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

ETHICS STATEMENT: All of the in vivo animal experiments described were performed according to an approved protocol and under the ethical guidelines of the Memorial Sloan Kettering Cancer Center Institutional Animal Care and Use Committee (IACUC).
1. Synthesis of Tz-Bn-NOTA
<ol>
	<li>In a small reaction vessel, dissolve 7 mg NH2-Bn-NOTA (1.25 x 10-2 mmol) in 600 &#181;l NaHCO3 buffer (0.1 M, pH 8.1). Check the pH of the solution. If needed, adjust the pH of the solution to 8.1 using small aliquots of 0.1 M Na2CO3.</li>
	<li>Add the NH2-Bn-NOTA solution to 0.5 mg Tz-NHS (1.25 x 10-3 mmol) in a 1.7 ml microcentrifuge tube. 
	NOTE: The Tz-NHS can either be weighed out dry or added from a stock solution of dry DMF or DMSO (&#60; 50 &#181;l).</li>
	<li>Allow the resulting reaction solution to react for 30 min at RT with mild agitation.</li>
	<li>After 30 min, purify the product using reversed-phase C18 HPLC chromatography to remove unreacted NH2-Bn-NOTA. The NH2-Bn-NOTA can be monitored at a wavelength of 254 nm, while the Tz-NHS and Tz-Bn-NOTA are best monitored at a wavelength of 525 nm. 
	NOTE: Retention times are obviously highly dependent on the HPLC equipment setup of each laboratory (pumps, columns, tubing, etc.). However, to present an example, if a gradient of 0:100 MeCN/H2O (both with 0.1% TFA) to 100:0 MeCN/H2O over 25 min and an analytical 4.6 x 250 mm C18 column is used, the retention times of Tz-Bn-NOTA, Tz-NHS, and NH2-Bn-NOTA will be around 15 min, 16.5 min, and 10 min, respectively. The product can be purified from the other reaction components in either a single run or multiple runs using a semi-preparative or preparative C18 HPLC column. 1H-NMR, analytical HPLC, and ESI-MS are all methods that can be used to verify the purity of the completed Tz-Bn-NOTA precursor.11</li>
	<li>Freeze the collected HPLC eluent using liquid nitrogen.</li>
	<li>Wrap the frozen collection tube in opaque aluminum foil.</li>
	<li>Place the frozen collection tube in a lyophilizing vessel O/N to remove the HPLC mobile phase.</li>
	<li>Store the purified product (a bright pink solid) in the dark at -80 &#176;C. 
	NOTE: This is an acceptable stopping point in the procedure. The completed Tz-Bn-NOTA precursor is stable for at least 1 year under these conditions.</li>
</ol>
2. Preparation of huA33-TCO Immunoconjugate
<ol>
	<li>In a 1.7 ml microcentrifuge tube, prepare a 1 mg/ml (2.7 mM) solution of TCO-NHS in dry DMF.</li>
	<li>In a 1.7 ml microcentrifuge tube, prepare a 2 mg/ml (13.3 &#181;M) solution of huA33 in 1 ml of phosphate buffered saline, pH 7.4 (0.01 M PO43-, 0.154 M NaCl).</li>
	<li>Using small aliquots (&#60; 5 &#181;l) of 0.1 M Na2CO3, adjust the pH of the antibody solution to 8.8-9.0. Use either pH paper or a pH meter with a microelectrode to monitor the pH, and be careful not to allow the pH to rise above pH 9.0.</li>
	<li>Once the antibody solution is at the correct pH, add a volume of the TCO-NHS solution corresponding to 8 molar equivalents of the activated ester. For example, add 7.9 &#181;l of the 1 mg/ml TCO-NHS solution (1.07 x 10-7 mol TCO-NHS) to the 1 ml of 2 mg/ml huA33 antibody solution (1.33 x 10-8 mol huA33). Do not exceed 5% DMF by volume in the solution.</li>
	<li>Gently mix the solution by inverting the microcentrifuge tube several times.</li>
	<li>Wrap the microcentrifuge tube in opaque aluminum foil.</li>
	<li>Allow the solution to incubate for 1 hr at RT with mild agitation.</li>
	<li>After 1 hr at RT, purify the resulting immunoconjugate using a pre-packed disposable size exclusion desalting column. First, rinse the size exclusion column as described by the supplier to remove any preservatives present on the column during storage. Then, add the reaction mixture to the size exclusion column, rinse the column with 1.5 ml 0.9% sterile saline, and subsequently collect the product using 2 ml of 0.9% sterile saline as the eluent. 
	NOTE: This step will yield the completed huA33-TCO as a 2 ml solution.</li>
	<li>Measure the concentration of the resultant huA33-TCO on a UV-Vis spectrophotometer.</li>
	<li>If a higher concentration is desired, concentrate the huA33-TCO solution using a centrifugal filter unit with a 50,000 molecular weight cut-off. 
	NOTE: It is important to note that while huA33 and a variety of other well-known antibodies (e.g., bevacizumab, trastuzumab, cetuximab, and J591) are very tolerant of being concentrated, aggregation and precipitation can occur upon concentration in other cases. Researchers attempting this procedure with a new antibody should trust the literature or their own knowledge of the antibody in question with regard to whether or not to concentrate the antibody.</li>
