Name: harnessing the bioorthogonal inverse electron demand diels-alder cycloaddition for pretargeted pet imaging
Uploaded: 2015-02-03T15:30:00
Description: Watch this Scientific Journal Video about Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging at JoVE.com

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

Etiske retningslinjer: Alle de in vivo dyreforsøg beskrevne blev udført efter en godkendt protokol og under de etiske retningslinjer for Memorial Sloan Kettering Cancer Center Institutional Animal Care og brug Udvalg (IACUC). 1. Syntese af Tz-Bn-NOTA <ol><li> I en lille reaktionsbeholder opløses 7 mg NH2 -Bn-NOTA (1,25 x 10 -2 mmol) i 600 pi NaHCO3-puffer (0,1 M, pH 8,1). Kontroller pH af opløsningen. Hvis det er nødvendigt, a...

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Den primære fordel ved denne pretargeted PET imaging strategi er, at det er i stand til at afgrænse tumorer med mål-til-baggrund billedets kontrast på kun en brøkdel af den baggrundsstråling dosis produceret af direkte mærkede antistoffer. For eksempel i kolorektal cancer imaging system her beskrevne data fra akutte biodistributionsmodificerende eksperimenter blev anvendt til at udføre dosimetri beregninger for 64 Cu-baserede pretargeting strategi sammen med direkte-mærkede 64 Cu-NOTA-huA3...

I løbet af de sidste tredive år har positronemissionstomografi (PET) blevet et uundværligt klinisk redskab i diagnosticering og behandling af kræft. Antistoffer har længe været anset for lovende vektorer til levering af positron-emitterende radioisotoper til tumorer på grund af deres udsøgte affinitet og specificitet for kræft biomarkører. 1,2 dog den relativt langsomme in vivo farmakokinetik af antistoffer bemyndiger anvendelse af radioaktive isotoper med flere dages fysiske halveringstider. Denne kombination kan give høj strålingsdoser til de ikke-målorganer patientorganisationer, en vigtig komplikation, der er af særlig klinisk betydning, da radioimmunoconjugates injiceres intravenøst, og derfor - i modsætning til delvis krop CT-scanninger - resultat i absorberede doser i alle dele af kroppen, uafhængigt af interrogerede væv. For at omgå dette problem, er betydelig indsats været dedikeret til udvikpment af PET-billeddannelse strategier, afkoble radioaktive isotop og målsøgende del, og derved udnytte de fordelagtige egenskaber af antistoffer samtidig fodpaneler deres iboende farmakokinetiske begrænsninger. Disse strategier - oftest betegnet pretargeting eller flertrins målretning - benytter typisk fire trin: (1) indgivelse af et antistof er i stand til at binde både et antigen og en radioligand; (2) akkumulering af antistoffet i målvævet og dets clearance fra blodet; (3) at behandling af et lille molekyle radioligand; og (4) in vivo ligering af radioligand til antistoffet efterfulgt af hurtig clearance af overskydende radioligand. 3-8 I nogle tilfælde er en yderligere klaringsmiddel injiceres mellem trin 2 og 3 for at fremskynde udskillelsen af hvilket som helst antistof , som endnu ikke binde tumoren og forbliver i blodet. 5 I store træk two typer pretargeting strategier er mest fremherskende i litteraturen. Mens begge har vist sig gode i prækliniske modeller, de har også vigtige begrænsninger, som har hindret deres kliniske anvendelighed. Den første strategi bygger på den høje affinitet mellem streptavidin-konjugerede antistoffer og biotinmodificerede radiomærker; har imidlertid immunogenicitet streptavidin-modificerede antistoffer vist sig at være et bekymrende problem med hensyn til translation. 5,6,9,10 Den anden strategi, i modsætning, beskæftiger bispecifikke antistoffer, der er blevet genetisk manipuleret til at binde både en cancer biomarkør antigen og et lille molekyle radiomærket hapten. 3,11-14 Mens sidstnævnte rute er bestemt kreativ, er dets brede anvendelighed begrænset af kompleksitet, omkostninger og manglende modulopbygning af systemet. For nylig har vi udviklet og udgivet en pretargeted PET imaging metode baseret på den inverse elektron efterspørgsel Diels-Alder (IEDDA) cycloadditionsreaktion mellem trans -cyclooctene (TCO) og tetrazin (Tz;. Figur 1) 11, medens selve reaktionen har været kendt i årtier, har IEDDA kemi oplevet en renæssance i de senere år som et klik kemi bioconjugation teknik, som illustreret ved den fascinerende gruppernes arbejde af Ralph Weissleder, Joseph Fox, og Peter Conti blandt andre. 12-15 Den IEDDA cycloaddition er blevet anvendt i en lang række indstillinger, herunder fluorescens billeddannelse med peptider, antistoffer og nanopartikler samt nuklear billeddannelse . med både radiohalogens og radiometaller 16-26 Ligeringen er højtydende, ren, hurtig (k 1&gt; 30.000 M-1 sek -1), selektiv, og - kritisk -. bioorthogonal 27 Og mens en række typer af klik kemi - herunder Cu-katalyseret azid-alkyn cycloadditioner, strain-forfremmet azid-alkyn cycloadditioner og Staudinger Ligtioner -. er bioorthogonal så godt, det er den unikke kombination af hurtige reaktionskinetik og bioorthogonality der gør IEDDA kemi så velegnet til pretargeting applikationer i hele organismer 28,29 Langs disse linjer, er det vigtigt at bemærke, at den seneste rapport fra vores laboratorier var ikke den første til at anvende IEDDA kemi pretargeting: den første rapport fra pretargeted billeddannelse med IEDDA opstået under arbejdet i Rossin, et al og fremhævede en SPECT metode anvender en 111 In-mærket tetrazin 30.. Som vi diskuteret ovenfor pretargeting metode har fire forholdsvis enkle trin (figur 2). I protokollen ved hånden, vil blive beskrevet en pretargeted strategi for PET-billeddannelse af kolorektal cancer, der anvender en 64 Cu-NOTA-mærket tetrazin radioligand og en TCO-modificeret konjugat af huA33 antistof. Men i sidste ende modulopbygning af denne metode er en af ​​sin GRspiser aktiver, som trans -cyclooctene del kan tilføjes til enhver ikke-internalisere antistof, og tetrazin kan knyttes til en bred vifte af radioaktive journalister.

<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.

De første tre trin af forsøget - syntesen af Tz-Bn-NOTA, konjugeringen af TCO til huA33 og radiomærkning af Tz-Bn-NOTA konstruktion (figur 3 og 4) - er meget pålidelige. I tilfælde af den ovennævnte procedure blev TZ-Bn-NOTA konstruktion syntetiseret i højt udbytte og renhed. Den huA33 antistof blev modificeret med 4,2 ± 0,6 TCO / mAb og Tz-Bn-NOTA blev radiomærket med 64 Cu til opnåelse af oprensede radioligand i&gt; 99% radiokemisk renhed,&gt; 85% henfald korriger...

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

Udnyttelse af 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.

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

Research

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Bioengineering

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