m锝标记的化合物的身份。 </li></ol></li><li>蒸发从用蒸发器（36℃，8毫巴，3分钟，6000转）的HPLC级分的溶剂。 </li><li>配制纯化的化合物在7.4 KBQ /微升在PBS中的浓度，含有0.5％BSA和0.01％吐温-80。 </li><li>为了确保标记的化合物是稳定的，执行的体外稳定性研究。孵育在37℃配制的化合物为1,4和6小时，在每个时间点注射该混合物在HPLC上少量（3.7活度）评估稳定性。 </li></ol></li></ol> 2.预靶向生物分布研究<ol><l我&gt; 动物的制备 <ol><li>使用7-9周龄，雌性BALB / c小鼠（N = 3），管理TCO-BP生理盐水（20毫克/公斤）（5微克/μL）制定的，经尾静脉注射。 </li><li>放置鼠标在物理约束装置，和识别位于尾部的横向表面上的静脉和用酒精棉签擦拭。在从尾部的端部大约2厘米，以小角度，平行于静脉插入一个30号针头。慢慢地压低柱塞注入，去掉针头和注射部位有轻微的压力施加干净的纱布海绵，直到出血停止。 </li><li>在1小时后注射的TCO-BP，辖〜99m锝四嗪0.74活度（20微居里）的100微升0.5％的BSA的配制，0.01％吐温80在PBS中，通过尾静脉注射。 </li></ol></li><li> 生物分布研究 <ol><li>在期望的时间点（T = 6小时），麻醉用3％异氟烷和2％的氧的气体混合物的小鼠。表现出以在麻醉鼠标ê捏撤离，以确保它们麻醉手术平面之下。 </li><li>通过使用注射器肝素预处理心脏穿刺收集血液（1mL）中。它与鼻子的鼻锥为继续回麻醉将鼠标和定位对动物剑突。 <ol><li>插入地下25针，略向剑突下动物的中线的左边，在一个20°的角度。完全插入针，慢慢向后拉柱塞看到血针毂如果心脏被刺破。略微调整针的同时保持柱塞如果必要，刺穿心脏。慢慢地抽取血液到注射器中。 </li></ol></li><li>通过颈脱位安乐死的动物，而在麻醉下。 </li><li>将每个动物在一个塑料袋，并使用剂量校准（99m锝设置）来测量全身活性水平。 </li><li>收集在下面的组织和体液的预称重ED计数管:血液，骨（膝盖和肩部），胆，肾，肝，胃（有内容），小肠（有内容），大肠和盲肠（有内容），甲状腺和气管，有尿膀胱和尾部。 </li><li>在冲洗PBS适当组织（不包括血液，胆囊和膀胱）将组织以适当的计数管之前去除血液和吸干。 </li><li>放置动物胴体在一个塑料袋中并使用剂量校准测量剩余全身活性。 </li><li>称量每个管含有组织样品。该管的减去初始重量以获得组织的质量。 </li><li>使用剂量校准（99m锝设置）在注射为每只小鼠的时间来测量酶活性的测试样品（100μL）中的量。 注意:此测试样本等于注射体积，从而在注射时给予活动计数。 </li><li>在组织的测量，一个时liquot5μL的以前使用的测试样品。使用多检测器γ计数器（99m锝设置）并计数，以获得每分钟（CPM）的计数为5微升试样。 </li><li>使用所获得的两个值中2.2.9和2.2.10计算使用等式1获得的换算因子（CPM微居里-1）的活性和CPM关系。 （1） <img alt="式（1）" src="/files/ftp_upload/55188/55188eq1.jpg" /></li><li>使用γ计数器测定在每个组织或流体样品的放射性的量。 </li><li>使用方程1在相对于总注射剂量的测量的时间来计算每个组织或流体的活动量。此值然后通过器官重量归一化并且报告为组织的每克百分比注射剂量（ 即，％ID /克）。 </li><li>按照步骤2.1.2 2.2.13在艾比森用99m锝标记的四嗪配体进行阴性对照实验TCO-BP的行政长官。牺牲小鼠（n = 3）为0.5，1,4和6小时后注射，将获得的组织或流体，如上所述。 </li></ol></li></ol>

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The authors declare they have no competing financial interests.

不同极性的四嗪联三齿螯合物的集合被制备，并且与在体内的TCO衍生物IEDDA反应其99m锝复合物的效用进行评价。一种有效的和可再现的99m锝标记方法是五四嗪螯合物，其中该配位体的浓度为10 -3 M的标记步骤之后是叔丁基基（化合物2-5）的脱保护的发展。配体的高浓度使用以提高放射化学产率，并减少反应时间，这减少了四嗪21的...

