Development of a 99mTc-labeled tetrazine for pretargeted SPECT imaging using an alendronic acid-based bone targeting model.

Pretargeting, which is the separation of target accumulation and the administration of a secondary imaging agent into two sequential steps, offers the potential to improve image contrast and reduce radiation burden for nuclear imaging. In recent years, the tetrazine ligation has emerged as a promising approach to facilitate covalent pretargeted imaging due to its unprecedented kinetics and bioorthogonality. Pretargeted bone imaging with TCO-modified alendronic acid (Aln-TCO) is an attractive model that allows the evaluation of tetrazines in healthy animals without the need for complex disease models or targeting regimens. Recent structure-activity relationship studies of tetrazines evaluated important parameters for the design of potent tetrazine-radiotracers for pretargeted imaging. However, limited information is available for 99mTc-labeled tetrazines. In this study, four tetrazines intended for labeling with fac-[99mTc(OH2)3 (CO)3]+ were synthesized and evaluated using an Aln-TCO mouse model. 3,6-bis(2-pyridyl)-1,2,4,5-Tz without additional linker showed higher pretargeted bone uptake and less background activity compared to the same scaffold with a PEG8 linker or 3-phenyl-1,2,4,5-Tz-based compounds. Additionally, improved bone/blood ratios were observed in pretargeted animals compared to animals receiving directly labeled Aln-TCO. The results of this study implicate 3,6-bis(2-pyridyl)-1,2,4,5-Tz as a promising scaffold for potential 99mTc-labeled tetrazines.

Investigation of imaging the somatostatin receptor by opening the blood-brain barrier with melittin - A feasibility study using positron emission tomography and [64Cu]Cu-DOTATATE.

DOTATATE is a somatostatin peptide analog used in the clinic to detect somatostatin receptors which are highly expressed on neuroendocrine tumors. Somatostatin receptors are found naturally in the intestines, pancreas, lungs, and brain (mainly cortex). In vivo measurement of the somatostatin receptors in the cortex has been challenging because available tracers cannot cross the blood-brain barrier (BBB) due to their intrinsic polarity. A peptide called melittin, a main component of honeybee venom, has been shown to disrupt plasma membranes and increase the permeability of biological membranes. In this study, we assessed the feasibility of using melittin to facilitate the passage of [64Cu]Cu-DOTATATE through the BBB and its binding to somatostatin receptors in the cortex. Evaluation included in vitro autoradiography on Long Evans rat brains to estimate the binding affinity of [64Cu]Cu-DOTATATE to the somatostatin receptors in the cortex and an in vivo evaluation of [64Cu]Cu-DOTATATE binding in NMRI mice after injection of melittin. This study found an in vitro Bmax = 89 ± 4 nM and KD = 4.5 ± 0.6 nM in the cortex, resulting in a theoretical binding potential (BP) calculated as Bmax/KD ≈ 20, which is believed suitable for in vivo brain PET imaging. However, the in vivo results showed no significant difference between the control and melittin injected mice, indicating that the honeybee venom failed to open the BBB. Additional experiments, potentially involving faster injection rates are required to verify that melittin can increase brain uptake of non-BBB permeable PET tracers. Furthermore, an evaluation of whether a venom with a narrow therapeutic range can be used for clinical purposes needs to be considered.

Development and Preclinical Evaluation of [211At]PSAt-3-Ga: An Inhibitor for Targeted α-Therapy of Prostate Cancer.

