Name: the bioconjugation and radiosynthesis of 89zr-dfo-labeled antibodies
Uploaded: 2015-02-12T14:00:00.000+00:00
Duration: 9 min 29 s
Description: Watch this Scientific Journal Video about The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies at JoVE.com

作者感谢教授托马斯·莱纳，雅各布博士霍顿，和Serge Lyaschenko博士有益的对话。

所有上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.共轭DFO-NCS来J591 <ol><li>在1.7 ml离心管，准备一个2-5毫克/毫升的溶液J591在1ml任1×磷酸盐缓冲盐水（pH 7.4）中或0.5M的HEPES缓冲液（pH 7.4）中的。 </li><li>溶解DFO-NCS在无水DMSO 5-10毫米（3.8-7.6毫克/毫升）间的浓度。声处理或涡流，以促进完全溶解的溶液彻底。 </li><li>通过加入小等分试样的0.1M的Na 2（&lt;10微升）CO 3调整J591溶液的pH至8.8-9.0。 </li><li>一旦抗体溶液是在正确的pH值，添加了对应于一个3-4倍摩尔过量的二官能螯合剂的DFO-NCS溶液的体积。 <ol><li>对于考试PLE，加4-5微升的10mM（7.6毫克/毫升）的DFO-NCS溶液（40.4纳摩尔DFO-NCS），以1ml的2mg / ml的J591抗体溶液（13.3纳摩尔J591）。 DMSO在最终含水反应混合物中的量应不超过2％的体积/体积。 </li></ol></li><li>温育30分钟，在37℃的搅拌加热块为350rpm对反应。 </li><li> 1小时，在37℃后，使用预填充的一次性尺寸排阻脱盐柱使用0.5M的HEPES缓冲液（pH为7.4）作为洗脱剂50,000分子量截止纯化所得免疫。这一步骤将产生完成J591-DFO构建体的溶液2ml。 </li><li>测量J591-DFO的浓度构造上的紫外可见分光光度计。 </li><li>如果构建体的浓度较高是理想的，集中使用离心过滤器单元具有50000分子量截止的J591-DFO溶液。 </li><li>存储完成的J591-DFO免疫偶联物在-20在黑暗中的溶液℃。 </ol> 2.放射性标记J591-DFO 89锆注意: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，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。应采取一切可能的措施，以尽量减少暴露于电离辐射。 注意:在适当的放射化学音符维持兴趣，放射​​性的样品中的量应使用的剂量校准器测量和记录前和步骤2.2-2.13在下面的协议之后。这将有助于准确测定放射化学产量和具体活动。 <ol><li>制备0.5-2.0毫克J591-DFO的溶液中加入200μl的0.5M HEPES缓冲液，pH值7.5。 </li><li>吸取的体积&lt;对应于1.0-6.0毫居里（37-222活度）至2ml塑料螺旋盖微量离心管SUP&gt; 89的Zr 4+原液（通常在1.0M的草酸提供）。调节该溶液的体积，以使用1.0M的草酸共300微升。 </li><li>调节89的Zr 4+溶液用的1.0M的Na 2 CO 3的pH值至6.8-7.5。通过加入250微升的1.0M的Na 2 CO 3至89的Zr 4+溶液开始，并随后添加小（&lt;10微升）基的等分试样以达到所需的pH值。 </li><li>加的pH调节的89的Zr 4+溶液所需量的在步骤2.1中制备的J591-DFO溶液。 </li><li>检查放射性标记反应混合物的pH，以确保它落入6.8-7.5的期望的范围内。 </li><li>孵育在室温60分钟的搅拌加热块为350rpm的放射性标记反应。 </li><li>孵育60分钟后，测量RADiolabeling使用无线电型TLC，反应的产率。 <ol><li>为此目的，在硅胶浸渍型TLC条斑的放射性标记反应混合物的1微居里。允许等分试样进行干燥，运行TLC使用50mM的DTPA（pH5.5）中的洗脱液并使用无线电型TLC扫描器分析薄层色谱条89的Zr 4+结合到J591-DFO构建体将出现在原点（R ˚F&lt;0.1），而自由89的Zr 4+阳离子将由DTPA被螯合并会与溶剂前沿（ 的 R F洗脱&gt; 0.9）。 </li><li>通过整合radiochromatogram，曲线下将该区域从R中的曲线在F下0.0-0.1通过的总面积，再乘以100计算出反 ​​应的放射性标记收率。 </li></ol></li><li>如果放射性标记收率是足够（通常&gt; 2毫居里/毫克的理论比活性），淬火用5微升50毫DTPA，pH 5.5的反应。 </li><li>净化产生的免疫USI纳克具有50000分子量截止使用任0.9％的无菌盐水与5毫克/毫升龙胆酸5mg / ml的龙胆酸或0.25M的乙酸钠（pH 5.5）洗脱剂预填充的一次性大小排阻柱脱盐。这一步骤将产生完成89锆DFO-J591放射免疫偶联的溶液2ml。 </li><li>净化后，确认89锆DFO-J591采用无线电TLC的放射化学纯度在2.7步描述。 </li><li>除以活性最初添加到抗体溶液通过放射性的分离与纯化的89的Zr-DFO-J591放射免疫的量的量计算出反 ​​应的总放射性标记收率。 </li><li>除以活性的DFO-J591中的放射性标记反应的初始质量分离与纯化的89的Zr-DFO-J591放射免疫偶联量计算最终比活性。 </li><li>如果浓度较高是理想的，集中第Ë89使用离心过滤器单元具有50000分子量截止锆DFO-J591溶液。 注:在最终纯化步骤中使用的龙胆酸是一个无线电防护剂用于抗体的降解最小化由于辐46虽然89锆DFO-J591放射免疫偶联长达48小时的贮存在4℃。是可能的，这是不推荐的。如果放射免疫偶联是要被存储，则使用0.25M的乙酸钠（pH 5.5）以5毫克/毫升龙胆酸如，以减少次氯酸盐介导的放射分解的风险的存储缓冲器。47 </li></ol> 3. 体内 PET显像与89锆DFO-J591 注意:由于协议第2条，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。所有possib应采取勒步骤，以尽量减少暴露于电离辐射。 <ol><li>在雄性无胸腺裸鼠皮下植入5×10 6的LNCaP前列腺癌细胞，并允许这些（在接种后3-4周），以生长到100-150毫米3异种移植物。44 </li><li>稀释89锆DFO-J591放射免疫，以1.0毫居里/ ml的0.9％无菌生理盐水的浓度。 </li><li>注入200微升89锆DFO-J591溶液（200微居里; 7.4活度）的。到尾静脉移植荷瘤小鼠的48 </li><li>在所需的成像时间点（ 例如，12，24，48，72，96，或120小时后喷射），麻醉的小鼠用2％异氟烷:氧混合气。 </li><li>放置在小动物PET扫描仪的床的鼠标，并用1％异氟烷在扫描期间保持麻醉:氧混合气。在此之前将动物放在扫描仪床上，用脚趾捏法和APPL验证麻醉Ÿ眼药膏鼠标，以防止在麻醉过程中干燥的眼睛。49 </li><li>通过用最少的使用350-700千电子伏的能量窗和6纳秒重合定时窗口40000000同步事件的静态扫描获得的小鼠的PET的数据。50 </li><li>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>

