Name: pretargeted radioimmunotherapy based on the inverse electron demand diels-alder reaction
Uploaded: 2019-01-29T14:30:00
Description: Watch this Scientific Journal Video about Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction at JoVE.com

The authors thank Dr. Jacob Houghton for helpful conversations.&#160;The authors would also like to thank the NIH for their generous funding (R00CA178205 and U01CA221046).

All in vivo animal experiments described in this work were performed according to approved protocols and executed under the ethical guidelines of the Memorial Sloan Kettering Cancer Center, Weill Cornell Medical Center, and Hunter College Institutional Animal Care and Use Committees (IACUC).
1. The preparation of huA33-TCO
NOTE: The synthesis of huA33-TCO has been previously reported29. However, for the ease of the reader, it is replic...

<ol>
	<li>Goldenberg, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+Therapy+of+Cancer+with+Radiolabeled+Antibodies.">Targeted Therapy of Cancer with Radiolabeled Antibodies.</a> Journal of Nuclear Medicine. 43 (5), 693-713 (2002).</li><li>Goldenberg, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioimmunotherapy+of+B-cell+Lymphomas+with+Iodine-131-labeled+LL2+Monoclonal+Antibody.">Radioimmunotherapy of B-cell Lymphomas with Iodine-131-labeled LL2 Monoclonal Antibody.</a> Journal of Clinical Oncology. 9 (4), 548-564 (1991).</li><li>Kaminski, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioimmunotherapy+with+131I-Tositumomab+for+Relapsed+or+Refractory+B-cell+non-Hodgkin+Lymphoma:+Updated+Results+and+Long-Term+Follow-Up+of+the+University+of+Michigan+Experience.">Radioimmunotherapy with 131I-Tositumomab for Relapsed or Refractory B-cell non-Hodgkin Lymphoma: Updated Results and Long-Term Follow-Up of the University of Michigan Experience.</a> Blood. 96 (4), 1259-1266 (2000).</li><li>Davies, A. 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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Step+Targeting+of+Xenografted+Colon+Carcinoma+Using+a+Bispecific+Antibody+and+188Re-Labeled+Bivalent+Hapten:+Biodistribution+and+Dosimetry+Studies.">Two-Step Targeting of Xenografted Colon Carcinoma Using a Bispecific Antibody and 188Re-Labeled Bivalent Hapten: Biodistribution and Dosimetry Studies.</a> Journal of Nuclear Medicine. 42 (1), 146-153 (2001).</li><li>Green, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Analysis+of+Bispecific+Antibody+and+Streptavidin-Targeted+Radioimmunotherapy+for+B-cell+Cancers.">Comparative Analysis of Bispecific Antibody and Streptavidin-Targeted Radioimmunotherapy for B-cell Cancers.</a> Cancer Research. 76 (22), 6669 (2016).</li><li>Sharkey, R. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=18F-Based+Pretargeted+PET+Imaging+Based+on+Bioorthogonal+Diels-Alder+Click+Chemistry.">18F-Based Pretargeted PET Imaging Based on Bioorthogonal Diels-Alder Click Chemistry.</a> Bioconjugate Chemistry. 27 (2), 298-301 (2016).</li><li>Rossin, R., L&#228;ppchen, T., vanden Bosch, S. M., Laforest, R., Robillard, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diels-Alder+Reaction+for+Tumor+Pretargeting:+In+Vivo+Chemistry+Can+Boost+Tumor+Radiation+Dose+Compared+with+Directly+Labeled+Antibody.">Diels-Alder Reaction for Tumor Pretargeting: In Vivo Chemistry Can Boost Tumor Radiation Dose Compared with Directly Labeled Antibody.</a> Journal of Nuclear Medicine. 54 (11), 1989-1995 (2013).</li><li>Zeglis, B. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+the+In+Vivo+Efficacy+of+Pretargeted+Radioimmunotherapy+Utilizing+Inverse+Electron+Demand+Diels-Alder+Click+Chemistry.">Establishment of the In Vivo Efficacy of Pretargeted Radioimmunotherapy Utilizing Inverse Electron Demand Diels-Alder Click Chemistry.</a> Molecular Cancer Therapeutics. 16 (1), 124-133 (2017).</li><li>Membreno, R., Cook, B. E., Fung, K., Lewis, J. S., Zeglis, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click-Mediated+Pretargeted+Radioimmunotherapy+of+Colorectal+Carcinoma.">Click-Mediated Pretargeted Radioimmunotherapy of Colorectal Carcinoma.</a> Molecular Pharmaceutics. 15 (4), 1729-1734 (2018).</li><li>Reiner, T., Lewis, J. S., Zeglis, B. 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One of the strengths of this approach to in vivo pretargeting &#8212; especially in relation to strategies predicated on bispecific antibodies and radiolabeled haptens &#8212; is its modularity: trans-cyclooctene moieties can be appended to any antibody, and tetrazine radioligands can be radiolabeled with an extraordinary variety of radionuclides without impairing their ability to react with their click partners. Yet the adaptation of this approach to other antibody/antigen system is not as simple as du...

