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

Summary

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.

Abstract

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.

Introduction

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 — unlike partial body CT scans — 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 — most often termed pretargeting or multistep targeting — 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 > 30,000 M^-1sec^-1), selective, and — critically — bioorthogonal.²⁷ And while a number of types of click chemistry — including Cu-catalyzed azide-alkyne cycloadditions, strain-promoted azide-alkyne cycloadditions, and Staudinger ligations — 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.

Protocol

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

1. In a small reaction vessel, dissolve 7 mg NH₂-Bn-NOTA (1.25 x 10^-2 mmol) in 600 µl 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.
2. 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 (< 50 µl).
3. Allow the resulting reaction solution to react for 30 min at RT with mild agitation.
4. 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
5. Freeze the collected HPLC eluent using liquid nitrogen.
6. Wrap the frozen collection tube in opaque aluminum foil.
7. Place the frozen collection tube in a lyophilizing vessel O/N to remove the HPLC mobile phase.
8. Store the purified product (a bright pink solid) in the dark at -80 °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.

2. Preparation of huA33-TCO Immunoconjugate

1. In a 1.7 ml microcentrifuge tube, prepare a 1 mg/ml (2.7 mM) solution of TCO-NHS in dry DMF.
2. In a 1.7 ml microcentrifuge tube, prepare a 2 mg/ml (13.3 µM) solution of huA33 in 1 ml of phosphate buffered saline, pH 7.4 (0.01 M PO₄^3-_, 0.154 M NaCl).
3. Using small aliquots (< 5 µl) 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.
4. 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 µl 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.
5. Gently mix the solution by inverting the microcentrifuge tube several times.
6. Wrap the microcentrifuge tube in opaque aluminum foil.
7. Allow the solution to incubate for 1 hr at RT with mild agitation.
8. 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.
9. Measure the concentration of the resultant huA33-TCO on a UV-Vis spectrophotometer.
10. 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.
11. Store the completed huA33-TCO immunoconjugate at 4 °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.

3. ⁶⁴Cu Radiolabeling of Tz-Bn-NOTA

NOTE: This step of the protocol involves the handling and manipulation of radioactivity. Before performing these steps — or performing any other work with radioactivity — researchers should consult with their home institution’s Radiation Safety Department. Take all possible steps to minimize exposure to ionizing radiation.

1. In a 1.7 ml microcentrifuge tube, prepare a 0.5 mg/ml (723 µM) solution of Tz-Bn-NOTA.
2. In a 1.7 ml microcentrifuge tube, add 10 µl of the Tz-Bn-NOTA solution (5 µg) to 400 µl of 0.2 M NH₄OAc pH 5.5 buffer_.
3. 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.
4. Add 2,000 µCi (74 MBq) of ⁶⁴Cu to the Tz-Bn-NOTA solution.
   NOTE: Typically, [⁶⁴Cu]CuCl₂ is supplied in a small volume (< 30 µl) of 0.1 N HCl, and thus only small volumes (< 10 µl) 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.
5. Allow the solution to incubate for 10 min at RT with mild agitation.
6. 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.
7. Using a rotary evaporator, remove the HPLC eluent.
8. 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.

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’s Radiation Safety Department. Take all possible steps to minimize exposure to ionizing radiation.

1. 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
2. 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.
3. Inject 200 µl of the huA33-TCO solution (100 µg) into the tail vein of the xenograft-bearing mouse.
4. Allow 24 hr for the accumulation of the huA33-TCO in the tumor of the mouse.
5. Dilute the ⁶⁴Cu-Tz-Bn-NOTA radioligand to a concentration of 1.5 mCi/ml in 0.9% sterile saline.
6. Inject 200 µl of the ⁶⁴Cu-Tz-Bn-NOTA radioligand solution (300 µCi; 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.
7. 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.
8. 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.
9. 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.

Results

The initial three steps of the experiment — 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) — 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 ± 0.6 TCO/mAb, and Tz-Bn-NOTA was radiolabeled with ⁶⁴Cu to yield the purified radioligand in >99% radio...

Discussion

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

Materials

Name	Company	Catalog Number	Comments
Tetrazine NHS Ester	Sigma-Aldrich	764701	Store at -80 °C
Trans-cyclooctene NHS Ester	Sigma-Aldrich	764523	Store at -80 °C
p-NH2-Bn-NOTA	Macrocyclics	B-601	Store at -80 °C