	<li>Store the completed huA33-TCO immunoconjugate at 4 &#176;C in the dark. 
	NOTE: This is an acceptable stopping point in the procedure. The completed mAb-TCO conjugate should be stable for at least 3 months under these storage conditions.</li>
</ol>
3. 64Cu Radiolabeling of Tz-Bn-NOTA
NOTE: This step of the protocol involves the handling and manipulation of radioactivity. Before performing these steps &#8212; or performing any other work with radioactivity &#8212; researchers should consult with their home institution&#8217;s Radiation Safety Department. Take all possible steps to minimize exposure to ionizing radiation.
<ol>
	<li>In a 1.7 ml microcentrifuge tube, prepare a 0.5 mg/ml (723 &#181;M) solution of Tz-Bn-NOTA.</li>
	<li>In a 1.7 ml microcentrifuge tube, add 10 &#181;l of the Tz-Bn-NOTA solution (5 &#181;g) to 400 &#181;l of 0.2 M NH4OAc pH 5.5 buffer. </li>
	<li>In the interest of proper radiochemical note-keeping, measure and record the amount of radioactivity in the sample using a dose calibrator before and after the ensuing steps in the protocol below (3.4-3.8). This will help with the accurate determination of radiochemical yields.</li>
	<li>Add 2,000 &#181;Ci (74 MBq) of 64Cu to the Tz-Bn-NOTA solution. 
	NOTE: Typically, [64Cu]CuCl2 is supplied in a small volume (&#60; 30 &#181;l) of 0.1 N HCl, and thus only small volumes (&#60; 10 &#181;l) of this stock solution are needed for the radiolabeling reaction. If larger volumes of the [64Cu]CuCl2 stock are needed, the radiolabeling reaction is tolerant of increasing the overall reaction volume. However, the pH of the radiolabeling reaction solution should be monitored carefully to ensure that it does not fall below pH 4.0.</li>
	<li>Allow the solution to incubate for 10 min at RT with mild agitation.</li>
	<li>After 10 min of incubation, purify the product using reversed-phase C18 HPLC chromatography. Retention times are obviously highly dependent on the HPLC equipment setup of each laboratory (pumps, columns, tubing, etc.). However, to present an example, if a gradient of 5:95 MeCN/H2O (both with 0.1% TFA) to 95:5 MeCN/H2O over 15 min is used, the retention time of 64Cu-Tz-Bn-NOTA should be around 9.8 min while free, uncomplexed 64Cu will elute with the solvent front at around 2-4 min.</li>
	<li>Using a rotary evaporator, remove the HPLC eluent.</li>
	<li>Redissolve the 64Cu-Tz-Bn-NOTA product in 0.9% sterile saline. 
	NOTE: Given the 12.7 hr physical half-life of 64Cu, this is not an acceptable stopping point in the procedure. Perform the synthesis of 64Cu-Tz-Bn-NOTA immediately prior to the injection of the radioligand, and follow Step 3.7 immediately by Step 4.5.</li>
</ol>
4. In Vivo Pretargeted PET Imaging
NOTE: As in Protocol Section 3, this step of the protocol involves the handling and manipulation of radioactivity. Before performing these steps researchers should consult with their home institution&#8217;s Radiation Safety Department. Take all possible steps to minimize exposure to ionizing radiation.
<ol>
	<li>In a female athymic nude mouse, subcutaneously implant 1 x 106 SW1222 colorectal cancer cells and allow these to grow into a 100-150 mm3 xenograft (9-12 days after inoculation).11</li>
	<li>Dilute an aliquot of the huA33-TCO solution from Protocol Section 2 to a concentration of 0.5 mg/ml in 0.9% sterile saline.</li>
	<li>Inject 200 &#181;l of the huA33-TCO solution (100 &#181;g) into the tail vein of the xenograft-bearing mouse.</li>
	<li>Allow 24 hr for the accumulation of the huA33-TCO in the tumor of the mouse.</li>
	<li>Dilute the 64Cu-Tz-Bn-NOTA radioligand to a concentration of 1.5 mCi/ml in 0.9% sterile saline.</li>
	<li>Inject 200 &#181;l of the 64Cu-Tz-Bn-NOTA radioligand solution (300 &#181;Ci; 11.1 MBq; 1.6 nmol of 64Cu-Tz-Bn-NOTA, assuming a specific activity of 6.7 MBq/nmol) into the tail vein of the xenograft-bearing mice.</li>
	<li>At the desired imaging time point (e.g., 2, 6, 12, or 24 hr post-injection), anesthetize the mouse with a 2% isoflurane:oxygen gas mixture.</li>
	<li>Place the mouse on the bed of the small animal PET scanner. Maintain anesthesia during the scan using a 1% isoflurane:oxygen gas mixture. Prior to placing the animal on the scanner bed, verify anesthesia using the toe-pinch method and apply veterinary ointment to the eyes of the mouse to prevent drying during anesthesia.</li>
	<li>Acquire the PET data for the mouse via a static scan with a minimum of 20 million coincident events using an energy window of 350-700 keV and a coincidence timing window of 6 nsec. After completing the acquisition of the image, do not leave the mouse unattended and do not place it in a cage with other mice until it has regained consciousness.</li>
</ol>

