This work supported by research grant funding from the Natural Sciences and Engineering Research Council (NSERC) of Canada, the Ontario Institute for Cancer Research (OICR, #P.SI.015.8), and the Canadian Cancer Society (CCS, #703857). The authors acknowledge the contributions of Dr. Denis Snider who provided assistance in preparing the manuscript.

Animal studies were approved by the Animal Research Ethics Board at McMaster University in accordance with Canadian Council on Animal Care (CCAC) guidelines.

1. Radiolabeling of Tz-tridentate Ligands with 99mTc
CAUTION: The following procedures require the use of radioactive compounds. Work should only be done in a licensed laboratory with adherence to safety and disposal regulations. Microwave reactions should be performed in a microwave specifically designed for chemical synthesis.
<ol>
	<li>Synthesis of [99mTc(CO)3(H2O)3]+ 19,20
	<ol>
		<li>In a microwave vial, combine 8 mg K2[BH3CO2], 15 mg Na2CO3, 20 mg Na2B4O7&#183;10H2O, and 25 mg KOCO[CH(OH)]2COONa&#183;4H2O. Purge the vial for 10 min with argon gas.</li>
		<li>Add 4 mL of 99mTcO4- (~1,100 MBq, ~30 mCi) in 0.9% saline to the vial.</li>
		<li>Heat the reaction in a microwave for 3.5 min at 110 &#176;C after 10 s of stirring to ensure thorough mixing of reagents.</li>
		<li>Adjust the pH of the solution to 3.5-4 using ~400 &#956;L of 1 M HCl. Verify using pH paper.</li>
	</ol>
	</li>
	<li>Radiolabeling of Tetrazine ligands 1-5
	<ol>
		<li>Dissolve 2 mg of each ligand (compounds 1-5) in 250 &#956;L MeOH21.</li>
		<li>Add 250 &#956;L of [99mTc(CO)3(H2O)3]+ (~74 MBq, ~2 mCi) to each solution.</li>
		<li>Heat the reaction mixture using a microwave for 20 min at 60 &#176;C. 
		NOTE: This step was identical for all 5 tetrazines.</li>
		<li>For compounds 2- 5, evaporate the solvent and re-dissolve the resulting products in 1 mL of 1:1 v/v DCM:TFA.</li>
		<li>Heat the dissolved reaction products (2-5) at 60 &#176;C in a microwave for 6 min (2-4) or 10 min (5).</li>
		<li>After cooling to room temperature, evaporate the solvent using an evaporator (36 &#176;C, 8 mbar, 3 min, 6,000 rpm) and dissolve the dried compound in 1:1 ACN:H2O or 1:1 MeOH:H2O, prior to HPLC purification.</li>
		<li>Purify the 99mTc-labeled compounds (1-5), including separating the labeled product from unlabeled tetrazine ligand, using HPLC (C18 reversed-phase). Typically, use an elution gradient of 30:70 ACN:H2O (both with 0.1% TFA) to 40:60 ACN:H2O over 20 min (18 min) and a C18 analytical 4.6 x 100 mm column. Use both UV (254 nm) and gamma detection.
		<ol>
			<li>Take a small sample of each labeled product and compare its HPLC retention time to that of a co-injected, non-radioactive, Re-labeled standard (0.125 mg in 20% methanol-H2O). The Re-labeled standard is identified in the UV HPLC trace, and will elute at the same time as the 99mTc-labeled compound in the &#947;-HPLC trace. This co-injection shows peaks at comparable retention times, confirming the identity of the 99mTc-labeled compound.</li>
		</ol>
		</li>
		<li>Evaporate the solvent from HPLC fractions using an evaporator (36 &#176;C, 8 mbar, 3 min, 6,000 rpm).</li>
		<li>Formulate the purified compound at a concentration of 7.4 kBq/&#181;L in PBS, containing 0.5% BSA and 0.01% Tween-80.</li>
		<li>To ensure the labelled compounds are stable, perform an in vitro stability study. Incubate the formulated compound at 37 &#176;C for 1, 4 and 6 h, injecting a small amount (3.7 MBq) of the mixture on the HPLC at each time point to assess stability.</li>
	</ol>
	</li>
</ol>
2. Pre-targeted Bio-distribution Studies
<ol>
	<li>Preparation of animals
	<ol>
		<li>Using 7-9 week old, female Balb/c mice (n=3), administer TCO-BP formulated in saline (20 mg/kg) (5 &#956;g/&#956;L), via tail-vein injection.</li>
