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.

Les études animales ont été approuvées par le Comité d&#39;éthique de la recherche animale à l&#39;Université McMaster en conformité avec Conseil canadien de protection des animaux (CCPA) Lignes directrices. 1. Ligands de radiomarquage Tz-tridentate avec 99mTc ATTENTION: Les procédures suivantes nécessitent l&#39;utilisation de composés radioactifs. Le travail ne doit être effectué dans un laboratoire agréé par le respect des règles de sécurité et d&#39;élimination. Les réactions à micro-ondes doivent être effectuées dans un four micro-ondes conçu spécifiquement pour la synthèse chimique. <ol><li> Synthèse du [99mTc (CO) 3 (H 2 O) 3] + 19, 20 <ol><li> Dans un flacon à micro - ondes, mélanger 8 mg de K 2 [BH 3 CO 2], 15 mg de Na 2 CO 3, 20 mg de Na 2 B 4 O 7 · 10H 2 O et 25 mg KOCO [CH (OH)]2 COONa · 4H 2 O. Purge le flacon pendant 10 minutes avec du gaz argon. </li><li> Ajouter 4 ml de 99m TcO 4 - (~ 1100 MBq, ~ 30 mCi) dans du sérum physiologique à 0,9% au flacon. </li><li> Chauffer le mélange réactionnel dans un micro-ondes pendant 3,5 minutes à 110 ° C, après 10 s d&#39;agitation pour assurer un mélange intime des réactifs. </li><li> Ajuster le pH de la solution à 3,5-4 à l&#39;aide ~ 400 ul de HCl 1 M. Vérifiez en utilisant du papier pH. </li></ol></li><li> Radiomarquage des ligands 1-5 tétrazine <ol><li> Dissoudre 2 mg de chaque ligand (composés 1-5) dans 250 ul de MeOH 21. </li><li> Ajouter 250 ul de [99mTc (CO) 3 (H 2 O) 3] + (~ 74 MBq ~ 2 mCi) à chaque solution. </li><li> Chauffer le mélange réactionnel à l&#39;aide d&#39;un four micro-ondes pendant 20 min à 60 ° C. REMARQUE: Cette étape est identique pour tous les 5 tétrazines. </li><li> Pour les composés 2 à 5, évaporer le solvant et re-dissolve les produits obtenus dans 1 ml de 1: 1 v / v DCM: TFA. </li><li> Chauffer les produits réactionnels dissous (2-5) à 60 ° C dans un four micro - ondes pendant 6 min (2-4) ou 10 min (5). </li><li> Après refroidissement à température ambiante, évaporer le solvant en utilisant un évaporateur (36 ° C, de 8 mbar, 3 min, 6000 rpm) et on dissout le composé séché dans 1: 1 d&#39; ACN: H 2 O ou 1: 1 MeOH: H 2 O, avant la purification par HPLC. </li><li> Purifier les 99m Tc-composés marqués (1-5), y compris la séparation du produit marqué du ligand tétrazine non marqué, par HPLC (C 18 en phase inverse). Typiquement, en utilisant un gradient d&#39;élution de 30:70 d&#39; ACN: H 2 O (tous deux avec 0,1% de TFA) à 40:60 d&#39; ACN: H 2 O de plus de 20 min (18 min) et une colonne 4,6 x 100 mm C 18 analytique. Utilisez les deux UV (254 nm) et la détection gamma. <ol><li> Prenez un petit échantillon de chaque produit étiqueté et de comparer son temps de rétention HPLC à celle d&#39;un co-injète, non radioactif, norme réétiquetée (0,125 mg dans 20% de méthanol-H 2 O). La norme de Re-marqué est identifié dans la trace HPLC UV, et éluer en même temps que le 99m Tc composé marqué dans la trace γ-HPLC. Cette co-injection présente des pics aux temps de rétention comparable, ce qui confirme l&#39;identité du composé 99m marqué par Tc. </li></ol></li><li> On évapore le solvant des fractions HPLC en utilisant un évaporateur (36 ° C, de 8 mbar, 3 min, 6000 rpm). </li><li> Formuler le composé purifié à une concentration de 7,4 kBq / pl dans du PBS contenant 0,5% de BSA et 0,01% de Tween-80. </li><li> Pour garantir que les composés marqués sont stables, effectuer une étude in vitro de la stabilité. Incuber le composé formulé à 37 ° C pendant 1, 4 et 6 h, en injectant une petite quantité (3,7 MBq) du mélange sur la HPLC à chaque point de temps pour évaluer la stabilité. </li></ol></li></ol> 2. Les études Bio-distribution de pré-ciblés <ol><li&gt; Préparation des animaux <ol><li> Utilisation de vieux 7-9 semaine souris Balb / c, femelles (n = 3), administrer TCO-BP formulé dans une solution saline (20 mg / kg) (5 ug / ul), par injection de queue veineuse. </li><li> Placez la souris dans le dispositif de contrainte physique, et d&#39;identifier les veines situées sur les surfaces latérales de la queue et essuyer avec un tampon imbibé d&#39;alcool. À environ 2 cm de l&#39;extrémité de la queue, à insérer une aiguille de calibre 30 à un angle faible, parallèlement