作者什么都没有透露。

而施工，放射性标记，成像和89锆DFO-标有放射免疫通常是一个相当简单的过程，它保持了几个重要的考虑因素记在该过程的每个步骤是重要的。例如，也许在该过程的缀合步骤关注最可能的原因是该抗体的缀合反应期间的聚合。此问题是最经常在加入DFO-NCS原液后的缀合反应的混合不良的产品。22当这种情况发生时，DFO-NCS的非均匀分布可能导致过高的水平与当地反应的抗体，其可以反过来导致聚集。这个问题可以相对容易地通过在。温度添加DFO-NCS原液在小等份（&lt;5微升），将反应混合物在加入DFO-NCS的后充分混合，并搅拌该反应混合物规避电子控制的振动筛。此外，DFO-单抗构建体的缀合和纯化后，就精确确定缀合每种mAb DFO的数量是非常重要的。可以使用辐射同位素稀释实验类似于由荷兰执行来实现每个抗体DFO螯合物的数目的充分表征， 等。和Anderson 等人 ，虽然MALDI-TOF质谱法是一种可行的选择。14,23 ，30,51,52在放射性标记的步骤，轻松地最常见的问题是低于预期的产量放射性标记。如果尽管刻苦按照上述的协议发生出乎意料低的产率，三种不同的故障排除的策略是可用的:（1）温育对较长的时间量的放射性标记反应（ 例如，2-3小时）; （2）重复使用抗体的更高浓度的放射性标记反应;或（3）重复使用较高摩尔过量次的初始DFO-NCS的缀合反应Ë双功能螯合剂。 而DFO-NCS偶联轻便和健壮，其不可否认的弱点之一是，它不是位点特异性:DFO-NCS形成一个与在该抗体可用赖氨酸而不论其位置的硫脲键。其结果是，它有可能是螯合剂可能成为附加到该抗体的抗原结合区，由此将89锆DFO标记偶联物的免疫反应性产生不利影响。因此，一个良好的平衡，必须在89锆标记的放射免疫建筑被击中:较高的数字每抗体螯合剂利于提高具体的活动，但高学历的标签也增加危及结构的免疫反应的风险。最后，我们的目标是简单的:附加必要而不损害免疫反应尽可能多螯合剂。获得纯化的89锆DFO-单克隆抗体放射免疫后，确定是关键<em&gt;体外免疫反应之前的任何体内实验的构建体。为此，建议使用由Lindmo公布的体外方法， 等。53,54如果构建体的免疫反应性低于80％-90％，这可能是必要的，返回到缀合反应并追加更少DFO部分每抗体。另外，如果将纯化的89的Zr-DFO-mAb的免疫反应性是高（&gt; 90％）和较高的特定活动需要的话，它可能会附加更多螯合剂与抗体不降低的免疫反应性。 最后，一个89锆DFO标记的抗体的体内行为是，当然，高度依赖于抗体的两个身份和所用的肿瘤模型。在这里提出的模型系统，在肿瘤的最大摄取值达到约60％ID /克;然而，报告在文献中获得最大的肿瘤摄取Values ​​范围从低至15％-20％的ID / g至高达80％-90％的ID / g的33,44,55-57同样，吸收在非靶组织的量-尤其是肝和脾 - 可以变化很大，取决于所研究的抗体/抗原系统。 89锆DFO标记的抗体的比活性是在体内实验中的一个重要的考虑因素。对于89锆DFO蛋白单克隆抗体的具体活动的文献值通常一般为1-6毫居里/毫克（37-222活度/毫克）。8,10，更高的具体活动是可取的，因为它们减少了无意中的可能性饱和的抗原（ 即，自阻塞）的。这成为在具有较低水平的抗原的表达系统中尤其如此。不管抗体/抗原系统，无体内调查89锆DFO标记的显像剂是完全没有选择性的演示。这可以通过使用阻断实验来实现大量未标记的生物分子，或使用细胞系不表达所讨论的抗原。在这里所描述的方法，前者使用的，但89锆DFO-J591的选择性也已使用PSMA阴性PC3前列腺癌异种移植物表现出23 要注意的是，尽管其明显的优点，这DFO-NCS基合成方法是不完美的是重要的。正如我们所讨论的，DFO不是一个理想的螯合剂89的Zr 4+，和缀合反应的非位点特异性性质可以证明繁琐。规避这些问题，令人兴奋的努力开发新的螯合剂89的Zr 4+和位点特异性放射性标记方法目前正在进行，但这些新的技术仍然需要被优化和验证在实验室和诊所。24,26,27， 29,44最终，DFO-NCS方法论建设89锆DFO标记的抗体已被证明是一种非常强大的工具，放射性免疫的合成和有潜力可用于创建多种临床上有用的放射性药物。