		<li>Place mouse in physical restraint device, and identify the veins located on the lateral surfaces of the tail and wipe with an alcohol swab. At approximately 2 cm from the end of the tail, insert a 30-gauge needle at a shallow angle, parallel to the vein. Slowly depress the plunger to inject, remove needle and apply clean gauze sponge at injection site with slight pressure until bleeding stops.</li>
		<li>At 1 h post injection of TCO-BP, administer ~0.74 MBq (20 &#956;Ci) of 99mTc-tetrazine formulated in 100 &#956;L of 0.5% BSA, 0.01% Tween-80 in PBS, via tail-vein injection.</li>
	</ol>
	</li>
	<li>Bio-distribution studies
	<ol>
		<li>At the desired time point (t = 6 h), anaesthetize the mice using 3% isoflurane and 2% oxygen gas mixture. Demonstrate a toe pinch withdrawal on the anesthetized mouse to ensure they are under surgical plane of anesthesia.</li>
		<li>Collect blood (1 mL) via cardiac puncture using a syringe pre-treated with heparin. Place mouse on its back with nose in the nose cone for continued anesthesia and locate the xiphoid process on the animal.
		<ol>
			<li>Insert a 25 G needle, slightly to the left of the animal&#39;s midline under the xiphoid process, at a 20&#176; angle. Fully insert the needle, and slowly pull back on the plunger to see blood in the needle hub if the heart was punctured. Slightly readjust the needle while holding the plunger if necessary, to puncture the heart. Slowly draw blood into the syringe.</li>
		</ol>
		</li>
		<li>Euthanize the animal by cervical dislocation, while under anesthesia.</li>
		<li>Place each animal in a plastic bag and use a dose calibrator (99mTc setting) to measure the whole body activity level.</li>
		<li>Collect the following tissues and fluids in pre-weighed counting tubes: blood, bone (knee and shoulder), gall bladder, kidneys, liver, stomach (with contents), small intestines (with contents), large intestines and caecum (with contents), thyroid and trachea, urinary bladder with urine, and tail.</li>
		<li>Rinse appropriate tissues (excluding blood, gall bladder, and urinary bladder) in PBS to remove blood and blot dry before placing the tissues in appropriate counting tubes.</li>
		<li>Place animal carcass in a plastic bag and measure residual whole body activity using a dose calibrator.</li>
		<li>Weigh each tube containing a tissue sample. Subtract initial weight of the tube to obtain mass of the tissue.</li>
		<li>Use a dose calibrator (99mTc setting) to measure the amount of activity in a test sample (100 &#181;L) at the time of injection for each mouse. 
		NOTE: This test sample is equal to the injection volume, thus giving the activity count at the time of injection.</li>
		<li>At the time of tissue measurement, aliquot 5 &#181;L of the test sample used previously. Use a multi-detector gamma counter (99mTc setting) and count to obtain the count per minute (CPM) for the 5 &#181;L test sample.</li>
		<li>Use the two values obtained in 2.2.9 and 2.2.10 to calculate the activity and CPM relationship using equation 1 to obtain a conversion factor (CPM &#181;Ci-1). 
		(1) <img alt="Equation 1" src="/files/ftp_upload/55188/55188eq1.jpg" /></li>
		<li>Use the gamma counter to measure the amount of radioactivity in each tissue or fluid sample.</li>
		<li>Use equation 1 to calculate the amount of activity in each tissue or fluid at the time of measurement relative to the total injected dose. This value is then normalized by organ weight and reported as percent injected dose per gram (i.e., %ID/g) of tissue.</li>
		<li>Follow steps 2.1.2 to 2.2.13 to conduct a negative control experiment using the 99mTc-labeled tetrazine ligands in the absence of TCO-BP. Sacrifice mice (n = 3) at 0.5, 1, 4 and 6 h post injection and obtain tissue or fluid as described above.</li>
	</ol>
	</li>
</ol>

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The authors declare they have no competing financial interests.

