This work was supported by grants from the National Research Foundation of Korea, funded by the government of the Republic of Korea, (Grant nos. 2012M2B2B1055245 and 2012M2A2A6011335) and by the RI-Biomics Center of Korea Atomic Energy Research Institute.

Attenzione: la forma ossidata di iodio radioattivo è molto volatile e deve essere maneggiato con adeguati schermi di piombo e fiale di piombo. Tutti i passaggi radiochimiche dovrebbero essere eseguite in una cappa carbone filtrata ben ventilata, e le procedure sperimentali devono essere controllati da dispositivi di rilevazione di radioattività. 1. Preparazione della Chimica e della cartuccia Reverse Phase per la sintesi del 125 Azide I-marcato <ol><li> Preparazione dei reagenti in soluzione <ol><li> Sciogliere 1 mg del precursore azide (2) in 150 ml di etanolo assoluto (Figura 1). NOTA: Una procedura di sintesi dettagliata per il precursore azide (2) è stato riportato nel precedente articolo 22. </li><li> Sciogliere 1 mg cloramina T in 20 ml di 1x tampone fosfato isotonico (pH = 7,4). </li><li> Sciogliere 2 mg di sodio metabisolfito in 20 ml di H 2 O. </li></ol></li><li> preparazione della cartuccia <ol><li> Lavare la cartuccia TC18 con 10 ml di etanolo assoluto seguito da 10 ml di H 2 O. Non asciugare la matrice della cartuccia con aria. </li></ol></li></ol> 2. radiosintesi del Azide Protesi Gruppo marcato 125 I <ol><li> Reazione Radioiodination del precursore <ol><li> Aggiungere la soluzione azide precursore (1 mg in 150 ml di etanolo assoluto) e acido acetico (10 ml) in una provetta da 1,5 ml microcentrifuga. </li><li> Aggiungere 150 MBq di [125 I] NaI in 0,1 M NaOH (50 ml) alla miscela di reazione. </li><li> Aggiungere una soluzione cloramina T (1 mg in 20 ml di 1x tampone fosfato salino) e chiudere la miscela di reazione provetta contenente. </li><li> Incubare la miscela di reazione a temperatura ambiente per 15 minuti fino a quando la reazione è completa radioiodination. </li><li> Aggiungere una soluzione di sodio metabisolfito (2 mg in 20 ml H 2 O) alla miscela di reazioneper placare la reazione radioiodination. </li><li> Prelevare 0,2 ml di prodotto grezzo e diluire con 100 ml di soluzione di (H 2 O / CH 3 CN, 1: 1) per l&#39;analisi HPLC. NOTA: Per tutti gli esperimenti HPLC, usa acido formico 0,1% contenente H 2 O (solvente A) e 0,1% di acido formico contenente acetonitrile (solvente B) come eluenti. </li><li> Analizzare il prodotto grezzo diluito utilizzando una fase inversa analitica radio-HPLC (C18 a fase inversa colonna; portata: 1 ml / min; eluente pendenza: 20% B solvente per 0-2 min, 20-80% di solvente B per 2-22 min, 80-100% B solvente per 22-23 min, e il 100% di solvente B per 23-28 min; tempo di ritenzione: 16,4 min) (Figura 2). </li></ol></li><li> La purificazione del prodotto grezzo con una HPLC preparativa NOTA: fornire abbastanza schermatura di piombo intorno parti HPLC quali l&#39;iniettore, colonne, rivelatore, fiale di raccolta, il contenitore in cui l&#39;effluente viene raccolto. <ol><li> ritirare °una e intera miscela di reazione in una fiala HPLC. Lavare il tubo di reazione con acetonitrile (0,5 ml) e aggiungere il risciacquo nello stesso flaconcino. Diluire la soluzione raccolta con H 2 O (1 ml). </li><li> Iniettare il prodotto grezzo su una preparativa radio-HPLC (C18 a fase inversa colonna; la portata: 10 ml / min; gradiente eluente: 20% di solvente B per 0-2 min, 20-80% di solvente B per 2-22 min, 80-100% B solvente per 22-23 min, e il 100% B solvente per 23-28 min). </li><li> Raccogliere il picco radioattivi rappresenta il azide marcato 125 I (1) (t R in queste condizioni HPLC è 17,8-18,8 min) in una provetta di vetro (Figura 2). </li><li> Misurare la resa radiochimica della frazione con un calibratore di dosi di radioattività secondo il protocollo del produttore. </li><li> Iniettare il prodotto purificato su una analisi radio-HPLC utilizzando le stesse condizioni HPLC per determinare la purezza radiochimica del prodotto. </li></ol></li><li> Estrazione in fase