Radioimmunotherapy (RIT) &#8212; the use of antibodies for the delivery of therapeutic radionuclides to tumors &#8212; has long been an enticing approach to the treatment of cancer1,2. Indeed, this promise has been underscored by the United States Food and Drug Administration&#8217;s approval of two radioimmunoconjugates for the treatment of Non-Hodgkin&#8217;s Lymphoma: 90Y-ibritumomab tiuxetan and 131I-tositumomab3,4. Yet even from its earliest days, the clinical prospects of RIT have been hampered by a critical complication: high radiation dose rates to healthy tissues5,6. Generally speaking, radioimmunoconjugates for RIT are labeled with long-lived radionuclides (e.g., 131I [t&#189; = 8.0 days] and 90Y [t&#189; = 2.7 days]) with physical half-lives that dovetail well with the long pharmacokinetic half-lives of immunoglobulins. This is essential, as it ensures that sufficient radioactivity remains once the antibody has reached its optimal biodistribution after several days of circulation. However, this combination of long residence times in the blood and long physical half-lives inevitably results in the irradiation of healthy tissues, thereby reducing therapeutic ratios and limiting the efficacy of therapy7. Several strategies have been explored to circumvent this problem, including the use of truncated antibody fragments such as Fab, Fab&#39;, F(ab&#39;)2, minibodies, and nanobodies8,9,10. One of the most promising and fascinating, yet undeniably complex, alternative approaches is in vivo pretargeting11.
In vivo pretargeting is an approach to nuclear imaging and therapy that seeks to harness the exquisite affinity and selectivity of antibodies while skirting their pharmacokinetic drawbacks11,12,13. To this end, the radiolabeled antibody used in traditional radioimmunotherapy is deconstructed into two components: a small molecule radioligand and an immunoconjugate that can bind both a tumor antigen and the aforementioned radioligand. The immunoconjugate is injected first and given a &#8216;head start&#8217;, often several days, during which it accumulates in the target tissue and clears from the blood. Subsequently, the small molecule radioligand is administered and either combines with the immunoconjugate at the tumor or rapidly clears from the body. In essence, in vivo pretargeting relies upon performing radiochemistry within the body itself. By reducing the circulation of the radioactivity, this approach simultaneously reduces radiation doses to healthy tissues and facilitates the use of radionuclides (e.g., 68Ga, t&#189; = 68 min211; As, t&#189; = 7.2 h) with half-lives that are typically considered incompatible with antibody-based vectors.
Starting in the late 1980s, a handful of different approaches to in vivo pretargeting have been developed, including strategies based on bispecific antibodies, the interaction between streptavidin and biotin, and the hybridization of complementary oligonucleotides14,15,16,17,18. Yet each has been held back to varying degrees by complications, most famously the potent immunogenicity of streptavidin-modified antibodies19,20. Over the last five years, our group and others have developed an approach to in vivo pretargeting based on the rapid and bioorthogonal inverse electron demand Diels-Alder ligation between trans-cyclooctene (TCO) and tetrazine (Tz)21,22,23,24. The most successful of these strategies have employed a TCO-modified antibody and a Tz-bearing radioligand, as TCO is typically more stable in vivo than its Tz partner (Figure 1)25,26. As in other pretargeting methodologies, the mAb-TCO immunoconjugate is administered first and given time to clear from circulation and accumulate in tumor tissue. Subsequently, the small molecule Tz radioligand is injected, after which it either clicks with the immunoconjugate within the target tissue or clears rapidly from the body. This in vivo pretargeting strategy has proven highly effective for PET and SPECT imaging with several different antibody/antigen systems, consistently producing images with high contrast and enabling the use of short-lived radionuclides such as 18F (t&#189; = 109 min) and 64Cu (t1/2 = 12.7 h)21,22,24. More recently, the efficacy of click-based pretargeted radioimmunotherapy (PRIT) has been demonstrated in murine models of pancreatic ductal adenocarcinoma (PDAC) and colorectal carcinoma27,28. To this end, the therapeutic radionuclide 177Lu (&#946;max = 498 keV, t1/2 = 6.7 days) was employed in conjunction with two different antibodies: 5B1, which targets carbohydrate antigen 19.9 (CA19.9) ubiquitously expressed in PDAC, and huA33, which targets A33, a transmembrane glycoprotein expressed in &#62;95% of colorectal cancers. In both cases, this approach to 177Lu-PRIT yielded high activity concentrations in tumor tissue, created a dose-dependent therapeutic effect, and simultaneously reduced activity concentrations in healthy tissues compared to traditional directly-labeled radioimmunoconjugates.
In this article, we describe protocols for PRIT using a 177Lu-DOTA-labeled tetrazine radioligand ([177Lu]Lu-DOTA-PEG7-Tz) and a TCO-modified variant of the huA33 antibody (huA33-TCO). More specifically, we describe the construction of huA33-TCO (Figure 2), the synthesis and radiolabeling of [177Lu]Lu-DOTA-PEG7-Tz (Figure 3 and Figure 4), and the performance of in vivo biodistribution and longitudinal therapy studies in murine models of colorectal carcinoma. Furthermore, in the representative results and discussion, we present a sample data set, address possible strategies for the optimization of this approach, and consider this strategy in the wider context of in vivo pretargeting and PRIT. Finally, it is important to note that while we have chosen to focus on pretargeting using huA33-TCO and [177Lu]Lu-DOTA-PEG7-Tz in this protocol, this strategy is highly modular and can be adapted to suit a wide range of antibodies and radionuclides.

