The authors thank Prof. Thomas Reiner, Dr. Jacob Houghton, and Dr. Serge Lyaschenko for helpful conversations.&#160;

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. Conjugation of DFO-NCS to J591
<ol>
	<li>In a 1.7 ml microcentrifuge tube, prepare a 2-5 mg/ml solution of J591 in 1 ml of either 1x phosphate-buffered saline (pH 7.4) or 0.5 M HEPES buffer (pH 7.4).</li>
	<li>Dissolve DFO-NCS in dry DMSO at a concentration between 5-10 mM (3.8-7.6 mg/ml). Sonicate or vortex the solution thoroughly in order to facilitate complete dissolution.</li>
	<li>Adjust the pH of the J591 solution to 8.8-9.0 by adding small aliquots (&#60; 10 &#181;l) of 0.1 M Na2CO3.</li>
	<li>Once the antibody solution is at the correct pH, add a volume of the DFO-NCS solution corresponding to a 3-4-fold molar excess of the bifunctional chelator.
	<ol>
		<li>For example, add 4-5 &#181;l of a 10 mM (7.6 mg/ml) DFO-NCS solution (40.4 nmol DFO-NCS) to 1 ml of a 2 mg/ml J591 antibody solution (13.3 nmol J591). The amount of DMSO in the final aqueous reaction mixture should not exceed 2% v/v.</li>
	</ol>
	</li>
	<li>Incubate the reaction for 30 min at 37 &#176;C on an agitating heating block at 350 rpm.</li>
	<li>After 1 hr at 37 &#176;C, purify the resulting immunoconjugate using a pre-packed disposable size exclusion desalting column with a 50,000 molecular weight cut-off using 0.5 M HEPES buffer (pH 7.4) as the eluent. This step will yield a 2 ml solution of the completed J591-DFO construct.</li>
	<li>Measure the concentration of the J591-DFO construct on a UV-Vis spectrophotometer.</li>
	<li>If a higher concentration of the construct is desired, concentrate the J591-DFO solution using a centrifugal filter unit with a 50,000 molecular weight cut-off.</li>
	<li>Store the solution of the completed J591-DFO immunoconjugate at -20 &#176;C in the dark. 
	NOTE: This is an acceptable stopping point in the procedure.</li>
</ol>
2. Radiolabeling J591-DFO with 89Zr
CAUTION: This step of the protocol involves the handling and manipulation of radioactivity. Before performing these steps or performing any other work with radioactivity researchers should consult with their home institution&#8217;s Radiation Safety Department. All possible steps should be taken to minimize exposure to ionizing radiation. &#160;
NOTE: In the interest of proper radiochemical note-keeping, the amount of radioactivity in the sample should be measured using a dose calibrator and recorded before and after Steps 2.2-2.13 in the protocol below. This will help with the accurate determination of radiochemical yields and specific activities.
<ol>
	<li>Prepare a solution of 0.5-2.0 mg of J591-DFO in 200 &#181;l of 0.5 M HEPES buffer, pH 7.5.</li>
	<li>Pipette a volume of the 89Zr4+ stock solution (typically supplied in 1.0 M oxalic acid) corresponding to 1.0-6.0 mCi (37-222 MBq) into a 2 ml plastic screw-cap microcentrifuge tube. Adjust the volume of this solution to a total 300 &#181;l using 1.0 M oxalic acid.</li>
	<li>Adjust the pH of the 89Zr4+ solution to 6.8-7.5 using 1.0 M Na2CO3. Begin by adding 250 &#181;l of 1.0 M Na2CO3 to the 89Zr4+ solution and subsequently add smaller (&#60; 10 &#181;l) aliquots of base to achieve the desired pH.</li>
	<li>Add the desired amount of pH-adjusted 89Zr4+ solution to the J591-DFO solution prepared in Step 2.1.</li>
	<li>Check the pH of the radiolabeling reaction mixture to ensure that it falls within the desired range of 6.8-7.5.</li>
	<li>Incubate the radiolabeling reaction for 60 min at RT on an agitating heating block at 350 rpm.</li>
	<li>After 60 min of incubation, measure the radiolabeling yield of the reaction using radio-TLC.
