This work was supported by grants from the National Research Foundation of Korea, funded by the government of the Republic of Korea, (Grant nos. 2012M2B2B1055245 and 2012M2A2A6011335) and by the RI-Biomics Center of Korea Atomic Energy Research Institute.

Caution: The oxidized form of radioactive iodine is quite volatile and must be handled with adequate lead shields and lead vials. All radiochemical steps should be carried out in a well-ventilated charcoal-filtered hood, and the experimental procedures need to be monitored by radioactivity detection devices.
1. Preparation of Chemicals and the Reverse Phase Cartridge for the Synthesis of the 125I-labeled Azide
<ol>
	<li>Preparation of reagents in solution
	<ol>
		<li>Dissolve 1 mg of the azide precursor (2) in 150 &#181;l absolute ethanol (Figure 1). 
		NOTE: A detailed synthetic procedure for the azide precursor (2) was reported in the previous paper22.</li>
		<li>Dissolve 1 mg chloramine T in 20 &#181;l of 1x phosphate buffer saline (pH = 7.4).</li>
		<li>Dissolve 2 mg sodium metabisulfite in 20 &#181;l H2O.</li>
	</ol>
	</li>
	<li>Preparation of the cartridge
	<ol>
		<li>Wash the tC18 cartridge with 10 ml absolute ethanol followed by 10 ml H2O. Do not dry the matrix of the cartridge with air.</li>
	</ol>
	</li>
</ol>
2. Radiosynthesis of the 125I-labeled Azide Prosthetic Group
<ol>
	<li>Radioiodination reaction of the precursor
	<ol>
		<li>Add the azide precursor solution (1 mg in 150 &#181;l of absolute ethanol) and acetic acid (10 &#181;l) to a 1.5 ml microcentrifuge tube.</li>
		<li>Add 150 MBq of [125I]NaI in 0.1 M NaOH (50 &#181;l) to the reaction mixture.</li>
		<li>Add a chloramine T solution (1 mg in 20 &#181;l of 1x phosphate buffer saline) and close the microcentrifuge tube containing reaction mixture.</li>
		<li>Incubate the reaction mixture at room temperature for 15 min until the radioiodination reaction is completed.</li>
		<li>Add a sodium metabisulfite solution (2 mg in 20 &#181;l H2O) to the reaction mixture to quench the radioiodination reaction.</li>
		<li>Withdraw 0.2 &#181;l of the crude product and then dilute it with 100 &#181;l of solution (H2O/CH3CN, 1:1) for HPLC analysis. 
		NOTE: For all HPLC experiments, use 0.1% formic acid containing H2O (solvent A) and 0.1% formic acid containing acetonitrile (solvent B) as eluents.</li>
		<li>Analyze the diluted crude product by using a reverse-phase analytical radio-HPLC (C18 reverse-phase column; flow rate: 1 ml/min; eluent gradient: 20% solvent B for 0-2 min, 20-80% solvent B for 2-22 min, 80-100% solvent B for 22-23 min, and 100% solvent B for 23-28 min; retention time: 16.4 min) (Figure 2).</li>
	</ol>
	</li>
	<li>Purification of the crude product with a preparative HPLC 
	NOTE: Provide enough lead shielding around HPLC parts such as the injector, column, detector, collection vials, and the container in which the effluent is collected.
	<ol>
		<li>Withdraw the entire reaction mixture into an HPLC vial. Rinse the reaction tube with acetonitrile (0.5 ml) and add the rinse into the same injection vial. Dilute the collected solution with H2O (1 ml).</li>
		<li>Inject the crude product onto a preparative radio-HPLC (C18 reverse-phase column; flow rate: 10 ml/min; eluent gradient: 20% solvent B for 0-2 min, 20-80% solvent B for 2-22 min, 80-100% solvent B for 22-23 min, and 100% solvent B for 23-28 min).</li>
		<li>Collect the radioactive peak representing the 125I-labeled azide (1) (tR under these HPLC conditions is 17.8-18.8 min) in a glass test tube (Figure 2).</li>
		<li>Measure the radiochemical yield of the fraction using a radioactivity dose calibrator according to the manufacturer&#39;s protocol.</li>
		<li>Inject the purified product onto an analytical radio-HPLC using the same HPLC conditions for determining the radiochemical purity of the product.</li>