由于其显着的敏感性，亲和力和选择性，抗体长期以来被认为是有前途的载体用于递送放射性同位素至肿瘤细胞。但是，它们在正电子发射断层扫描（PET）成像中的应用受到阻碍，缺乏适当的正电子发射放射性同位素为其标签1-3之一在放射性免疫的设计中最关键的考虑因素是匹配的物理衰变半寿命放射性同位素与抗体的体内药动学。更具体地说，抗体通常具有相对长的，多天的生物半衰期，因此必须用放射性同位素标记具有可比的物理半衰期。用于PET成像的应用中，抗体历来放射性标记的64铜（叔1/2 = 12.7小时），86 Y（吨1/2 = 14.7小时），或124 I（叔1/2 = 4.18 d）所示。4， 5然而，每个这些放射性同位素拥有妨碍其适用于临床影像显著的局限性。而放射免疫标记86 Y和64铜已被证明有希望的临床前调查，既同位素具有物理半衰期太短是有效用于成像在人类。124 I，与此相反，具有接近理想的物理半衰期为成像用的抗体，但它是昂贵的，并且具有导致相对较低的分辨率临床图像欠佳衰减特性。此外，124 I标记的放射性免疫能受到脱卤体内 ，一种能降低肿瘤-背景活动比率的过程。6,7 该驱动器找到一个正电子发射放射性同位素来取代64铜，86 Y，124我放射免疫起到了推波助澜的最近激增的研究89锆标记的抗体。8-12牛逼他原因89锆问世是直截了当:放射性金属具有近乎理想的化学和物理性质用于诊断的PET放射性免疫使用13 89锆经由89 Y（P，N）上使用一个回旋加速器89 Zr的反应而产生。市售和100％自然丰富89ÿ目标。14,15的放射性金属具有23％的正电子产率，衰变78.4小时的半衰期，并且发射正电子与395.5千电子伏（ 图1）的相对低的能量。 13,16,17要注意的是89锆还发射较高能量是很重要的，909千电子伏γ射线具有99％的效率。虽然这种发射不使劲与发射的511keV光子干扰，但是它对于运输，处理，和剂量需要额外考虑。尽管这样的警告，这些衰减特性最终意味着89锆不仅拥有更有利的ħ用于成像ALF-寿命的抗体比86 Y和64铜，但也可以比124 I产生更高分辨率的图像，其发射正电子与687和975 keV的更高的能量，以及一个数的光子能量与内100-150千电子伏的在511千电子伏的正电子创建的光子。13此外，89的Zr也更安全地处理，以产生更便宜，并且residualizes肿瘤更有效地比其对应物的放射性碘。18,19的一个潜在的89 Zr的限制是，它不具有治疗isotopologue， 比如 ，86 Y（PET）与90 Y（治疗）。这就排除了可以用作剂量球探对他们的治疗对口化学性质相同，替代显像剂的建设。尽管如此，调查结果表明，89锆标记的抗体是有潜力的成像代理人90 Y-和177路标记免疫。20,21 从化学的观点来看，作为一种Ⅳ族金属，89 Zr的存在是为4阳离子在水溶液。所述的Zr 4+离子被高度带电的，相对大的（有效离子半径= 0.84埃），并且可以被分类为一"硬"阳离子。因此，它表现出偏爱配体承载多达八个硬的，阴离子的氧供体。容易在89锆标记的放射性免疫中使用的最常见的螯合剂是去铁胺（DFO），一铁载体衍生的，无环螯合剂轴承3异羟肟酸基团。配位体稳定地协调的Zr 4+阳离子迅速干净在RT在生物相关的pH水平，并且将所得的Zr-DFO络合物保持稳定的在盐水中，血清多天的过程中，和全血。22计算研究强烈提示该DFO形成六配位配合物的Zr 4+，其中金属中心配位的三NEUT拉尔和配体的3阴离子氧供体，以及两个外源性配体的水 （ 图2）。23,24放射免疫采用89锆DFO结合支架的体内行为已普遍出色。然而，在一些情况下，成像和急性生物分布研究显示升高的活性水平在注射89锆标记的抗体的小鼠的骨，该数据表明，osteophilic 89的Zr 4+阳离子选自螯合剂释放在体内并且随后矿化在骨。25最近，一些调查到的新的89锆4+螯合剂的发展尤其是配体与八氧捐助者已经出现在文献中。24,26,27不过，目前，DFO是最广泛采用的螯合剂在89大幅锆标记的放射免疫。各种不同生物结合策略已被用来DFO附加到抗体，包括生物正交点击化学，硫醇反应性DFO构建体与抗体的半胱氨酸反应，和活化酯承载DFO构建体与抗体赖氨酸反应。4,28- 30轻松最常见的策略，然而，已使用DFO，DFO-NCS的异硫氰酸酯轴承衍生物（ 图2）。22本市售双功能螯合剂鲁棒且可靠地形成稳定的，共价的硫脲用的赖氨酸键抗体（ 图3）。 在过去的几年中，各种各样的89锆DFO标记放射性免疫已在文献中报道。临床前研究已经特别丰富，具有抗体不等的较为知名的西妥昔单抗，贝伐单抗，曲妥珠单抗和更深奥的抗体，如CD105靶向牛逼RC105和游离PSA靶向5A10 30-36最近，少数使用89锆DFO标记抗体早期临床试验已经出现在文献中。具体而言，在荷兰团体已经发表的试验采用89锆DFO-政制及内地事务U36，89锆DFO-替伊莫单抗，和89锆DFO -曲妥单抗。21,32,37此外，一系列的89其他临床试验锆标记的放射免疫目前正在进行，其中包括使用PSMA靶向89锆DFO-J591前列腺癌成像和HER2靶向89锆DFO，曲妥珠单抗对乳腺癌影像学检查在这里纪念Sloan Kettering癌症中心23， 30此外，虽然放射性标记的抗体仍然是最常用的89锆标记的放射性药物，放射性金属，也越来越多地被使用与其他载体，包括肽，蛋白质，和纳米材料。38-43 </sup&gt; 这89锆DFO标签方法的模块化是 ​​一笔巨大的财富。生物标志物靶向抗体的剧目是不断扩展的，并且在使用这些结构进行体内 PET成像的兴趣日益高涨。因此，我们认为，更加规范的做法和协议的制定可能受益的领域。优异的书面实验方案为DFO-NCS的共轭和89 Zr的放射性标记已经公布由Vosjan 等人 22，我们认为，通过这项工作所提供的视觉演示可进一步帮助调查新到这些技术。在协议中，在另一方面，前列腺特异性膜抗原靶向抗体J591将被用作模型系统，以说明（1）的双功能螯合剂的生物缀DFO-异硫氰酸酯的抗体，89（2）的放射合成和纯化ZR-DFO-单克隆抗体放射免疫，及（3） 在体内 PET成像与89锆DFO-单抗放射免疫偶联在癌症的小鼠模型。23,44,45