	<ol>
		<li>To this end, spot 1 &#181;Ci of the radiolabeling reaction mixture on a silica-impregnated TLC strip. Allow the aliquot to dry, run the TLC using an eluent of 50 mM DTPA (pH 5.5) and analyze the TLC strip using a radio-TLC scanner. 89Zr4+ bound to the J591-DFO construct will appear at the origin (Rf &#60; 0.1), while free 89Zr4+ cations will be chelated by DTPA and will elute with the solvent front (Rf &#62; 0.9).</li>
		<li>Calculate the radiolabeling yield of the reaction by integrating the radiochromatogram, dividing the area under the curve from Rf 0.0-0.1 by the total area under the curve, and multiplying by 100.</li>
	</ol>
	</li>
	<li>If the radiolabeling yield is sufficient (typically a theoretical specific activity of &#62;2 mCi/mg), quench the reaction with 5 &#181;l of 50 mM DTPA, pH 5.5.</li>
	<li>Purify the resulting immunoconjugate using a pre-packed disposable size exclusion desalting column with a 50,000 molecular weight cut-off using an eluent of either 0.9% sterile saline with 5 mg/ml gentisic acid or 0.25 M sodium acetate (pH 5.5) with 5 mg/ml gentisic acid. This step will yield a 2 ml solution of the completed 89Zr-DFO-J591 radioimmunoconjugate.</li>
	<li>After purification, verify the radiochemical purity of the 89Zr-DFO-J591 using radio-TLC as described in Step 2.7.</li>
	<li>Calculate the overall radiolabeling yield of the reaction by dividing the amount of activity initially added to the antibody solution by the amount of radioactivity isolated with the purified 89Zr-DFO-J591 radioimmunoconjugate.</li>
	<li>Calculate the final specific activity by dividing the amount of activity isolated with the purified 89Zr-DFO-J591 radioimmunoconjugate by the initial mass of DFO-J591 in the radiolabeling reaction.</li>
	<li>If a higher concentration is desired, concentrate the 89Zr-DFO-J591 solution using a centrifugal filter unit with a 50,000 molecular weight cut-off. 
	NOTE: The gentisic acid used in the final purification step is a radio-protectant employed to minimize the degradation of the antibody due to radiolysis.46 While the storage of the 89Zr-DFO-J591 radioimmunoconjugate for up to 48 hr at 4 &#176;C is possible, it is not recommended. If the radioimmunoconjugate is to be stored, use 0.25 M sodium acetate (pH 5.5) with 5 mg/ml gentisic acid as the storage buffer in order to minimize the risk of hypochlorite-mediated radiolysis.47</li>
</ol>
3. In Vivo PET Imaging with 89Zr-DFO-J591
CAUTION: As in Protocol Section 2, 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. All possible steps should be taken to minimize exposure to ionizing radiation.
<ol>
	<li>In male athymic nude mice, subcutaneously implant 5 x 106 LNCaP prostate cancer cells and allow these to grow to a 100-150 mm3 xenograft (3-4 weeks after inoculation).44</li>
	<li>Dilute the 89Zr-DFO-J591 radioimmunoconjugate to a concentration of 1.0 mCi/ml in 0.9% sterile saline.</li>
	<li>Inject 200 &#181;l of the 89Zr-DFO-J591 solution (200 &#181;Ci; 7.4 MBq) into the lateral tail vein of the xenograft-bearing mice.48</li>
	<li>At the desired imaging time point (e.g., 12, 24, 48, 72, 96, or 120 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, and 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 ophthalmic&#160;ointment to the eyes of the mouse to prevent drying during anesthesia.49</li>
	<li>Acquire the PET data for the mouse via a static scan with a minimum of 40 million coincident events using an energy window of 350-700 keV and a coincidence timing window of 6 nsec.50</li>
	<li>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>