The application of prostate-specific membrane antigen (PSMA)-targeted α-therapy is a promising alternative to ß--particle-based treatments. 211At is among the potential α-emitters that are favorable for this concept. Herein, 211At-based PSMA radiopharmaceuticals were designed, developed, and evaluated. Methods: To identify a 211At-labeled lead, a surrogate strategy was applied. Because astatine does not exist as a stable nuclide, it is commonly replaced with iodine to mimic the pharmacokinetic behavior of the corresponding 211At-labeled compounds. To facilitate the process of structural design, iodine-based candidates were radiolabeled with the PET radionuclide 68Ga to study their preliminary in vitro and in vivo properties before the desired 211At-labeled lead compound was formed. The most promising candidate from this evaluation was chosen to be 211At-labeled and tested in biodistribution studies. Results: All 68Ga-labeled surrogates displayed affinities in the nanomolar range and specific internalization in PSMA-positive LNCaP cells. PET imaging of these compounds identified [68Ga]PSGa-3 as the lead compound. Subsequently, [211At]PSAt-3-Ga was synthesized in a radiochemical yield of 35% and showed tumor uptake of 19 ± 8 percentage injected dose per gram of tissue (%ID/g) at 1 h after injection and 7.6 ± 2.9 %ID/g after 24 h. Uptake in off-target tissues such as the thyroid (2.0 ± 1.1 %ID/g), spleen (3.0 ± 0.6 %ID/g), or stomach (2.0 ± 0.4 %ID/g) was low, indicating low in vivo deastatination of [211At]PSAt-3-Ga. Conclusion: The reported findings support the use of iodine-based and 68Ga-labeled variants as a convenient strategy for developing astatinated compounds and confirm [211At]PSAt-3 as a promising radiopharmaceutical for targeted α-therapy.

A facile synthesis of precursor for the σ-1 receptor PET radioligand [18 F]FTC-146 and its radiofluorination.

The σ-1 receptor is a non-opioid transmembrane protein involved in various human pathologies including neurodegenerative diseases, inflammation, and cancer. The previously published ligand [18 F]FTC-146 is among the most promising tools for σ-1 molecular imaging by positron emission tomography (PET), with a potential for application in clinical diagnostics and research. However, the published six- or four-step synthesis of the tosyl ester precursor for its radiosynthesis is complicated and time-consuming. Herein, we present a simple one-step precursor synthesis followed by a one-step fluorine-18 labeling procedure that streamlines the preparation of [18 F]FTC-146. Instead of a tosyl-based precursor, we developed a one-step synthesis of the precursor analog AM-16 containing a chloride leaving group for the SN 2 reaction with 18 F-fluoride. 18 F-fluorination of AM-16 led to a moderate decay-corrected radiochemical yield (RCY = 7.5%) with molar activity (Am ) of 45.9 GBq/µmol. Further optimization of this procedure should enable routine radiopharmaceutical production of this promising PET tracer.

Next generation fibroblast activation protein (FAP) targeting PET tracers - The tetrazine ligation allows an easy and convenient way to 18F-labeled (4-quinolinoyl)glycyl-2-cyanopyrrolidines.

Small-molecular fibroblast activation protein inhibitor (FAPI)-based tracer have been shown to be promising Positron Emission Tomography (PET) 68Ga-labeled radiopharmaceuticals to image a variety of tumors including pancreatic, breast, and colorectal cancers, among others. In this study, we developed a novel 18F-labeled FAPI derivative. [18F]6 was labeled using a synthon approach based on the tetrazine ligation. It showed subnanomolar affinity for the FAP protein and a good selectivity profile against known off-target proteases. Small animal PET studies revealed high tumor uptake and good target-to-background ratios. [18F]6 was excreted via the liver. Overall, [18F]6 showed promising characteristics to be used as a PET tracer and could serve as a lead for further development of halogen-based theranostic FAPI radiopharmaceuticals.

Quantitative Evaluation of a Multimodal Aptamer-Targeted Long-Circulating Polymer for Tumor Targeting.

