The authors thank Prof. Ralph Weissleder, Dr. Pat Zanzonico, and Dr. NagaVaraKishore Pillarsetty for helpful conversations and the NIH for funding (BMZ: 1K99CA178205-01A1)

ETHIK STATEMENT: Alle beschriebenen In-vivo-Tierversuchen wurden nach einer genehmigten Protokoll und unter den ethischen Richtlinien des Memorial Sloan Kettering Cancer Center Institutional Animal Care und Verwenden Committee (IACUC) durchgeführt. 1. Synthese von Tz-Bn-NOTA <ol><li> In einem kleinen Reaktionsgefß löst man 7 mg NH 2 -Bn-NOTA (1,25 x 10 -2 mmol) in 600 &amp; mgr; l NaHCO 3 Puffer (0,1 M, pH 8,1). Überprüfen Sie den pH-Wert der Lösung. Falls erforderlich, den pH-Wert der Lösung einzustellen, um 8.1 mit kleinen Aliquots von 0,1 M Na 2 CO 3. </li><li> Füge das NH 2 -Bn-NOTA Lösung auf 0,5 mg Tz-NHS (1,25 x 10 -3 mmol) in einem 1,7 ml Mikrozentrifugenröhrchen. HINWEIS: Die Tz-NHS kann entweder trocken gewogen oder aus einer Stammlösung trockenem DMF oder DMSO (&lt;50 ul) hinzugefügt werden. </li><li> Ermöglichen die erhaltene Reaktionslösung für 30 min bei RT reagierenunter leichtem Rühren. </li><li> Nach 30 min reinigen Sie das Gerät mit Umkehrphasen-C18-HPLC-Chromatographie, um nicht umgesetztes NH 2 Bn-NOTA entfernen. Die NH 2 -Bn-NOTA kann bei einer Wellenlänge von 254 nm überwacht werden, während die Tz-NHS und Tz-Bn-NOTA werden am besten bei einer Wellenlänge von 525 nm überwacht. HINWEIS: Die Retentionszeiten sind natürlich stark abhängig von der HPLC-Geräte-Setup jedes Labor (Pumpen, Säulen, Schläuche, etc.). Jedoch, um ein Beispiel zu präsentieren, wenn ein Gradient von 0: 100 MeCN / H 2 O (beide mit 0,1% TFA) bis 100: 0 MeCN / H 2 O über 25 min und einer Analysen 4,6 x 250 mm C18-Säule verwendet wird die Retentionszeiten von Tz-Bn-NOTA, Tz-NHS und NH 2 -Bn-NOTA wird etwa 15 min, 16,5 min und 10 min, betragen. Das Produkt kann von den anderen Reaktionskomponenten entweder in einem einzigen Durchgang oder mehreren Durchläufen unter Verwendung einer semi-präparativen oder präparative C18-HPLC-Säule 1 H-NMR, anal, gereinigt werden.ytical HPLC und ESI-MS sind Methoden, die verwendet werden können, um die Reinheit des abgeschlossenen Tz-Bn-NOTA Vorläufer verifizieren. 11 </li><li> Frieren Sie die gesammelten HPLC Eluenten mit flüssigem Stickstoff. </li><li> Wickeln Sie das gefrorene Sammelröhrchen in undurchsichtigen Aluminiumfolie. </li><li> Das tiefgefrorene Sammelröhrchen in einer Gefriertrocknung Schiff O / N, um das HPLC mobilen Phase zu entfernen. </li><li> Lagern Sie das gereinigte Produkt (ein helles rosa Feststoff) im Dunkeln bei -80 ° C. Anmerkung: Dies ist eine akzeptable Anhaltepunkt in dem Verfahren. Die fertige Tz-Bn-NOTA Vorstufe für mindestens 1 Jahr unter diesen Bedingungen stabil. </li></ol> 2. Herstellung huA33-TCO Immunkonjugat <ol><li> In einem 1,7 ml Mikrozentrifugenröhrchen, bereiten eine 1 mg / ml (2,7 mM) Lösung von TCO-NHS in trockenem DMF. </li><li> In einem 1,7 ml-Mikrozentrifugenröhrchen, bereiten eine 2 mg / ml (13,3 &amp; mgr; M) Lösung von huA33 in 1 ml phosphatgepufferter Salzlösung, pH 7,4 (0,01 M PO 4 3-, </sub&gt; 0,154 M NaCl). </li><li> Mit Hilfe von kleinen Aliquoten (&lt;5 &amp; mgr; l) von 0,1 M Na 2 CO 3, pH-Wert der Antikörperlösung auf 8,8-9,0 einzustellen. Verwenden Sie entweder pH-Papier oder ein pH-Meter mit einer Mikroelektrode, um den pH-Wert zu überwachen und darauf achten, nicht, damit der pH-Wert auf pH-Wert über 9,0 steigen. </li><li> Nachdem die Antikörperlösung in der richtigen pH, einen Volumen der TCO-NHS-Lösung entsprechend 8 Moläquivalente des aktivierten Esters. Fügen Sie beispielsweise 7,9 ul der 1 mg / ml TCO-NHS-Lösung (1,07 x 10 -7 mol TCO-NHS) an die 1 ml 2 mg / ml huA33 Antikörperlösung (1,33 x 10 -8 mol huA33). 