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

Summary

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.

Abstract

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.

Introduction

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 cells^2,3. The interaction between PD....

Protocol

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

1. Swell 100 mg of 4-methylbenzhydrylamine (MBHA) resin (loading capacity of 0.37 mmol/g) in 1 mL of N-methyl-2-pyr.......

Discussion

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 tumor^6,7. Peptides were c.......

Materials

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