solida del prodotto <ol><li> Diluire la frazione contenente il prodotto desiderato (1) con 40 ml puro H 2 O. </li><li> Aggiungere la soluzione diluita in una cartuccia TC18 condizionato. </li><li> Lavare la cartuccia con un ulteriore 15 ml H 2 O. </li><li> Eluire il prodotto (1) intrappolato nella cartuccia con 2 ml di acetone in una fiala di vetro da 10 ml che è protetto da uno scudo piombo. Misurare la radioattività del prodotto eluito utilizzando un calibratore di dosi di radioattività secondo il protocollo del produttore. NOTA: dimetilsolfossido (DMSO) o etanolo assoluto può essere utilizzato anche per eluizione del prodotto dalla cartuccia. Circa il 5-10% della radioattività attacca normalmente alla cartuccia, e il prodotto radiomarcato rimanente non può essere completamente eluito utilizzando quantità in eccesso di solvente organico. </li><li> Evaporare l&#39;acetone con un flusso di azoto o gas argon. </li><li> Sciogliere il residue con DMSO (100-200 ml) per il prossimo passo radiomarcatura. </li></ol></li></ol> 3. Sintesi di DBCO-gruppo-coniugato oro Nanoparticelle <ol><li> Modifica della superficie di oro 13-nm di dimensioni nanoparticelle con DBCO-gruppo contenente glicole polietilenico <ol><li> Nanoparticelle d&#39;oro Preparare sodio-citrato-stabilizzato (3) (dimensione media = 13 nm), secondo un rapporto precedente 24. </li><li> Aggiungere una soluzione acquosa di Tween 20 (1 mM, 1,5 ml) per le nanoparticelle di oro citrato-stabilizzato (10 nM, 15 ml). Agitare la soluzione per 20 minuti in un agitatore orbitale. </li><li> Aggiungere una soluzione acquosa di DBCO-contenente gruppi tiolo polietilenglicole (peso molecolare medio = 5000, 100 mM, 1,5 ml). Agitare la soluzione per 2 ore su un agitatore orbitale. </li></ol></li><li> Purificazione delle nanoparticelle di oro DBCO-gruppo modificati <ol><li> Purificare il nanoparticelle d&#39;oro DBCO-gruppo-modificato (4) &lt;/ Strong&gt; per centrifugazione successivi (11.400 xg, 15 min x 3). </li><li> Decantare il surnatante e aggiungere acqua pura per la risospensione del pellet delle nanoparticelle d&#39;oro. </li></ol></li></ol> 4. radiomarcatura di DBCO-gruppo-modificato oro nanoparticelle tramite la libera-Rame Clicca Reazione <ol><li> Sintesi di nanoparticelle d&#39;oro 125 I-etichettati utilizzando l&#39;azide marcato 125 I (1) <ol><li> Preparare una soluzione concentrata di nanoparticelle di oro DBCO-gruppo-modificato usando centrifugazione (11.400 xg, 15 min) e regolare la concentrazione delle nanoparticelle di oro a 2 mM. </li><li> Aggiungere 4,1 MBq del azide I-marcato 125 (1) in DMSO (5 ml) ad una sospensione di nanoparticelle di oro (4) (2 mM, 50 microlitri). </li><li> Incubare la miscela di reazione risultante a 40 ° C per 60 min. </li><li> Prelevare un&#39;aliquota (0,2 ml) dal prodotto grezzo e applicarlo su una silice-coated cromatografia su strato sottile (TLC) piatto. </li><li> Sviluppare la piastra TLC usando acetato di etile come fase mobile. </li><li> Posizionare la piastra TLC su uno scanner radio TLC ed eseguire lo scanner per monitorare la reazione radiomarcatura (figura 3) secondo il protocollo del produttore. </li></ol></li><li> La purificazione del prodotto grezzo <ol><li> Purificare la miscela di reazione contenente 125 nanoparticelle I marcato con oro (4) mediante centrifugazione (11.400 xg, 15 min). </li><li> Decantare il surnatante e aggiungere acqua pura per la risospensione del pellet delle nanoparticelle d&#39;oro. </li><li> Prelevare un&#39;aliquota (0,2 ml) dal prodotto purificato e applicarlo su una piastra TLC rivestiti di silice. </li><li> Sviluppare la piastra TLC usando acetato di etile come fase mobile. </li><li> Posizionare la piastra TLC su uno scanner radio TLC ed eseguire lo scanner per determinare la resa radiochimica e purezza radiochimica del gol marcato 125 Id nanoparticelle (4) (Figura 3) secondo il protocollo del produttore. </li></ol></li></ol>

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The authors have nothing to disclose.