5% DMF nicht überschreiten Volumenkonzentration in der Lösung. </li><li> Vorsichtig mischen Sie die Lösung durch Invertierung der Mikrozentrifugenröhrchen mehrmals. </li><li> Wickeln Sie das Reaktionsgefäß in undurchsichtigen Aluminiumfolie. </li><li> Lassen Sie die Lösung für 1 h bei RT unter leichtem Rühren inkubiert. </li><li> Nach 1 Stunde bei Raumtemperatur, reinigen das resultierende Immunkonjugat mit einem abgepackten Einweg size Ausschluss Entsalzungssäule. Zuerst spülen Sie die Größenausschlusssäule, wie vom Hersteller beschrieben, welche die über die Spalte vorhanden Konservierungsmittel während der Lagerung zu entfernen. Fügen Sie dann das Reaktionsgemisch auf die Größenausschlusssäule, spülen Sie die Säule mit 1,5 ml 0,9% steriler Kochsalzlösung, und anschließend das Produkt mit 2 ml 0,9% steriler Kochsalzlösung als Laufmittel zu sammeln. HINWEIS: Dieser Schritt wird das fertige huA33-TCO als 2 ml Lösung zu erhalten. </li><li> Messung der Konzentration des resultierenden huA33-TCO auf einem UV-Vis-Spektrophotometers. </li><li> Wenn eine höhere Konzentration gewünscht wird, konzentrieren den huA33-TCO-Lösung unter Verwendung einer Zentrifugalfiltereinheit mit einer 50.000 Molekulargewicht-Cut-off. HINWEIS: Es ist wichtig zu beachten, dass, während huA33 und eine Vielzahl von anderen bekannten Antikörpern (zB Bevacizumab, Trastuzumab, Cetuximab und J591) sind sehr tolerant gegenüber, die konzentriert, Aggregation und Präzipitation kann nach Konzentration in anderen Fällen auftreten. Forscher versuchen diese procedure mit einem neuen Antikörper sollte die Literatur oder ihrer eigenen Kenntnisse des fraglichen Antikörpers mit Bezug darauf, ob oder nicht, um den Antikörper zu konzentrieren vertrauen. </li><li> Lagern Sie das ausgefüllte huA33-TCO Immunkonjugat bei 4 ° C im Dunkeln. Anmerkung: Dies ist eine akzeptable Anhaltepunkt in dem Verfahren. Die fertige mAb-TCO-Konjugat sollte für mindestens 3 Monate unter diesen Lagerbedingungen stabil sein. </li></ol> 3. 64 Cu Radiomarkierung von Tz-Bn-NOTA Hinweis: dieser Schritt des Protokolls umfaßt die Handhabung und Manipulation von Radioaktivität. Vor der Durchführung dieser Schritte - oder bei anderen Arbeiten mit Radioaktivität - Forscher sollten mit Strahlenschutzabteilung ihrer Heimateinrichtung konsultieren. Alle möglichen Maßnahmen, um die Exposition gegenüber ionisierender Strahlung zu minimieren. <ol><li> In einem 1,7 ml Mikrozentrifugenröhrchen, bereiten Sie eine 0,5 mg / ml (723 &amp; mgr; M) Lösung von Tz-Bn-NOTA. </li><li> In einem 1,7 ml MicroCentrifuge Rohr, mit 10 ul des Tz-Bn-NOTA Lösung (5 ug) und 400 ul 0,2 M NH 4 OAc, pH 5,5-Puffer. </li><li> Im Interesse einer ordnungsgemäßen radiochemische Hinweis Haltung, Messung und Aufzeichnung der Menge an Radioaktivität in der Probe unter Verwendung einer Dosiskalibrator vor und nach den folgenden Schritten in der folgenden Protokoll (3,4-3,8). Dies wird mit der genauen Bestimmung der radiochemischen Ausbeuten helfen. </li><li> In 2000 &amp; mgr; Ci (74 MBq) von 64 Cu auf die Tz-Bn-NOTA Lösung. HINWEIS: In der Regel wird [64 Cu] CuCl 2 in einem kleinen Volumen (&lt;30 ul) 0,1 N HCl zugeführt wird, und damit nur geringe Mengen (&lt;10 ul) dieser Stammlösung werden für die Radiomarkierungsreaktion benötigt. Wenn größere Mengen des [64 Cu] CuCl 2 Lager benötigt werden, ist der Radiomarkierungsreaktion tolerant Erhöhung der Gesamtreaktionsvolumen. Jedoch sollte der pH-Wert der Radiomarkierungsreaktion Lösung sorgfältig überwacht, um sicherzustellen,dass er nicht unter pH 4,0 fallen. </li><li> Die Lösung wird für 10 min bei RT unter leichtem Rühren inkubiert. </li><li> Nach 10 min Inkubation, reinigen Sie das Gerät mit Umkehrphasen-C18-HPLC-Chromatographie. Die Retentionszeiten sind natürlich stark abhängig von der HPLC-Geräte-Setup jedes Labor (Pumpen, Säulen, Schläuche, etc.). Jedoch, um ein Beispiel zu präsentieren, wenn ein Gradient von 5:95 MeCN / H 2 O (beide mit 0,1% TFA) bis 95: 5 MeCN / H 2 O über 15 min verwendet wird, die Rückhaltezeit von 64 Cu-TZ Bn-NOTA sollte etwa 9,8 min, während freie, nicht komplexierte Cu 64 wird mit der Lösungsmittelfront bei etwa 2-4 min zu eluieren. </li><li> Mit einem Rotationsverdampfer, entfernen Sie die HPLC Eluenten. </li><li> Abgeblasen, und der 64-Cu Tz-Bn-NOTA Produkt in 0,9% steriler Kochsalzlösung. HINWEIS: In Anbetracht der 12,7 hr physikalische Halbwertszeit von 64 Cu, ist dies kein akzeptables Anhaltepunkt in dem Verfahren. Führen Sie die Synthese von 64 Cu-Tz-Bn-NICHTEin unmittelbar vor der Injektion des Radioliganden, und folgt nach Schritt 3.7 Schritt 4.5. </li></ol> 4. In Vivo Pretargeted PET Imaging HINWEIS: Wie in Protocol Abschnitt 3, wird dieser Schritt des Protokolls umfaßt die Handhabung und Manipulation von Radioaktivität. Vor der Durchführung dieser Schritte Forscher sollten mit Strahlenschutzabteilung ihrer Heimateinrichtung konsultieren. Alle möglichen Maßnahmen, um die Exposition gegenüber ionisierender Strahlung zu minimieren. <ol><li> In einem weiblichen athymischen nackten Maus, subkutan Implantat 1 x 10 6 SW1222 Kolorektalkrebszellen und ermöglichen, dass diese in einem 100-150 mm 3 Xenograft (9-12 Tage nach der Beimpfung) wachsen. 11 </li><li> Ein Aliquot des huA33-TCO-Lösung von Protocol Abschnitt 2 auf eine Konzentration von 0,5 mg / ml in 0,9% steriler Kochsalzlösung. </li><li> Injizieren 200 ul der huA33-TCO-Lösung (100 &amp; mgr; g) in die Schwanzvene der Xenotransplantat-tragenden Mäusen. </li><li> Lassen Sie 24 Stunden für die Akkumulation des huA33-TCO im Tumor der Maus. </li><li> Verdünne die 64 Cu-Tz-Bn-NOTA Radioligand in einer Konzentration von 1,5 mCi / ml in 0,9% steriler Kochsalzlösung. </li><li> Injizieren 200 ul des 64 Cu-Tz-Bn-NOTA Radioligand-Lösung (300 &amp; mgr; Ci; 11,1 MBq; 1,6 nmol von 64 Cu-Tz-Bn-NOTA, unter der Annahme einer spezifischen Aktivität von 6,7 MBq / nmol) in die Schwanzvene der Xenotransplantat-tragenden Mäusen. </li><li> An der gewünschten Abbildungs ​​Zeitpunkt (zB 2, 6, 12, oder 24 Stunden nach der Injektion), betäuben die Maus mit einer 2% Isofluran: Sauerstoff-Gasgemisch. </li><li> Platzieren Sie die Maus auf dem Bett des Kleintier-PET-Scanner. Aufrechterhaltung einer Narkose bei der Untersuchung mit einem 1% Isofluran: Sauerstoff-Gasgemisch. Vor Inbetriebnahme des Tieres auf das Scanner-Bett, überprüfen Anästhesie mit dem Zehe-Pinch-Methode und wenden Tier Salbe auf den Augen der Maus, um das Trocknen während der Narkose zu verhindern. </li><li> Erwerben Sie die PET-Daten für die Maus über einen statischenScannen mit einem Minimum von 20 Millionen koinzident Ereignisse unter Verwendung eines Energiefensters von 350-700 keV und eine Koinzidenzzeitfenster von 6 nsec. Nach Abschluss der Akquisition des Bildes, darf die Maus nicht unbeaufsichtigt zu lassen, und stellen Sie sie nicht in einem Käfig mit anderen Mäusen, bis sie wieder zu sich kam. </li></ol>