The authors have nothing to disclose.

While the construction, radiolabeling, and imaging of 89Zr-DFO-labled radioimmunoconjugates is generally a rather straightforward procedure, it is important to keep a few key considerations in mind during each step of the process. For example, perhaps the most likely cause for concern during the conjugation step of the procedure is the aggregation of the antibody during the conjugation reaction. This problem is most often a product of poor mixing of the conjugation reaction after the addition of the DFO-NCS stock solution.22 When this happens, the non-homogenous distribution of the DFO-NCS can cause excessively high levels of local reaction with the antibody, which can in turn lead to aggregation. This issue can be relatively easily circumvented by adding the DFO-NCS stock solution in small aliquots (&#60; 5 &#181;l), thoroughly mixing the reaction mixture after the addition of the DFO-NCS, and agitating the reaction mixture on a temperature-controlled shaker. In addition, after the conjugation and purification of the DFO-mAb construct, it is important to precisely determine the number of DFO conjugated to each mAb. The full characterization of the number of DFO chelates per antibody can be achieved using radiometric isotopic dilution experiments similar to those performed by Holland, et al. and Anderson, et al., though MALDI-TOF mass spectrometry is a viable alternative.14,23,30,51,52 During the radiolabeling step, easily the most common problem is lower-than-expected radiolabeling yields. If unexpectedly low yields occur despite assiduously following the protocol above, three different troubleshooting strategies are available: (1) incubating the radiolabeling reaction for longer amounts of time (e.g., 2-3 hr); (2) repeating the radiolabeling reaction using a higher concentration of antibody; or (3) repeating the initial DFO-NCS conjugation reaction using a higher molar excess of the bifunctional chelator.
While the DFO-NCS conjugation is facile and robust, one of its undeniable weaknesses is that it is not site-specific: DFO-NCS forms thiourea linkages with available lysines in the antibody regardless of their position. As a result, it is possible that the chelators may become appended to the antigen-binding region of the antibody, thereby adversely affecting the immunoreactivity of the 89Zr-DFO-labeled conjugate. Therefore, a fine balance must be struck in the construction of 89Zr-labeled radioimmunoconjugates: higher numbers of chelators per antibody facilitate higher specific activities, but higher degrees of labeling also increase the risk of compromising the immunoreactivity of the construct. In the end, the goal is simple: attach as many chelators as necessary without compromising immunoreactivity. After obtaining the purified 89Zr-DFO-mAb radioimmunoconjugate, it is critical to determine the in vitro immunoreactivity of the construct prior to any in vivo experimentation. To this end, we recommend using the in vitro methods published by Lindmo, et al.53,54 If the immunoreactivity of the construct is lower than 80-90%, it may be necessary to return to the conjugation reaction and append fewer DFO moieties per antibody. Alternatively, if the immunoreactivity of the purified 89Zr-DFO-mAb is high (&#62; 90%) and higher specific activities are desired, it may be possible to attach more chelators to the antibody without decreasing immunoreactivity.
Finally, the in vivo behavior of a 89Zr-DFO-labeled antibody is, of course, highly dependent on both the identity of the antibody and the tumor model employed. In the model system presented here, the maximum uptake value in the tumor reaches approximately 60% ID/g; however, reports in the literature for maximum tumor uptake values range from as low as 15-20% ID/g to as high as 80-90% ID/g.33,44,55-57 Likewise, the amount of uptake in non-target tissues &#8212; in particular the liver and spleen &#8212; can vary widely depending on the antibody/antigen system being studied. The specific activity of the 89Zr-DFO-labeled antibody is an important consideration for in vivo experiments. Literature values for the specific activities of 89Zr-DFO-mAbs typically range from 1-6 mCi/mg (37-222 mBq/mg).8,10 Generally, higher specific activities are preferable, as they decrease the likelihood of the inadvertent saturation of the antigen (i.e., self-blocking). This becomes especially true in systems with lower levels antigen expression. Regardless of the antibody/antigen system, no in vivo investigation of a 89Zr-DFO-labeled imaging agent is complete without a demonstration of selectivity. This can be achieved via blocking experiments using large amounts of unlabeled biomolecule or the use of a cell line that does not express the antigen in question. In the procedure described herein, the former was employed, but the selectivity of 89Zr-DFO-J591 has also been demonstrated using PSMA-negative PC3 prostate cancer xenografts.23
It is important to note that despite its clear advantages, this DFO-NCS-based synthetic methodology is not perfect. As we have discussed, DFO is not an ideal chelator for 89Zr4+, and the non-site-specific nature of the conjugation reaction can prove cumbersome. To circumvent these issues, exciting efforts to develop new chelators for 89Zr4+ and site-specific radiolabeling methodologies are currently underway, yet these new technologies still need to be optimized and validated in both the laboratory and clinic.24,26,27,29,44 Ultimately, the DFO-NCS methodology for the construction of 89Zr-DFO-labeled antibodies has proven to be an extremely powerful tool for the synthesis of radioimmunoconjugates and has the potential to be used to create a wide variety of clinically useful radiopharmaceuticals.

