Name: development of a 68gallium-labeled d-peptide pet tracer for imaging programmed death-ligand 1 expression
Uploaded: 2023-02-03T14:30:00
Description: Watch this Scientific Journal Video about Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression at JoVE.com

This study was supported by the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (no. 2022-RC350-04) and the CAMS Innovation Fund for Medical Sciences (nos. 2021-I2M-1-026, 2022-I2M-1-026-1, 02120101, 02130101, and 2022-I2M-2-002).

The animal experimental procedures were approved by the Animal Ethics Committee of Nanjing Medical University or the National Institutes of Quantum Science and Technology. Mice experiments were strictly performed in compliance with the institutional guidelines of the Committee for the Care and Use of Laboratory Animals.
1. Peptide synthesis
<ol>
	<li>Swell 100 mg of 4-methylbenzhydrylamine (MBHA) resin (loading capacity of 0.37 mmol/g) in 1 mL of&#160;N-methyl-2-pyr...

The critical steps described in this method include the efficient labeling of 68Ga to DPA and choosing a suitable time window for PET imaging, which must perfectly match the pharmacodynamic pattern of DPA in the tumor.
In contrast to IHC, PET imaging enables real-time detection of whole-body PD-L1 expression in a noninvasive manner, allowing the visualization of each positive area in a heterogeneous tumor6,7. Peptides were c...