à la veine. Appuyer lentement sur le piston pour injecter, retirer l&#39;aiguille et appliquer éponge propre et de gaze sur le site d&#39;injection avec une légère pression jusqu&#39;à ce que le saignement cesse. </li><li> À 1 h après l&#39; injection de TCO-BP, administrer ~ 0,74 MBq (20 uCi) de 99mTc-tétrazine formulés dans 100 ul de 0,5% de BSA, 0,01% de Tween-80 dans du PBS, par injection de la veine de queue. </li></ol></li><li> Études Bio de distribution <ol><li> Au point de temps désiré (t = 6 h), anesthésier les souris en utilisant 3% d&#39;isoflurane et le mélange de gaz d&#39;oxygène de 2%. Démontrer àe pincée retrait sur la souris anesthésiée pour assurer qu&#39;ils sont sous plan chirurgical de l&#39;anesthésie. </li><li> Recueillir du sang (1 ml) par ponction cardiaque en utilisant une seringue pré-traitée avec de l&#39;héparine. Placez la souris sur le dos avec le nez dans le cône de nez pour l&#39;anesthésie continue et de localiser le processus xiphoïde sur l&#39;animal. <ol><li> Insérez une aiguille G 25, légèrement à gauche de la ligne médiane de l&#39;animal dans le cadre du processus xiphoïde, à un angle de 20 °. Insérez complètement l&#39;aiguille et tirez lentement sur le piston pour voir le sang dans le moyeu de l&#39;aiguille si le cœur a été perforé. Légèrement réajuster l&#39;aiguille tout en maintenant le piston si nécessaire, pour percer le cœur. Aspirez lentement le sang dans la seringue. </li></ol></li><li> Euthanasier l&#39;animal par dislocation cervicale, alors que sous anesthésie. </li><li> Placez chaque animal dans un sac en plastique et utiliser un calibrateur de dose (99m Tc de réglage) pour mesurer l&#39;ensemble du niveau d&#39;activité du corps. </li><li> Recueillir les tissus et les fluides suivants en pré-peséeed compter tubes: le sang, les os (genou et de l&#39;épaule), la vésicule biliaire, les reins, le foie, l&#39;estomac (avec le contenu), l&#39;intestin grêle (avec contenu), gros intestin et le caecum (avec contenu), de la thyroïde et de la trachée, de la vessie avec de l&#39;urine , et la queue. </li><li> Rincer les tissus appropriés (à l&#39;exclusion du sang, de la vésicule biliaire et de la vessie) dans PBS pour éliminer le sang et le sécher avant de placer les tissus dans des tubes de comptage appropriés. </li><li> Placez la carcasse de l&#39;animal dans un sac en plastique et mesurer l&#39;activité du corps entier résiduel en utilisant un calibrateur de dose. </li><li> Peser chaque tube contenant un échantillon de tissu. Soustraire poids initial du tube pour obtenir la masse du tissu. </li><li> Utiliser un calibreur de dose (99m de réglage Tc) pour mesurer la quantité d&#39;activité dans un échantillon d&#39;essai (100 ul) au moment de l&#39; injection pour chaque souris. REMARQUE: Cet échantillon d&#39;essai est égal au volume d&#39;injection, ce qui donne le compte de l&#39;activité au moment de l&#39;injection. </li><li> Au moment de la mesure de tissu, unliquot 5 ul de l&#39;échantillon d&#39;essai utilisé précédemment. Utilisez un compteur gamma multi-détecteur (99m de réglage Tc) et compter pour obtenir le nombre par minute (CPM) pour l&#39;échantillon d&#39;essai de 5 pi. </li><li> Utilisez les deux valeurs obtenues en 2.2.9 et 2.2.10 pour calculer l&#39;activité et la relation CPM en utilisant l&#39; équation 1 pour obtenir un facteur de conversion (CPM uCi -1). (1) <img alt="L&#39;équation 1" src="/files/ftp_upload/55188/55188eq1.jpg" /></li><li> Utiliser le compteur de rayons gamma pour mesurer la quantité de radioactivité dans chaque échantillon de tissu ou de fluide. </li><li> Utiliser l&#39;équation 1 pour calculer la quantité d&#39;activité dans chaque tissu ou de liquide au moment de la mesure par rapport à la dose totale injectée. Cette valeur est ensuite normalisée en fonction du poids des organes et rapportée comme dose injectée par gramme pour cent ( par exemple,% ID / g) de tissu. </li><li> Suivez les étapes 2.1.2 à 2.2.13 pour mener une expérience de contrôle négatif en utilisant les 99m Tc marqué ligands tétrazine dans le Absence du TCO-BP. Sacrifice des souris (n = 3) après l&#39;injection de 0,5, 1, 4 et 6 h, et d&#39;obtenir un tissu ou d&#39;un fluide tel que décrit ci-dessus. </li></ol></li></ol>