<table>
	<tbody>
		<tr>
			<td>(E)-Cyclooct-4-enyl 2,5-dioxo-1-pyrrolidinyl carbonate (TCO-NHS)</td>
			<td>Sigma-Aldrich</td>
			<td>764523</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr>
			<td>2,5-Dioxo-1-pyrrolidinyl 5-[4-(1,2,4,5-tetrazin-3-yl)benzylamino]-5-oxopentanoate (Tz-NHS)</td>
			<td>Sigma-Aldrich</td>
			<td>764701</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Acetonitrile (MeCN)</td>
			<td>Fisher Scientific</td>
			<td>A998-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate (NH4OAc)</td>
			<td>Fisher Scientific</td>
			<td>A639-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Boc-PEG7-amine (O-(2-Aminoethyl)-O&#8242;-[2-(Boc-amino)ethyl]hexaethylene glycol)</td>
			<td>Sigma-Aldrich</td>
			<td>70023</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Fisher Scientific</td>
			<td>D143-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO), anhydrous</td>
			<td>Fisher Scientific</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>EMD Millipore Amicon Ultra-2 Centrifugal Filter Unit</td>
			<td>Fisher Scientific</td>
			<td>UFC205024</td>
			<td></td>
		</tr>
		<tr>
			<td>GE Healthcare Disposable PD-10 Desalting Columns</td>
			<td>Fisher Scientific</td>
			<td>45-000-148</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide (DMF), anhydrous</td>
			<td>Fisher Scientific</td>
			<td>AC610941000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>70-011-044</td>
			<td>10x Concentrated</td>
		</tr>
		<tr>
			<td>p-SCN-Bn-DOTA</td>
			<td>Macrocyclics</td>
			<td>B-205</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Triethylamine (TEA)</td>
			<td>Fisher Scientific</td>
			<td>AC157911000</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic Acid (TFA)</td>
			<td>Fisher Scientific</td>
			<td>A116-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Tumor measuring device</td>
			<td>Peira TM900</td>
			<td>Peira TM900</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The conjugation of TCO to huA33 is predicated on the coupling between the amine-reactive TCO-NHS and the lysine residues on the surface of the immunoglobulin. This method is highly robust and reproducible and reliably yields a degree-of-labeling of 2-4 TCO/mAb. In this case, MALDI-ToF mass spectrometry was employed to confirm a degree of labeling of approximately 4.0 TCO/mAb; a similar value was obtained using a fluorophore-modified tetrazine as a reporter24. The s...

Watch this Scientific Journal Video about Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction at JoVE.com

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

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 ...