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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=Evaluation+of+strained+alkynes+for+Cu-free+click+reaction+in+live+mice.">Evaluation of strained alkynes for Cu-free click reaction in live mice.</a> Nuclear Medicine and Biology. 40, 415-423 (2013).</li><li>Rossin, R., 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=In+vivo+chemisry+for+pretargeted+tumor+imaging+in+live+mice.">In vivo chemisry for pretargeted tumor imaging in live mice.</a> Angewandte Chemie International Edition. 49, 3375-3378 (2010).</li><li>Ackerman, M. E., 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=A33+antigen+displays+persistent+surface+expression.">A33 antigen displays persistent surface expression.</a> Cancer Immunology and Immunotherapy. 57, 1017-1027 (2008).</li><li>Carrasquillo, J. A., 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=124I-huA33+antibody+PET+of+colorectal+cancer.">124I-huA33 antibody PET of colorectal cancer.</a> Journal of Nuclear Medicine. 52, 1173-1180 (2011).</li><li>Sakamoto, 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=A+phase+I+radioimmunolocalization+trial+of+humanized+monoclonal+antibody+huA33+in+patients+with+gastric+carcinoma.">A phase I radioimmunolocalization trial of humanized monoclonal antibody huA33 in patients with gastric carcinoma.</a> Cancer Science. 97, 1248-1254 (2006).</li><li>Rossin, R., Lappchen, R., 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, 1989-1995 (2013).</li><li>Rossin, R., 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=Highly+reactive+trans-cyclooctene+tags+with+improved+stability+for+Diels-Alder+chemistry+in+living+systems.">Highly reactive trans-cyclooctene tags with improved stability for Diels-Alder chemistry in living systems.</a> Bioconjugate Chemistry. 34, 1210-1217 (2014).</li><li>Emmetiere, 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=18F-labeled-bioorthogonal+liposomes+for+in+vivo+targeting.">18F-labeled-bioorthogonal liposomes for in vivo targeting.</a> Bioconjugate Chemistry. 24, 1784-1789 (2013).</li></ol>