	</ol>
	</li>
	<li>Solid phase extraction of the product
	<ol>
		<li>Dilute the fraction containing the desired product (1) with 40 ml pure H2O.</li>
		<li>Add the diluted solution into a preconditioned tC18 cartridge.</li>
		<li>Wash the cartridge with an additional 15 ml H2O.</li>
		<li>Elute the product (1) trapped in the cartridge with 2 ml acetone into a 10-ml glass vial that is protected by a lead shield. Measure the radioactivity of the eluted product using a radioactivity dose calibrator according to the manufacturer&#39;s protocol. 
		NOTE: Dimethyl sulfoxide (DMSO) or absolute ethanol can also be used for elution of the product from the cartridge. Approximately 5-10% of the radioactivity normally sticks to the cartridge, and the remaining radiolabeled product cannot be fully eluted by using excess amounts of organic solvent.</li>
		<li>Evaporate the acetone with a stream of nitrogen or argon gas.</li>
		<li>Dissolve the residue with DMSO (100-200 &#181;l) for the next radiolabeling step.</li>
	</ol>
	</li>
</ol>
3. Synthesis of DBCO-group-conjugated Gold Nanoparticles
<ol>
	<li>Surface modification of 13-nm-sized gold nanoparticles with DBCO-group-containing polyethylene glycol
	<ol>
		<li>Prepare sodium-citrate-stabilized gold nanoparticles (3) (average size = 13 nm) according to a previous report24.</li>
		<li>Add an aqueous solution of Tween 20 (1 mM, 1.5 ml) to the citrate-stabilized gold nanoparticles (10 nM, 15 ml). Shake the solution for 20 min on an orbital shaker.</li>
		<li>Add an aqueous solution of DBCO-group-containing polyethylene glycol thiol (average molecular weight = 5,000, 100 &#956;M, 1.5 ml). Shake the solution for 2 hr on an orbital shaker.</li>
	</ol>
	</li>
	<li>Purification of the DBCO-group-modified gold nanoparticles
	<ol>
		<li>Purify the DBCO-group-modified gold nanoparticles (4) by successive centrifugation (11,400 x g, 15 min x 3).</li>
		<li>Decant the supernatant and add pure water for resuspension of the gold nanoparticle pellets.</li>
	</ol>
	</li>
</ol>
4. Radiolabeling of DBCO-group-modified Gold Nanoparticles via the Copper-free Click Reaction
<ol>
	<li>Synthesis of 125I-labeled gold nanoparticles using the 125I-labeled azide (1)
	<ol>
		<li>Prepare a concentrated solution of DBCO-group-modified gold nanoparticles by using centrifugation (11,400 x g, 15 min), and adjust the concentration of the gold nanoparticles to 2 &#181;M.</li>
		<li>Add 4.1 MBq of the 125I-labeled azide (1) in DMSO (5 &#181;l) to a suspension of gold nanoparticles (4) (2 &#956;M, 50 &#181;l).</li>
		<li>Incubate the resulting reaction mixture at 40 &#176;C for 60 min.</li>
		<li>Withdraw an aliquot (0.2 &#181;l) from the crude product and apply it onto a silica-coated thin-layer chromatography (TLC) plate.</li>
		<li>Develop the TLC plate using ethyl acetate as a mobile phase.</li>
		<li>Place the TLC plate on a radio-TLC scanner and run the scanner to monitor the radiolabeling reaction (Figure 3) according to the manufacturer&#39;s protocol.</li>
	</ol>
	</li>
	<li>Purification of the crude product
	<ol>
		<li>Purify the reaction mixture containing the 125I-labeled gold nanoparticles (4) by centrifugation (11,400 x g, 15 min).</li>
		<li>Decant the supernatant and add pure water for resuspension of the gold nanoparticle pellets.</li>
		<li>Withdraw an aliquot (0.2 &#181;l) from the purified product and apply it onto a silica-coated TLC plate.</li>
		<li>Develop the TLC plate using ethyl acetate as the mobile phase.</li>
		<li>Place the TLC plate on a radio-TLC scanner and run the scanner to determine the radiochemical yield and radiochemical purity of the 125I-labeled gold nanoparticles (4) (Figure 3) according to the manufacturer&#39;s protocol.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