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The authors declare they have no competing financial interests.

Une collection de tridentes chélates tétrazine liés de différentes polarités a été préparé, et l&#39;utilité de leurs complexes 99m Tc dans la réaction IEDDA avec un dérivé TCO in vivo a été évaluée. Un procédé de marquage 99mTc efficace et reproductible a été développée pendant cinq tétrazine chelates, où la concentration de ligand était de 10 -3 M. L&#39;étape de marquage est suivie par une déprotection des groupes butyle T- (pour les compo...

99m Tc reste le radio - isotope dominant utilisé en médecine nucléaire diagnostique, avec plus de 50 millions de procédures d&#39;imagerie effectuées par an dans le monde entier 1, 2, 3. La majorité des agents 99m Tc utilisés cliniquement sont radiopharmaceutiques de type perfusion. Il y a un nombre limité de composés activement ciblés dans lesquels 99m Tc est dirigé pour lier un biomarqueur spécifique à travers la ligature à une construction de ciblage. La création de 99m ciblée radiopharmaceutiques Tc est souvent entravée par l&#39;influence de 99m complexes Tc-ligand sur la capacité de la molécule de ciblage pour lier le biomarqueur d&#39;intérêt, ou les isotopes demi-vie est pas assez long pour une utilisation avec des biomolécules de poids moléculaire élevé telles que des anticorps. Ce dernier nécessite généralement plusieurs jours avant que les images sont acquises pour que la biomolécule pour effacer de la non-cible tiss<html> ues. Pré-ciblage propose une approche alternative pour surmonter ces défis. Pré-ciblage combinée avec la chimie bioorthogonal a été montré pour être un moyen efficace de développer de nouvelles sondes d&#39;imagerie moléculaire à la fois pour la fluorescence et la radio-imagerie 4, 5, 6, 7, 8. La réaction de la demande d&#39;électrons inverse de Diels-Alder (IEDDA) entre 1,2,4,5-tétrazine (Tz) et -cyclooctene trans (TCO) , ses dérivés, comme le montre la figure 1, a été montré comme étant particulièrement efficace 6. La réaction IEDDA avec ces composants peut présenter une cinétique rapide dans du PBS (k 2 ≈ 6.000 M -1 s -1) et une sélectivité élevée, le rendant idéal pour des applications in vivo pré-ciblage 9, 10. e_content &quot;&gt; L&#39;approche la plus couramment utilisée consiste à administrer un vecteur de ciblage TCO dérivés et à la suite d&#39; une période de retard suffisant, un tétrazine radiomarqué est administré. tétrazines radiomarqués sur la base de 11 C, 18 F, 64 Cu, 89 Zr, et 111 en ont été rapporté 11, 12, 13, 14, 15. en revanche, il n&#39;y a qu&#39;un seul rapport d&#39;une 99mTc marqué Tz, qui a été préparé en utilisant un ligand de type HYNIC nécessitant l&#39;utilisation de co-ligands pour éviter la liaison aux protéines et à la dégradation in vivo , 16. a titre d&#39;alternative, nous rapportons ici la synthèse de 99mTc (I) marqué tétrazines en utilisant une famille de ligands qui forment des complexes stables avec un tridentate [99mTc (CO) 3] + noyau. Figure 1: La réaction IEDDA bioorthogonal entre tétrazine et -cyclooctene trans. <a href="http://ecsource.jove.com/files/ftp_upload/55188/55188fig1large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a> La famille de ligands préparés contiennent des chélates tridentés qui varient en polarité et de la nature du groupe de liaison entre la région de liaison métallique et la Tz (figure 2). L&#39;objectif était d&#39;identifier un 99m Tc-tétrazine construire qui pourrait efficacement localiser et de réagir avec des sites TCO marqués in vivo et rapidement clair lorsqu&#39;ils ne sont pas liés, afin de produire une valeur cible élevée à non-cible ratios. Pour tester les ligands, un TCO-dérivé d&#39;un bisphosphonate (TCO-BP) a été utilisé 17. Nous avons montré précédemment que le TCO-BP se localise dans les zones du métabolisme osseux actif et peut réagir avectétrazines radiomarqués in vivo 18. Il est un réactif commode pour tester de nouveaux tétrazines, car il peut être préparé en une seule étape et les expériences peuvent être effectuées chez des souris normales lorsque la localisation a lieu principalement au niveau des articulations (genoux et des épaules).