The discovery of immune checkpoint proteins was a breakthrough in tumor therapy, and has led to major advances in the development of immune checkpoint blockade therapy1. Programmed cell death-protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) are potential drug targets with several antibodies approved by the Food and Drug Administration (FDA). PD-1 is expressed by tumor-infiltrating immune cells, such as CD4+, CD8+ T cells, and regulatory T cells. PD-L1 is one of the PD-1 ligands, which is overexpressed in a variety of tumor cells2,3. The interaction between PD-1 and PD-L1 inactivates PD-1, thus suppressing the antitumor immune response4. These findings suggest that the inhibition of PD-L1 can improve the killing effect of immune cells and eliminate tumor cells5. Currently, chromogenic immunohistochemistry (IHC) is the most commonly used approach to identify patients who are most likely to respond to immune checkpoint therapy6,7. However, due to the heterogeneous expression of PD-L1 in tumor cells, IHC results from biopsies cannot provide accurate information about PD-L1 expression in patients8. Previous studies have reported that only 20%-40% of patients gain long-term benefits from immune checkpoint blockade therapy1,9,10. There is, therefore, an urgent need to develop a new method to circumvent the false-negative results caused by the heterogeneous expression of these immune checkpoint proteins.
Molecular imaging technology, such as positron emission tomography (PET), enables real-time visualization of the whole body in a noninvasive way, and thus can outperform the conventional IHC method11,12,13. Radiolabeled antibodies, peptides, and small molecules are promising tracers for monitoring PD-L1 expression in cancer patients14,15,16,17,18,19,20,21,22,23,24,25. The FDA has approved three PD-L1 therapeutic monoclonal antibodies: avelumab, atezolizumab, and durvalumab26. Immuno-PET tracers based on these antibodies have been well documented27,28,29,30,31,32. Early-phase clinical trials have revealed limited value for clinical application, because of the unfavorable pharmacokinetics30. Compared with antibodies, peptides exhibit faster blood and organ clearance from healthy organs, and can be easily chemically modified33. Multiple peptides with high affinities for PD-1/PD-L1 have been reported2; WL12 is a reported peptide that shows specific binding to PD-L134. Radiolabeled tracers, [64Cu]WL12, [68Ga]WL12, and [18F]FPyWL12, have been reported to show high in vivo specific tumor-targeting ability, which allows for the harvest of high-quality images of PD-L1 expression in tumors26,35,36,37. Moreover, the first in-human evaluation of radiolabeled WL12 has demonstrated that [68Ga]WL12 (chelated by NOTA) has a safe and efficient potential for clinical tumor imaging38. Due to its high hydrophobicity and high uptake in the healthy liver, WL12 has limited clinical use. Other radiolabeling peptides, such as TPP1 and SETSKSF, which specifically bind to PD-L1, have also showed potential stability and specificity to visualize whole-body PD-L1 expression39,40. However, unmodified peptides are easily degraded by proteases, and are rapidly metabolized by the kidney. Dextrorotary(D)-peptides have been widely used as effective mediators, due to the poor stability of left-handed (L)-peptides41,42,43. D-peptides are hyper-resistant to proteolytic degradation and have remarkably prolonged metabolic half-lives. Compared with their L-counterparts, D-peptides mostly show specific binding abilities44,45,46.
This study designed a new method to detect PD-L1 expression, based on PET imaging of a 68Ga-labeled PD-L1-targeted D-peptide, D-dodecapeptide antagonist (DPA), in a tumor-bearing mouse model47. The stability of [68Ga]DPA was first studied in phosphate-buffered saline (PBS) and mouse serum, after which the binding affinity of [68Ga]DPA in PD-L1-overexpressing tumors was tested. Thereafter, PET imaging was performed in glioblastoma xenograft models to confirm whether [68Ga]DPA was an ideal PET tracer to monitor PD-L1 expression in tumors. The combination of PET imaging and DPA not only provides a new approach to overcome challenges associated with the heterogeneous expression of PD-L1, but also lays the basis for the development of D-peptide-based radiotracers.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)</td>
			<td>Merck</td>
			<td>60239-18-1</td>
			<td>68Ga&#160; chelation</td>
		</tr>
		<tr>
			<td>3,3-diaminobenzidine (DAB) Kit</td>
			<td>Sigma-Aldrich</td>
			<td>D7304-1SET</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr>
			<td>anti-PD-L1 monoclonal antibody</td>
			<td>Wuhan Proteintech</td>
			<td>17952-1-ap</td>
			<td>Immunohistochemistry: primary antibody</td>
		</tr>
		<tr>
			<td>BMS202</td>
			<td>Selleck</td>
			<td>1675203-84-5</td>
			<td>Competitive binding assay: inhibitor</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Merck</td>
			<td>V900933</td>
			<td>Immunofluorescent : blocking&#160;</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Merck</td>