Aptamers are promising targeting agents for imaging and therapy of numerous diseases, including cancer. However, a significant shortcoming of aptamers is their poor stability and fast excretion, limiting their application in vivo. Common strategies to overcome these challenges is to chemically modify aptamers in order to increase their stability and/or to apply formulation technologies such as conjugating them to polymers or nanocarriers in order to increase their circulation half-life. This is expected to result in improved cellular uptake or retention to passively targeted nanomedicines. Herein, we report a modular conjugation strategy based on click chemistry between functionalized tetrazines and trans-cyclooctene (TCO), for the modification of high molecular weight hyperbranched polyglycerol (HPG) with sgc8 aptamer, fluorescent dyes, and 111In. Our data indicate strong affinity of sgc8 against a range of solid tumor-derived cell lines that have previously not been tested with this aptamer. Nevertheless, nonspecific uptake of scrambled ssDNA-functionalized HPG in cells highlights inherent challenges of aptamer-targeted probes that remain to be solved for clinical translation. We validate HPG-sgc8 as a nontoxic nanoprobe with high affinity against MDA-MB-468 breast and A431 lung cancer cells and show significantly increased plasma stability compared to free sgc8. In vivo quantitative SPECT/CT imaging indicates EPR-mediated tumor uptake of HPG-sgc8 and nontargeted or scrambled ssDNA-conjugated HPG but no statistically significant difference between these formulations in terms of total tumor uptake or retention. Our study emphasizes the need for stringent controls and quantification in the evaluation of aptamer-targeted probes. For this purpose, our versatile synthesis strategy provides a simple approach for the design and evaluation of long-circulating aptamer-conjugated nanoformulations.

Synthesis and radiolabeling of a polar [125 I]I-1,2,4,5-tetrazine.

Pretargeting imaging has gained a lot of prominence, due to its excellent bioorthogonality and improved imaging contrast compared to conventional imaging. A new iodo tetrazine (Tz) derivative has been synthesized and further developed into the corresponding iodine-125 (125 I) analog (12), via the trimethylstannane precursor. Radiolabeling with either N-chlorosuccinimide or chloramine-T, in either MeCN or MeOH proceeded with a radiochemical conversion (RCC) of >80%. Subsequent deprotection only proved successful, among the tested conditions, when the radiolabeled Tz was stirred in 6-M HCl(aq.) at 60°C for 2.5 h. To the best of our knowledge, this is the first H-tetrazine labeled with iodine. In vivo investigations on the pretargeting ability of 12 are currently under way.

Synthesis and in vivo evaluation of [11C]tucatinib for HER2-targeted PET imaging.

Tucatinib is a selective human epidermal growth factor receptor 2 (HER2) tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration (FDA) in April 2020 for HER2-positive lesions in metastatic breast cancer patients, including CNS metastases. In this article, we attempted to develop the first small molecule, blood-brain-barrier (BBB) penetrant HER2 PET imaging probe based on tucatinib. [11C]tucatinib was synthesized via a Stille-coupling from the respective trimethylstannyl precursor and its biodistribution was evaluated in NMRI nude mice bearing HER2-overexpressing human ovarian cancer cells (SKOV-3). No significant tumor accumulation was observed despite its high affinity for HER-2 receptors (IC50 = 6.9 nM). High liver and intestinal uptake indicate that [11C]tucatinib is too lipophilic to be used as a tumor targeting PET tracer. Therefore, chemical modifications of [11C]tucatinib are needed to increase the polarity for tumor imaging. Tucatinib as an FDA approved drug is still an interesting platform to develop the first small molecule HER2-selective PET tracer. The study highlights the differences between a drug, which needs to be effective, and an imaging agent, which is dependent on contrast.

Radiolabeled albumin through SNAr of cysteines as a potential pretargeting theranostic agent.