In generale, la resa radiochimica osservato del purificato 125 azide I-marcato (1) è stato 75 ± 10% (n = 8). La radiomarcatura è stata compiuta con 50-150 MBq di radioattività, ed i risultati radiochimiche sono abbastanza coerenti. Se [125 I] NaI (t 1/2 = 59,4 d) sottoposto decadimento radioattivo per più di un mese è stato utilizzato nella reazione radioiodination, la resa radiochimica 1 è stata osservata essere leggermente diminuita (53...

The strain-promoted copper-free click reaction between azides and cyclooctynes has been extensively applied to the efficient bioorthogonal labeling of a wide range of biomolecules, nanomaterials, and living subjects1-7. Due to the excellent site-specificity and rapid reaction rate of this conjugation reaction, it has also been used to synthesize radiolabeled tracers. A few 18F-labeled azide or DBCO prosthetic groups have been prepared for in vitro labeling of various cancers targeting peptides and antibodies, as well as for in vivo pre-targeted imaging of tumors8-13. In addition to these examples, the same conjugation reaction was applied to the metal-radioisotope-labeling of nanomaterials for positron emission tomography (PET) imaging studies14-16.
For several decades, radioactive iodines have been used for biomedical research and clinical trials through PET imaging (124I), single-photon emission computed tomography (SPECT) imaging (123I, 125I), and thyroid cancer treatment (131I)17-21. Therefore, an efficient method for radioactive iodine labeling is fundamentally important for various investigations, including molecular imaging studies, analysis of organ distribution of biomolecules, biomarker identification, and drug development. A copper-free click reaction strategy could be used in radioactive iodine labeling. However, this application has not been investigated as extensively as 18F-labeled biomolecules22-23. Here, we will provide a step-by-step protocol for the synthesis of an 125I-labeled azide for radiolabeling of DBCO-group-derived molecules. The procedures in the present report will include radioiodination of the stannylated precursor, purification steps with HPLC, and solid phase extraction. We also demonstrate efficient radiolabeling of DBCO-group-modified 13-nm-sized gold nanoparticles using the 125I-labeled azide. The detailed protocol in this report will help synthetic chemists understand a new radiolabeling methodology for the synthesis of radiolabeled products.

<table><tbody><tr><td>Chloramine T trihydrate</td><td>Sigma</td><td>402869</td><td></td></tr><tr><td>[&lt;sup&gt;125&lt;/sup&gt;I]NaI in aq. NaOH</td><td>Perkin-Elmer</td><td>NEZ033A010MC</td><td></td></tr><tr><td>Sodium metabisulfite&amp;nbsp;</td><td>Sigma</td><td>S9000</td><td></td></tr><tr><td>Formic acid</td><td>Sigma</td><td>251364</td><td></td></tr><tr><td>Sep-Pak tC18 plus cartridge</td><td>Waters</td><td>WAT036800</td><td></td></tr><tr><td>Dimethyl sulfoxide&amp;nbsp;</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Acetone</td><td>Sigma</td><td>650501</td><td></td></tr><tr><td>Ethanol</td><td>Sigma</td><td>459844</td><td></td></tr><tr><td>Gold(III) chloride trihydrate</td><td>Sigma</td><td>520918</td><td></td></tr><tr><td>Tween 20&amp;nbsp;</td><td>Sigma</td><td>P1379</td><td></td></tr><tr><td>DBCO PEG SH (MW 5,000)</td><td>NANOCS</td><td>PG2-DBTH-5k</td><td></td></tr><tr><td>TLC silica gel 60 F&lt;sub&gt;254&lt;/sub&gt;</td><td>Merck</td><td></td><td></td></tr><tr><td>Analytical HPLC</td><td>Agilent</td><td>1290 Infinity</td><td>Model number</td></tr><tr><td>Preparative HPLC</td><td>Agilent</td><td>1260 Infinity</td><td>Model number</td></tr><tr><td>Analytical C18 reverse-phase column</td><td>Agilent</td><td>Zorbax Eclipse XDB-C18</td><td></td></tr><tr><td>Preparative C18 reverse-phase column</td><td>Agilent</td><td>PrepHT XDB-C18</td><td></td></tr><tr><td>Radio TLC scanner</td><td>Bioscan</td><td>AR-2000</td><td>Model number</td></tr><tr><td>Radioisotope dose calibrator</td><td>Capintec, Inc</td><td>CRC -25R dose calibrator</td><td>Model number</td></tr></tbody></table>

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.

