Name: pretargeted radioimmunotherapy based on the inverse electron demand diels-alder reaction
Uploaded: 2019-01-29T14:30:00
Description: Watch this Scientific Journal Video about Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction at JoVE.com

作者感谢雅各布·霍顿博士的有益对话。作者还要感谢国家卫生研究院的慷慨资助 (r00ca178205 和 U01CA221046)。

本研究中描述的所有体内动物实验都是根据批准的协议进行的, 并根据纪念斯隆·凯特林癌症中心、威尔·康奈尔大学医疗中心和亨特学院的道德准则进行机构动物护理和使用委员会 (iacuc)。
1. hua33-tco 的制备
注: hua33-tco 的合成以前曾报告过 29。但是, 为了便于读取器, 它在这里复制, 并进行调整以获得最佳条件。
<ol><li>在 1.7 ml 微?...

<ol>
	<li>Goldenberg, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+Therapy+of+Cancer+with+Radiolabeled+Antibodies.">Targeted Therapy of Cancer with Radiolabeled Antibodies.</a> Journal of Nuclear Medicine. 43 (5), 693-713 (2002).</li><li>Goldenberg, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioimmunotherapy+of+B-cell+Lymphomas+with+Iodine-131-labeled+LL2+Monoclonal+Antibody.">Radioimmunotherapy of B-cell Lymphomas with Iodine-131-labeled LL2 Monoclonal Antibody.</a> Journal of Clinical Oncology. 9 (4), 548-564 (1991).</li><li>Kaminski, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioimmunotherapy+with+131I-Tositumomab+for+Relapsed+or+Refractory+B-cell+non-Hodgkin+Lymphoma:+Updated+Results+and+Long-Term+Follow-Up+of+the+University+of+Michigan+Experience.">Radioimmunotherapy with 131I-Tositumomab for Relapsed or Refractory B-cell non-Hodgkin Lymphoma: Updated Results and Long-Term Follow-Up of the University of Michigan Experience.</a> Blood. 96 (4), 1259-1266 (2000).</li><li>Davies, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioimmunotherapy+for+B-cell+Lymphoma:+Y90+Ibritumomab+Tiuxetan+and+I131+Tositumomab.">Radioimmunotherapy for B-cell Lymphoma: Y90 Ibritumomab Tiuxetan and I131 Tositumomab.</a> Oncogene. 26, 3614 (2007).</li><li>Rajendran, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Radiation+Dose+Estimation+for+Myeloablative+Radioimmunotherapy+for+Relapsed+or+Recurrent+Mantle+Cell+Lymphoma+Using+131I+Tositumomab+to+That+of+Other+Types+of+Non-Hodgkin's+Lymphoma.">Comparison of Radiation Dose Estimation for Myeloablative Radioimmunotherapy for Relapsed or Recurrent Mantle Cell Lymphoma Using 131I Tositumomab to That of Other Types of Non-Hodgkin's Lymphoma.</a> Cancer Biotherapy and Radiopharmaceuticals. 19 (6), 738-745 (2004).</li><li>Rajendran, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Dose+131I-Tositumomab+(Anti-CD20)+Radioimmunotherapy+for+Non-Hodgkin's+Lymphoma:+Adjusting+Radiation+Absorbed+Dose+to+Actual+Organ+Volumes.">High-Dose 131I-Tositumomab (Anti-CD20) Radioimmunotherapy for Non-Hodgkin's Lymphoma: Adjusting Radiation Absorbed Dose to Actual Organ Volumes.</a> Journal of Nuclear Medicine. 45 (6), 1059-1064 (2004).</li><li>Larson, S. M., Carrasquillo, J. A., Cheung, N. -. K. V., Press, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioimmunotherapy+of+Human+tumours.">Radioimmunotherapy of Human tumours.</a> Nature Reviews Cancer. 15 (6), 347-360 (2015).</li><li>Kelly, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Targeting+by+a+Multivalent+Single-Chain+Fv+(scFv)+Anti-Lewis+Y+Antibody+Construct.">Tumor Targeting by a Multivalent Single-Chain Fv (scFv) Anti-Lewis Y Antibody Construct.</a> Cancer Biotherapy &#38; Radiopharmaceuticals. 23 (4), 411-423 (2008).</li><li>Yazaki, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Targeting+of+Radiometal+Labeled+Anti-CEA+Recombinant+T84.66+Diabody+and+T84.66+Minibody:+Comparison+to+Radioiodinated+Fragments.">Tumor Targeting of Radiometal Labeled Anti-CEA Recombinant T84.66 Diabody and T84.66 Minibody: Comparison to Radioiodinated Fragments.</a> Bioconjugate Chemistry. 12 (2), 220-228 (2001).</li><li>van Duijnhoven, S. M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabody+Pretargeting+with+Click+Chemistry+In+Vivo.">Diabody Pretargeting with Click Chemistry In Vivo.</a> Journal of Nuclear Medicine. 56 (9), 1422-1428 (2015).</li><li>Altai, M., Membreno, R., Cook, B., Tolmachev, V., Zeglis, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretargeted+Imaging+and+Therapy.">Pretargeted Imaging and Therapy.</a> Journal of Nuclear Medicine. 58 (10), 1553-1559 (2017).</li><li>Goldenberg, D. M., Chatal, J. -. F., Barbet, J., Boerman, O., Sharkey, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Imaging+and+Therapy+with+Bispecific+Antibody+Pretargeting.">Cancer Imaging and Therapy with Bispecific Antibody Pretargeting.