In general, the observed radiochemical yield of the purified 125I-labeled azide (1) was 75 &#177; 10% (n = 8). The radiolabeling was accomplished with 50-150 MBq of radioactivity, and the radiochemical results are quite consistent. If [125I]NaI (t1/2 = 59.4 d) that underwent radioactive decay for more than a month was used in the radioiodination reaction, the radiochemical yield of 1 was observed to be slightly decreased (53-65%). Therefore, it is recommended that [125I]NaI be utilized as soon as it is produced or is delivered to the lab in order to obtain optimized radiochemical yield. In addition, a freshly prepared chloramine T solution should also be used in the reaction to obtain the desired radiochemical yield.
Because the precursor (2) was quite hydrophobic, 150 &#181;l absolute ethanol should be added to dissolve 1 mg of 2 in the radioiodination reaction. Otherwise, the whole reaction mixture could become turbid after adding an aqueous solution of the oxidant and [125I]NaI. Decreased solubility of the precursor often results in low radiochemical yield of 1. DMSO can also be used for dissolving 2 in the radiolabeling step. In addition, acetic acid should be added to the precursor solution to obtain high radiochemical yield in the radioiodination step.
Before using preparative HPLC for purification of the crude product containing 1, the reverse phase HPLC column needs to be washed with solvents A and B (flow rate: 10 ml/min; eluent gradient: 100% solvent B for 0-10 min, 100-0% solvent B for 10-25 min, and 0% solvent B for 25-30 min) to remove trace amount of impurities from the system. Next, the reverse phase HPLC column is equilibrated with 20% solvent B in 80% solvent A for at least 20 min to obtain the consistent retention time of 1.
The fraction containing purified 1 should be diluted with more than 4 times the volume of H2O in the solid phase extraction procedure. Otherwise, some of the purified product cannot be trapped in the tC18 cartridge. When acetone is used to elute purified 1 from the cartridge, the final volume can be reduced by evaporation of acetone with a stream of nitrogen or argon gas at ambient temperature.
Among several radioactive iodines, 125I was selected and used in the current research. Different kinds of iodine radioisotopes need to be tested using the present method in other biological and medical studies (e.g.,124I for PET imaging, 131I for therapeutic purposes).
As far as we understand, the present radiolabeling protocol is the first report describing detailed synthetic steps for a radioiodine-labeled azide group. Recently, we published another azide prosthetic group, which has a different structure23. However, the radiolabeled azide (1) in the current method provided slightly better radiochemical results than the other in terms of radiolabeling efficiency with DBCO-group-containing molecules. Existing prosthetic groups (i.e., N-hydroxysuccinimide and maleimide) for the labeling of radioactive iodine could not provide site-specificity. However, the present method demonstrates straightforward radiolabeling efficiency along with excellent bioorthogonality. Since the azide functional group is known to be highly stable in physiological conditions and in vivo environments, the radiolabeled product (1) can be utilized in pre-targeted in vivo imaging studies. We anticipate that this method will be efficiently applied to both in vitro and in vivo iodine radioisotope labeling of biomolecules and nanomaterials that contain a strained cyclooctyne structure.
Based on the specific radioactivity of 1, the calculated molar ratio of 125I and gold nanoparticles is ~1:1. 125I-labeled gold nanoparticles (4) can be used in molecular imaging and biodistribution studies of nanomaterials. The current method can also be applied to radioactive iodine labeling of different sizes and shapes of gold nanomaterials.