La reazione radioiodination del precursore stannylated (2) è stata effettuata utilizzando 150 MBq di [125 I] NaI, acido acetico, e cloramina T a temperatura ambiente per 15 minuti per fornire il prodotto radiomarcato (1). Dopo la purificazione preparativa HPLC della miscela grezza, il prodotto desiderato è stato ottenuto con 75 ± 10% (n = 8) della resa radiochimica. Analytical HPLC ha rivelato che la purezza radiochimica del prodotto 1...

Watch this Scientific Journal Video about An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group at JoVE.com

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Una procedura dettagliata per la sintesi di un azide 125 I-etichettati e la radiomarcatura di dibenzocyclooctyne (DBCO) -gruppo-coniugati, nanoparticelle d&#39;oro 13-nm dimensioni utilizzando una reazione click senza il rame è descritto.

Un protocollo ottimizzato per la radiomarcatura efficiente di oro nanoparticelle utilizzando un<sup&gt; 125</sup&gt; Azide gruppo I-etichettati protesico

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

an-optimized-protocol-for-efficient-radiolabeling-gold-nanoparticles

Universidad San Sebastian

Research

JoVE Journal

Chemistry

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

8.1K Views.  Korea Atomic Energy Research Institute.  Una procedura dettagliata per la sintesi di un azide 125 I-etichettati e la radiomarcatura di dibenzocyclooctyne (DBCO) -gruppo-coniugati, nanoparticelle d'oro 13-nm dimensioni utilizzando una reazione click senza il rame è descritto. 

Video: An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

Efficiente e anticorpi etichettatura per cicloaddizione azide-alchino Strain-promosso site-specific

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

Ricerca

Biochimica

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.

Il bioconjugation e radiosintesi di<sup&gt; 89</sup&gt; Anticorpi Zr-DFO-etichettati

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET ...

Chimica

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.

Sfruttando la domanda Bioorthogonal Inverse Electron Diels-Alder cicloaddizione per 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 ...

Bioingegneria

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 Radioimmunotherapy basato sulla reazione di Diels-Alder domanda inversa Electron

While radioimmunotherapy (RIT) is a promising approach for the treatment of cancer, the long pharmacokinetic half-life of radiolabeled antibodies can ...

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized cancer therapies in recent years. However, only a fraction of patients responds to PD-1/PD-L1 inhibitors, owing to the heterogeneous expression of PD-L1 in tumor cells. This heterogeneity presents a challenge in the precise detection of tumor cells by the commonly used immunohistochemistry (IHC) approach. This situation calls for better methods to stratify patients who will benefit from immune checkpoint blockade therapy, to improve treatment efficacy. Positron emission tomography (PET) enables real-time visualization of the whole-body PD-L1 expression in a noninvasive way. Therefore, there is a need for the development of radiolabeled tracers to detect PD-L1 distribution in tumors through PET imaging.
Compared to their L-counterparts, dextrorotary (D)-peptides have properties such as proteolytic resistance and remarkably prolonged metabolic half-lives. This study designed a new method to detect PD-L1 expression based on PET imaging of 68Ga-labeled PD-L1-targeted D-peptide, a D-dodecapeptide antagonist (DPA), in tumor-bearing mice. The results showed that the [68Ga]DPA can specifically bind to PD-L1-overexpressing tumors in vivo, and showed favorable stability as well as excellent imaging ability, suggesting that [68Ga]DPA-PET is a promising approach for the assessment of PD-L1 status in tumors.

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

This study developed a noninvasive and real-time method to evaluate the distribution of programmed death-ligand 1 in the whole body, based on positron emission tomographic imaging of [68Ga] D-dodecapeptide antagonist. This technique has advantages over conventional immunohistochemistry and improves the efficiency of identifying appropriate patients who will benefit from immune checkpoint blockade therapy.

Sviluppo di un tracciante PET del peptide D marcato con 68gallio per l'imaging dell'espressione programmata del ligando di morte 1

The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized ...

Ricerca sul cancro

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.

Tomografia a emissione di positroni Imaging del traffico cellulare: un metodo di radiomarcatura cellulare

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

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

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).

Murino linfociti Etichettatura da<sup&gt; 64</sup&gt; Cu-anticorpo recettore Targeting per<em&gt; In Vivo</em&gt; Cell Traffico da PET / CT

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

Immunologia e infezioni

<meta charset="utf8"></head><body>Sintesi efficiente di radiotraccianti al carbonio-11 utilizzando il metodo della metilazione ad anello

Enhancing Efficiency and Radiolabeling Yields of Carbon-11 Radioligands for Clinical Research Using the Loop Method

A successful positron emission tomography imaging program involving carbon-11 radiotracers demands fast, efficient, and reliable synthesis methods, requiring an on-site cyclotron and radiochemistry group, as well as clinical staff trained to operate under the unique constraints of the carbon-11 radionuclide. This study examines the merits and advantages of a captive solvent &#39;loop method&#39; of radiolabeling four tracers with the carbon-11 radionuclide, producing the radioligands [11C]ER-176, [11C]MRB, [11C]mHED, and [11C]PiB.
The &#39;loop method&#39; is compared against the traditional reactor-based method of carbon-11 methylation in the course of synthesizing the same radiotracers on the identical automated platform. Further, a complete overview of the clinical research preparation of the [11C]ER-176 radiotracer is presented. As demonstrated by the production of [11C]ER-176, the captive solvent &#39;loop method&#39; of heterogeneous alkylation proved to be more efficient, with excellent radiochemical purity (99.6 &#177; 0.6%, n = 25), higher and more consistent radiochemical yield (end of synthesis (EOS) = 5.4 &#177; 2.2 GBq, n = 25) compared to the reactor method (EOS = 1.6 &#177; 0.5 GBq, n = 6), increased molar activity (loop method = 194 &#177; 66 GBq/&#181;mol, n = 25; reactor method = 132 &#177; 78 GBq/&#181;mol, n = 6), along with an average 5 min shorter reaction sequence.

<meta charset="utf8"></head><body>Miglioramento dell'efficienza e della resa di radiomarcatura dei radioligandi al carbonio-11 per la ricerca clinica utilizzando il metodo Loop

This protocol presents a detailed procedure to prepare four carbon-11 radiotracers by way of the &#8220;loop method&#8221; [11C]methylation. We describe the procedure to synthesize [11C]ER-176, &#160;with data for three additional radiotracers: [11C]MRB, [11C]mHED, and [11C]PiB. The loop method provides an efficient synthesis with increased radiochemical yields against traditional reaction vessel [11C]methylation.

Miglioramento dell'efficienza e della resa di radiomarcatura dei radioligandi al carbonio-11 per la ricerca clinica utilizzando il metodo Loop

A successful positron emission tomography imaging program involving carbon-11 radiotracers demands fast, efficient, and reliable synthesis methods, ...

Un protocollo ottimizzato per la radiomarcatura efficiente di oro nanoparticelle utilizzando un

Riepilogo

Esplora Altri Video

Capitoli in questo video

Efficiente e anticorpi etichettatura per cicloaddizione azide-alchino Strain-promosso site-specific

Il bioconjugation e radiosintesi di

Sfruttando la domanda Bioorthogonal Inverse Electron Diels-Alder cicloaddizione per Pretargeted PET Imaging

Pretargeted Radioimmunotherapy basato sulla reazione di Diels-Alder domanda inversa Electron

Sviluppo di un tracciante PET del peptide D marcato con ⁶⁸gallio per l'imaging dell'espressione programmata del ligando di morte 1

Tomografia a emissione di positroni Imaging del traffico cellulare: un metodo di radiomarcatura cellulare

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of ⁶⁸Ga-Labelled Nano Molecules

Murino linfociti Etichettatura da

Miglioramento dell'efficienza e della resa di radiomarcatura dei radioligandi al carbonio-11 per la ricerca clinica utilizzando il metodo Loop

Miglioramento dell'efficienza e della resa di radiomarcatura dei radioligandi al carbonio-11 per la ricerca clinica utilizzando il metodo Loop