<ol>
	<li>Wu, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+and+antimatter:+The+resurgence+of+immuno-PET.">Antibodies and antimatter: The resurgence of immuno-PET.</a> Journal of Nuclear Medicine. 50, 2-5 (2009).</li><li>Zeglis, B. M., Lewis, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+the+construction+of+radiometallated+bioconjugates+for+positron+emission+tomography.">A practical guide to the construction of radiometallated bioconjugates for positron emission tomography.</a> Dalton Transactions. 40, 6168-6195 (2011).</li><li>Hollander, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bispecific+antibodies+for+cancer+therapy.">Bispecific antibodies for cancer therapy.</a> Immunotherapy. 1, 211-222 (2009).</li><li>Liu, 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=Tumor+pretargeting+in+mice+using+99mTc-labeled+morpholino,+a+DNA+analog.">Tumor pretargeting in mice using 99mTc-labeled morpholino, a DNA analog.</a> Journal of Nuclear Medicine. 43, 384-391 (2002).</li><li>Boerman, O. C., van Schaijk, F. G., Oyen, W. J. G., Corstens, F. H. 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+radioimmunotherapy+of+cancer:+Progress+step+by+step.">Pretargeted radioimmunotherapy of cancer: Progress step by step.</a> Journal of Nuclear Medicine. 44, 400-411 (2003).</li><li>Goldenberg, D. M., Sharkey, R. M., Paganelli, G., Barbet, J., Chatal, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+pretargeting+advances+cancer+radioimmunodetection+and+radioimmunotherapy.">Antibody pretargeting advances cancer radioimmunodetection and radioimmunotherapy.</a> Journal of Clinical Oncology. 24, 823-834 (2006).</li><li>Sharkey, R. M., Chang, C. H., Rossi, E. A., McBride, W. J., 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=Pretargeting:+taking+an+alternate+route+for+localizing+radionuclides.">Pretargeting: taking an alternate route for localizing radionuclides.</a> Tumor Biology. 33, 591-600 (2012).</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=Improving+the+delivery+of+radionuclides+for+imaging+and+therapy+of+cancer+using+pretargeting+methods.">Improving the delivery of radionuclides for imaging and therapy of cancer using pretargeting methods.</a> Clinical Cancer Research. 11, 7109-7121 (2005).</li><li>Schultz, 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=A+tetravalent+single-chain+antibody-streptavidin+fusion+protein+for+pretargeted+lymphoma+therapy.">A tetravalent single-chain antibody-streptavidin fusion protein for pretargeted lymphoma therapy.</a> Cancer Research. 60, 6663-6669 (2000).</li><li>Lewis, M. 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+evaluation+of+pretargeted+64Cu+for+tumor+imaging+and+therapy.">In vivo evaluation of pretargeted 64Cu for tumor imaging and therapy.</a> Journal of Nuclear Medicine. 44, 1284-1292 (2003).</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+bioorthgonal+Diels-Alder+click+chemistry.">A pretargeted PET imaging strategy based on bioorthgonal Diels-Alder click chemistry.</a> Journal of Nuclear Medicine. 54, 1389-1396 (2013).</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-Alder+reactivity.">Tetrazine ligation: fast bioconjugation based on inverse electron demand Diels-Alder reactivity.</a> Journal of the American Chemical Society. 130, 13518-13519 (2008).</li><li>Devaraj, N. K., Upadhyay, R., Hatin, J. B., Hilderbrand, S. A., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+sensitive+pretargeted+labeling+of+cancer+cells+through+a+tetrazine/trans-cyclooctene+cycloaddition.">Fast and sensitive pretargeted labeling of cancer cells through a tetrazine/trans-cyclooctene cycloaddition.