</a> Update on cancer therapeutics. 2 (1), 19-31 (2007).</li><li>Rossin, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-Vivo+Chemistry+for+Pretargeted+Tumor+Imaging+in+Live+Mice.">In-Vivo Chemistry for Pretargeted Tumor Imaging in Live Mice.</a> Angewandte Chemie International Edition. 49 (19), 3375-3378 (2010).</li><li>Gestin, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Step+Targeting+of+Xenografted+Colon+Carcinoma+Using+a+Bispecific+Antibody+and+188Re-Labeled+Bivalent+Hapten:+Biodistribution+and+Dosimetry+Studies.">Two-Step Targeting of Xenografted Colon Carcinoma Using a Bispecific Antibody and 188Re-Labeled Bivalent Hapten: Biodistribution and Dosimetry Studies.</a> Journal of Nuclear Medicine. 42 (1), 146-153 (2001).</li><li>Green, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Analysis+of+Bispecific+Antibody+and+Streptavidin-Targeted+Radioimmunotherapy+for+B-cell+Cancers.">Comparative Analysis of Bispecific Antibody and Streptavidin-Targeted Radioimmunotherapy for B-cell Cancers.</a> Cancer Research. 76 (22), 6669 (2016).</li><li>Sharkey, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Streptavidin&#8722;Anti-Carcinoembryonic+Antigen+Antibody,+Radiolabeled+Biotin+Pretargeting+Method+for+Radioimmunotherapy+of+Colorectal+Cancer.+Studies+in+a+Human+Colon+Cancer+Xenograft+Model.">Development of a Streptavidin&#8722;Anti-Carcinoembryonic Antigen Antibody, Radiolabeled Biotin Pretargeting Method for Radioimmunotherapy of Colorectal Cancer. Studies in a Human Colon Cancer Xenograft Model.</a> Bioconjugate Chemistry. 8 (4), 595-604 (1997).</li><li>Knox, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+II+Trial+of+Yttrium-90-DOTA-Biotin+Pretargeted+by+NR-LU-10+Antibody/Streptavidin+in+Patients+with+Metastatic+Colon+Cancer.">Phase II Trial of Yttrium-90-DOTA-Biotin Pretargeted by NR-LU-10 Antibody/Streptavidin in Patients with Metastatic Colon Cancer.</a> Clinical Cancer Research. 6 (2), 406 (2000).</li><li>Schubert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Tumor+Pretargeting+System+Based+on+Complementary+L-Configured+Oligonucleotides.">Novel Tumor Pretargeting System Based on Complementary L-Configured Oligonucleotides.</a> Bioconjugate Chemistry. 28 (4), 1176-1188 (2017).</li><li>Forero, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+1+Trial+of+a+Novel+Anti-CD20+Fusion+Protein+in+Pretargeted+Radioimmunotherapy+for+B-cell+Non-Hodgkin+Lymphoma.">Phase 1 Trial of a Novel Anti-CD20 Fusion Protein in Pretargeted Radioimmunotherapy for B-cell Non-Hodgkin Lymphoma.</a> Blood. 104 (1), 227 (2004).</li><li>Kalofonos, H. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+Tumor+in+Patients+with+Indium-111-Labeled+Biotin+and+Streptavidin-Conjugated+Antibodies:+Preliminary+Communication.">Imaging of Tumor in Patients with Indium-111-Labeled Biotin and Streptavidin-Conjugated Antibodies: Preliminary Communication.</a> Journal of Nuclear Medicine. 31 (11), 1791-1796 (1990).</li><li>Zeglis, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pretargeted+PET+Imaging+Strategy+Based+on+Bioorthogonal+Diels-Alder+Click+Chemistry.">A Pretargeted PET Imaging Strategy Based on Bioorthogonal Diels-Alder Click Chemistry.</a> Journal of Nuclear Medicine. 54 (8), 1389-1396 (2013).</li><li>Meyer, J. -. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=18F-Based+Pretargeted+PET+Imaging+Based+on+Bioorthogonal+Diels-Alder+Click+Chemistry.">18F-Based Pretargeted PET Imaging Based on Bioorthogonal Diels-Alder Click Chemistry.</a> Bioconjugate Chemistry. 27 (2), 298-301 (2016).</li><li>Rossin, R., L&#228;ppchen, T., vanden Bosch, S. M., Laforest, R., Robillard, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diels-Alder+Reaction+for+Tumor+Pretargeting:+In+Vivo+Chemistry+Can+Boost+Tumor+Radiation+Dose+Compared+with+Directly+Labeled+Antibody.">Diels-Alder Reaction for Tumor Pretargeting: In Vivo Chemistry Can Boost Tumor Radiation Dose Compared with Directly Labeled Antibody.</a> Journal of Nuclear Medicine. 54 (11), 1989-1995 (2013).</li><li>Zeglis, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+Pretargeted+Strategy+for+the+PET+Imaging+of+Colorectal+Carcinoma+via+the+Modulation+of+Radioligand+Pharmacokinetics.">Optimization of a Pretargeted Strategy for the PET Imaging of Colorectal Carcinoma via the Modulation of Radioligand Pharmacokinetics.