利用生物正交反电子需求的Diels-Alder环的预靶向PET成像
Summary
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.
Abstract
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.
Introduction
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> 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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank Prof. Ralph Weissleder, Dr. Pat Zanzonico, and Dr. NagaVaraKishore Pillarsetty for helpful conversations and the NIH for funding (BMZ: 1K99CA178205-01A1)
Materials
| Tetrazine NHS Ester | Sigma-Aldrich | 764701 | Store at -80 °C |
| Trans-cyclooctene NHS Ester | Sigma-Aldrich | 764523 | Store at -80 °C |
| p-NH2-Bn-NOTA | Macrocyclics | B-601 | Store at -80 °C |
References
- Wu, A. M. Antibodies and antimatter: The resurgence of immuno-PET. Journal of Nuclear Medicine. 50, 2-5 (2009).
- Zeglis, B. M., Lewis, J. S. A practical guide to the construction of radiometallated bioconjugates for positron emission tomography. Dalton Transactions. 40, 6168-6195 (2011).
- Hollander, N. Bispecific antibodies for cancer therapy. Immunotherapy. 1, 211-222 (2009).
- Liu, G., et al. Tumor pretargeting in mice using 99mTc-labeled morpholino, a DNA analog. Journal of Nuclear Medicine. 43, 384-391 (2002).
- Boerman, O. C., van Schaijk, F. G., Oyen, W. J. G., Corstens, F. H. M. Pretargeted radioimmunotherapy of cancer: Progress step by step. Journal of Nuclear Medicine. 44, 400-411 (2003).
- Goldenberg, D. M., Sharkey, R. M., Paganelli, G., Barbet, J., Chatal, J. F. Antibody pretargeting advances cancer radioimmunodetection and radioimmunotherapy. Journal of Clinical Oncology. 24, 823-834 (2006).
- Sharkey, R. M., Chang, C. H., Rossi, E. A., McBride, W. J., Goldenberg, D. M. Pretargeting: taking an alternate route for localizing radionuclides. Tumor Biology. 33, 591-600 (2012).
- Sharkey, R. M., et al. Improving the delivery of radionuclides for imaging and therapy of cancer using pretargeting methods. Clinical Cancer Research. 11, 7109-7121 (2005).
- Schultz, J., et al. A tetravalent single-chain antibody-streptavidin fusion protein for pretargeted lymphoma therapy. 암 연구학. 60, 6663-6669 (2000).
- Lewis, M. R., et al. In vivo evaluation of pretargeted 64Cu for tumor imaging and therapy. Journal of Nuclear Medicine. 44, 1284-1292 (2003).
- Zeglis, B. M., et al. A pretargeted PET imaging strategy based on bioorthgonal Diels-Alder click chemistry. Journal of Nuclear Medicine. 54, 1389-1396 (2013).
- Blackman, M. L., Royzen, M., Fox, J. M. Tetrazine ligation: fast bioconjugation based on inverse electron demand Diels-Alder reactivity. Journal of the American Chemical Society. 130, 13518-13519 (2008).
- Devaraj, N. K., Upadhyay, R., Hatin, J. B., Hilderbrand, S. A., Weissleder, R. Fast and sensitive pretargeted labeling of cancer cells through a tetrazine/trans-cyclooctene cycloaddition. Angewandte Chemie-International Edition. 48, 7013-7016 (2009).
- Devaraj, N. K., Weissleder, R. Biomedical applications of tetrazine cycloadditions. Accounts of Chemical Research. 44, 816-827 (2011).
- Devaraj, N. K., Weissleder, R., Hilderbrand, S. A. Tetrazine-based cycloadditions: application to pretargeted live cell imaging. Bioconjugate Chemistry. 19, 2297-2299 (2008).
- Keliher, E. J., Reiner, T., Turetsky, A., Hilderbrand, S., Weinberg, R. A. High-yielding, two-step 18F labeling strategy for 18F-PARP1 inhibitors. ChemMedChem. 6, 424-427 (2011).
- Reiner, T., Earley, S., Turetsky, A., Weissleder, R. Bioorthogonal small-molecule ligands for PARP1 imaging in living cells. ChemBioChem. 11, 2375-2377 (2010).