The strain-promoted copper-free click reaction between azides and cyclooctynes has been extensively applied to the efficient bioorthogonal labeling of a wide range of biomolecules, nanomaterials, and living subjects1-7. Due to the excellent site-specificity and rapid reaction rate of this conjugation reaction, it has also been used to synthesize radiolabeled tracers. A few 18F-labeled azide or DBCO prosthetic groups have been prepared for in vitro labeling of various cancers targeting peptides and antibodies, as well as for in vivo pre-targeted imaging of tumors8-13. In addition to these examples, the same conjugation reaction was applied to the metal-radioisotope-labeling of nanomaterials for positron emission tomography (PET) imaging studies14-16.
For several decades, radioactive iodines have been used for biomedical research and clinical trials through PET imaging (124I), single-photon emission computed tomography (SPECT) imaging (123I, 125I), and thyroid cancer treatment (131I)17-21. Therefore, an efficient method for radioactive iodine labeling is fundamentally important for various investigations, including molecular imaging studies, analysis of organ distribution of biomolecules, biomarker identification, and drug development. A copper-free click reaction strategy could be used in radioactive iodine labeling. However, this application has not been investigated as extensively as 18F-labeled biomolecules22-23. Here, we will provide a step-by-step protocol for the synthesis of an 125I-labeled azide for radiolabeling of DBCO-group-derived molecules. The procedures in the present report will include radioiodination of the stannylated precursor, purification steps with HPLC, and solid phase extraction. We also demonstrate efficient radiolabeling of DBCO-group-modified 13-nm-sized gold nanoparticles using the 125I-labeled azide. The detailed protocol in this report will help synthetic chemists understand a new radiolabeling methodology for the synthesis of radiolabeled products.

<table><tbody><tr><td>Chloramine T trihydrate</td><td>Sigma</td><td>402869</td><td></td></tr><tr><td>[&lt;sup&gt;125&lt;/sup&gt;I]NaI in aq. NaOH</td><td>Perkin-Elmer</td><td>NEZ033A010MC</td><td></td></tr><tr><td>Sodium metabisulfite&amp;nbsp;</td><td>Sigma</td><td>S9000</td><td></td></tr><tr><td>Formic acid</td><td>Sigma</td><td>251364</td><td></td></tr><tr><td>Sep-Pak tC18 plus cartridge</td><td>Waters</td><td>WAT036800</td><td></td></tr><tr><td>Dimethyl sulfoxide&amp;nbsp;</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Acetone</td><td>Sigma</td><td>650501</td><td></td></tr><tr><td>Ethanol</td><td>Sigma</td><td>459844</td><td></td></tr><tr><td>Gold(III) chloride trihydrate</td><td>Sigma</td><td>520918</td><td></td></tr><tr><td>Tween 20&amp;nbsp;</td><td>Sigma</td><td>P1379</td><td></td></tr><tr><td>DBCO PEG SH (MW 5,000)</td><td>NANOCS</td><td>PG2-DBTH-5k</td><td></td></tr><tr><td>TLC silica gel 60 F&lt;sub&gt;254&lt;/sub&gt;</td><td>Merck</td><td></td><td></td></tr><tr><td>Analytical HPLC</td><td>Agilent</td><td>1290 Infinity</td><td>Model number</td></tr><tr><td>Preparative HPLC</td><td>Agilent</td><td>1260 Infinity</td><td>Model number</td></tr><tr><td>Analytical C18 reverse-phase column</td><td>Agilent</td><td>Zorbax Eclipse XDB-C18</td><td></td></tr><tr><td>Preparative C18 reverse-phase column</td><td>Agilent</td><td>PrepHT XDB-C18</td><td></td></tr><tr><td>Radio TLC scanner</td><td>Bioscan</td><td>AR-2000</td><td>Model number</td></tr><tr><td>Radioisotope dose calibrator</td><td>Capintec, Inc</td><td>CRC -25R dose calibrator</td><td>Model number</td></tr></tbody></table>

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.