The authors have nothing to disclose.

Der Hauptvorteil dieser pretargeted PET Strategie ist, dass es in der Lage ist die Abgrenzung von Tumoren mit Ziel-zu-Hintergrund-Bildkontrast bei nur einem Bruchteil der direkt markierten Antikörpern produziert Hintergrund Strahlungsdosis. Zum Beispiel wird in dem hier beschriebenen Darmkrebs Abbildungssystem, Daten von akuten Bioverteilung Experimente wurden verwendet, um die Dosimetrie Berechnungen für die 64 Cu-Basis Pretargeting Strategie zusammen mit direkt markiertem 64 Cu-NOTA-huA33 und <...

Im Laufe der letzten dreißig Jahre, Positronenemissionstomographie (PET) ist ein unverzichtbares klinisches Werkzeug bei der Diagnose und Behandlung von Krebs. Antikörper werden seit langem als vielversprechende Vektoren für die Abgabe von Positronen emittierende Radioisotope zu Tumoren durch ihre vorzügliche Affinität und Spezifität für-Biomarkern. 1,2 jedoch die relativ langsame in vivo pharmakokinetischen Eigenschaften von Antikörpern sieht die Verwendung von Radioisotopen mit mehrtägiges physikalische Halbwertszeiten. Im Gegensatz zu Teilkörper-CT-Scans - - in Energiedosen in jedem Teil des Körpers Ergebnis Diese Kombination kann hohe Strahlendosen auf die Nichtzielorgane von Patienten, eine wichtige Komplikation, die von besonderer klinischer Bedeutung ist, da Radioimmunkonjugate intravenös injiziert und damit ergeben unabhängig von den abgefragten Geweben. Um dieses Problem zu umgehen, hat erhebliche Anstrengungen unternommen worden, um die develo gewidmetpment der PET-Bildgebung Strategien, die die Radioisotops und der Targeting-Einheit zu entkoppeln, wodurch die Nutzung der vorteilhaften Eigenschaften von Antikörpern und gleichzeitig Sockel ihrer intrinsischen pharmakokinetischen Einschränkungen. Diese Strategien - am häufigsten genannt Pretargeting oder mehrstufige Targeting - typischerweise vier Schritte: (1) die Verabreichung eines Antikörpers, der zur Bindung sowohl ein Antigen und ein Radioligand; (2) die Anreicherung des Antikörpers in das Zielgewebe und seine Clearance aus dem Blut; (3) die Verabreichung eines kleinen Moleküls Radioligand; und (4) die in vivo-Ligation des Radioliganden an den Antikörper, gefolgt durch die schnelle Clearance von überschüssigem Radioligand. 3-8 In einigen Fällen kann ein zusätzlicher Klärungsmittel zwischen den Schritten 2 und 3, um die Ausscheidung von jedem Antikörper beschleunigen spritzt Dazu ist es noch, den Tumor zu binden und im Blut bleibt. 5 Im Großen und Ganzen two Arten von Pretargeting Strategien sind besonders häufig in der Literatur. Während beide erfolgreich in präklinischen Modellen nachgewiesen, sie besitzen auch Schlüssel Einschränkungen, die ihre klinische Anwendbarkeit behindert haben. Die erste Strategie beruht auf der hohen Affinität zwischen Streptavidin-konjugierte Antikörper und Biotin-modifizierten Radiomarkierungen; jedoch die Immunogenität der Streptavidin-modifizierten Antikörper erwies sich eine beunruhigende Problem hinsichtlich der Übersetzung sein. 5,6,9,10 Die zweite Strategie, die im Gegensatz dazu verwendet bispezifischen Antikörpern, die genetisch manipuliert wurden, um sowohl eine Krebs binden Biomarker Antigen und ein kleines Molekül radiomarkierten Haptens. 3,11-14 Während dieser letztere Weg ist sicherlich kreativ, wird ihre breite Anwendbarkeit durch die Komplexität, die Kosten und die fehlende Modularität des Systems begrenzt. In letzter Zeit entwickelt und veröffentlicht eine pretargeted PET Methodik, die auf dem inversem Elektronenbedarf Diels-Alder (I wirEDDA) Cycloaddition zwischen trans -Cycloocten (TCO) und Tetrazin (Tz;. Abbildung 1) 11 Während der Reaktion selbst ist seit Jahrzehnten bekannt, hat IEDDA Chemie eine Renaissance in den letzten Jahren als Klick-Chemie Biokonjugation Technik erlebt, wie dargestellt die faszinierende Arbeit der Gruppen von Ralph Weissleder, Joseph Fox und Peter Conti unter anderem. 12-15 Die IEDDA Cycloaddition wurde in einer Vielzahl von Einstellungen angewendet wurden, einschließlich Fluoreszenz-Imaging mit Peptiden, Antikörpern, und Nanopartikel sowie die nuklearmedizinische Bildgebung . mit beiden Radiohalogene und Radiometalle 16-26 Die Ligation ist ertragreiche, saubere, schnelle (k 1&gt; 30000 M -1 s -1), selektiver und - entscheidend -. bioorthogonale 27 Und während eine Reihe von Arten von Klick-Chemie - einschließlich Cu-katalysierte Azid-Alkin-Cycloaddition, spannungskatalysierten Azid-Alkin-Cycloaddition und Staudinger ligtionen -. sind bioorthogonale als gut, es ist die einzigartige Kombination von schnellen Reaktionskinetik und Bioorthogonalität die IEDDA Chemie macht so gut geeignet, um Pretargeting Anwendungen in ganzen Organismen 28,29 In diesem Sinne ist es wichtig zu beachten, dass dem jüngsten Bericht von unserem Labors war nicht der erste, IEDDA Chemie Pretargeting gelten: der erste Bericht über pretargeted Bildgebung mit IEDDA entstand aus der Arbeit von Rossin, et al und enthielt eine SPECT Methodik unter Verwendung einer 111 In-markierten Tetrazin 30.. Wie wir oben diskutiert, hat der Pretargeting Methodik vier ziemlich einfachen Schritten (Abbildung 2). In dem Protokoll zur Hand, wird ein pretargeted Strategie für die PET-Bildgebung von Darmkrebs, der eine 64 Cu-NOTA-markierten Tetrazin Radioliganden und einem TCO-modifizierte Konjugat des Antikörpers huA33 beschäftigt beschrieben werden. Ist einer seiner gr jedoch letztendlich die Modularität dieser Methodikißt Vermögen als trans -Cycloocten Rest kann ein beliebiges nicht-internalisierenden Antikörper angehängt werden, und das Tetrazin kann auf eine Vielzahl von radioaktiven Reporter befestigt werden.

