Aiming the magic bullet: targeted delivery of imaging and therapeutic agents to solid tumors by pHLIP peptides.

The family of pH (Low) Insertion Peptides (pHLIP) comprises a tumor-agnostic technology that uses the low pH (or high acidity) at the surfaces of cells within the tumor microenvironment (TME) as a targeted biomarker. pHLIPs can be used for extracellular and intracellular delivery of a variety of imaging and therapeutic payloads. Unlike therapeutic delivery targeted to specific receptors on the surfaces of particular cells, pHLIP targets cancer, stromal and some immune cells all at once.

Targeted intracellular delivery of dimeric STINGa by two pHLIP peptides for treatment of solid tumors.

We have developed a delivery approach that uses two pHLIP peptides that collaborate in the targeted intracellular delivery of a single payload, dimeric STINGa (dMSA). dMSA was conjugated with two pHLIP peptides via S-S cleavable self-immolating linkers to form 2pHLIP-dMSA. Biophysical studies were carried out to confirm pH-triggered interactions of the 2pHLIP-dMSA with membrane lipid bilayers.

pHLIP targeted intracellular delivery of calicheamicin.

Calicheamicin is a potent, cell-cycle independent enediyne antibiotic that binds and cleaves DNA. Toxicity has led to its use in a targeted form, as an antibody-drug conjugate approved for the treatment of liquid tumors. We used a reduced calicheamicin to conjugate it to a single cysteine residue at the membrane-inserting end of a pH Low Insertion Peptide (pHLIP) that targets imaging and therapeutic agents to tumors.

Tumor treatment by pHLIP-targeted antigen delivery.

Targeted antigen delivery allows activation of the immune system to kill cancer cells. Here we report the targeted delivery of various epitopes, including a peptide, a small molecule, and a sugar, to tumors by pH Low Insertion Peptides (pHLIPs), which respond to surface acidity and insert to span the membranes of metabolically activated cancer and immune cells within tumors. Epitopes linked to the extracellular ends of pH Low Insertion Peptide peptides were positioned at the surfaces of tumor cells and were recognized by corresponding anti-epitope antibodies.

Targeting Bladder Urothelial Carcinoma with pHLIP-ICG and Inhibition of Urothelial Cancer Cell Proliferation by pHLIP-amanitin.

Acidity is a useful biomarker for the targeting of metabolically active-cells in tumors. pH Low Insertion Peptides (pHLIPs) sense the pH at the surfaces of tumor cells and can facilitate intracellular delivery of cell-permeable and cell-impermeable cargo molecules. In this study we have shown the targeting of malignant lesions in human bladders by fluorescent pHLIP agents, intracellular delivery of amanitin toxin by pHLIP for the inhibition of urothelial cancer cell proliferation, and enhanced potency of pHLIP-amanitin for cancer cells with 17p loss, a mutation frequently present in urothelial cancers.

Eradication of tumors and development of anti-cancer immunity using STINGa targeted by pHLIP.

Despite significant progress in the development of novel STING agonists (STINGa), applications appear to be challenged by the low efficiency and poor selectivity of these agents. A pH Low Insertion Peptide (pHLIP) extends the lifetime of a STINGa in the blood and targets it to acidic cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), myeloid derived suppressor cells (mMDSCs) and dendritic cells (DCs). CAFs constitute 25% of all live cells within CT26 tumors, and M2-type TAMs and mMDSCs are the most abundant among the immune cells.

Taking out the trash: How misfolded proteins are removed from the endoplasmic reticulum.

Proteins that are expressed on membrane surfaces or secreted are involved in all aspects of cellular and organismal life, and as such require extremely high fidelity during their synthesis and maturation. These proteins are synthesized at the endoplasmic reticulum (ER) where a dedicated quality control system (ERQC) ensures only properly matured proteins reach their destinations. An essential component of this process is the identification of proteins that fail to pass ERQC and their retrotranslocation to the cytosol for proteasomal degradation.

pHLIP Peptides Target Acidity in Activated Macrophages.

