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>

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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><tbody><tr><td>Tetrazine NHS Ester</td><td>Sigma-Aldrich</td><td>764701</td><td>Store at -80 &amp;deg;C</td></tr><tr><td>Trans-cyclooctene NHS Ester</td><td>Sigma-Aldrich</td><td>764523</td><td>Store at -80 &amp;deg;C</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-NH&lt;sub&gt;2&lt;/sub&gt;-Bn-NOTA</td><td>Macrocyclics</td><td>B-601</td><td>Store at -80 &amp;deg;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 ...

harnessing-bioorthogonal-inverse-electron-demand-diels-alder

Research

JoVE Journal

Bioengineering

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. 

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

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

Chemistry

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 ...

Automation of a Positron-emission Tomography (PET) Radiotracer Synthesis Protocol for Clinical Production

The development of new positron-emission tomography (PET) tracers is enabling researchers and clinicians to image an increasingly wide array of biological targets and processes. However, the increasing number of different tracers creates challenges for their production at radiopharmacies. While historically it has been practical to dedicate a custom-configured radiosynthesizer and hot cell for the repeated production of each individual tracer, it is becoming necessary to change this workflow. Recent commercial radiosynthesizers based on disposable cassettes/kits for each tracer simplify the production of multiple tracers with one set of equipment by eliminating the need for custom tracer-specific modifications. Furthermore, some of these radiosynthesizers enable the operator to develop and optimize their own synthesis protocols in addition to purchasing commercially-available kits. In this protocol, we describe the general procedure for how the manual synthesis of a new PET tracer can be automated on one of these radiosynthesizers and validated for the production of clinical-grade tracers. As an example, we use the ELIXYS radiosynthesizer, a flexible cassette-based radiochemistry tool that can support both PET tracer development efforts, as well as routine clinical probe manufacturing on the same system, to produce [18F]Clofarabine ([18F]CFA), a PET tracer to measure in vivo deoxycytidine kinase (dCK) enzyme activity. Translating a manual synthesis involves breaking down the synthetic protocol into basic radiochemistry processes that are then translated into intuitive chemistry &#34;unit operations&#34; supported by the synthesizer software. These operations can then rapidly be converted into an automated synthesis program by assembling them using the drag-and-drop interface. After basic testing, the synthesis and purification procedure may require optimization to achieve the desired yield and purity. Once the desired performance is achieved, a validation of the synthesis is carried out to determine its suitability for the production of the radiotracer for clinical use.

Automation of a Positron-emission Tomography PET Radiotracer Synthesis Protocol for Clinical Production

Positron-emission tomography (PET) imaging sites that are involved in multiple early clinical research trials need robust and versatile radiotracer manufacturing capabilities. Using the radiotracer [18F]Clofarabine as an example, we illustrate how to automate the synthesis of a radiotracer using a flexible, cassette-based radiosynthesizer and validate the synthesis for clinical use.

The development of new positron-emission tomography (PET) tracers is enabling researchers and clinicians to image an increasingly wide array of biological ...

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 ...

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized cancer therapies in recent years. However, only a fraction of patients responds to PD-1/PD-L1 inhibitors, owing to the heterogeneous expression of PD-L1 in tumor cells. This heterogeneity presents a challenge in the precise detection of tumor cells by the commonly used immunohistochemistry (IHC) approach. This situation calls for better methods to stratify patients who will benefit from immune checkpoint blockade therapy, to improve treatment efficacy. Positron emission tomography (PET) enables real-time visualization of the whole-body PD-L1 expression in a noninvasive way. Therefore, there is a need for the development of radiolabeled tracers to detect PD-L1 distribution in tumors through PET imaging.
Compared to their L-counterparts, dextrorotary (D)-peptides have properties such as proteolytic resistance and remarkably prolonged metabolic half-lives. This study designed a new method to detect PD-L1 expression based on PET imaging of 68Ga-labeled PD-L1-targeted D-peptide, a D-dodecapeptide antagonist (DPA), in tumor-bearing mice. The results showed that the [68Ga]DPA can specifically bind to PD-L1-overexpressing tumors in vivo, and showed favorable stability as well as excellent imaging ability, suggesting that [68Ga]DPA-PET is a promising approach for the assessment of PD-L1 status in tumors.

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

This study developed a noninvasive and real-time method to evaluate the distribution of programmed death-ligand 1 in the whole body, based on positron emission tomographic imaging of [68Ga] D-dodecapeptide antagonist. This technique has advantages over conventional immunohistochemistry and improves the efficiency of identifying appropriate patients who will benefit from immune checkpoint blockade therapy.

The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized ...