Human serum albumin (HSA) has been shown to be a promising tumor targeting vector and target for generating theranostics by bioconjugation. Unstable chemical conjugation to HSA via a cysteine (Cys34) by reversible Michael additions is most commonly applied for this purpose. Herein, we describe utilization of our recently developed site-selective irreversible SNAr conjugation to Cys34 using perfluorobenzene sulfonyl derivatives to introduce a trans-cyclooctene (TCO) handle. The TCO could then be bioorthogonally ligated within minutes through an inverse-electron demand Diels-Alder reaction (IEDDA) to tetrazines (Tzs) containing a radionuclide. The methodology opens up a wide range of chemistries including pretargeting, 'click-to-release' tumor selective drug delivery or ultra-fast and complete conjugation of any drug. The proof-of-principle study demonstrated that the conjugation chemistry is feasible, robust and easy to carry out, being promising for pretargeted imaging and therapy studies as well as selective drug delivery using HSA.

Covalent core-radiolabeling of polymeric micelles with 125I/211At for theranostic radiotherapy.

Astatine-211 (211At) is one of the most promising α-emitters for targeted alpha therapy, especially of cancer metastases. However, the lack of a stable isotope, frequent in vivo deastatination, and limited radiochemical knowledge makes it challenging to apply. Here, we report a new strategy for radiolabeling the lipophilic core of polymeric micelles (PMs) with covalently bound 211At. The PMs were radiolabeled via either an indirect synthon-based method or directly on the amphipathic block copolymer. The radiochemistry was optimized with iodine-125 (125I) and then adapted for 211At, enabling the use of both elements as a potential theranostic pair. PMs that were core-radiolabeled with both 125I or 211At were prepared and characterized, based on a PEG(5k)-PLGA(10k) co-polymer. The stability of the radiolabeled PMs was evaluated in mouse serum for 21 h, showing radiochemical stability above 85%. After in vivo evaluation of the 211At- labeled PMs, 4-5 % ID/g of the 211At could still be detected in the blood, showing a promising in vivo stability of the PMs. Further, 211At-labeled PMs accumulated in the spleen (20-30 %ID/g) and the liver (2.5- 5.5 %ID/g), along with some detection of 211At in the thyroid (3.5-9 %ID/g). This led to the hypothesis that deastatination takes place in the liver, whereas good stability of the 211At core-radiolabel was observed in the blood.

Bioorthogonal Click of Colloidal Gold Nanoparticles to Antibodies In vivo.

Combining nanotechnology and bioorthogonal chemistry for theranostic strategies offers the possibility to develop next generation nanomedicines. These materials are thought to increase therapeutic outcome and improve current cancer management. Due to their size, nanomedicines target tumors passively. Thus, they can be used for drug delivery purposes. Bioorthogonal chemistry allows for a pretargeting approach. Higher target-to-background drug accumulation ratios can be achieved. Pretargeting can also be used to induce internalization processes or trigger controlled drug release. Colloidal gold nanoparticles (AuNPs) have attracted widespread interest as drug delivery vectors within the last decades. Here, we demonstrate for the first time the possibility to successfully ligate AuNPs in vivo to pretargeted monoclonal antibodies. We believe that this possibility will facilitate the development of AuNPs for clinical use and ultimately, improve state-of-the-art patient care.

Development of 18F-Labeled Bispyridyl Tetrazines for In Vivo Pretargeted PET Imaging.

Pretargeted PET imaging is an emerging and fast-developing method to monitor immuno-oncology strategies. Currently, tetrazine ligation is considered the most promising bioorthogonal reaction for pretargeting in vivo. Recently, we have developed a method to 18F-label ultrareactive tetrazines by copper-mediated fluorinations. However, bispyridyl tetrazines-one of the most promising structures for in vivo pretargeted applications-were inaccessible using this strategy. We believed that our successful efforts to 18F-label H-tetrazines using low basic labeling conditions could also be used to label bispyridyl tetrazines via aliphatic nucleophilic substitution. Here, we report the first direct 18F-labeling of bispyridyl tetrazines, their optimization for in vivo use, as well as their successful application in pretargeted PET imaging. This strategy resulted in the design of [18F]45, which could be labeled in a satisfactorily radiochemical yield (RCY = 16%), molar activity (Am = 57 GBq/µmol), and high radiochemical purity (RCP > 98%). The [18F]45 displayed a target-to-background ratio comparable to previously successfully applied tracers for pretargeted imaging. This study showed that bispyridyl tetrazines can be developed into pretargeted imaging agents. These structures allow an easy chemical modification of 18F-labeled tetrazines, paving the road toward highly functionalized pretargeting tools. Moreover, bispyridyl tetrazines led to near-instant drug release of iTCO-tetrazine-based 'click-to-release' reactions. Consequently, 18F-labeled bispyridyl tetrazines bear the possibility to quantify such release in vivo in the future.