<table border="0" cellpadding="0" cellspacing="0" width="565">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrazine NHS Ester</td>
			<td>Sigma-Aldrich</td>
			<td>764701</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trans-cyclooctene NHS Ester</td>
			<td>Sigma-Aldrich</td>
			<td>764523</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p-NH2-Bn-NOTA</td>
			<td>Macrocyclics</td>
			<td>B-601</td>
			<td>Store at -80 &#176;C</td>
		</tr>
	</tbody>
</table>

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.

Die ersten drei Schritte des Experiments - die Synthese von Tz-Bn-NOTA, die Konjugation von TCO zu huA33 und der radioaktiven Markierung des Tz-Bn-NOTA konstruieren (Abbildungen 3 und 4) - sind sehr zuverlässig. Im Fall der oben beschriebenen Verfahren wurde die Tz-Bn-NOTA Konstrukt in hoher Ausbeute und Reinheit hergestellt. Die huA33 Antikörper wurde mit 4,2 ± 0,6 TCO / mAb modifiziert und Tz-Bn-NOTA wurde radioaktiv markiert und mit 64 Cu, um das gereinigte Radioligand ...

Watch this Scientific Journal Video about Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging at JoVE.com

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

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.

Die Nutzung der Bioorthogonale inversem Elektronenbedarf Diels-Alder-Cycloaddition für 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 ...

harnessing-bioorthogonal-inverse-electron-demand-diels-alder

Research

JoVE Journal

Bioengineering

8.7K Aufrufe.  Memorial Sloan Kettering Cancer Center. 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. 

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

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

Mikrofluidik-basierten electrotaxis für On-Demand-Quantitative Analyse von<em&gt; Caenorhabditis elegans</em&gt; &#39;Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Forschung

Biotechnik

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Erstellen Adhesive und löslichen Farbverläufe for Imaging Cell Migration mit Fluoreszenz-Mikroskopie

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Fluoreszenz-Abschrecken eines Liposomenverkapseltes NIR-Fluorophore als Instrument zur<em&gt; In-vivo-</em&gt; Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

Rapid-Scan-Elektronenspinresonanz eröffnet neue Wege für Imaging Physiologisch Wichtige Parameter<em&gt; In Vivo</em

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Korrelative Licht Elektronenmikroskopie (CLEM) für die Verfolgung und Imaging virales Protein verbundenen Strukturen in den Cryo-immobilisierten Zellen