<table><tbody><tr><td>&lt;em&gt;p&lt;/em&gt;-SCN-Bn-DFO</td><td>Macrocyclics</td><td>B-705</td><td>Store at -80 &amp;deg;C</td></tr><tr><td>[&lt;sup&gt;89&lt;/sup&gt;Zr]Zr-oxalate</td><td>Various, including Perkin-Elmer</td><td></td><td>Caution: Radioactive material</td></tr><tr><td>PD-10 Desalting Columns</td><td>GE Healthcare</td><td>17-0851-01&amp;nbsp;</td><td>Store at room temperature</td></tr><tr><td>Amicon Ultra-4 Centrifugal Filter Units</td><td>EMD Millipore</td><td>UFC805024</td><td>Store at room temperature</td></tr><tr><td>Silica Gel Impregnated RadioTLC Paper</td><td>Agilent Technologies</td><td>SGI0001</td><td>Cut into strips 0.5 cm wide</td></tr></tbody></table>

the bioconjugation and radiosynthesis of 89zr-dfo-labeled antibodies

特殊的亲和力，特异性和选择性的抗体使其成为肿瘤靶向PET放射性药物非常有吸引力的载体。由于其多天的生物半衰期，抗体必须贴有正电子发射放射性核素具有相对长的物理衰变半衰期。传统上，正电子发射同位素124 I（叔1/2 = 4.18 d）中，86 Y（吨1/2 = 14.7小时），和64的Cu（叔1/2 = 12.7小时），已被用于标记抗体PET成像。最近，然而，该领域已经见证了在基于抗体的PET成像剂的使用的正电子发射放射性金属89 Zr的显着增加。89锆是一种近乎理想的放射性同位素用于PET成像的免疫，因为它具有一个物理半-life（吨1/2 = 78.4小时），它与抗体的体内药动学相容并发射相对低的烯产生高分辨率的图像红绿黄正电子。此外，抗体可直接地使用铁载体衍生的螯合剂去铁胺（DFO）标记89锆。在这个协议中，前列腺特异性膜抗原靶向抗体J591将被用作模型系统，以说明（1）的双功能螯合剂的生物缀DFO-异硫氰酸酯的抗体，（2）的放射和89 ZR-纯化DFO-单抗放射免疫偶联，和（3） 在体内 PET成像与89锆DFO-单抗放射免疫偶联中的癌症的小鼠模型。