Radiolabeling of a polypeptide polymer for intratumoral delivery of alpha-particle emitter, 225Ac, and beta-particle emitter, 177Lu.

INTRODUCTION: Radiotherapy of cancer requires both alpha- and beta-particle emitting radionuclides, as these radionuclide types are efficient at destroying different types of tumors. Both classes of radionuclides require a vehicle, such as an antibody or a polymer, to be delivered and retained within the tumor. Polyglutamic acid (pGlu) is a polymer that has proven itself effective as a basis of drug-polymer conjugates in the clinic, while its derivatives have been used for pretargeted tumor imaging in a research setup. trans-Cyclooctene (TCO) modified pGlu is suitable for pretargeted imaging or therapy, as well as for intratumoral radionuclide therapy. In all cases, it becomes indirectly radiolabeled via the bioorthogonal click reaction with the tetrazine (Tz) molecule carrying the radionuclide. In this study, we report the radiolabeling of TCO-modified pGlu with either lutetium-177 (177Lu), a beta-particle emitter, or actinium-225 (225Ac), an alpha-particle emitter, using the click reaction between TCO and Tz. METHODS: A panel of Tz derivatives containing a metal ion binding chelator (DOTA or macropa) connected to the Tz moiety directly or through a polyethylene glycol (PEG) linker was synthesized and tested for their ability to chelate 177Lu and 225Ac, and click to pGlu-TCO. Radiolabeled 177Lu-pGlu and 225Ac-pGlu were isolated by size exclusion chromatography. The retention of 177Lu or 225Ac by the obtained conjugates was investigated in vitro in human serum. RESULTS: All DOTA-modified Tzs efficiently chelated 177Lu resulting in average radiochemical conversions (RCC) of >75%. Isolated radiochemical yields (RCY) for 177Lu-pGlu prepared from 177Lu-Tzs ranged from 31% to 55%. TLC analyses detected <5% unchelated 177Lu for all 177Lu-pGlu preparations over six days in human serum. For 225Ac chelation, optimized RCCs ranged from 61 ± 34% to quantitative for DOTA-Tzs and were quantitative for the macropa-modified Tz (>98%). Isolated radiochemical yields (RCY) for 225Ac-pGlu prepared from 225Ac-Tzs ranged from 28% to 51%. For 3 out of 5 225Ac-pGlu conjugates prepared from DOTA-Tzs, the amount of unchelated 225Ac stayed below 10% over six days in human serum, while 225Ac-pGlu prepared from macropa-Tz showed a steady release of up to 37% 225Ac. CONCLUSION: We labeled TCO-modified pGlu polymers with alpha- and beta-emitting radionuclides in acceptable RCYs. All 177Lu-pGlu preparations and some 225Ac-pGlu preparations showed excellent stability in human plasma. Our work shows the potential of pGlu as a vehicle for alpha- and beta-radiotherapy of tumors and demonstrated the usefulness of Tz ligation for indirect radiolabeling.

Lipophilicity and Click Reactivity Determine the Performance of Bioorthogonal Tetrazine Tools in Pretargeted In Vivo Chemistry.

