The pH (low) insertion peptides (pHLIPs) target acidity at the surfaces of cancer cells and show utility in a wide range of applications, including tumor imaging and intracellular delivery of therapeutic agents. Here we report pHLIP constructs that significantly improve the targeted delivery of agents into tumor cells. The investigated constructs include pHLIP bundles (conjugates consisting of two or four pHLIP peptides linked by polyethylene glycol) and Var3 pHLIPs containing either the nonstandard amino acid, γ-carboxyglutamic acid, or a glycine-leucine-leucine motif. The performance of the constructs in vitro and in vivo was compared with previous pHLIP variants. A wide range of experiments was performed on nine constructs including () biophysical measurements using steady-state and kinetic fluorescence, circular dichroism, and oriented circular dichroism to study the pH-dependent insertion of pHLIP variants across the membrane lipid bilayer; () cell viability assays to gauge the pH-dependent potency of peptide-toxin constructs by assessing the intracellular delivery of the polar, cell-impermeable cargo molecule amanitin at physiological and low pH (pH 7.4 and 6.0, respectively); and () tumor targeting and biodistribution measurements using fluorophore-peptide conjugates in a breast cancer mouse model. The main principles of the design of pHLIP variants for a range of medical applications are discussed.

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.

Acidity is a biomarker of cancer that is not subject to the blunting clonal selection effects that reduce the efficacy of other biomarker technologies, like antibody targeting. The pH (Low) Insertion Peptides (pHLIPs) provide new opportunities for targeting acidic tissues. Through the physical mechanism of membrane-associated folding, pHLIPs are triggered by the acidic microenvironment to insert and span the membranes of tumor cells. The pHLIP platform can be applied to imaging acidic tissues, delivering cell-permeable and impermeable molecules to the cytoplasm, and promoting the cellular uptake of nanoparticles. Since acidosis is a hallmark of tumor development, progression, and aggressiveness, the pHLIP technology may prove useful in targeting cancer cells and metastases for tumor diagnosis, imaging, and therapy.

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Introduction: Although heterogeneous, the extracellular tumor environment is generally marked by decreased pH relative to nontumor tissues due to cancer cell metabolism. Using a pH-sensitive peptide to deliver diagnostic PET isotopes can provide clinicians with improved diagnostic tools for cancer-specific imaging and an alternative to receptor-based cancer targeting. pH (low) Insertion Peptides (pHLIPs) have previously been shown to target cancerous tissues and are able to deliver PET metals to the cancer site utilizing a NOTA-based chelator. Methods: The thiol-containing cysteine moiety on the pHLIP peptide sequence was conjugated to the NOTA chelator. The NOTA-derivatized peptides were labeled with 64CuCl2 in 100 mM NH4OAc (pH 5) solution; the [18F]-fluoride was reacted with AlCl3 before forming the [18F]-AlF-NOTA-pHLIP complexes in NH4OAc buffered reaction mixtures (pH ∼ 4.1). After purification and formulation, the radiolabeled peptides were injected intravenously into 4T1 breast, PC-3 prostate, LNCaP prostate, or B16-F10 melanoma tumor-bearing mice. Results: The 4T1 tumor-bearing mice showed significant uptake > 10%ID/g by 4 h p.i. and continued to show increased uptake of the 64Cu-NOTA-cysVar3 over 20 h. Tumors were visible in the 4T1 tumor-bearing mice as early as 2 h, but had the greatest tumor-to-background ratios at 24 h. Initial studies with B16-F10 tumor-bearing mice showed > 9%ID/g uptake in the tumor by 4 h p.i. with slight increase of 64Cu-NOTA-cysVar3 uptake over 24 h. Due to the placement of the melanoma tumors away from internal organs, tumors were visible by 1 h p.i. Additionally, the size of the melanoma tumors did not affect the%ID/g of radiotracer uptake. Conclusions: Our studies indicate that the NOTA-cys(pHLIP), specifically NOTA-cysVar3, radiolabeled with 64Cu or 18F is a viable imaging tool for detecting highly metabolic tumors in preclinical mouse models. Funding: NIH F32 CA186721 (D.W.D.), NIH R01 CA138468 (J.S.L.), NIH MSKCC Center Grant (P30-CA08748). Citation Format: Dustin W. Demoin, Kimberly J. Edwards, Linden C. Wyatt, Mirkka Sarparanta, Jacob Pourat, Oleg A. Andreev, Yana K. Reshetnyak, Nerissa Viola-Villegas, Jason S. Lewis. Targeting a variety of cancers with NOTA-derivatized pH (low) insertion peptide (pHLIP) complexes with 64Cu and 18F: What cancers are targetable. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4205.