在这个协议中DFO-NCS的与抗体缀合的第一个步骤通常是相当坚固和可靠的。一般地，纯化的，螯合剂改性的免疫可以在&gt; 90％的产率获得，并且使用DFO-NCS的3摩尔当量在最初的缀合反应将产生一定程度的-的标记的约1.0-1.5螯合剂DFO的/单克隆抗体。 89锆放射性标记和纯化步骤的过程中也同样简单。在上述协议中列出的浓度&gt; 80％的放射性标记的产量和&gt; 2.0毫居里/毫克因此具体活动是在室温下60分钟后的典型。将粗混合物放射性标记的无线电型TLC色谱将可能揭示一些DTPA结合89的Zr 4+洗脱的在溶剂前沿（ 图4A）。然而，骤冷与DTPA反应和纯化通过大小排阻层析的89锆DFO-单抗构建体，所述radioc后纯化的，分离的89锆DFO-单抗缀合物hemical纯度应&gt; 95％（ 图4B）。在事件的孤立89锆DFO-单抗偶联物的放射化学纯度是95％以上，该纯化步骤应重复执行任何体外之前或在体内实验。 移动到体内实验，在协议中如上所述，无胸腺裸鼠的PSMA表达，将LNCaP前列腺癌异种移植物用于研究的89锆DFO-J591的体内行为。急性生物分布和PET成像实验表明89锆DFO-J591清楚地描述了前列腺癌异种移植物具有优良的图像的对比度和高肿瘤-背景活动率（ 图5）。在肿瘤的放射免疫偶联的摄取是显而易见的，早在24小时（20.9％±5.6％ID / g）和活性浓度在肿瘤增加到最大值的57.5％±5.3％ID / g的在96小时后注射。作为是典型的放射免疫，相对高浓度的放射性示踪剂的是存在于血液中，在早期时间点（9.1％±5.3％ID / g的为24小时），随后缓慢降低放射性的量在血液过当然实验。非靶组织具有最高的活性浓度为骨骼，其在整个实验有10％左右的ID / g的显示摄取值，这可能是由于在体内释放的osteophilic阳离子89的Zr 4+的结果。所有其它器官包括心脏，肺，肝，脾，胃，大肠和小肠，肾脏，并显示相对低的活性浓度肌肉，往往远低于5％的ID / g以下。作为对照，小鼠的额外队列注射共注射300微克未标记DFO-J591以饱和抗原，从而示出了选择性阻断。 Criti<html>美云，阻挡实验降低了肿瘤的放射免疫偶联摄取从48.9％±9.3％ID / g至23.5％±11.1％ID / g的在72小时后注射，清楚地表明，有89锆DFO-J591选择性靶向其抗原。 <img alt="图1" src="/files/ftp_upload/52521/52521fig1highres.jpg" width="700px" /> 图1（A）的简化衰减方案和（B）的89一些显着衰减特性锆13,16,17 IT =异构转变。 EC =电子捕获。修改，转载来自DERI 等权限。核医学和生物学 。40，3-14（2013年）。 <a href="https://www.jove.com/files/ftp_upload/52521/52521fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html>重2"SRC ="/文件/ ftp_upload / 52521 / 52521fig2highres.jpg"WIDTH ="700像素"/&gt; 图2.（A）DFO-NCS与红色的协调氧原子的结构; （B）的锆DFO配合物的DFT衍生结构。修改，转载来自DERI 等权限。药物化学杂志，57，4849-4860（2014）。版权所有2014年美国化学学会。 <a href="https://www.jove.com/files/ftp_upload/52521/52521fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52521/52521fig3highres.jpg" width="700px" /> 图3.计划的89锆DFO-J591的生物耦合和放射性标记。等="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/52521/52521fig4highres.jpg" width="700px" /> 89锆DFO-J591粗放射性标记混合物（A）和纯化的产物（B）的图4代表无线电薄层色谱图。无线电薄层色谱扫描法进行了关于使用50mM的DTPA，pH 5.0的洗脱剂的二氧化硅条运行。 <a href="https://www.jove.com/files/ftp_upload/52521/52521fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52521/52521fig5highres.jpg" width="700px" /> 89锆DFO-J591图5.冠状PET图像（11.1-12.9活度[300-345微居里]经尾静脉200微升0.9％的无菌生理盐水注射）的无胸腺裸鼠皮下轴承，PSMA表达24和120小时后注射之间的LNCaP前列腺癌裸鼠（白色箭头）。修改和Zeglis 等许可转载。生物共轭化学 。24，1057至1067年（2013年）。版权所有2013美国化学学会。 <a href="https://www.jove.com/files/ftp_upload/52521/52521fig5highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies at JoVE.com