99m锝保留在核医学诊断中使用的主导放射性同位素，每年全球1，2，3进行超过5000万的成像程序。大多数临床使用99m锝剂是灌流型的放射性药物。有在其中99m锝涉及通过结扎特定生物标志物结合至靶向构建积极靶向化合物的数量有限。靶向99m锝放射性药物的创建通常是由99m锝-配体复合的对靶向分子的能力的影响阻碍了对感兴趣的生物标记物结合，或同位素半衰期不够长为具有更高分子量的生物分子用如抗体。后者通常需要数天的图像，以获得前的生物分子从非目标清除做卷烟<html>的UE。预靶向提供了一种替代的方法来克服这些挑战。 预靶向结合生物正交化学已被证明是开发用于两个荧光和放射成像4，5，6，7，8个新的分子成像探针的有效方法。 -1,2,4,5-四（TZ）和反式 -cyclooctene（TCO）的衍生物之间的逆电子需求狄尔斯-阿尔德（IEDDA）反应， 如图1，已被证明是特别有效的6。这些组件的IEDDA反应可在PBS呈现快速动力学（K 2≈6000 M -1秒-1）和高选择性，因此非常适合在体内预先指定的应用9,10。 e_content"&gt;使用的最常见的方法包括施用的TCO源性靶向载体和以下足够长的延迟时间，放射性标记的四嗪是基于11℃，（18）F，64的Cu，89 Zr和111在已经施用。放射性标记的四嗪报告11，12，13，14，15，相反，仅存在一个一个99m锝标记Tz的，其使用一个HYNIC型配位体需要使用共配体的，以防止在体内蛋白结合和降解制备的报告16。作为替代方案，我们在这里报告99m锝（Ⅰ）的合成中使用的是家族，其形成具有稳定的三齿络合物的配体的标记的四嗪[99m锝（CO）3] +核心。 图1:四嗪和反 -cyclooctene之间的生物正交反应IEDDA。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55188/55188fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 制备配体的家族包含三齿螯合物，在极性和连接基团的金属结合区和Tz的（ 图2）之间的性质不同而不同。该目标是确定一个99m锝-四嗪构造，可以有效地定位和在体内的TCO标记位点和当未结合迅速明确反应，以产生高靶-非靶比率。为了测试配位体，二膦酸盐的TCO-衍生物（TCO-BP），使用17。之前我们已经表明，TCO-BP定位于活跃的骨代谢领域，可以与反应放射性四嗪在体内 18。它是一种方便的试剂测试新四嗪，因为它可以在一个单一的步骤进行制备和实验可以在本地化主要发生在关节（膝和肩）正常小鼠进行。

<table><tbody><tr><td>Argon gas</td><td>Alphagaz</td><td>---</td><td>---</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;</td><td>EMD Millipore</td><td>106395</td><td>---</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;B&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;&amp;#183;10H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Anachemia</td><td>S9640</td><td>---</td></tr><tr><td>KNaC&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;&amp;#183;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Anachemia</td><td>217255</td><td>---</td></tr><tr><td>Technelite &lt;sup&gt;99m&lt;/sup&gt;Tc generator</td><td>Lantheus medical imaging</td><td>---</td><td>Source of &lt;sup&gt;99m&lt;/sup&gt;TcO&lt;sup&gt;4-&lt;/sup&gt;</td></tr><tr><td>0.9% Saline</td><td>Lantheus medical imaging</td><td>---</td><td>To elute generator</td></tr><tr><td>1 M HCl</td><td>Lab Chem</td><td>---</td><td>---</td></tr><tr><td>MeOH</td><td>Caledon</td><td>---</td><td>---</td></tr><tr><td>ACN</td><td>Caledon</td><td>---</td><td>HPLC grade</td></tr><tr><td>Millipore H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Thermo Fisher Scientific</td><td>Barnstead Nanopure</td><td>---</td></tr><tr><td>DCM</td><td>Caledon</td><td>---</td><td>---</td></tr><tr><td>TFA</td><td>Caledon</td><td>---</td><td>---</td></tr><tr><td>PBS</td><td>Thermo Fisher Scientific</td><td>10010023</td><td>pH 7.4 1x</td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A7906</td><td>---</td></tr><tr><td>Tween80</td><td>Sigma Aldrich</td><td>P8047</td><td>---</td></tr><tr><td>Isoflurane</td><td>CDMV</td><td>108737</td><td>Supplier: Fresenius Kabi Animal Health</td></tr><tr><td>HPLC</td><td>Waters</td><td>1525 Binary Pump, 2998 Photodiodde Array Detector, E-SAT/IN, Bioscan Flowcount PMT detector (item # 15590)</td><td>---</td></tr><tr><td>HPLC column for analysis and purification of compounds 2-4</td><td>Phenomenex</td><td>00G-4435-E0</td><td>Gemini&amp;reg; 5 &amp;micro;m C18 110&amp;nbsp;&amp;Aring;, LC Column 250 x 4.6 mm</td></tr><tr><td>HPLC column for analysis and purification of compounds 1 and 5</td><td>Waters&amp;nbsp;</td><td>186003115</td><td>XBridge BEH C18 Column, 130 &amp;Aring;, 5 &amp;micro;m, 4.6 mm x 100 mm</td></tr><tr><td>Microwave Reactor&amp;nbsp;</td><td>Biotage&amp;nbsp;</td><td>Initiator 8</td><td>---</td></tr><tr><td>Biotage V10 Evaporator</td><td>Biotage&amp;nbsp;</td><td>Serial # V1041</td><td>---</td></tr><tr><td>Dose calibrator</td><td>Capintec, Inc.&amp;nbsp;</td><td>CRC-25R</td><td>---</td></tr><tr><td>Gamma counter</td><td>Perkin Elmer</td><td>Wizard 1470 Automatic Gamma Counter</td><td>---</td></tr><tr><td>Animal room scale&amp;nbsp;</td><td>Mettler Toledo</td><td>XP105 Delta Range</td><td>---</td></tr><tr><td>Microwave vials&amp;nbsp;</td><td>Biotage&amp;nbsp;</td><td>355629</td><td>0.5-2 mL&amp;nbsp;</td></tr></tbody></table>