</a> Molecular Pharmaceutics. 12 (10), 3575-3587 (2015).</li><li>Agard, N. J., Prescher, J. A., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Strain-Promoted+[3+++2]+Azide&#8722;Alkyne+Cycloaddition+for+Covalent+Modification+of+Biomolecules+in+Living+Systems.">A Strain-Promoted [3 + 2] Azide&#8722;Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems.</a> Journal of the American Chemical Society. 126 (46), 15046-15047 (2004).</li><li>Blackman, M. L., Royzen, M., Fox, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrazine+Ligation:+Fast+Bioconjugation+Based+on+Inverse-Electron-Demand+Diels&#8722;Alder+Reactivity.">Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels&#8722;Alder Reactivity.</a> Journal of the American Chemical Society. 130 (41), 13518-13519 (2008).</li><li>Houghton, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+the+In+Vivo+Efficacy+of+Pretargeted+Radioimmunotherapy+Utilizing+Inverse+Electron+Demand+Diels-Alder+Click+Chemistry.">Establishment of the In Vivo Efficacy of Pretargeted Radioimmunotherapy Utilizing Inverse Electron Demand Diels-Alder Click Chemistry.</a> Molecular Cancer Therapeutics. 16 (1), 124-133 (2017).</li><li>Membreno, R., Cook, B. E., Fung, K., Lewis, J. S., Zeglis, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click-Mediated+Pretargeted+Radioimmunotherapy+of+Colorectal+Carcinoma.">Click-Mediated Pretargeted Radioimmunotherapy of Colorectal Carcinoma.</a> Molecular Pharmaceutics. 15 (4), 1729-1734 (2018).</li><li>Reiner, T., Lewis, J. S., Zeglis, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+Bioorthogonal+Inverse+Electron+Demand+Diels-Alder+Cycloaddition+for+Pretargeted+PET+Imaging.">Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging.</a> Journal of Visualized Experiments. (96), e52335 (2015).</li><li>Cook, B. E., Membreno, R., Zeglis, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrimer+Scaffold+for+the+Amplification+of+In+Vivo+Pretargeting+Ligations.">Dendrimer Scaffold for the Amplification of In Vivo Pretargeting Ligations.</a> Bioconjugate Chemistry. 29 (8), 2734-2740 (2018).</li><li>Cheal, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theranostic+Pretargeted+Radioimmunotherapy+of+Colorectal+Cancer+Xenografts+in+Mice+Using+Picomolar+Affinity+Y-86-+or+Lu-177-DOTA-Bn+Binding+scFv+C825/GPA33+IgG+Bispecific+Immunoconjugates.">Theranostic Pretargeted Radioimmunotherapy of Colorectal Cancer Xenografts in Mice Using Picomolar Affinity Y-86- or Lu-177-DOTA-Bn Binding scFv C825/GPA33 IgG Bispecific Immunoconjugates.</a> European journal of nuclear medicine and molecular imaging. 43 (5), 925-937 (2016).</li><li>Meyer, J. -. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+Masking+of+Circulating+Antibody-TCO+Groups+Using+Tetrazine-Functionalized+Dextran+Polymers.">Bioorthogonal Masking of Circulating Antibody-TCO Groups Using Tetrazine-Functionalized Dextran Polymers.</a> Bioconjugate Chemistry. 29 (2), 538-545 (2018).</li><li>Houghton, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretargeted+Immuno-PET+of+Pancreatic+Cancer:+Overcoming+Circulating+Antigen+and+Internalized+Antibody+to+Reduce+Radiation+Doses.">Pretargeted Immuno-PET of Pancreatic Cancer: Overcoming Circulating Antigen and Internalized Antibody to Reduce Radiation Doses.</a> Journal of Nuclear Medicine. 57 (3), 453-459 (2016).</li><li>Kein&#228;nen, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretargeting+of+Internalizing+Trastuzumab+and+Cetuximab+with+a+(18)F-Tetrazine+Tracer+in+Xenograft+Models.">Pretargeting of Internalizing Trastuzumab and Cetuximab with a (18)F-Tetrazine Tracer in Xenograft Models.</a> European Journal of Nuclear Medicine and Molecular Imaging Research. 7, 95 (2017).</li><li>Billaud, E. M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-flow+Photosynthesis+of+New+Dienophiles+for+Inverse-Electron-Demand+Diels-Alder+Reactions.+Potential+Applications+for+Pretargeted+In+Vivo+PET+Imaging.">Micro-flow Photosynthesis of New Dienophiles for Inverse-Electron-Demand Diels-Alder Reactions. Potential Applications for Pretargeted In Vivo PET Imaging.</a> Chemical Science. 8 (2), 1251-1258 (2017).</li></ol>