Due to their remarkable sensitivity, affinity, and selectivity, antibodies have long been considered promising vectors for the delivery of radioisotopes to cancer cells. However, their application in positron emission tomography (PET) imaging has been hampered by the lack of a suitable positron-emitting radioisotope for their labeling.1-3 One of the most critical considerations in the design of radioimmunoconjugates is matching the physical decay half-life of the radioisotope to the in vivo pharmacokinetics of the antibody. More specifically, antibodies often have relatively long, multi-day biological half-lives and therefore must be labeled with radioisotopes with comparable physical half-lives. For PET imaging applications, antibodies have traditionally been radiolabeled with 64Cu (t1/2 = 12.7 hr), 86Y (t1/2 = 14.7 hr), or 124I (t1/2 = 4.18 d).4,5 However, each of these radioisotopes possesses significant limitations that hamper their suitability for clinical imaging. While radioimmunoconjugates labeled with 86Y and 64Cu have proven promising in preclinical investigations, both isotopes possess physical half-lives that are too short to be effective for imaging in humans. 124I, in contrast, has a nearly ideal physical half-life for imaging with antibodies, but it is expensive and has suboptimal decay characteristics that lead to relatively low resolution clinical images. Furthermore, 124I-labeled radioimmunoconjugates can be subject to dehalogenation in vivo, a process which can lower tumor-to-background activity ratios.6,7
The drive to find a positron-emitting radioisotope to supplant 64Cu, 86Y, and 124I in radioimmunoconjugates has fueled the recent surge in research on 89Zr-labeled antibodies.8-12 The reason for the advent of 89Zr is straightforward: the radiometal possesses near-ideal chemical and physical properties for use in diagnostic PET radioimmunoconjugates.13 89Zr is produced via the 89Y(p,n)89Zr reaction on a cyclotron using a commercially available and 100% naturally abundant 89Y target.14,15 The radiometal has a positron yield of 23%, decays with a half-life of 78.4 hr, and emits positrons with the relatively low energy of 395.5 keV (Figure 1).13,16,17 It is important to note that 89Zr also emits a high energy, 909 keV &#947;-ray with 99% efficiency. While this emission does not interfere energetically with the emitted 511 keV photons, it does require extra consideration with regard to transport, handling, and dosimetry. Despite this caveat, these decay characteristics ultimately mean that 89Zr not only has a more favorable half-life for imaging with antibodies than 86Y and 64Cu but can also produce higher resolution images than 124I, which emits positrons with higher energies of 687 and 975 keV as well as a number of photons with energies within 100-150 keV of the 511 keV positron-created photons.13 Moreover, 89Zr is also safer to handle, less expensive to produce, and residualizes in tumors more effectively than its radioiodine counterpart.18,19 One potential limitation of 89Zr is that it does not have a therapeutic isotopologue, e.g., 86Y (PET) vs. 90Y (therapy). This precludes the construction of chemically identical, surrogate imaging agents that can be employed as dosimetric scouts for their therapeutic counterparts. That said, investigations suggest that 89Zr-labeled antibodies do have potential as imaging surrogates for 90Y- and 177Lu-labeled immunoconjugates.20,21