The radioiodination reaction of the stannylated precursor (2) was carried out using 150 MBq of [125I]NaI, acetic acid, and chloramine T at room temperature for 15 min to provide the radiolabeled product (1). After preparative HPLC purification of the crude mixture, the desired product was obtained with 75 &#177; 10% (n = 8) of radiochemical yield. Analytical HPLC revealed that the radiochemical purity of the 125I-labeled product was more than 99% (Figure 2), and the observed specific radioactivity of product 1 is 40.7 MBq/&#181;mol. Solid phase extraction of the fraction containing the purified product by using the cartridge provided an acetone solution of 1. Using a stream of nitrogen or argon gas, the organic solvent can be evaporated, and then the residue can be dissolved again in DMSO or absolute ethanol for the next step.
For 125I-labeling of polyethylene-glycol-modified gold nanoparticles, the DBCO-group-modified gold nanoparticles were prepared by the procedure shown in Figure 1. An excess amount of polyethylene glycol thiol (MW 5,000) with DBCO groups was reacted with the citrated, stabilized 13-nm gold nanoparticles. After the modification step, the product was purified by successive centrifugation to give the DBCO-functionalized gold nanoparticles (3). In the radiolabeling step, 3.7 MBq of 1 was added to 2 &#181;M of 3 (~400 &#181;M of the DBCO groups), and the labeling reaction was carried out at 40 &#176;C for 1 hr. Radio-TLC analysis showed that more than 95% of 1 was reacted with the DBCO-group-functionalized gold nanoparticles (3) within 60 min. The reaction was carried out for 60 min, and then the crude product was purified by centrifugation. 125I-labeled gold nanoparticles (4) were obtained with &#62;99% (n = 4) radiochemical yield as determined by radio TLC (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/54759/54759fig1.jpg" /> 
Figure 1. Radiosynthesis of the 125I-labeled azide (1) and 125I-labeled gold nanoparticles (4). Reagents and conditions: (a) [125I]NaI, acetic acid, chloramine T, RT, 15 min, 75 &#177; 10% (n = 8) radiochemical yield; (b) DBCO-PEG-SH (MW 5,000), H2O, RT, 2 hr; ~40 &#176;C, 60 min, &#62;99% radiochemical yield. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54759/54759fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54759/54759fig2.jpg" /> 
Figure 2. Analytical HPLC chromatogram of the 125I-labeled azide (1). (a) Radiochromatogram of the crude product. (b) Radiochromatogram of the purified product. (c) UV chromatogram (254 nm) of the purified product. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54759/54759fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54759/54759fig3.jpg" /> 
Figure 3. Radio-TLC results of the 125I-labeled gold nanoparticles (4) (Rf of 4 = 0.05, Rf of 1 = 0.45, eluent: ethyl acetate) (a) after a 60 min reaction and (b) after purification. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54759/54759fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group at JoVE.com

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.

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 ...

an-optimized-protocol-for-efficient-radiolabeling-gold-nanoparticles

Nanyang Technological University

Research

JoVE Journal

Chemistry

8.1K Views.  Korea Atomic Energy Research Institute. 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.

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

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

Biochemistry

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 ...

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 ...

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

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 ...

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

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, ...

Automated Preparation of [68Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe targeting the fibroblast activation protein for positron emission tomography imaging of the tumor microenvironment. The reaction conditions involved acetate buffer (final concentration: 0.1 M), methionine as an anti-radiolysis agent (final concentration: 5.4 mg/mL), and 30 &#181;g of 3BP-3940, with heating for 8 min at 98 &#176;C. A final purification step on a C18 cartridge was necessary to obtain a radiolabeled product of high purity. In contrast, the generator-produced 68Ga was used directly without a concentration step on a cation exchange cartridge. The production of three validation batches confirmed the method&#39;s reliability, allowing the synthesis of [68Ga]Ga-3BP-3940 in 22.3 &#177; 0.6 min with high radiochemical purity (RCP), as determined by both radio-HPLC (99.1% &#177; 0.1%) and radio-TLC (99.2% &#177; 0.1%). The average radiochemical yield, based on RCP values measured by radio-HPLC, was 74.4% &#177; 3.3%. The stability of the radiolabeled product was demonstrated for up to 4 h after preparation. This protocol provides a reliable, rapid, and efficient methodology for the preparation of [68Ga]Ga-3BP-3940, which can easily be transposed to a clinical setting.

This research describes the automated process for [68Ga]Ga-3BP-3940 production with the GAIA V2 synthesizer, for PET imaging of fibroblast activation protein. The results of quality control tests performed on three test batches are also presented.

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe ...

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

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Chapters in this video

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

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

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

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

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

Positron Emission Tomography Imaging of Cell Trafficking: A Method of Cell Radiolabeling

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of ⁶⁸Ga-Labelled Nano Molecules

Murine Lymphocyte Labeling by ⁶⁴Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

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

Automated Preparation of [⁶⁸Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment