PET Imaging of Extracellular pH in Tumors with⁶⁴Cu- and¹⁸F-Labeled pHLIP Peptides: A Structure–Activity Optimization Study

Abstract

pH (low) insertion peptides (pHLIP peptides) target acidic extracellular environments in vivo due to pH-dependent cellular membrane insertion. Two variants (Var3 and Var7) and wild-type (WT) pHLIP peptides have shown promise for in vivo imaging of breast cancer. Two positron emitting radionuclides (64Cu and 18F) were used to label the NOTA- and NO2A-derivatized Var3, Var7, and WT peptides for in vivo biodistribution studies in 4T1 orthotopic tumor-bearing BALB/c mice. All of the constructs were radiolabeled with 64Cu or [18F]-AlF in good yield. The in vivo biodistribution of the 12 constructs in 4T1 orthotopic allografted female BALB/c mice indicated that NO2A-cysVar3, radiolabeled with either 18F (4T1 uptake; 8.9 ± 1.7%ID/g at 4 h p.i.) or 64Cu (4T1 uptake; 8.2 ± 0.9%ID/g at 4 h p.i. and 19.2 ± 1.8% ID/g at 24 h p.i.), shows the most promise for clinical translation. Additional studies to investigate other tumor models (melanoma, prostate, and brain tumor models) indicated the universality of tumor targeting of these tracers. From this study, future clinical translation will focus on 18F- or 64Cu-labeled NO2A-cysVar3.

pHLIP Peptide Interaction with a Membrane Monitored by SAXS

“…The pHLIP peptides find a wide range of applications in biomedical sciences 16,20–22,43,44 and, also, they prove to be a very convenient model system for the investigation of polypeptide insertion into the lipid bilayer of a membrane triggered by pH. 12,28 Whereas various spectroscopic methods applied previously probed changes in the conformation of peptides, SAXS employed in this study allowed us, for the first time, to monitor the changes in the liposome and lipid bilayer structure.…”

Applications of pHLIP Technology for Cancer Imaging and Therapy

“…Variation of the WT pHLIP sequence led to novel pHLIPs, such as Variant 3 (Var3), with significantly improved tumor targeting properties [18, 28, 33–35]. The overall features of the pHLIP peptide sequences are still present in all variants: a middle region interspersed with a combination of hydrophobic residues and residues that are negatively charged at physiological pH but become neutrally charged at low pH, and hydrophilic flanking regions, with the membrane-inserting C-terminus (in most sequences) containing a few additional protonatable residues (Box 1) [9, 36–38].…”

“…One of the main challenges in PET/SPECT imaging is to obtain images of target tissue that contrast highly with the image background, motivating the use of a targeting agent. When a radionuclide is conjugated to the non-inserting terminus of a pHLIP (Figure 3A), the resulting construct not only targets tumor tissue very well, but also remains securely in the tumor long enough to allow the excess, non-inserted construct to clear from normal tissues, resulting in higher contrast images [17, 23, 25, 29, 33]. Success has been seen using the radioisotopes 18 F (9 ± 2 %ID/g at 4 h p.i.)…”

“…and 64 Cu (19 ± 2%ID/g at 24 h p.i.) conjugated to Var3 [33]. These constructs are in the process of clinical translation as novel nuclear imaging markers of diseased tissue acidity.…”

Multiplexed imaging for diagnosis and therapy

“…CT reinforces the clinical utility of PET in several ways that enhance diagnostic accuracy: the spatio-anatomic reference given by CT assists in the generation of quantitative PET data by providing a tissue-density map to facilitate appropriate correction for attenuation effects 47 ; it assists clinicians in recognition of false-positive tracer uptake 48 ; and it assists in identifying precise tumour margins for optimal subsequent care 49 (for example, biopsy, resection or monitoring). The availability of both fluorinated small molecules, and more recently macromolecules labelled with a variety of radiometals, has promoted further applications in PET-CT (Table 4; SI Table 1 50,51 ).…”