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 ...

the-bioconjugation-and-radiosynthesis-of-89zr-dfo-labeled-antibodies

Nanyang Technological University

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Unit 1

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

Fundamentals of Chemistry

SCIENTISTS IN ACTION

17.1K 次观看.  Memorial Sloan Kettering Cancer Center. 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. 

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

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 ...

科研

化学

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.

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 ...

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

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 ...

免疫与感染

Determining Binding Affinity (KD) of Radiolabeled Antibodies to Immobilized Antigens

Determining binding affinity (KD) is an important aspect of the characterization of radiolabeled antibodies (rAb). Typically, binding affinity is represented by the equilibrium dissociation constant, KD, and can be calculated as the concentration of antibody at which half the antibody binding sites are occupied at equilibrium. This method can be generalized to any radiolabeled antibody or other protein and peptide scaffolds. In contrast to cell-based methods, the choice of immobilized antigens is particularly useful for validating binding affinities after long-term storage of antibodies, distinguishing binding affinities of fragment antigen-binding region (Fab) arms in bispecific antibody constructs, and determining if there is variability in antigen expression between different cell lines. This method involves immobilizing a fixed amount of antigen to specified wells on a breakable 96-well plate. Then, nonspecific binding was blocked in all wells with bovine serum albumin (BSA). Subsequently, the rAb was added in a concentration gradient to all wells. A range of concentrations was chosen to allow the rAb to reach saturation, i.e., a concentration of antibody at which all antigens are continuously bound by the rAb. In designated wells without immobilized antigen, nonspecific binding of the rAb can be determined. By subtracting nonspecific binding from total binding in the wells with immobilized antigen, specific binding of the rAb to the antigen can be determined. The KD of the rAb was calculated from the resulting saturation binding curve. As an example, binding affinity was determined using radiolabeled amivantamab, a bispecific antibody for epidermal growth factor receptor (EGFR) and cytoplasmic mesenchymal-epithelial transition (cMET) proteins.

Determining Binding Affinity KD of Radiolabeled Antibodies to Immobilized Antigens

Here, a method is described to determine the binding affinity (KD) of radiolabeled antibodies to immobilized antigens. KD is the equilibrium dissociation constant that can be determined from a saturation binding experiment by measuring the total, specific, and nonspecific binding of a radiolabeled antibody at various concentrations to its antigen.

确定放射性标记抗体与固定抗原的结合亲和力（KD）

Determining binding affinity (KD) is an important aspect of the characterization of radiolabeled antibodies (rAb). Typically, binding affinity is ...

医学

Pretargeted Radioimmunotherapy: A Method for Targeted Delivery of Therapeutic Radionuclides to Tumors

Source: Membreno, R., et al. <a href="https://www.jove.com/v/59041"> Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction.</a> J. Vis. Exp. (2019).
This video describes the technique of pretargeted radioimmunotherapy or PRIT, in which immunoconjugates followed by radioligands are administered for treating tumors. This radioimmunotherapy reduces radiation doses to healthy tissues and utilizes radionuclides with very low half-lives incompatible with antibody-based vectors.

靶向前放射免疫治疗:一种将治疗性放射性核素靶向递送到肿瘤的方法

Source: Membreno, R., et al.  Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction. J. Vis. Exp. (2019).
This video ...

该生物耦合和重新布置

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一个优化的协议通过使用金纳米粒子的放射性标记效率

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