This protocol describes how to apply a pretargeted methodology to the targeted radiotherapy of cancer xenographs in the mouse model. Pretargeted radioimmunotherapy or PRIT allows us to inject the antibody and radioligand separately, and let the two combine in vivo using click chemistry. While we present PRIT using a xenograph model of colorectal cancer, this technique can be applied to any surface antigen targeting antibody.It is important to select an antigen that does not internalize rapidly or shed extensively as these can reduce the efficacy of the approach. To synthesize huA33-TCO first prepare a 125 microliter solution of 40 milligrams per milliliter TCO-NHS in dry methylformamide in a 1.7 milliliter microcentrifuge tube. This solution can be aliquoted and frozen at 80 degree celsius for use in future experiments.In a separate 1.7 milliliter microcentrifuge tube prepare a five milligram per milliliter solution of huA33 in one milliliter of phosphate buffered saline. Using small aliquots of 0.1 molar sodium carbonate, adjust the pH of the antibody solution to between 8.8 and 9.0. Use either pH paper or a pH meter with a microelectrode to monitor the pH, and exercise care that the pH does not exceed 9.0.Add a volume corresponding to 40 molar equivalence of TCO-NHS to the antibody solution. To prevent precipitation of the hydrophobic TCO-NHS, add it slowly and with agitation. Allow the reaction to incubate at 25 degrees celsius on a thermal mixer for one hour with mild agitation.Before purifying the huA33-TCO immunoconjugate using a pre-packed disposable size exclusion desalting column, equilibrate the size exclusion column as described by the supplier to remove any preservatives present in the column during storage which usually involves washing the column five times with a volume of PBS that corresponds to the volume of the column. Add the reaction mixture to the size exclusion column, noting the volume of the reaction mixture. After the reaction mixture has entered the column add an appropriate amount of PBS to bring the total volume of solution added to the column up to 2.5 milliliters.Collect the product using two milliliters of PBS as the eluent. Measure the concentration of the huA33-TCO using a UV vis spectrophotometer monitoring the wavelength at 280 nanometers. If a solution with a higher concentration of immunoconjugate is desired concentrate the huA33-TCO solution using a centrifugal filter unit with a 50, 000 molecular weight cutoff following manufacturer's instructions.Store the completed huA33-TCO immunoconjugate at 4 degree celsius in the dark. If it is to be used more than four days in the future store it at 80 degrees celsius in the dark. In a 1.7 milliliters centrifuge tube add 200 microliters of 0.25 molar ammonium acetate buffer adjusted to pH 5.5.Add the desired amount of Lutetium-177 chloride to the buffer solution. The amount added will be dependent on the number of subjects in the experiment, and the radioactive doses being administered. Add Tetrazine PEG7-DOTA in DMSO to the radioactive mixture.The amount added is dependent on the number of subjects being tested. Allow the solution to incubate at 37 degrees celsius for 20 minutes. Verify the radiolabeling is complete using radio-instant-thin-layer chromatography with 50 millimolar EDTA pH 5.5 as the mobile phase.The labeled Lutetium-177-DOTA-PEG7-Tetrazine will remain at the baseline while free Lutetium-177 will be coordinated by the EDTA and travel with the solvent front. It is critical that the placement of the subcutaneous tumors does not interfere with your restraint technique. I recommend placing them on the flank side that is opposite to your restraining hand.Since I restrain with my left hand I have chosen to place the tumors on the right flank. Sort female athymic nude mice with colorectal cancer xenograph, so that the average tumor volume in each cohort is roughly equal. To do so, list the animal identification numbers, ear notch pattern and tumor volume in three separate columns of a spreadsheet program.Sort the data from smallest to largest tumor volume. In a fourth column assign each animal a cage number. Cycle through the cages in a snake-like pattern until all the animals are assigned a cage.Once the cages are assigned sort the data by cage number. To inject the immunoconjugate draw doses of 150 microliters of huA33-TCO solution in syringes pretreated with 1%bovine serum albumin in PBS, and store these syringes on ice. To inject the radioligand, first draw doses in 150 microliters of 0.9%sterile saline containing 1.1 molar equivalence of the radiolabeled Tetrazine-PEG7-DOTA relative to the amount of huA33-TCO administered.Use calipers to measure the longest side of the oblong tumor as well as the width which is perpendicular to the length. Weigh each mouse on a balance to track weight gain or weight loss over time. Finally, calculate the volume using the formula for volume of an ellipsoid assuming that the height is approximately equal to the width.Shown here is the biodistribution of an in vivo pretargeting with huA33-TCO and Lutetium-177-DOTA-PEG7-Tetrazine in athymic nude mice bearing subcutaneous SW1222 human colorectal caner tumors. All injection intervals produce high activity concentrations in the tumor tissue as well as low activity concentrations in healthy organs. The 24 hour injection interval affords the highest tumoral uptake at 120 hours post injection.Based on these findings a 24 hour interval was chosen for the subsequent longitudinal therapy study. Shown here is longitudinal therapy study of five groups of mice bearing subcutaneous SW1222 tumors depicted in average tumor volume as a function of time, and tumor volume normalized to initial volume as a function of time. There is a stark difference in the response of the experimental cohorts as compared to the control groups.While the tumors in the mice receiving only one component of the pretargeted radioimmunotherapy strategy continued to grow unchecked, the tumors of the mice receiving the full pretargeted radioimmunotherapy regimen stopped growing and ultimately shrank. Importantly, no toxic side-effects were observed, and all animals maintained a weight within 20%of their initial mass. Strikingly, the mice of the experimental cohorts had a perfect record of survival at the end of the investigation.When attempting this procedure it is of the utmost importance to accurately and reproducibly measure the tumors. When working with radioactivity consult your institution's radiation safety officer to develop safe protocols and facilities. Having proper safety controls in place will limit your exposure and avoid contamination.Pretargeting affords a lower radiation dose to background organs than traditional radioimmunotherapy. By sharing this protocol we hope that others may be able to test their own models and ideas that will lead to new and exciting advances for patient treatment.

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