A collection of tetrazine-linked tridentate chelates of varying polarities was prepared, and the utility of their 99mTc complexes in the IEDDA reaction with a TCO derivative in vivo was assessed. An effective and reproducible 99mTc labeling method was developed for five tetrazine-chelates, where the ligand concentration was 10-3 M. The labeling step was followed by deprotection of t-butyl groups (for compounds 2-5). The high concentration of ligand was u...

99mTc remains the dominant radioisotope used in diagnostic nuclear medicine, with over 50 million imaging procedures conducted per year worldwide1,2,3. The majority of 99mTc agents used clinically are perfusion type radiopharmaceuticals. There are a limited number of actively targeted compounds in which 99mTc is directed to bind a specific biomarker through ligation to a targeting construct. The creation of targeted 99mTc radiopharmaceuticals is often hindered by the influence of 99mTc-ligand complexes on the ability of the targeting molecule to bind the biomarker of interest, or the isotopes half-life is not long enough for use with higher molecular weight biomolecules such as antibodies. The latter typically requires several days before images are acquired in order for the biomolecule to clear from non-target tissues. Pre-targeting offers an alternative approach to overcome these challenges.
Pre-targeting combined with bioorthogonal chemistry has been shown to be an effective way to develop new molecular imaging probes for both fluorescence and radio-imaging4,5,6,7,8. The inverse electron demand Diels-alder (IEDDA) reaction between 1,2,4,5-tetrazine (Tz) and trans-cyclooctene (TCO) derivatives, as shown in Figure 1, has been shown to be particularly effective6. The IEDDA reaction with these components can exhibit fast kinetics in PBS (k2 &#8776; 6,000 M-1 s-1) and high selectivity, making it ideal for in vivo pre-targeting applications9,10.
The most common approach used involves administering a TCO-derived targeting vector and following a sufficient delay period, a radiolabeled tetrazine is administered. Radiolabeled tetrazines based on 11C, 18F, 64Cu, 89Zr, and 111In have been reported11,12,13,14,15. In contrast, there is only one report of a 99mTc-labeled Tz, which was prepared using a HYNIC type ligand requiring the use of co-ligands to prevent protein binding and degradation in vivo16. As an alternative, we report here the synthesis of 99mTc(I) labeled tetrazines using a family of ligands which form stable tridentate complexes with a [99mTc(CO)3]+ core.
<img alt="Figure 1" src="/files/ftp_upload/55188/55188fig1.jpg" /> 
Figure 1: The bioorthogonal IEDDA reaction between tetrazine and trans-cyclooctene. <a href="http://cloudflare.jove.com/files/ftp_upload/55188/55188fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The family of ligands prepared contain tridentate chelates that vary in polarity and the nature of the linker group between the metal binding region and the Tz (Figure 2). The goal was to identify a 99mTc-Tetrazine construct that could effectively localize and react with TCO-labeled sites in vivo and rapidly clear when not bound, in order to yield high target-to-non-target ratios. To test the ligands, a TCO-derivative of a bisphosphonate (TCO-BP) was used17. We have shown previously that TCO-BP localizes to areas of active bone metabolism and can react with radiolabeled tetrazines in vivo18. It is a convenient reagent to test new tetrazines, because it can be prepared in a single step and experiments can be performed in normal mice where localization occurs primarily in the joints (knees and shoulders).