From a chemical standpoint, as a Group IV metal, 89Zr exists as a +4 cation in aqueous solution. The Zr4+ ion is highly charged, relatively large (effective ionic radius = 0.84 &#197;), and can be classified as a &#8220;hard&#8221; cation. As such, it exhibits a preference for ligands bearing up to eight hard, anionic oxygen donors. Easily the most common chelator used in 89Zr-labeled radioimmunoconjugates is desferrioxamine (DFO), a siderophore-derived, acyclic chelator bearing three hydroxamate groups. The ligand stably coordinates the Zr4+ cation quickly and cleanly at RT at biologically relevant pH levels, and the resulting Zr-DFO complex remains stable over the course of multiple days in saline, blood serum, and whole blood.22 Computational studies strongly suggest that DFO forms a hexacoordinate complex with Zr4+ in which the metal center is coordinated to the three neutral and three anionic oxygen donors of the ligand as well as two exogenous water ligands (Figure 2).23,24 The in vivo behavior of radioimmunoconjugates employing the 89Zr-DFO conjugation scaffold has generally been excellent. However, in some cases, imaging and acute biodistribution studies have revealed elevated activity levels in the bones of mice injected with 89Zr-labeled antibodies, data that suggests that the osteophilic 89Zr4+ cation is released from the chelator in vivo and subsequently mineralizes in the bone.25 Recently, a number of investigations into the development of novel 89Zr4+ chelators particularly ligands with eight oxygen donors have appeared in the literature.24,26,27 Nevertheless, at present, DFO is the most widely employed chelator in 89Zr-labeled radioimmunoconjugates by a wide margin. A variety of different bioconjugation strategies have been employed to attach DFO to antibodies, including bioorthogonal click chemistry, the reaction of thiol-reactive DFO constructs with cysteines in the antibody, and the reaction of activated ester-bearing DFO constructs with lysines in the antibody.4,28-30 Easily the most common strategy, however, has been the use of an isothiocyanate-bearing derivative of DFO, DFO-NCS (Figure 2).22 This commercially available bifunctional chelator robustly and reliably forms stable, covalent thiourea linkages with the lysines of the antibody (Figure 3).
Over the past few years, a wide variety of 89Zr-DFO-labeled radioimmunoconjugates have been reported in the literature. Preclinical investigations have been especially abundant, featuring antibodies ranging from the more well-known cetuximab, bevacizumab, and trastuzumab to more esoteric antibodies such as the CD105-targeting TRC105 and fPSA-targeting 5A10.30-36 More recently, a small number of early-phase clinical trials using 89Zr-DFO-labeled antibodies have emerged in the literature. Specifically, groups in the Netherlands have published trials employing 89Zr-DFO-cmAb U36, 89Zr-DFO-ibritumomab tiuxetan, and 89Zr-DFO-trastuzumab.21,32,37 In addition, a range of other clinical trials with 89Zr-labeled radioimmunoconjugates are currently underway, including investigations here at Memorial Sloan Kettering Cancer Center using the PSMA-targeting 89Zr-DFO-J591 for prostate cancer imaging and the HER2-targeting 89Zr-DFO-trastuzumab for breast cancer imaging.23,30 In addition, while radiolabeled antibodies remain the most common 89Zr-labeled radiopharmaceuticals, the radiometal has also increasingly been employed with other vectors, including peptides, proteins, and nanomaterials.38-43
The modularity of this 89Zr-DFO labeling methodology is a tremendous asset. The repertoire of biomarker-targeting antibodies is ever-expanding, and the interest in performing in vivo PET imaging using these constructs is growing apace. As a result, we believe that the development of more standardized practices and protocols could benefit the field. An excellent written experimental protocol for DFO-NCS conjugation and 89Zr radiolabeling has already been published by Vosjan, et al.22 We feel that the visual demonstration provided by this work could further help investigators new to these techniques. In the protocol at hand, the prostate-specific membrane antigen targeting antibody J591 will be used as a model system to illustrate (1) the bioconjugation of the bifunctional chelator DFO-isothiocyanate to an antibody, (2) the radiosynthesis and purification of the 89Zr-DFO-mAb radioimmunoconjugate, and (3) in vivo PET imaging with a 89Zr-DFO-mAb radioimmunoconjugate in a murine model of cancer.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