The development of highly selective and fast biocompatible reactions for ligation and cleavage has paved the way for new diagnostic and therapeutic applications of pretargeted in vivo chemistry. The concept of bioorthogonal pretargeting has attracted considerable interest, in particular for the targeted delivery of radionuclides and drugs. In nuclear medicine, pretargeting can provide increased target-to-background ratios at early time-points compared to traditional approaches. This reduces the radiation burden to healthy tissue and, depending on the selected radionuclide, enables better imaging contrast or higher therapeutic efficiency. Moreover, bioorthogonally triggered cleavage of pretargeted antibody-drug conjugates represents an emerging strategy to achieve controlled release and locally increased drug concentrations. The toolbox of bioorthogonal reactions has significantly expanded in the past decade, with the tetrazine ligation being the fastest and one of the most versatile in vivo chemistries. Progress in the field, however, relies heavily on the development and evaluation of (radio)labeled compounds, preventing the use of compound libraries for systematic studies. The rational design of tetrazine probes and triggers has thus been impeded by the limited understanding of the impact of structural parameters on the in vivo ligation performance. In this work, we describe the development of a pretargeted blocking assay that allows for the investigation of the in vivo fate of a structurally diverse library of 45 unlabeled tetrazines and their capability to reach and react with pretargeted trans-cyclooctene (TCO)-modified antibodies in tumor-bearing mice. This study enabled us to assess the correlation of click reactivity and lipophilicity of tetrazines with their in vivo performance. In particular, high rate constants (>50 000 M-1 s-1) for the reaction with TCO and low calculated logD 7.4 values (below -3) of the tetrazine were identified as strong indicators for successful pretargeting. Radiolabeling gave access to a set of selected 18F-labeled tetrazines, including highly reactive scaffolds, which were used in pretargeted PET imaging studies to confirm the results from the blocking study. These insights thus enable the rational design of tetrazine probes for in vivo application and will thereby assist the clinical translation of bioorthogonal pretargeting.

Evaluation of [64Cu]Cu-NOTA-PEG7-H-Tz for Pretargeted Imaging in LS174T Xenografts-Comparison to [111In]In-DOTA-PEG11-BisPy-Tz.

Pretargeted nuclear imaging for the diagnosis of various cancers is an emerging and fast developing field. The tetrazine ligation is currently considered the most promising reaction in this respect. Monoclonal antibodies are often the preferred choice as pretargeting vector due to their outstanding targeting properties. In this work, we evaluated the performance of [64Cu]Cu-NOTA-PEG7-H-Tz using a setup we previously used for [111In]In-DOTA-PEG11-BisPy-Tz, thereby allowing for comparison of the performance of these two promising pretargeting imaging agents. The evaluation included a comparison of the physicochemical properties of the compounds and their performance in an ex vivo blocking assay. Finally, [64Cu]Cu-NOTA-PEG7-H-Tz was evaluated in a pretargeted imaging study and compared to [111In]In-DOTA-PEG11-BisPy-Tz. Despite minor differences, this study indicated that both evaluated tetrazines are equally suited for pretargeted imaging.

In Vitro and In Vivo Characterization of Dibenzothiophene Derivatives [125I]Iodo-ASEM and [18F]ASEM as Radiotracers of Homo- and Heteromeric α7 Nicotinic Acetylcholine Receptors.