Purpose: Acidity can be a useful alternative biomarker for the targeting of metabolically active cells in certain diseased tissues, as in acute inflammation or aggressive tumors. We investigated the targeting of activated macrophages by pH low insertion peptides (pHLIPs), an established technology for targeting cell-surface acidity.

Procedures: The uptake of fluorescent pHLIPs by activated macrophages was studied in cell cultures, in a mouse model of lung inflammation, and in a mouse tumor model.

Correction to 'Tumor-selective, antigen-independent delivery of a pH sensitive peptide-topoisomerase inhibitor conjugate suppresses tumor growth without systemic toxicity'.

[This corrects the article DOI: 10.1093/narcan/zcab021.].

MicroRNA function can be reversed by altering target gene expression levels.

Paradoxically, many microRNAs appear to exhibit entirely opposite functions when placed in different contexts. For example, miR-125b has been shown to be pro-apoptotic in some studies, but anti-apoptotic in others. To investigate this phenomenon, we combine computational modeling with experimental approaches to examine how the function of miR-125b in apoptosis varies with respect to the expression levels of its pro-apoptotic and anti-apoptotic targets.

Tumor-selective, antigen-independent delivery of a pH sensitive peptide-topoisomerase inhibitor conjugate suppresses tumor growth without systemic toxicity.

Topoisomerase inhibitors are potent DNA damaging agents which are widely used in oncology, and they demonstrate robust synergistic tumor cell killing in combination with DNA repair inhibitors, including poly(ADP)-ribose polymerase (PARP) inhibitors. However, their use has been severely limited by the inability to achieve a favorable therapeutic index due to severe systemic toxicities. Antibody-drug conjugates address this issue via antigen-dependent targeting and delivery of their payloads, but this approach requires specific antigens and yet still suffers from off-target toxicities.

Pharmacokinetic modeling reveals parameters that govern tumor targeting and delivery by a pH-Low Insertion Peptide (pHLIP).

A pH-Low Insertion Peptide (pHLIP) is a pH-sensitive peptide that undergoes membrane insertion, resulting in transmembrane helix formation, on exposure to acidity at a tumor cell surface. As a result, pHLIPs preferentially accumulate within tumors and can be used for tumor-targeted imaging and drug delivery. Here we explore the determinants of pHLIP insertion, targeting, and delivery through a computational modeling approach.

pHLIP ICG for delineation of tumors and blood flow during fluorescence-guided surgery.

Fluorescence imaging has seen enduring use in blood flow visualization and is now finding a new range of applications in image-guided surgery. In this paper, we report a translational study of a new fluorescent agent for use in surgery, pHLIP ICG, where ICG (indocyanine green) is a surgical fluorescent dye used widely for imaging blood flow. We studied pHLIP ICG interaction with the cell membrane lipid bilayer, the pharmacology and toxicology in vitro and in vivo (mice and dogs), and the biodistribution and clearance of pHLIP ICG in mice.

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

The advantages of targeted therapy have motivated many efforts to find distinguishing features between the molecular cell surface landscapes of diseased and normal cells. Typically, the features have been proteins, lipids or carbohydrates, but other approaches are emerging. In this discussion, we examine the use of cell surface acidity as a feature that can be exploited by using pH-sensitive peptide folding to target agents to diseased cell surfaces or cytoplasms.

Kinetics of pHLIP peptide insertion into and exit from a membrane.

To advance mechanistic understanding of membrane-associated peptide folding and insertion, we have studied the kinetics of three single tryptophan pHLIP (pH-Low Insertion Peptide) variants, where tryptophan residues are located near the N terminus, near the middle, and near the inserting C-terminal end of the pHLIP transmembrane helix. Single-tryptophan pHLIP variants allowed us to probe different parts of the peptide in the pathways of peptide insertion into the lipid bilayer (triggered by a pH drop) and peptide exit from the bilayer (triggered by a rise in pH). By using pH jumps of different magnitudes, we slowed down the processes and established the intermediates that helped us to understand the principles of insertion and exit.