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1-13C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3&#176;-5&#176;). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

Messung der Spin-Lattice Relaxation Magnetfeldabhängigkeit von hyperpolarisiertem [1-13C]pyruvat

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

Blutfluss-Bildgebung mit ultraschnellem Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different species. The octopus is one of these exceptional animals that has given scientists new insights into the fields of neuroscience, robotics, regenerative medicine, and prosthetics. Research with this species of cephalopods requires the set-up of complex facilities and intensive care for both the octopus and its ecosystem that is critical for the project&#39;s success. This system requires multiple mechanical and biological filtering systems to provide a safe and clean environment for the animal. Along with the control system, specialized routine maintenance and cleaning are required to effectively keep the facility operating long term. It is advised to provide an enriched environment to these intelligent animals by changing the tank&#39;s landscape, incorporating a variety of prey, and introducing challenging tasks for them to work through. Our results include MRI and a whole-body autofluorescence imaging as well as behavioral studies to better understand their nervous system. Octopuses possess unique physiology that can impact many areas of biomedical research. Providing them with a sustainable ecosystem is the first crucial step in uncovering their distinct capabilities.

Understanding the unique physiological and anatomical structures of octopuses can greatly impact biomedical research. This guide demonstrates how to set-up and maintain a marine environment to accommodate this species and includes state-of-the-art imaging and analytical approaches to visualize octopus' nervous system anatomy and function.

Etablierung eines Octopus-Ökosystems für die biomedizinische und biotechnologische Forschung

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different ...

Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, an abundant supply of AAAs is important for cancer cells to maintain their rapid growth and division. With this, there is a rising demand for a highly specific, noninvasive imaging approach with minimal sample preparation to directly visualize how cells harness AAAs for their metabolism in situ. Here, we develop an optical imaging platform that combines deuterium oxide (D2O) probing with stimulated Raman scattering (DO-SRS) and integrates DO-SRS with two-photon excitation fluorescence (2PEF) into a single microscope to directly visualize the metabolic activities of HeLa cells under AAA regulation. Collectively, the DO-SRS platform provides high spatial resolution and specificity of newly synthesized proteins and lipids in single HeLa cell units. In addition, the 2PEF modality can detect autofluorescence signals of nicotinamide adenine dinucleotide (NADH) and Flavin in a label-free manner. The imaging system described here is compatible with both in vitro and in vivo models, which is flexible for various experiments. The general workflow of this protocol includes cell culture, culture media preparation, cell synchronization, cell fixation, and sample imaging with DO-SRS and 2PEF modalities.

We present a protocol to directly visualize metabolic activities in cells regulated by amino acids using deuterium-oxide (heavy water D2O) probed stimulated Raman scattering (DO-SRS) microscopy, which is integrated with two-photon excitation fluorescence microscopy (2PEF).

Bioorthogonale chemische Bildgebung des Zellstoffwechsels, der durch aromatische Aminosäuren reguliert wird

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, ...

Analyse der Herzkontraktion von Zebrafischen mit Virtual-Reality-Interaktion

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for gaining insights into structural and functional changes in response to injuries. However, obtaining high-resolution and high-speed 4-dimensional (4D, 3D spatial + 1D temporal) images of the zebrafish heart to assess cardiac architecture and contractility remains challenging. In this context, an in-house light-sheet microscope (LSM) and customized computational analysis are&#160;used to overcome these technical limitations. This strategy, involving LSM system construction, retrospective synchronization, single cell tracking, and user-directed analysis, enables one to investigate the micro-structure and contractile function across the entire heart at the single-cell resolution in the transgenic Tg(myl7:nucGFP) zebrafish larvae. Additionally, we are able to further incorporate microinjection of small molecule compounds to induce cardiac injury in a precise and controlled manner. Overall, this framework allows one to track physiological and pathophysiological changes, as well as the regional mechanics at the single-cell level during cardiac morphogenesis and regeneration.

Autor im Rampenlicht: Hochauflösende 4D-Lichtblatt-Bildgebung und Virtual Reality im Zebrafisch zur Einzelzellanalyse der Herzfunktion

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

4D-Lichtblatt-Bildgebung der Herzkontraktion von Zebrafischen

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for ...