Antiproliferative Effect of pHLIP-Amanitin

Toxins could be effective anticancer drugs, if their selective delivery into cancer cells could be achieved. We have shown that the energy of membrane-associated folding of water-soluble membrane peptides of the pHLIP (pH low insertion peptide) family could be used to move cell-impermeable cargo across the lipid bilayer into the cytoplasm of cancer cells. Here we present the results of a study of pHLIP-mediated cellular delivery of a polar cell-impermeable toxin, α-amanitin, an inhibitor of RNA polymerase II. We show that pHLIP can deliver α-amanitin into cells in a pH-dependent fashion and induce cell death within 48 h. Translocation capability could be tuned by conjugating amanitin to the C-terminus of pHLIP via linkers of different hydrophobicities that could be cleaved in the cytoplasm. pHLIP-SPDP-amanitin, which exhibits 4–5 times higher antiproliferative ability at pH 6 than at pH 7.4, was selected as the best construct. The major mechanism of amanitin delivery is direct translocation (flip) across a membrane by pHLIP and cleavage of the S–S bond in the cytoplasm. The antiproliferative effect was monitored on four different human cancer cell lines. pHLIP-mediated cytoplasmic delivery of amanitin could create great opportunities to use the toxin as a potent pH-selective anticancer agent, which predominantly targets highly proliferative cancer cells at low extracellular pH values

Family of pH (Low) Insertion Peptides for Tumor Targeting

Cancer is a complex disease with a range of genetic and biochemical markers within and among tumors, but a general tumor characteristic is extracellular acidity, which is associated with tumor growth and development. Acidosis could be a universal marker for cancer imaging and the delivery of therapeutic molecules, but its promise as a cancer biomarker has not been fully realized in the clinic. We have discovered a unique approach for the targeting of acidic tissue using the pH-sensitive folding and transmembrane insertion of pH (low) insertion peptide (pHLIP). The essence of the molecular mechanism has been elucidated, but the principles of design need to be understood for optimal clinical applications. Here, we report on a library of 16 rationally designed pHLIP variants. We show how the tuning of the biophysical properties of peptide–lipid bilayer interactions alters tumor targeting, distribution in organs, and blood clearance. Lead compounds for PET/single photon emission computed tomography and fluorescence imaging/MRI were identified, and targeting specificity was shown by use of noninserting variants. Finally, we present our current understanding of the main principles of pHLIP design

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

pH (Low) Insertion Peptide (pHLIP) Targets Ischemic Myocardium

The pH (low) insertion peptide (pHLIP) family enables targeting of cells in tissues with low extracellular pH. Here, we show that ischemic myocardium is targeted, potentially opening a new route to diagnosis and therapy. The experiments were performed using two murine ischemia models: regional ischemia induced by coronary artery occlusion and global low-flow ischemia in isolated hearts. In both models, pH-sensitive pHLIPs [wild type (WT) and Var7] or WT-pHLIP–coated liposomes bind ischemic but not normal regions of myocardium, whereas pH-insensitive, kVar7, and liposomes coated with PEG showed no preference. pHLIP did not influence either the mechanical or the electrical activity of ischemic myocardium. In contrast to other known targeting strategies, the pHLIP-based binding does not require severe myocardial damage. Thus, pHLIP could be used for delivery of pharmaceutical agents or imaging probes to the myocardial regions undergoing brief restrictions of blood supply that do not induce irreversible changes in myocytes

pHLIP peptide targets nanogold particles to tumors

Progress in nanomedicine depends on the development of nanomaterials and targeted delivery methods. In this work, we describe a method for the preferential targeting of gold nanoparticles to a tumor in a mouse model. The method is based on the use of the pH Low Insertion Peptide (pHLIP), which targets various imaging agents to acidic tumors. We compare tumor targeting by nonfunctionalized nanogold particles with nanogold–pHLIP conjugates, where nanogold is covalently attached to the N terminus of pHLIP. Our most important finding is that both intratumoral and i.v. administration demonstrated a significant enhancement of tumor uptake of gold nanoparticles conjugated with pHLIP. Statistically significant reduction of gold accumulation was observed in acidic tumors and kidney when pH-insensitive K-pHLIP was used as a vehicle, suggesting an important role of pH in the pHLIP-mediated targeting of gold nanoparticles. The pHLIP technology can substantially improve the delivery of gold nanoparticles to tumors by providing specificity of targeting, enhancing local concentration in tumors, and distributing nanoparticles throughout the entire tumor mass where they remain for an extended period (several days), which is beneficial for radiation oncology and imaging

Targeting Pancreatic Ductal Adenocarcinoma Acidic Microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the USA, accounting for ~40,000 deaths annually. The dismal prognosis for PDAC is largely due to its late diagnosis. Currently, the most sensitive diagnosis of PDAC requires invasive procedures, such as endoscopic ultrasonography, which has inherent risks and accuracy that is highly operator dependent. Here we took advantage of a general characteristic of solid tumors, the acidic microenvironment that is generated as a by-product of metabolism, to develop a novel approach of using pH (Low) Insertion Peptides (pHLIPs) for imaging of PDAC. We show that fluorescently labeled pHLIPs can localize and specifically detect PDAC in human xenografts as well as PDAC and PanIN lesions in genetically engineered mouse models. This novel approach may improve detection, differential diagnosis and staging of PDAC