preparation and evaluation of 99mtc-labeled tridentate chelates for pre-targeting using bioorthogonal chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

该配体通过一个简单的还原胺化的策略（ 图2），随后将产物的耦合到市售四嗪22,23使用不同的连接体和螯合剂合成。放射性标记物使用的所有化合物中相同的方法进行，并且是高度可重复的。 83％（1），45％（2），31％:该过程通过改变pH值，配体，反应时间和温度的量，随后在99m?...

Watch this Scientific Journal Video about Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry at JoVE.com

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

制备和评价<sup&gt;99米</sup&gt;锝标记的三齿螯合物预靶向使用生物正交化学

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

preparation-evaluation-99mtc-labeled-tridentate-chelates-for-pre

Universidad San Sebastian

Research

JoVE Journal

Chemistry

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

8.1K 次观看.  McMaster University. Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry. 

视频: Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

微流控芯片上捕获环加成反应可逆地固定小分子或多组分结构的生物传感器应用

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

科研

化学

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET radiopharmaceuticals. Due to their multi-day biological half-life, antibodies must be labeled with positron-emitting radionuclides with relatively long physical decay half-lives. Traditionally, the positron-emitting isotopes 124I (t1/2 = 4.18 d), 86Y (t1/2 = 14.7 hr), and 64Cu (t1/2 = 12.7 hr) have been used to label antibodies for PET imaging. More recently, however, the field has witnessed a dramatic increase in the use of the positron-emitting radiometal 89Zr in antibody-based PET imaging agents. 89Zr is a nearly ideal radioisotope for PET imaging with immunoconjugates, as it possesses a physical half-life (t1/2 = 78.4 hr) that is compatible with the in vivo pharmacokinetics of antibodies and emits a relatively low energy positron that produces high resolution images. Furthermore, antibodies can be straightforwardly labeled with 89Zr using the siderophore-derived chelator desferrioxamine (DFO). In this protocol, the prostate-specific membrane antigen targeting antibody J591 will be used as a model system to illustrate (1) the bioconjugation of the bifunctional chelator DFO-isothiocyanate to an antibody, (2) the radiosynthesis and purification of a 89Zr-DFO-mAb radioimmunoconjugate, and (3) in vivo PET imaging with an 89Zr-DFO-mAb radioimmunoconjugate in a murine model of cancer.

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

Due to its multi-day radioactive half-life and favorable decay properties, the positron-emitting radiometal 89Zr is extremely well-suited for use in antibody-based radiopharmaceuticals for PET imaging. In this protocol, the bioconjugation, radiosynthesis, and preclinical application of 89Zr-labeled antibodies will be described.

该生物耦合和重新布置<sup&gt; 89</sup&gt;锆DFO标记的抗体

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

一个优化的协议通过使用金纳米粒子的放射性标记效率<sup&gt; 125</sup&gt;我标记的叠氮辅基

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

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.

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 ...

生物工程

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

前列腺癌与小分子肽CLT1靶向造影剂的磁共振分子成像

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

医学

Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction

While radioimmunotherapy (RIT) is a promising approach for the treatment of cancer, the long pharmacokinetic half-life of radiolabeled antibodies can result in high radiation doses to healthy tissues. Perhaps not surprisingly, several different strategies have been developed to circumvent this troubling limitation. One of the most promising of these approaches is pretargeted radioimmunotherapy (PRIT). PRIT is predicated on decoupling the radionuclide from the immunoglobulin, injecting them separately, and then allowing them to combine in vivo at the target tissue. This approach harnesses the exceptional tumor-targeting properties of antibodies while skirting their pharmacokinetic drawbacks, thereby lowering radiation doses to non-target tissues and facilitating the use of radionuclides with half-lives that are considered too short for use in traditional radioimmunoconjugates. Over the last five years, our laboratory and others have developed an approach to in vivo pretargeting based on the inverse electron-demand Diels-Alder (IEDDA) reaction between trans-cyclooctene (TCO) and tetrazine (Tz). This strategy has been successfully applied to pretargeted positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging with a variety of antibody-antigen systems. In a pair of recent publications, we have demonstrated the efficacy of IEDDA-based PRIT in murine models of pancreatic ductal adenocarcinoma and colorectal carcinoma. In this protocol, we describe protocols for PRIT using a 177Lu-DOTA-labeled tetrazine radioligand ([177Lu]Lu-DOTA-PEG7-Tz) and a TCO-modified variant of the colorectal cancer targeting huA33 antibody (huA33-TCO). More specifically, we will describe the construction of huA33-TCO, the synthesis and radiolabeling of [177Lu]Lu-DOTA-PEG7-Tz, and the performance of in vivo biodistribution and longitudinal therapy studies in murine models of colorectal carcinoma.

This protocol describes the synthesis and characterization of a trans-cyclooctene (TCO)-modified antibody and a 177Lu-labeled tetrazine (Tz) radioligand for pretargeted radioimmunotherapy (PRIT). In addition, it details the use of these two constructs for in vivo biodistribution and longitudinal therapy studies in a murine model of colorectal cancer.

基于逆电子需求函数-alder 反应的隐形放射免疫治疗

While radioimmunotherapy (RIT) is a promising approach for the treatment of cancer, the long pharmacokinetic half-life of radiolabeled antibodies can ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

硫醇反应试剂的合成与生物共轭作用--用于原位选择性修饰免疫共轭的生成

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

Positron Emission Tomography Imaging of Cell Trafficking: A Method of Cell Radiolabeling

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for various cancers. Despite significant progress having been made in these areas, there is still more to be learned to better understand the pharmacokinetics and pharmacodynamics of the administered therapeutic cells in the living system. For noninvasive, in vivo tracking of cells with positron emission tomography (PET), a novel [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN)-mediated cell radiolabeling method has been developed utilizing 89Zr (t1/2 78.4 h). The present protocol describes a [89Zr]Zr-DBN-mediated, ready-to-use, radiolabeling synthon for direct radiolabeling of variety of cells, including mesenchymal stem cells, lineage-guided cardiopoietic stem cells, liver regenerating hepatocytes, white blood cells, melanoma cells, and dendritic cells. The developed methodology enables noninvasive PET imaging of cell trafficking for up to 7 days post-administration without affecting the nature or the function of the radiolabeled cells. Additionally, this protocol describes a stepwise method for the radiosynthesis of [89Zr]Zr-DBN, biocompatible formulation of [89Zr]Zr-DBN, preparation of cells for radiolabeling, and finally the radiolabeling of cells with [89Zr]Zr-DBN, including all the intricate details needed for the successful radiolabeling of cells.

Presented here is a protocol to radiolabel cells with a positron emission tomography (PET) radioisotope, 89Zr (t1/2 78.4 h), using a ready-to-use radiolabeling synthon, [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN). Radiolabeling cells with [89Zr]Zr-DBN allows noninvasive tracking and imaging of administered radiolabeled cells in the body with PET for up to 7 days post-administration.

细胞运输的正电子发射断层扫描成像:一种细胞放射性标记方法

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for ...

癌症研究

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

病毒衍生肽修饰抗体结合物对增强细胞积累和改善肿瘤靶向性的初步评价

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

小鼠淋巴细胞通过标签<sup&gt; 64</sup&gt;的Cu-抗体受体靶向用于<em&gt;体内</em&gt;由PET / CT细胞运输

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

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