这种方法在体内预瞄准的优点之一--特别是在基于双特异性抗体和放射性标记的浸渍的策略方面--是它的模块化:反周期细胞分子可以附加到任何抗体上,四氮体辐射寡核苷酸可以用非常多的放射性核素进行放射性标记, 而不会损害它们与点击伙伴发生反应的能力。然而, 这种方法对其他抗体抗原系统的适应并不像复制这里描述的协议那么简单。当然, 任何试图创建一个新的 mAb-TCO 免疫共?...

放射免疫治疗 (rit)--使用抗体将治疗性放射性核素输送到肿瘤--长期以来一直是治疗癌症1,2的一种诱人方法。事实上, 美国食品药品监督管理局批准了治疗非霍奇金淋巴瘤的两种放射免疫共轭物: 90Y-ibritumomab tiuxetan 和131i-to质他莫单 3.,4. 然而, 即使从一开始, rit 的临床前景也受到一个严重并发症的阻碍: 对健康组织的高辐射剂量率5、6.一般来说, rit 的放射免疫共轭标记为长寿命放射性核素 (例如, 131i [t半= 8.0] 和90y [t-学期 = 2.7天]), 物理半衰期与免疫球蛋白的半衰期长。这一点至关重要, 因为它确保一旦抗体在循环几天后达到最佳生物分布, 就会保持足够的放射性。然而, 这种在血液中停留时间长和物理半衰期长的组合不可避免地导致健康组织的照射, 从而降低治疗率,限制治疗7的疗效。已经探索了几种策略来规避这个问题, 包括使用截断的抗体片段, 如 fab、fab '、f (ab')2、微型体和纳米棒8, 9,10。最有希望的、最引人入胜的、但不可否认的复杂的替代方法之一是在体内预先针对11。
在体内预定位是一种方法, 核成像和治疗, 寻求利用微妙的亲和力和选择性的抗体, 同时绕过其药代动力学的缺点 11,12,13。为此, 传统放射免疫治疗中使用的放射性标记抗体被解构为两个组成部分: 一个是小分子的放射性寡核苷酸, 另一个是免疫结合, 既能结合肿瘤抗原, 又能结合上述放射性寡聚 and。免疫共轭首先注射, 并给予 "头部启动", 通常几天, 在此期间, 它积累在目标组织中, 并清除血液。随后, 小分子的放射寡核苷酸给药, 或者与肿瘤的免疫共轭结合, 或者迅速清除身体。从本质上讲,在体内, 预瞄准依赖于在体内进行放射性化学。通过减少放射性循环, 这种方法同时减少了对健康组织的辐射剂量, 并促进了放射性核素的使用 (例如, 68ga, t半= 68min211;由于半衰期通常被认为与基于抗体的向量不兼容, 因为 t 半 = 7.2 h)。
从20世纪80年代末开始, 开发了几种不同的体内预定位方法, 包括基于双特异性抗体的策略、链霉素与生物素的相互作用以及互补的杂交寡核苷酸14,15,16,17,18。然而, 每一种都被并发症不同程度地牵制住了, 最著名的是链霉素修饰抗体的强效免疫原性 19,20。在过去的五年里, 我们的团队和其他人已经开发出一种基于快速和生物正交逆电子需求 diels-alder 结扎的体内预定位方法。21,22,23,24。这些策略中最成功的是使用了 tco 修饰抗体和含 z 半径, 因为总体拥有成本通常比 tz 伙伴 (图 1)25、26更稳定. 与其他预靶向方法一样, mAb-TCO 免疫共轭首先被给予, 并给予时间从循环中清除并积累在肿瘤组织中。随后, 小分子 tz 半径低聚糖被注射, 之后它点击与免疫共轭在目标组织内或迅速地从身体清除。这种体内预瞄准策略已被证明是非常有效的 pet 和 spect 成像与几种不同的抗体抗原系统, 不断产生高对比度的图像, 并能够使用短命放射性核素, 如18f (t半= 109分钟) 和64铜 (t半= 12.7 小时)21,22,24。最近, 点击靶向放射免疫治疗 (prit) 的有效性已在胰导管腺癌 (pdac) 和结直肠癌 27,28的小鼠模型中得到了证明。为此, 结合两种不同的抗体使用了治疗性放射性核素177lu (β最大值 = 498 kev, t 半 = 6.7天): 5b1, 针对碳水化合物抗原 19.9 (c.9) 在 pdac 中普遍表示 和 hua33, 它的目标是 a33, 一种跨膜糖蛋白, 表达于 & gt;95 的结直肠癌。在这两种情况下, 这种方法对177luprit 在肿瘤组织中产生了较高的活性浓度, 产生了剂量依赖性的治疗效果, 与传统相比, 健康组织中的活性浓度降低。直接标记的辐射免疫共轭。
在本文中, 我们描述了使用177lu-dota 标记的四氨酸半径和 ([177 路] ludat-peg 7-tz) 和 hua33抗体 (hua33-tco) 的 tco 修饰变种的 prit 协议。更具体地说, 我们描述了 hua33-tco 的构造 (图 2)、[177lu] ludat-peg 7-tz 的合成和径向标记 (图 3和图 4), 以及在体内的性能。大肠癌小鼠模型的生物分布和纵向治疗研究。此外, 在具有代表性的结果和讨论中, 我们提出了一个样本数据集, 讨论了优化这种方法的可能策略, 并在体内预定位和 prit 的更广泛背景下考虑了这一策略。最后, 需要注意的是, 虽然我们选择在本协议中专注于使用 hua33-tco 和 [177lu] ludat-peg 7-tz进行预瞄准, 但这一策略是高度模块化的, 可以适应广泛的抗体和放射性 核素。

<table>
	<tbody>
		<tr>
			<td>(E)-Cyclooct-4-enyl 2,5-dioxo-1-pyrrolidinyl carbonate (TCO-NHS)</td>
			<td>Sigma-Aldrich</td>
			<td>764523</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr>
			<td>2,5-Dioxo-1-pyrrolidinyl 5-[4-(1,2,4,5-tetrazin-3-yl)benzylamino]-5-oxopentanoate (Tz-NHS)</td>
			<td>Sigma-Aldrich</td>
			<td>764701</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Acetonitrile (MeCN)</td>
			<td>Fisher Scientific</td>
			<td>A998-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate (NH4OAc)</td>
			<td>Fisher Scientific</td>
			<td>A639-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Boc-PEG7-amine (O-(2-Aminoethyl)-O&#8242;-[2-(Boc-amino)ethyl]hexaethylene glycol)</td>
			<td>Sigma-Aldrich</td>
			<td>70023</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Fisher Scientific</td>
			<td>D143-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO), anhydrous</td>
			<td>Fisher Scientific</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>EMD Millipore Amicon Ultra-2 Centrifugal Filter Unit</td>
			<td>Fisher Scientific</td>
			<td>UFC205024</td>
			<td></td>
		</tr>
		<tr>
			<td>GE Healthcare Disposable PD-10 Desalting Columns</td>
			<td>Fisher Scientific</td>
			<td>45-000-148</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide (DMF), anhydrous</td>
			<td>Fisher Scientific</td>
			<td>AC610941000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>70-011-044</td>
			<td>10x Concentrated</td>
		</tr>
		<tr>
			<td>p-SCN-Bn-DOTA</td>
			<td>Macrocyclics</td>
			<td>B-205</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Triethylamine (TEA)</td>
			<td>Fisher Scientific</td>
			<td>AC157911000</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic Acid (TFA)</td>
			<td>Fisher Scientific</td>
			<td>A116-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Tumor measuring device</td>
			<td>Peira TM900</td>
			<td>Peira TM900</td>
			<td></td>
		</tr>
	</tbody>
</table>

pretargeted radioimmunotherapy based on the inverse electron demand diels-alder reaction

虽然放射免疫治疗 (rit) 是治疗癌症的一种有前途的方法, 但放射性标记抗体的长的药代动力学半衰期可能导致对健康组织的高辐射剂量。也许并不奇怪, 已经制定了几种不同的策略来规避这一令人不安的限制。其中最有希望的方法之一是预定目标放射免疫治疗 (prit)。prit 的前提是将放射性核素与免疫球蛋白脱钩, 分别注射, 然后允许它们在目标组织的体内结合。这种方法利用抗体的特殊肿瘤靶向特性, 同时规避其药代动力学缺陷, 从而降低对非目标组织的辐射剂量, 并促进使用半衰期为被认为太短, 可用于传统的辐射免疫共轭物。在过去的五年里, 我们的实验室和其他实验室开发了一种基于反式环环素 (tco) 和四氮素 (tz) 之间的逆随需电-diels-alder (iedda) 反应的体内预瞄准方法。该策略已成功地应用于具有多种抗体抗原系统的预定目标正电子发射断层扫描 (pet) 和单光子发射计算机断层扫描 (spect) 成像。在最近的两篇文章中, 我们已经证明了基于 iedda 的 prit 在胰腺导管腺癌和结直肠癌的小鼠模型中的有效性。在本协议中, 我们描述了使用 177lu-dota 标记的四氨酸半径和 ([177路] ludat-peg 7-tz) 和针对 hua33 抗体 (hua-tco) 的结直肠癌 tco 修饰变种的 prit 协议.更具体地说, 我们将描述 hua33-tco 的结构, [177lu] lu-dota-pg7-z 的合成和放射状标记, 以及在小鼠模型中的体内生物分布和纵向治疗研究的性能。肠癌。

tco 与 hua33 的结合是基于氨基反应 tco-nhs 与免疫球蛋白表面赖氨酸残基之间的耦合。该方法具有很强的鲁棒性和可重复性, 并能可靠地产生 2-4 tco/mab 的标签。在这种情况下, 使用了 aldi-tof 质谱法, 以确认约 4.0 tco/mab 的标记程度;以氟改性四嗪为记者24, 得到了类似的值。四氮合配体的合成分三个步骤进行: (1) tz-nhs 与单博保护的 peg 链接器的耦合 (2) 这种中间?...

Watch this Scientific Journal Video about Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction at JoVE.com

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

该协议描述了反式环磷 (tco) 修饰抗体和177个lu-tazine (tz) 辐射寡核苷酸的合成和表征, 用于预定目标放射治疗 (prit)。此外, 它还详细介绍了这两种结构在结直肠癌小鼠模型中的应用, 用于体内生物分布和纵向治疗研究。

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 ...

该协议描述了如何应用预先瞄准的方法在小鼠模型中的癌症异种仪的靶向放射治疗。预靶点放射性免疫疗法或PRIT允许我们分别注射抗体和放射性利甘，并允许两者使用点击化学在体内结合。当我们使用结肠直肠癌的Xenograph模型来呈现PRIT时，该技术可以应用于任何表面抗原靶向抗体。重要的是要选择一种抗原，不迅速内化或广泛脱落，因为这些可以降低方法的功效。合成 huA33-TCO 首先在 1.7 毫升微离心管中，在干甲基甲酰胺中准备 125 微升溶液，每毫升 TCO-NHS 为 40 毫克。该溶液可等值并冷冻在80摄氏度，供将来实验使用。在另外一个1.7毫升的微离心管中，每毫升5毫克的huA33溶液在一毫升磷酸盐缓冲盐水中。使用0.1摩尔碳酸钠的小等分，将抗体溶液的pH调至8.8至9.0之间。使用 pH 纸或带微电子的 pH 计来监控 pH 值，并注意 pH 值不超过 9.0。在抗体溶液中加入与 TCO-NHS 40 摩尔等价对应的体积。为了防止疏水性 TCO-NHS 的降水，请缓慢地加入，并搅拌。允许在热混合器上以25摄氏度的温度下孵育一小时，并轻微搅拌。在使用预包装的一次性尺寸排除脱盐柱净化 huA33-TCO 免疫结合之前，请平衡供应商描述的尺寸排除柱，以去除储存过程中柱中存在的任何防腐剂，这通常涉及用与柱体积对应的 PBS 体积清洗柱五次。将反应混合物添加到大小排除列中，注意反应混合物的体积。反应混合物进入柱后，添加适当量的PBS，使向柱中添加溶液的总体积达到2.5毫升。使用两毫升 PBS 作为吸水器收集产品。使用紫外光对分光光度计监测波长为 280 纳米，测量 huA33-TCO 的浓度。如果需要具有较高浓度免疫结合物的溶液，则使用离心过滤单元（按照制造商的指示切断 50，000 分子量）浓缩 huA33-TCO 溶液。将已完成的 huA33-TCO 免疫结合储存在黑暗中 4 摄氏度。如果将来使用超过四天，就把它储存在黑暗中80摄氏度。在1.7毫升离心管中加入200微升0.25摩尔铵醋酸铵缓冲液调整为pH5.5。将所需量的卢泰因-177氯化物添加到缓冲液中。增加的量将取决于实验中受试者的数量和被施用的放射性剂量。在DMSO中加入特特拉津PEG7-DOTA加入放射性混合物中。添加的金额取决于要测试的受试者的数量。让溶液在37摄氏度下孵育20分钟。使用无线电即时薄层色谱（以 50 毫摩尔 EDTA pH 5.5 作为移动相）验证无线电标记是否完整。标有 Luteteium-177-DOTA-PEG7-Tetrazine 的标签将保留在基线上，而免费的 Luteteium-177 将由 EDTA 协调，并带溶剂前部旅行。皮下肿瘤的放置不干扰您的约束技术至关重要。我建议把它们放在侧翼侧，这是对面的你约束的手。自从我用左手约束，我选择把肿瘤放在右侧。用结肠直肠癌Xenograph对雌性裸体小鼠进行排序，使每个队列的平均肿瘤体积大致相等。为此，请将动物识别号、耳口图案和肿瘤体积列在电子表格程序的三列中。将数据从最小到最大的肿瘤体积进行排序。在第四列中为每个动物分配一个笼子编号。以蛇形图案循环通过笼子，直到所有动物都被分配一个笼子。分配保持架后，按保持架编号对数据进行排序。在PBS中用1%牛血清白蛋白预处理的注射器中注射150微升huA33-TCO溶液的免疫结合抽发剂量，并将其储存在冰上。注射放射性利甘，首先在150微升0.9%无菌盐水中抽出剂量，其中含有放射性标记的Tetrazine-PEG7-DOTA的1.1摩尔等价，相对于huA33-TCO施用量。使用卡钳测量长方形肿瘤最长的一侧以及垂直于长度的宽度。在平衡上称量每只鼠标，以跟踪体重增加或体重减轻。最后，使用椭圆体体积公式计算体积，假设高度与宽度大致相等。这里展示的是体内预距与huA33-TCO和Luteteium-177-DOTA-PEG7-Tetrazine在携带皮下SW1222人类结肠直肠肿瘤的生物分布。所有注射间隔在肿瘤组织中产生高活性浓度，在健康器官中产生低活性浓度。24小时注射间隔提供注射后120小时的最高肿瘤吸收。根据这些发现，为随后的纵向治疗研究选择了24小时的间隔。这里展示的是五组携带皮下SW1222肿瘤的小鼠的纵向治疗研究，这些小鼠以平均肿瘤体积描述为时间函数，肿瘤体积与初始体积一样作为时间函数而正常化。与对照组相比，实验组的反应有明显差异。虽然只接受目标前放射性免疫治疗策略一个组成部分的小鼠的肿瘤继续无节制地生长，但接受完全目标前放射性免疫疗法方案的小鼠的肿瘤停止生长并最终缩小。重要的是，没有观察到有毒的副作用，所有动物的重量保持在20%的初始质量。令人吃惊的是，实验组的实验鼠在调查结束时有完美的存活记录。当尝试这个程序时，它是最重要的，准确和可重复测量肿瘤。与放射性合作时，请咨询您机构的辐射安全官员，以制定安全的协议和设施。适当的安全控制将限制您的暴露并避免污染。与传统的放射免疫疗法相比，预目标对背景器官的辐射剂量更低。通过分享这个协议，我们希望其他人能够测试他们自己的模型和想法，这将导致新的和令人兴奋的进展，病人治疗。

Research

JoVE Journal

Chemistry

Microwave-assisted Intramolecular Dehydrogenative Diels-Alder Reactions for the Synthesis of Functionalized Naphthalenes/Solvatochromic Dyes

微波辅助分子内Diels-Alder反应的功能化萘/变色染料的合成脱氢

Functionalized naphthalenes have applications in a variety of research fields ranging from the synthesis of natural or biologically active molecules to ...

科研

化学

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

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 ...

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

通过荧光基冰平面亲和力确定防冻蛋白的冰结合平面

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

Amide Coupling Reaction for the Synthesis of Bispyridine-based Ligands and Their Complexation to Platinum as Dinuclear Anticancer Agents

酰胺偶联反应为双吡啶基配体及其络合合成，以白金为双核抗癌药物

Amide coupling reactions can be used to synthesize bispyridine-based ligands for use as bridging linkers in multinuclear platinum anticancer drugs. ...

An Inverse Analysis Approach to the Characterization of Chemical Transport in Paints

一个反分析方法化工运输的油漆表征

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in ...

1,3,5-Triphenylbenzene and Corannulene as Electron Receptors for Lithium Solvated Electron Solutions

1,3,5-三苯和碗烯作为电子受体锂溶剂化电子解决方案

The authors report on conductivity studies carried out on lithium solvated electron solutions (LiSES) prepared using two types of polyaromatic ...

The Preparation and Properties of Thermo-reversibly Cross-linked Rubber Via Diels-Alder Chemistry

热可逆交联橡胶通过的Diels-Alder反应的化学制备与性能

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ...

A Rapid Synthesis Method for Au, Pd, and Pt Aerogels Via Direct Solution-Based Reduction

直接溶液还原法快速合成金、钯、铂气凝胶的研究

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various ...

Quantitative Atomic-Site Analysis of Functional Dopants/Point Defects in Crystalline Materials by Electron-Channeling-Enhanced Microanalysis

通过电子引导增强微分析对晶体材料中功能性多裤/点缺陷的定量原子场分析

A novel elemental and chemical analysis scheme based on electron-channeling phenomena in crystalline materials is introduced, where the incident ...

Picometer-Precision Atomic Position Tracking through Electron Microscopy

通过电子显微镜进行精确原子位置跟踪

The modern aberration-corrected scanning transmission electron microscopes (AC-STEM) have successfully achieved direct visualization of atomic columns ...