Ku80-Targeted pH-Sensitive Peptide-PNA Conjugates Are Tumor Selective and Sensitize Cancer Cells to Ionizing Radiation.

The development of therapeutic agents that specifically target cancer cells while sparing healthy tissue could be used to enhance the efficacy of cancer therapy without increasing its toxicity. Specific targeting of cancer cells can be achieved through the use of pH-low insertion peptides (pHLIP), which take advantage of the acidity of the tumor microenvironment to deliver cargoes selectively to tumor cells. We developed a pHLIP-peptide nucleic acid (PNA) conjugate as an antisense reagent to reduce expression of the otherwise undruggable DNA double-strand break repair factor, KU80, and thereby radiosensitize tumor cells.

Tumor-Targeted, Cytoplasmic Delivery of Large, Polar Molecules Using a pH-Low Insertion Peptide.

Tumor-targeted drug delivery systems offer not only the advantage of an enhanced therapeutic index, but also the possibility of overcoming the limitations that have largely restricted drug design to small, hydrophobic, "drug-like" molecules. Here, we explore the ability of a tumor-targeted delivery system centered on the use of a pH-low insertion peptide (pHLIP) to directly deliver moderately polar, multi-kDa molecules into tumor cells. A pHLIP is a short, pH-responsive peptide capable of inserting across a cell membrane to form a transmembrane helix at acidic pH.

Mapping pH at Cancer Cell Surfaces.

Purpose: To develop a tool to measure the pH at the surfaces of individual cells.

Procedures: The SNARF pH-sensitive dye was conjugated to a pHLIP® peptide (pH-Low Insertion Peptide) that binds cellular membranes in tumor spheroids. A beam splitter allows simultaneous recording of two images (580 and 640 nm) by a CCD camera.

Bilayer Thickness and Curvature Influence Binding and Insertion of a pHLIP Peptide.

The physical properties of lipid bilayers, such as curvature and fluidity, can affect the interactions of polypeptides with membranes, influencing biological events. Additionally, given the growing interest in peptide-based therapeutics, understanding the influence of membrane properties on membrane-associated peptides has potential utility. pH low insertion peptides (pHLIPs) are a family of water-soluble peptides that can insert across cell membranes in a pH-dependent manner, enabling the use of pH to follow peptide-lipid interactions.

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

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.

Two transmembrane dimers of the bovine papillomavirus E5 oncoprotein clamp the PDGF β receptor in an active dimeric conformation.

The dimeric 44-residue E5 protein of bovine papillomavirus is the smallest known naturally occurring oncoprotein. This transmembrane protein binds to the transmembrane domain (TMD) of the platelet-derived growth factor β receptor (PDGFβR), causing dimerization and activation of the receptor. Here, we use Rosetta membrane modeling and all-atom molecular dynamics simulations in a membrane environment to develop a chemically detailed model of the E5 protein/PDGFβR complex.

Applications of pHLIP Technology for Cancer Imaging and Therapy.

Acidity is a biomarker of cancer that is not subject to the blunting clonal selection effects that reduce the efficacy of other biomarker technologies, such as 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.

Targeted imaging of urothelium carcinoma in human bladders by an ICG pHLIP peptide ex vivo.

Bladder cancer is the fifth most common in incidence and one of the most expensive cancers to treat. Early detection greatly improves the chances of survival and bladder preservation. The pH low insertion peptide (pHLIP) conjugated with a near-infrared fluorescent dye [indocyanine green (ICG)] targets low extracellular pH, allowing visualization of malignant lesions in human bladder carcinoma ex vivo.

PET Imaging of Extracellular pH in Tumors with (64)Cu- and (18)F-Labeled pHLIP Peptides: A Structure-Activity Optimization Study.

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 ((64)Cu and (18)F) 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.