</a> Angewandte Chemie-International Edition. 48, 7013-7016 (2009).</li><li>Devaraj, N. K., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+applications+of+tetrazine+cycloadditions.">Biomedical applications of tetrazine cycloadditions.</a> Accounts of Chemical Research. 44, 816-827 (2011).</li><li>Devaraj, N. K., Weissleder, R., Hilderbrand, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrazine-based+cycloadditions:+application+to+pretargeted+live+cell+imaging.">Tetrazine-based cycloadditions: application to pretargeted live cell imaging.</a> Bioconjugate Chemistry. 19, 2297-2299 (2008).</li><li>Keliher, E. J., Reiner, T., Turetsky, A., Hilderbrand, S., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yielding,+two-step+18F+labeling+strategy+for+18F-PARP1+inhibitors.">High-yielding, two-step 18F labeling strategy for 18F-PARP1 inhibitors.</a> ChemMedChem. 6, 424-427 (2011).</li><li>Reiner, T., Earley, S., Turetsky, A., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+small-molecule+ligands+for+PARP1+imaging+in+living+cells.">Bioorthogonal small-molecule ligands for PARP1 imaging in living cells.</a> ChemBioChem. 11, 2375-2377 (2010).</li><li>Reiner, T., Keliher, E. J., Earley, S., Marinelli, B., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+in+vivo+imaging+of+a+18F-labeled+PARP1+inhibitor+using+a+chemically+orthogonal+scavenger-assisted+high-performance+method.">Synthesis and in vivo imaging of a 18F-labeled PARP1 inhibitor using a chemically orthogonal scavenger-assisted high-performance method.</a> Angewandte Chemie International Edition. 50, 1922-1925 (2011).</li><li>Taylor, M. T., Blackman, M., Dmitrenko, O., 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=Design+and+synthesis+of+highly+reactive+dienophiles+for+the+tetrazine-trans-cyclooctene+ligation.">Design and synthesis of highly reactive dienophiles for the tetrazine-trans-cyclooctene ligation.</a> Journal of the American Chemical Society. 133, 9646-9649 (2011).</li><li>Selvaraj, 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=Tetrazine-trans-cyclooctene+ligation+for+the+rapid+construction+of+integrin+alpha(v)beta(3)+targeted+PET+tracer+based+on+a+cyclic+RGD+peptide.">Tetrazine-trans-cyclooctene ligation for the rapid construction of integrin alpha(v)beta(3) targeted PET tracer based on a cyclic RGD peptide.</a> Bioorganic and Medicinal Chemistry Letters. 21 (3), 5011-5014 (2011).</li><li>Liu, 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=Efficient+18F+labeling+of+cysteine-containing+peptides+and+proteins+using+tetrazine-trans-cyclooctene+ligation.">Efficient 18F labeling of cysteine-containing peptides and proteins using tetrazine-trans-cyclooctene ligation.</a> Molecular Imaging. 12, 121-128 (2013).</li><li>Han, H. 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=Development+of+a+bioorthogonal+and+highly+efficient+conjugation+method+for+quantum+dots+using+tetrazine-norbornene+cycloaddition.">Development of a bioorthogonal and highly efficient conjugation method for quantum dots using tetrazine-norbornene cycloaddition.</a> Journal of the American Chemical Society. 132, 7838-7839 (2010).</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=Modular+strategy+for+the+construction+of+radiometalated+antibodies+for+positron+emission+tomography+based+on+inverse+electron+demand+Diels-Alder+click+chemistry.">Modular strategy for the construction of radiometalated antibodies for positron emission tomography based on inverse electron demand Diels-Alder click chemistry.