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

The first step in this protocol the conjugation of DFO-NCS to the antibody is typically quite robust and reliable. Generally, the purified, chelator-modified immunoconjugate can be obtained in &#62; 90% yield, and using 3 molar equivalents of DFO-NCS in the initial conjugation reaction will yield a degree-of-labeling of the chelator of approximately 1.0-1.5 DFO/mAb. The 89Zr radiolabeling and purification steps of the procedure are likewise straightforward. At the concentrations outlined in the protocol above, radiolabeling yields of &#62; 80% and thus specific activities of &#62; 2.0 mCi/mg are typical after 60 min at RT. The radio-TLC chromatogram of the crude radiolabeling mixture will likely reveal some DTPA-bound 89Zr4+ that elutes at the solvent front (Figure 4A). However, after quenching the reaction with DTPA and purifying the 89Zr-DFO-mAb construct via size exclusion chromatography, the radiochemical purity of the purified, isolated 89Zr-DFO-mAb conjugate should be &#62; 95% (Figure 4B). In the event that the radiochemical purity of the isolated 89Zr-DFO-mAb conjugate is less than 95%, the purification procedure should be repeated prior to performing any in vitro or in vivo experiments.
Moving on to the in vivo experiments, in the protocol described above, athymic nude mice bearing PSMA-expressing, LNCaP prostate cancer xenografts were employed to investigate the in vivo behavior of 89Zr-DFO-J591. Both acute biodistribution and PET imaging experiments revealed that 89Zr-DFO-J591 clearly delineates the prostate cancer xenografts with excellent image contrast and high tumor-to-background activity ratios (Figure 5). The uptake of the radioimmunoconjugate in the tumor is evident as early as 24 hr (20.9% &#177; 5.6% ID/g), and the activity concentration in the tumor increases to a maximum of 57.5% &#177; 5.3% ID/g at 96 hr post-injection. As is typical for radioimmunoconjugates, a relatively high concentration of radiotracer is present in the blood at early time points (9.1% &#177; 5.3% ID/g at 24 hr), followed by a slow decrease in the amount of radioactivity in the blood over the course of the experiment. The non-target tissue with the highest activity concentration was the bone, which displayed uptake values around 10% ID/g throughout the experiment, presumably as a result of the in vivo release of the osteophilic cation 89Zr4+. All other organs including heart, lung, liver, spleen, stomach, large and small intestine, kidney, and muscle displayed relatively low activity concentrations, often well below 5% ID/g. As a control, an additional cohort of mice were injected co-injected 300 &#181;g unlabeled DFO-J591 in order to saturate the antigen and thus illustrate selective blocking. Critically, the blocking experiment lowered uptake of the radioimmunoconjugate in the tumor from 48.9% &#177; 9.3% ID/g to 23.5%&#160;&#177; 11.1% ID/g at 72 hr post-injection, clearly indicating that 89Zr-DFO-J591 selectively targets its antigen.
<img alt="Figure 1" src="/files/ftp_upload/52521/52521fig1highres.jpg" width="700px" /> 
Figure 1. (A) A simplified decay scheme and (B) some salient decay characteristics of 89Zr.13,16,17 IT = isomeric transition; EC = electron capture. Modified and reprinted with permission from Deri, et al. Nuclear Medicine and Biology. 40, 3-14 (2013). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52521/52521fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52521/52521fig2highres.jpg" width="700px" /> 
Figure 2. (A) The structure of DFO-NCS with the coordinating oxygen atoms colored red; (B) A DFT-derived structure of the Zr-DFO coordination complex. Modified and reprinted with permission from Deri, et al. Journal of Medicinal Chemistry. 57, 4849-4860 (2014). Copyright 2014 American Chemical Society. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52521/52521fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52521/52521fig3highres.jpg" width="700px" /> 
&#160;&#160;&#160;&#160;&#160;&#160; 
Figure 3. Scheme of the bioconjugation and radiolabeling of 89Zr-DFO-J591. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52521/52521fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52521/52521fig4highres.jpg" width="700px" /> 
Figure 4. Representative radio-TLC chromatograms of the crude radiolabeling mixture (A) and purified product (B) of 89Zr-DFO-J591. Radio-TLCs were run on silica strips using an eluent of 50 mM DTPA, pH 5.0. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52521/52521fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52521/52521fig5highres.jpg" width="700px" /> 
Figure 5. Coronal PET images of 89Zr-DFO-J591 (11.1-12.9 MBq [300-345 &#956;Ci] injected via tail vein in 200 &#956;l 0.9% sterile saline) in athymic nude mice bearing subcutaneous, PSMA-expressing LNCaP prostate cancer xenografts (white arrows) between 24 and 120 hr post-injection. Modified and reprinted with permission from Zeglis, et al. Bioconjugate Chemistry. 24, 1057-1067 (2013). Copyright 2013 American Chemical Society. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52521/52521fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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.

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

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

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17.1K Views.  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. 

Video: 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.

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

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.

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.

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

Bioengineering

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.

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.

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.

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

Cancer Research

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.

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).

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

Immunology and Infection

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.

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

Medicine

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

Source: Membreno, R., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/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.

Radioimmunoterapia pretargetowana: metoda celowanego dostarczania terapeutycznych radionuklidów do nowotworów

Source: Membreno, R., et al.  Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction. J. Vis. Exp. (2019).
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The Bioconjugation and Radiosynthesis of ⁸⁹Zr-DFO-labeled Antibodies

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

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