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. The pHLIP ICG tumor targeting and imaging efficacy studies were carried out in several murine and human mouse tumor models. Blood vessels were imaged in mice and pigs. Clinical Stryker imaging instruments for endoscopy and open surgery were used in the study. Intravenously administered pHLIP ICG exhibits a multi-hour circulation half-life, offering protracted delineation of vasculature. As it clears from the blood, pHLIP ICG targets tumors and tumor stroma, marking them for surgical removal. pHLIP ICG is non-toxic, marks blood flow for hours after injection, and effectively delineates tumors for improved resection on the day after administration

Novel pH-Sensitive Cyclic Peptides

A series of cyclic peptides containing a number of tryptophan (W) and glutamic acid (E) residues were synthesized and evaluated as pH-sensitive agents for targeting of acidic tissue and pH-dependent cytoplasmic delivery of molecules. Biophysical studies revealed the molecular mechanism of peptides action and localization within the lipid bilayer of the membrane at high and low pHs. The symmetric, c[(WE)4WC], and asymmetric, c[E4W5C], cyclic peptides translocated amanitin, a polar cargo molecule of similar size, across the lipid bilayer and induced cell death in a pH- and concentration-dependent manner. Fluorescently-labelled peptides were evaluated for targeting of acidic 4T1 mammary tumors in mice. The highest tumor to muscle ratio (5.6) was established for asymmetric cyclic peptide, c[E4W5C], at 24 hours after intravenous administration. pH-insensitive cyclic peptide c[R4W5C], where glutamic acid residues (E) were replaced by positively charged arginine residues (R), did not exhibit tumor targeting. We have introduced a novel class of cyclic peptides, which can be utilized as a new pH-sensitive tool in investigation or targeting of acidic tissue

The pH Low Insertion Peptide pHLIP Variant 3 as a Novel Marker of Acidic Malignant Lesions

Current strategies for early detection of breast and other cancers are limited in part because some lesions identified as potentially malignant do not develop into aggressive tumors. Acid pH has been suggested as a key characteristic of aggressive tumors that might distinguish aggressive lesions from more indolent pathology. We therefore investigated the novel class of molecules, pH low insertion peptides (pHLIPs), as markers of low pH in tumor allografts and of malignant lesions in a mouse model of spontaneous breast cancer, BALB/neu-T. pHLIP Variant 3 (Var3) conjugated with fluorescent Alexa546 was shown to insert into tumor spheroids in a sequence-specific manner. Its signal reflected pH in murine tumors. It was induced by carbonic anhydrase IX (CAIX) overexpression and inhibited by acetazolamide (AZA) administration. By using 31P magnetic resonance spectroscopy (MRS), we demonstrated that pHLIP Var3 was retained in tumors of pH equal to or less than 6.7 but not in tissues of higher pH. In BALB/neu-T mice at different stages of the disease, the fluorescent signal from pHLIP Var3 marked cancerous lesions with a very low false-positive rate. However, only ∼60% of the smallest lesions retained a pHLIP Var3 signal, suggesting heterogeneity in pH. Taken together, these results show that pHLIP can identify regions of lower pH, allowing for its development as a theranostic tool for clinical applications

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. Special attention was devoted to the targeted delivery of the nine residue HA peptide epitope from the Flu virus hemagglutinin. The HA sequence is not present in the human genome, and immunity is readily developed during viral infection or immunization with KLH-HA supplemented with adjuvants. We tested and refined a series of double-headed HA-pHLIP agents, where two HA epitopes were linked to a single pH Low Insertion Peptide peptide via two Peg12 or Peg24 polymers, which enable HA epitopes to engage both antibody binding sites. HA-epitopes positioned at the surfaces of tumor cells remain exposed to the extracellular space for 24–48 h and are then internalized. Different vaccination schemes and various adjuvants, including analogs of FDA approved adjuvants, were tested in mice and resulted in a high titer of anti-HA antibodies. Anti-HA antibody binds HA-pHLIP in blood and travels as a complex leading to significant tumor targeting with no accumulation in organs and to hepatic clearance. HA-pHLIP agents induced regression of 4T1 triple negative breast tumor and B16F10 MHC-I negative melanoma tumors in immunized mice. The therapeutic efficacy potentially is limited by the drop of the level of anti-HA antibodies in the blood to background level after three injections of HA-pHLIP. We hypothesize that additional boosts would be required to keep a high titer of anti-HA antibodies to enhance efficacy. pH Low Insertion Peptide-targeted antigen therapy may provide an opportunity to treat tumors unresponsive to T cell based therapies, having a small number of neo-antigens, or deficient in MHC-I presentation at the surfaces of cancer cells either alone or in combination with other approaches