The α7 nicotinic acetylcholine receptor (α7 nAChR) is involved in several cognitive and physiologic processes; its expression levels and patterns change in neurologic and psychiatric diseases, such as schizophrenia and Alzheimer's disease, which makes it a relevant drug target. Development of selective radioligands is important for defining binding properties and occupancy of novel molecules targeting the receptor. We tested the in vitro binding properties of [125I]Iodo-ASEM [(3-(1,4-diazabycyclo[3.2.2]nonan-4-yl)-6-(125I-iododibenzo[b,d]thiopentene 5,5-dioxide)] in the mouse, rat and pig brain using autoradiography. The in vivo binding properties of [18F]ASEM were investigated using positron emission tomography (PET) in the pig brain. [125I]Iodo-ASEM showed specific and displaceable high affinity (~1 nM) binding in mouse, rat, and pig brain. Binding pattern overlapped with [125I]α-bungarotoxin, specific binding was absent in α7 nAChR gene-deficient mice and binding was blocked by a range of α7 nAChR orthosteric modulators in an affinity-dependent order in the pig brain. Interestingly, relative to the wild-type, binding in ß2 nAChR gene-deficient mice was lower for [125I]Iodo-ASEM (58% ± 2.7%) than [125I]α-bungarotoxin (23% ± 0.2%), potentially indicating different binding properties to heteromeric α7ß2 nAChR. [18F]ASEM PET in the pig showed high brain uptake and reversible tracer kinetics with a similar spatial distribution as previously reported for α7 nAChR. Blocking with SSR-180,711 resulted in a significant decrease in [18F]ASEM binding. Our findings indicate that [125I]Iodo-ASEM allows sensitive and selective imaging of α7 nAChR in vitro, with better signal-to-noise ratio than previous tracers. Preliminary data of [18F]ASEM in the pig brain demonstrated principal suitable kinetic properties for in vivo quantification of α7 nAChR, comparable to previously published data.

Evaluation of a 68Ga-Labeled DOTA-Tetrazine as a PET Alternative to 111In-SPECT Pretargeted Imaging.

The bioorthogonal reaction between a tetrazine and strained transcyclooctene (TCO) has garnered success in pretargeted imaging. This reaction was first validated in nuclear imaging using an 111In-labeled 1,4,7,10tetraazacyclododecane1,4,7,10tetraacetic acid (DOTA)-linked bispyridyl tetrazine (Tz) ([111In]In-DOTA-PEG11-Tz) and a TCO functionalized CC49 antibody. Given the initial success of this Tz, it has been paired with TCO functionalized small molecules, diabodies, and affibodies for in vivo pretargeted studies. Furthermore, the single photon emission tomography (SPECT) radionuclide, 111In, has been replaced with the ß-emitter, 177Lu and α-emitter, 212Pb, both yielding the opportunity for targeted radiotherapy. Despite use of the 'universal chelator', DOTA, there is yet to be an analogue suitable for positron emission tomography (PET) using a widely available radionuclide. Here, a 68Ga-labeled variant ([68Ga]Ga-DOTA-PEG11-Tz) was developed and evaluated using two different in vivo pretargeting systems (Aln-TCO and TCO-CC49). Small animal imaging and ex vivo biodistribution studies were performed and revealed target specific uptake of [68Ga]Ga-DOTA-PEG11-Tz in the bone (3.7 %ID/g, knee) in mice pretreated with Aln-TCO and tumor specific uptake (5.8 %ID/g) with TCO-CC49 in mice bearing LS174 xenografts. Given the results of this study, [68Ga]Ga-DOTA-PEG11-Tz can serve as an alternative to [111In]In-DOTA-PEG11-Tz.

Trans-Cyclooctene-Functionalized PeptoBrushes with Improved Reaction Kinetics of the Tetrazine Ligation for Pretargeted Nuclear Imaging.

Tumor targeting using agents with slow pharmacokinetics represents a major challenge in nuclear imaging and targeted radionuclide therapy as they most often result in low imaging contrast and high radiation dose to healthy tissue. To address this challenge, we developed a polymer-based targeting agent that can be used for pretargeted imaging and thus separates tumor accumulation from the imaging step in time. The developed targeting agent is based on polypeptide-graft-polypeptoid polymers (PeptoBrushes) functionalized with trans-cyclooctene (TCO). The complementary 111In-labeled imaging agent is a 1,2,4,5-tetrazine derivative, which can react with aforementioned TCO-modified PeptoBrushes in a rapid bioorthogonal ligation. A high degree of TCO loading (up to 30%) was achieved, without altering the physicochemical properties of the polymeric nanoparticle. The highest degree of TCO loading resulted in significantly increased reaction rates (77-fold enhancement) compared to those with small molecule TCO moieties when using lipophilic tetrazines. Based on computer simulations, we hypothesize that this increase is a result of hydrophobic effects and significant rearrangements within the polymer framework, in which hydrophobic patches of TCO moieties are formed. These patches attract lipophilic tetrazines, leading to increased reaction rates in the bioorthogonal ligation. The most reactive system was evaluated as a targeting agent for pretargeted imaging in tumor-bearing mice. After the setup was optimized, sufficient tumor-to-background ratios were achieved as early as 2 h after administration of the tetrazine imaging agent, which further improved at 22 h, enabling clear visualization of CT-26 tumors. These findings show the potential of PeptoBrushes to be used as a pretargeting agent when an optimized dose of polymer is used.

