Therapeutic Efficacy of a Family of pHLIP–MMAF Conjugates in Cancer Cells and Mouse Models

Abstract

The targeting of therapeutics specifically to diseased tissue is crucial for the development of successful cancer treatments. The approach here is based on the pH(low) insertion peptide (pHLIP) for the delivery of a potent mitotic inhibitor monomethyl auristatin F (MMAF). We investigated six pHLIP variants conjugated to MMAF to compare their efficacy in vitro against cultured cancer cells. While all pHLIP–MMAF conjugates exhibit potent pH- and concentration-dependent killing, their cytotoxicity profiles are remarkably different. We also show that the lead conjugate exhibits significant therapeutic efficacy in mouse models without overt toxicities. This study confirms pHLIP–monomethyl auristatin conjugates as possible new therapeutic options for cancer treatment and supports their further development.

Peptides of pHLIP family for targeted intracellular and extracellular delivery of cargo molecules to tumors

“…The thermodynamic parameters of pK and cooperativity of pH-dependent transition from State II at pH 8 to State III at pH < 5 can be taken as predictors of the performance of a pHLIP for drug delivery and tumor targeting (17,28,29). While pK is a rather stable fitting parameter, the cooperativity parameter (Hill coefficient) might vary over a wide range resulting from different fittings which are within the level of accuracy of the experimental measurements.…”

“…The pHLIPs can be used in a wide variety of applications, so it is desirable to have a range of options for specific applications. Some examples of these applications include (i) fluorescence imaging (8)(9)(10) and fluorescence image-guided surgery (11); (ii) nuclear imaging including PET and SPECT (single-photon emission computed tomography) (12,13); (iii) therapeutic applications such as the targeted delivery of polar toxins that cannot cross cell membranes (14,15), drug-like molecules that inherently diffuse across cell membranes (16,17), and gene therapy (18); and (iv) nanotechnology for enhancing the delivery of gold nanoparticles (19,20) or liposome-encapsulated payloads to cancer cells (21).…”

Comparison of lipid-dependent bilayer insertion of pHLIP and its P20G variant

“…pHLIP-WT (H 2 N-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGG-CONH 2 ) and pHLIP-P20G (H 2 N-AAEQNPIYWARYADWLFTTGLLLLDLALLVDADEGG-CONH 2 ) were prepared by Fmoc solid-phase synthesis as described previously [10–13], using H-Rink Amide-ChemMatrix resin affording an amidated C-terminus and purified via reverse-phase high performance liquid chromatography (RP-HPLC) (Phenomenex Luna prep 10 μ 250 × 21.20 mm C8; flow rate 10mL/min; phase A: water 0.1% trifluoroacetic acid (TFA); phase B: acetonitrile 0.1% TFA; gradient 60 min from 95/5 A/B to 0/pure A/B). The purity of the peptides was determined by RP-HPLC (Agilent Zorbax Eclipse 5 μm 4.6 × 50 mm XDB-C8; flow rate 1 mL/min; phase A: water 0.01% TFA; phase B: acetonitrile 0.01% TFA; gradient 45 min from 95/5 A/B to 0/pure A/B) and their identity was confirmed via matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry.…”

“…The discovery of pHLIP (pH-Low Insertion Peptide) is a compelling example of how a basic-science study can be translated into a very useful and practical biomedical tool. While originally designed from the sequence of the C-helix of bacteriorhodopsin to study membrane protein folding [1], pHLIP is currently and most often used for imaging tumors and other acidic diseased tissues [2–9], as well as for targeted drug delivery [10–13]. The underlying mechanism of pHLIP’s pH-dependent action is associated with the protonation of Asp residues, allowing for membrane insertion at acidic pH [14–16].…”

Targeting Acidic Diseased Tissues by pH-Triggered Membrane-Associated Peptide Folding

“…Targeted pHLIP-mediated delivery of moderately hydrophobic small molecule drugs has proven to be successful as well. Among the drugs that have been delivered are potent inhibitors of tubulin, such as monomethyl auristatin E, poly (ADP-ribose) polymerase inhibitors (PARPi's) including rucaparib and talazoparib, and other molecules (Burns et al, 2015(Burns et al, , 2017Song et al, 2016). A notable property is that pHLIP delivery has been shown to reduce bone marrow accumulation and toxicity, which is a significant issue in the use of many potent cytotoxic molecules.…”