Cancer Research

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

Efficient Synthesis of Carbon-11 Radiotracers Using the Loop Method Methylation

Enhancing Efficiency and Radiolabeling Yields of Carbon-11 Radioligands for Clinical Research Using the Loop Method

A successful positron emission tomography imaging program involving carbon-11 radiotracers demands fast, efficient, and reliable synthesis methods, requiring an on-site cyclotron and radiochemistry group, as well as clinical staff trained to operate under the unique constraints of the carbon-11 radionuclide. This study examines the merits and advantages of a captive solvent &#39;loop method&#39; of radiolabeling four tracers with the carbon-11 radionuclide, producing the radioligands [11C]ER-176, [11C]MRB, [11C]mHED, and [11C]PiB.
The &#39;loop method&#39; is compared against the traditional reactor-based method of carbon-11 methylation in the course of synthesizing the same radiotracers on the identical automated platform. Further, a complete overview of the clinical research preparation of the [11C]ER-176 radiotracer is presented. As demonstrated by the production of [11C]ER-176, the captive solvent &#39;loop method&#39; of heterogeneous alkylation proved to be more efficient, with excellent radiochemical purity (99.6 &#177; 0.6%, n = 25), higher and more consistent radiochemical yield (end of synthesis (EOS) = 5.4 &#177; 2.2 GBq, n = 25) compared to the reactor method (EOS = 1.6 &#177; 0.5 GBq, n = 6), increased molar activity (loop method = 194 &#177; 66 GBq/&#181;mol, n = 25; reactor method = 132 &#177; 78 GBq/&#181;mol, n = 6), along with an average 5 min shorter reaction sequence.

This protocol presents a detailed procedure to prepare four carbon-11 radiotracers by way of the &#8220;loop method&#8221; [11C]methylation. We describe the procedure to synthesize [11C]ER-176, &#160;with data for three additional radiotracers: [11C]MRB, [11C]mHED, and [11C]PiB. The loop method provides an efficient synthesis with increased radiochemical yields against traditional reaction vessel [11C]methylation.

A successful positron emission tomography imaging program involving carbon-11 radiotracers demands fast, efficient, and reliable synthesis methods, ...

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB)

Biomolecules, including peptides,1-9 proteins,10,11 and antibodies and their engineered fragments,12-14 are gaining importance as both potential therapeutics and molecular imaging agents. Notably, when labeled with positron-emitting radioisotopes (e.g., Cu-64, Ga-68, or F-18), they can be used as probes for targeted imaging of many physiological and pathological processes.15-18 Therefore, significant effort has devoted to the synthesis and exploration of 18F-labeled biomolecules. Although there are elegant examples of the direct 18F-labeling of peptides,19-22 the harsh reaction conditions (i.e., organic solvent, extreme pH, high temperature) associated with direct radiofluorination are usually incompatible with fragile protein samples. To date, therefore, the incorporation of radiolabeled prosthetic groups into biomolecules remains the method of choice.23,24
N-Succinimidyl-4-[18F]fluorobenzoate ([18F]SFB),25-37 a Bolton-Hunter type reagent that reacts with the primary amino groups of biomolecules, is a very versatile prosthetic group for the 18F-labeling of a wide spectrum of biological entities, in terms of its evident in vivo stability and high radiolabeling yield. After labeling with [18F]SFB, the resulting [18F]fluorobenzoylated biomolecules could be explored as potential PET tracers for in vivo imaging studies.1 Most [18F]SFB radiosyntheses described in the current literatures require two or even three reactors and multiple purifications by using either solid phase extraction (SPE) or high-performance liquid chromatography (HPLC). Such lengthy processes hamper its routine production and widespread applications in the radiolabeling of biomolecules. Although several module-assisted [18F]SFB syntheses have been reported,29-32, 41-42 they are mainly based on complicated and lengthy procedures using costly commercially-available radiochemistry boxes (Table 1). Therefore, further simplification of the radiosynthesis of [18F]SFB using a low-cost setup would be very beneficial for its adaption to an automated process.
Herein, we report a concise preparation of [18F]SFB, based on a simplified one-pot microwave-assisted synthesis (Figure 1). Our approach does not require purification between steps or any aqueous reagents. In addition, microwave irradiation, which has been used in the syntheses of several PET tracers,38-41 can gives higher RCYs and better selectivity than the corresponding thermal reactions or they provide similar yields in shorter reaction times.38 Most importantly, when labeling biomolecules, the time saved could be diverted to subsequent bioconjugation or PET imaging step.28,43 The novelty of our improved [18F]SFB synthesis is two-fold: (1) the anhydrous deprotection strategy requires no purification of intermediate(s) between each step and (2) the microwave-assisted radiochemical transformations enable the rapid, reliable production of [18F]SFB.

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate [18F]SFB

A facile, one-pot synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) was developed based on a non-aqueous, three-step radiochemical process. Using microwave heating, the entire procedure can be completed in less than 30 min, or 60 min with further purification by preparative HPLC. The decay-corrected radiochemical yields (RCYs) were 35-5% (n > 30).

Biomolecules, including peptides,1-9 proteins,10,11 and antibodies and their engineered fragments,12-14 are gaining importance as both potential ...

Biology

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

Medicine

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...

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

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