Multifunctional Clickable Reagents for Rapid Bioorthogonal Astatination and Radio-Crosslinking.

In the past decade, several developments have expanded the chemical toolbox for astatination and the preparation of 211At-labeled radiopharmaceuticals. However, there is still a need for advanced methods for the synthesis of astatinated (bio)molecules to address challenges such as limited in vivo stability. Herein, we report the development of multifunctional 211At-labeled reagents that can be prepared by applying a modular and versatile click approach for rapid assembly. The introduction of tetrazines as bioorthogonal tags enables rapid radiolabeling and radio-crosslinking, which is demonstrated by steric shielding of 211At to significantly increase label stability in human blood plasma.

Evaluation of the inverse electron demand Diels-Alder reaction in rats using a scandium-44-labelled tetrazine for pretargeted PET imaging.

BACKGROUND: Pretargeted imaging allows the use of short-lived radionuclides when imaging the accumulation of slow clearing targeting agents such as antibodies. The biotin-(strept)avidin and the bispecific antibody-hapten interactions have been applied in clinical pretargeting studies; unfortunately, these systems led to immunogenic responses in patients. The inverse electron demand Diels-Alder (IEDDA) reaction between a radiolabelled tetrazine (Tz) and a trans-cyclooctene (TCO)-functionalized targeting vector is a promising alternative for clinical pretargeted imaging due to its fast reaction kinetics. This strategy was first applied in nuclear medicine using an 111In-labelled Tz to image TCO-functionalized antibodies in tumour-bearing mice. Since then, the IEDDA has been used extensively in pretargeted nuclear imaging and radiotherapy; however, these studies have only been performed in mice. Herein, we report the 44Sc labelling of a Tz and evaluate it in pretargeted imaging in Wistar rats. RESULTS: 44Sc was obtained from an in house 44Ti/44Sc generator. A 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-functionalized tetrazine was radiolabelled with 44Sc resulting in radiochemical yields of 85-95%, a radiochemical purity > 99% at an apparent molar activity of 1 GBq/mmol. The 44Sc-labelled Tz maintained stability in solution for up to 24 h. A TCO-functionalized bisphosphonate, which accumulates in skeletal tissue, was used as a targeting vector to evaluate the 44Sc-labelled Tz. Biodistribution data of the 44Sc-labelled Tz showed specific uptake (0.9 ± 0.3% ID/g) in the bones (humerus and femur) of rats pre-treated with the TCO-functionalized bisphosphonate. This uptake was not present in rats not receiving pre-treatment (< 0.03% ID/g). CONCLUSIONS: We have prepared a 44Sc-labelled Tz and used it in pretargeted PET imaging with rats treated with TCO-functionalized bisphosponates. This allowed for the evaluation of the IEDDA reaction in animals larger than a typical mouse. Non-target accumulation was low, and there was a 30-fold higher bone uptake in the pre-treated rats compared to the non-treated controls. Given its convenient half-life and the ability to perform positron emission tomography with a previously studied DOTA-functionalized Tz, scandium-44 (t1/2 = 3.97 h) proved to be a suitable radioisotope for this study.