			<td>D9542</td>
			<td>Immunofluorescent: staining of nucleus</td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Merck</td>
			<td>34856</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>DIPEA</td>
			<td>Merck</td>
			<td>3439</td>
			<td>Peptide coupling</td>
		</tr>
		<tr>
			<td>EDC&#183;HCl</td>
			<td>Merck</td>
			<td>E6383</td>
			<td>Activation of DOTA</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10099</td>
			<td>Cell culture: supplement</td>
		</tr>
		<tr>
			<td>FITC-conjugated anti-human IgG Fc Antibody</td>
			<td>Biolegend</td>
			<td>409310</td>
			<td>Immunofluorescent: secondary antibody</td>
		</tr>
		<tr>
			<td>FITC-conjugated anti PD-L1 antibody</td>
			<td>Biolegend</td>
			<td>393606</td>
			<td>Flow cytometry: direct antibody</td>
		</tr>
		<tr>
			<td>HCTU</td>
			<td>Energy Chemical</td>
			<td>E070004-25g</td>
			<td>Peptide coupling</td>
		</tr>
		<tr>
			<td>HRP labeled goat anti-rabbit antibody</td>
			<td>Servicebio</td>
			<td>GB23303</td>
			<td>Immunohistochemistry: secondary antibody</td>
		</tr>
		<tr>
			<td>Hydroxysuccinimide (NHS)</td>
			<td>Merck</td>
			<td>130672</td>
			<td>Activation of DOTA</td>
		</tr>
		<tr>
			<td>MeCN</td>
			<td>Merck</td>
			<td>PHR1551</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Morpholine</td>
			<td>Merck</td>
			<td>8.06127</td>
			<td>Fmoc- deprotection</td>
		</tr>
		<tr>
			<td>NMP</td>
			<td>Merck</td>
			<td>8.06072</td>
			<td>Solevent</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck</td>
			<td>30525-89-4</td>
			<td>Fixation of tissues</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>Cell culture: buffer</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin&#160;</td>
			<td>Gibco</td>
			<td>10378016</td>
			<td>Cell culture: supplement</td>
		</tr>
		<tr>
			<td>RIA tube</td>
			<td>PolyLab</td>
			<td>P10301A</td>
			<td>As tissue sample container</td>
		</tr>
		<tr>
			<td>RPMI-1640 medium</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td>Cell culture: basic medium</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Merck</td>
			<td>1.06264</td>
			<td>Salt for buffer</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td>Cell culture: dissociation agent</td>
		</tr>
		<tr>
			<td>U87MG cell line</td>
			<td>Procell Life Science &#38; Technology Co</td>
			<td>CL-0238</td>
			<td>Cell model</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>68Ge/68Ga generator</td>
			<td>Isotope Technologies Munich, ITM</td>
			<td>Not applicable</td>
			<td>Generation of [68Ga]</td>
		</tr>
		<tr>
			<td>Autogamma counter</td>
			<td>Perkin Elmer&#160;</td>
			<td>Wizard2</td>
			<td>Detection of radioactivity</td>
		</tr>
		<tr>
			<td>Confocal fluorescent microscopy</td>
			<td>Keyence</td>
			<td></td>
			<td>Observation of immunofluorescent results</td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>Becton Dickinson, BD</td>
			<td>LSRII</td>
			<td>Monitoring the PD-L1 positive cells</td>
		</tr>
		<tr>
			<td>High-performance liquid chromatography (HPLC)</td>
			<td>SHIMAZU</td>
			<td>LC-20AT&#160;</td>
			<td>Purification of DPA peptide</td>
		</tr>
		<tr>
			<td>PET scanner</td>
			<td>Siemens Medical Solutions</td>
			<td>Inveon MultiModality System</td>
			<td>PET imaging</td>
		</tr>
		<tr>
			<td>Optical microscopy</td>
			<td>Nikon&#160;</td>
			<td>Eclipse E100</td>
			<td>Observation of immunohistochemistry results</td>
		</tr>
		<tr>
			<td>Solid phase peptide synthesizer</td>
			<td>Promega Vac-Man Laboratory Vacuum Manifold</td>
			<td>LOT#11101</td>
			<td>Synthesis of DPA-DOTA peptide</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ASIPro</td>
			<td>Siemens Medical Solutions</td>
			<td>Not applicable</td>
			<td>Analysis of PET-CT results</td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>Becton Dickinson, BD</td>
			<td>FlowJo 7.6.1</td>
			<td>Analysis of the flow cytometer results</td>
		</tr>
		<tr>
			<td>Inveon Acquisition Workplace (IAW)</td>
			<td>Siemens Medical Solutions</td>
			<td>Not applicable</td>
			<td>Management of PET mechine</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>Graphpad</td>
			<td>Prism 8.0</td>
			<td>Analysis of the data&#160;</td>
		</tr>
	</tbody>
</table>

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.

[68Ga]DPA radiolabeling and stability 
The model peptide, DPA, is an effective PD-L1 antagonist. DOTA-DPA was obtained with &#62;95% purity and a yield of 68%. The mass of DOTA-DPA is experimentally observed at 1,073.3 ([M+2H]2+). 68Gallium is considered a suitable radionuclide to label peptides for PET imaging, and therefore was chosen for this study. To radiolabel DPA with 68Ga (half-life: 68 min), DOTA-PEG3-DPA was synt...

Watch this Scientific Journal Video about Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression at JoVE.com

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.

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 ...

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