<table><tbody><tr><td>Argon gas</td><td>Alphagaz</td><td>---</td><td>---</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;</td><td>EMD Millipore</td><td>106395</td><td>---</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;B&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;&amp;#183;10H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Anachemia</td><td>S9640</td><td>---</td></tr><tr><td>KNaC&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;&amp;#183;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Anachemia</td><td>217255</td><td>---</td></tr><tr><td>Technelite &lt;sup&gt;99m&lt;/sup&gt;Tc generator</td><td>Lantheus medical imaging</td><td>---</td><td>Source of &lt;sup&gt;99m&lt;/sup&gt;TcO&lt;sup&gt;4-&lt;/sup&gt;</td></tr><tr><td>0.9% Saline</td><td>Lantheus medical imaging</td><td>---</td><td>To elute generator</td></tr><tr><td>1 M HCl</td><td>Lab Chem</td><td>---</td><td>---</td></tr><tr><td>MeOH</td><td>Caledon</td><td>---</td><td>---</td></tr><tr><td>ACN</td><td>Caledon</td><td>---</td><td>HPLC grade</td></tr><tr><td>Millipore H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Thermo Fisher Scientific</td><td>Barnstead Nanopure</td><td>---</td></tr><tr><td>DCM</td><td>Caledon</td><td>---</td><td>---</td></tr><tr><td>TFA</td><td>Caledon</td><td>---</td><td>---</td></tr><tr><td>PBS</td><td>Thermo Fisher Scientific</td><td>10010023</td><td>pH 7.4 1x</td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A7906</td><td>---</td></tr><tr><td>Tween80</td><td>Sigma Aldrich</td><td>P8047</td><td>---</td></tr><tr><td>Isoflurane</td><td>CDMV</td><td>108737</td><td>Supplier: Fresenius Kabi Animal Health</td></tr><tr><td>HPLC</td><td>Waters</td><td>1525 Binary Pump, 2998 Photodiodde Array Detector, E-SAT/IN, Bioscan Flowcount PMT detector (item # 15590)</td><td>---</td></tr><tr><td>HPLC column for analysis and purification of compounds 2-4</td><td>Phenomenex</td><td>00G-4435-E0</td><td>Gemini&amp;reg; 5 &amp;micro;m C18 110&amp;nbsp;&amp;Aring;, LC Column 250 x 4.6 mm</td></tr><tr><td>HPLC column for analysis and purification of compounds 1 and 5</td><td>Waters&amp;nbsp;</td><td>186003115</td><td>XBridge BEH C18 Column, 130 &amp;Aring;, 5 &amp;micro;m, 4.6 mm x 100 mm</td></tr><tr><td>Microwave Reactor&amp;nbsp;</td><td>Biotage&amp;nbsp;</td><td>Initiator 8</td><td>---</td></tr><tr><td>Biotage V10 Evaporator</td><td>Biotage&amp;nbsp;</td><td>Serial # V1041</td><td>---</td></tr><tr><td>Dose calibrator</td><td>Capintec, Inc.&amp;nbsp;</td><td>CRC-25R</td><td>---</td></tr><tr><td>Gamma counter</td><td>Perkin Elmer</td><td>Wizard 1470 Automatic Gamma Counter</td><td>---</td></tr><tr><td>Animal room scale&amp;nbsp;</td><td>Mettler Toledo</td><td>XP105 Delta Range</td><td>---</td></tr><tr><td>Microwave vials&amp;nbsp;</td><td>Biotage&amp;nbsp;</td><td>355629</td><td>0.5-2 mL&amp;nbsp;</td></tr></tbody></table>

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.

The ligands were synthesized using different linkers and chelators via a simple reductive amination strategy (Figure 2), followed by coupling of the product to a commercially available tetrazine22,23. Radiolabeling was performed using the same method for all compounds and was highly reproducible. The process was optimized by varying the pH, amount of ligand, reaction time and temperature whereupon the 99m</su...

Watch this Scientific Journal Video about Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry at JoVE.com

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.

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 ...

preparation-evaluation-99mtc-labeled-tridentate-chelates-for-pre

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

8.1K Views.  McMaster University. Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry. 

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

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

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

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 ...

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 ...

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

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

Medicine

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 ...

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

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