</a> Bioconjugate Chemistry. 22, 2048-2059 (2011).</li><li>Zeng, D., Zeglis, B. M., Lewis, J. S., Anderson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+impact+of+bioorthogonal+click+chemistry+on+the+development+of+radiopharmaceuticals.">The growing impact of bioorthogonal click chemistry on the development of radiopharmaceuticals.</a> Journal of Nuclear Medicine. 54, 829-832 (2013).</li><li>Reiner, T., 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=The+inverse+electron+demand+Diels-Alder+reaction+in+radiochemistry.">The inverse electron demand Diels-Alder reaction in radiochemistry.</a> Journal of Labeled Compounds and Radiopharmaceuticals. 57, 285-290 (2014).</li><li>Li, Z., 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=Tetrazine-trans-cyclooctene+ligation+for+the+rapid+construction+of+18-F+labeled+probes.">Tetrazine-trans-cyclooctene ligation for the rapid construction of 18-F labeled probes.</a> Chemical Communications. 46, 8043-8045 (2010).</li><li>Karver, M. R., Weissleder, R., Hilderbrand, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+evaluation+of+a+series+of+1,2,4,5-tetrazines+for+bioorthogonal+conjugation.">Synthesis and evaluation of a series of 1,2,4,5-tetrazines for bioorthogonal conjugation.</a> Bioconjugate Chemistry. 22, 2263-2270 (2011).</li><li>Sletten, E. M., 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=Bioorthogonal+chemistry:+fishing+for+selectivity+in+a+sea+of+functionality.">Bioorthogonal chemistry: fishing for selectivity in a sea of functionality.</a> Angewandte Chemie International Edition. 48, 6973-6998 (2009).</li><li>Bosch, 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=Evaluation+of+strained+alkynes+for+Cu-free+click+reaction+in+live+mice.">Evaluation of strained alkynes for Cu-free click reaction in live mice.</a> Nuclear Medicine and Biology. 40, 415-423 (2013).</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+chemisry+for+pretargeted+tumor+imaging+in+live+mice.">In vivo chemisry for pretargeted tumor imaging in live mice.</a> Angewandte Chemie International Edition. 49, 3375-3378 (2010).</li><li>Ackerman, M. E., 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=A33+antigen+displays+persistent+surface+expression.">A33 antigen displays persistent surface expression.</a> Cancer Immunology and Immunotherapy. 57, 1017-1027 (2008).</li><li>Carrasquillo, J. 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=124I-huA33+antibody+PET+of+colorectal+cancer.">124I-huA33 antibody PET of colorectal cancer.</a> Journal of Nuclear Medicine. 52, 1173-1180 (2011).</li><li>Sakamoto, 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=A+phase+I+radioimmunolocalization+trial+of+humanized+monoclonal+antibody+huA33+in+patients+with+gastric+carcinoma.">A phase I radioimmunolocalization trial of humanized monoclonal antibody huA33 in patients with gastric carcinoma.</a> Cancer Science. 97, 1248-1254 (2006).</li><li>Rossin, R., Lappchen, R., 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, 1989-1995 (2013).</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=Highly+reactive+trans-cyclooctene+tags+with+improved+stability+for+Diels-Alder+chemistry+in+living+systems.">Highly reactive trans-cyclooctene tags with improved stability for Diels-Alder chemistry in living systems.</a> Bioconjugate Chemistry. 34, 1210-1217 (2014).</li><li>Emmetiere, 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=18F-labeled-bioorthogonal+liposomes+for+in+vivo+targeting.">18F-labeled-bioorthogonal liposomes for in vivo targeting.</a> Bioconjugate Chemistry. 24, 1784-1789 (2013).</li></ol>

The authors have nothing to disclose.

The principal advantage of this pretargeted PET imaging strategy is that it is capable of delineating tumors with target-to-background image contrast at only a fraction of the background radiation dose produced by directly labeled antibodies. For example, in the colorectal cancer imaging system described here, data from acute biodistribution experiments were employed to perform dosimetry calculations for the 64Cu-based pretargeting strategy along with directly-labeled 64Cu-NOTA-huA33 and 89</su...

Over the last thirty years, positron emission tomography (PET) has become an indispensable clinical tool in the diagnosis and management of cancer. Antibodies have long been considered promising vectors for the delivery of positron-emitting radioisotopes to tumors due to their exquisite affinity and specificity for cancer biomarkers.1,2 However, the relatively slow in vivo pharmacokinetics of antibodies mandates the use of radioisotopes with multi-day physical half-lives. This combination can yield high radiation doses to the non-target organs of patients, an important complication that is of particular clinical significance since radioimmunoconjugates are injected intravenously and therefore &#8212; unlike partial body CT scans&#160;&#8212; result in absorbed doses in every part of the body, irrespective of the interrogated tissues.
In order to bypass this issue, significant effort has been dedicated to the development of PET imaging strategies that decouple the radioisotope and the targeting moiety, thereby leveraging the advantageous properties of antibodies while simultaneously skirting their intrinsic pharmacokinetic limitations. These strategies &#8212; most often termed pretargeting or multistep targeting &#8212; typically employ four steps: (1) the administration of an antibody capable of binding both an antigen and a radioligand; (2) the accumulation of the antibody in the target tissue and its clearance from the blood; (3) the administration of a small molecule radioligand; and (4) the in vivo ligation of the radioligand to the antibody followed by the rapid clearance of excess radioligand.3-8 In some cases, an additional clearing agent is injected between steps 2 and 3 in order to accelerate the excretion of any antibody that has yet to bind the tumor and remains in the blood.5
Broadly speaking, two types of pretargeting strategies are most prevalent in the literature. While both have proven successful in preclinical models, they also possess key limitations that have impeded their clinical applicability. The first strategy relies on the high affinity between streptavidin-conjugated antibodies and biotin-modified radiolabels; however, the immunogenicity of the streptavidin-modified antibodies has proven to be a worrisome problem with regard to translation.5,6,9,10 The second strategy, in contrast, employs bispecific antibodies that have been genetically-engineered to bind both a cancer biomarker antigen and a small molecule radiolabeled hapten.3,11-14 While this latter route is certainly creative, its broad applicability is limited by the complexity, expense, and lack of modularity of the system.
Recently, we developed and published a pretargeted PET imaging methodology based on the inverse electron demand Diels-Alder (IEDDA) cycloaddition reaction between trans-cyclooctene (TCO) and tetrazine (Tz; Figure 1).11 While the reaction itself has been known for decades, IEDDA chemistry has experienced a renaissance in recent years as a click chemistry bioconjugation technique, as illustrated by the fascinating work of the groups of Ralph Weissleder, Joseph Fox, and Peter Conti among others.12-15 The IEDDA cycloaddition has been applied in a wide range of settings, including fluorescence imaging with peptides, antibodies, and nanoparticles as well as nuclear imaging with both radiohalogens and radiometals.16-26 The ligation is high yielding, clean, rapid (k1 &#62; 30,000 M-1sec-1), selective, and &#8212; critically &#8212; bioorthogonal.27 And while a number of types of click chemistry &#8212; including Cu-catalyzed azide-alkyne cycloadditions, strain-promoted azide-alkyne cycloadditions, and Staudinger ligations &#8212; are bioorthogonal as well, it is the unique combination of fast reaction kinetics and bioorthogonality that makes IEDDA chemistry so well suited to pretargeting applications in whole organisms.28,29 Along these lines, it is important to note that the recent report from our laboratories was not the first to apply IEDDA chemistry to pretargeting: the first report of pretargeted imaging with IEDDA arose from the work of Rossin, et al. and featured a SPECT methodology employing an 111In-labeled tetrazine.30
As we discussed above, the pretargeting methodology has four fairly simple steps (Figure 2). In the protocol at hand, a pretargeted strategy for the PET imaging of colorectal cancer that employs a 64Cu-NOTA-labeled tetrazine radioligand and a TCO-modified conjugate of the huA33 antibody will be described. However, ultimately the modularity of this methodology 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 radioactive reporters.

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

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

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

The initial three steps of the experiment&#160;&#8212; the synthesis of Tz-Bn-NOTA, the conjugation of TCO to huA33, and the radiolabeling of the Tz-Bn-NOTA construct (Figures 3 and 4) &#8212;&#160;are highly reliable. In the case of the procedure above, the Tz-Bn-NOTA construct was synthesized in high yield and purity. The huA33 antibody was modified with 4.2&#160;&#177; 0.6 TCO/mAb, and Tz-Bn-NOTA was radiolabeled with 64Cu to yield the purified radioligand in &#62;99% radio...

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

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

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

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

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