<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.

Les ligands ont été synthétisés en utilisant différents agents de liaison et des chélateurs via une stratégie simple d&#39;amination réductrice (figure 2), suivie par le couplage du produit à un tétrazine disponible dans le commerce 22, 23. Le radiomarquage a été réalisé en utilisant la même méthode que pour tous les composés et a été hautement reproductible. Le processus a été optimisé en f...

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.

Préparation et évaluation des<sup&gt; 99m</sup&gt; Tc marquée tridenté chélates de pré-ciblage utilisant 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

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

8.1K vues.  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. 

Vidéo: 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.

Microfluidique Capture-cyclo-réaction sur puce pour immobiliser de façon réversible de petites molécules ou structures multi-composants pour applications biocapteurs

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

Recherche

Chimie

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.

La bioconjugaison et radiosynthèse de<sup&gt; 89</sup&gt; Anticorps Zr-DFO-étiquetés

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.

Un protocole optimisé pour le radiomarquage efficace de nanoparticules d&#39;or à l&#39;aide d&#39;un<sup&gt; 125</sup&gt; I-étiqueté Azide prothétique Groupe

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.

Exploiter la demande Bioorthogonal inverse Electron Diels-Alder Cycloaddition pour préalablement visées PET Imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for ...

Bio-ingénierie

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.

M. imagerie moléculaire du cancer de la prostate avec un agent de contraste ciblé Petit moléculaire CLT1 Peptide

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

Médecine

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.

Pretargeted radio-immunothérapie basée sur la réaction de Diels-Alder demande Inverse d’électrons

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.

Synthèse et bioconjugaison de Thiol-réactifs pour la création de Site-sélectivement modifiés immunoconjugués

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.

Imagerie par tomographie par émission de positons du trafic cellulaire : une méthode de radiomarquage cellulaire

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for ...

Recherche en cancérologie

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.

Évaluation initiale des anticorps-conjugués modifié avec Peptides viraux dérivés pour accroître l’Accumulation cellulaire et améliorer le ciblage tumoral

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).

Murine lymphocytaire par étiquetage<sup&gt; 64</sup&gt; Cu-anticorps du récepteur de ciblage pour<em&gt; In Vivo</em&gt; Le trafic cellulaire par PET / CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Préparation et évaluation des

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