- Reiner, T., Keliher, E. J., Earley, S., Marinelli, B., Weissleder, R. Synthesis and in vivo imaging of a 18F-labeled PARP1 inhibitor using a chemically orthogonal scavenger-assisted high-performance method. Angewandte Chemie International Edition. 50, 1922-1925 (2011).
- Taylor, M. T., Blackman, M., Dmitrenko, O., Fox, J. M. Design and synthesis of highly reactive dienophiles for the tetrazine-trans-cyclooctene ligation. Journal of the American Chemical Society. 133, 9646-9649 (2011).
- Selvaraj, R., et al. Tetrazine-trans-cyclooctene ligation for the rapid construction of integrin alpha(v)beta(3) targeted PET tracer based on a cyclic RGD peptide. Bioorganic and Medicinal Chemistry Letters. 21 (3), 5011-5014 (2011).
- Liu, S., et al. Efficient 18F labeling of cysteine-containing peptides and proteins using tetrazine-trans-cyclooctene ligation. Molecular Imaging. 12, 121-128 (2013).
- Han, H. S., et al. Development of a bioorthogonal and highly efficient conjugation method for quantum dots using tetrazine-norbornene cycloaddition. Journal of the American Chemical Society. 132, 7838-7839 (2010).
- Zeglis, B. M., et al. Modular strategy for the construction of radiometalated antibodies for positron emission tomography based on inverse electron demand Diels-Alder click chemistry. Bioconjugate Chemistry. 22, 2048-2059 (2011).
- Zeng, D., Zeglis, B. M., Lewis, J. S., Anderson, C. J. The growing impact of bioorthogonal click chemistry on the development of radiopharmaceuticals. Journal of Nuclear Medicine. 54, 829-832 (2013).
- Reiner, T., Zeglis, B. M. The inverse electron demand Diels-Alder reaction in radiochemistry. Journal of Labeled Compounds and Radiopharmaceuticals. 57, 285-290 (2014).
- Li, Z., et al. Tetrazine-trans-cyclooctene ligation for the rapid construction of 18-F labeled probes. Chemical Communications. 46, 8043-8045 (2010).
- Karver, M. R., Weissleder, R., Hilderbrand, S. A. Synthesis and evaluation of a series of 1,2,4,5-tetrazines for bioorthogonal conjugation. Bioconjugate Chemistry. 22, 2263-2270 (2011).
- Sletten, E. M., Bertozzi, C. R. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angewandte Chemie International Edition. 48, 6973-6998 (2009).
- Bosch, S. M., et al. Evaluation of strained alkynes for Cu-free click reaction in live mice. Nuclear Medicine and Biology. 40, 415-423 (2013).
- Rossin, R., et al. In vivo chemisry for pretargeted tumor imaging in live mice. Angewandte Chemie International Edition. 49, 3375-3378 (2010).
- Ackerman, M. E., et al. A33 antigen displays persistent surface expression. Cancer Immunology and Immunotherapy. 57, 1017-1027 (2008).
- Carrasquillo, J. A., et al. 124I-huA33 antibody PET of colorectal cancer. Journal of Nuclear Medicine. 52, 1173-1180 (2011).
- Sakamoto, J., et al. A phase I radioimmunolocalization trial of humanized monoclonal antibody huA33 in patients with gastric carcinoma. Cancer Science. 97, 1248-1254 (2006).
- Rossin, R., Lappchen, R., vanden Bosch, S. M., LaForest, R., Robillard, M. S. Diels-Alder reaction for tumor pretargeting: In vivo chemistry can boost tumor radiation dose compared with directly labeled antibody. Journal of Nuclear Medicine. 54, 1989-1995 (2013).
- Rossin, R., et al. Highly reactive trans-cyclooctene tags with improved stability for Diels-Alder chemistry in living systems. Bioconjugate Chemistry. 34, 1210-1217 (2014).
- Emmetiere, F., et al. 18F-labeled-bioorthogonal liposomes for in vivo targeting. Bioconjugate Chemistry. 24, 1784-1789 (2013).
