Arginine Residues Modulate the Membrane Interactions of pHLIP Peptides.

Most processes at the water-membrane interface often involve protonation events in proteins or peptides that trigger important biological functions and events. This is the working principle behind the pHLIP peptide technology. A key titrating aspartate (Asp14 in wt) is required to protonate to induce the insertion process, increase its thermodynamic stability when membrane-embedded, and trigger the peptide's overall clinical functionality. At the core of pHLIP properties, the aspartate pKa and protonation are a consequence of the residue side chain sensing the changing surrounding environment. In this work, we characterized how the microenvironment of the key aspartate residue (Asp13 in the investigated pHLIP variants) can be modulated by a simple point mutation of a cationic residue (ArgX) at distinct sequence positions (R10, R14, R15, and R17). We carried out a multidisciplinary study using pHRE simulations and experimental measurements. Fluorescence and circular dichroism measurements were carried out to establish the stability of pHLIP variants in state III and establish the kinetics of the insertion and exit of the peptide from the membrane. We estimated the contribution of the arginine to the local electrostatic microenvironment, which promotes or hinders other electrostatic players from coexisting in the Asp interaction shell. Our data indicate that the stability and kinetics of the peptide insertion and exit from the membrane are altered when Arg is topologically available for a direct salt-bridge formation with Asp13. Hence, the position of arginine contributes to fine-tuning the pH responses of pHLIP peptides, which finds wide applications in clinics.

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. The cytoplasmic reduction of the disulfide releases the calicheamicin, and activation, DNA binding, and strand scission ensue. We studied the interaction of pHLIP-calicheamicin with liposomal and cellular membranes and demonstrated that the agent exhibits cytotoxic activity both in highly proliferative cancer cells and in non-proliferative immune cells, such as polarized M2 macrophages. In vivo, the agent was effective in inhibiting tumor growth in mice with no signs of toxicity. Biodistribution studies confirmed tumor targeting with no accumulation of the agent in organs and tissues. The agent was found within the tumor mass and tumor-stroma interface. Treatment of tumors led to the depletion of CD206+ M2- tumor-associated macrophages within the tumor core. pHLIP-calicheamicin could be pursued as an effective therapeutic for the treatment of solid tumors.

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

Introduction: 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). Methods: dMSA was conjugated with two pHLIP peptides via S-S cleavable self-immolating linkers to form 2pHLIP-dMSA. Results: Biophysical studies were carried out to confirm pH-triggered interactions of the 2pHLIP-dMSA with membrane lipid bilayers. The kinetics of linker self-immolation and dMSA release, the pharmacokinetics, the binding to plasma proteins, the stability of the agent in plasma, the targeting and resulting cytokine activation in tumors, and the biodistribution of the construct was investigated. This is the first study demonstrating that combining the energy of the membrane-associated folding of two pHLIPs can be utilized to enhance the targeted intracellular delivery of large therapeutic cargo payloads. Discussion: Linking two pHLIPs to the cargo extends blood half-life, and targeted delivery of dimeric STINGa induces tumor eradication and the development of robust anti-cancer immunity.

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. The resulting activation of cytokines within the tumor microenvironment (TME) triggers the eradication of small (100 mm3) and large (400-700 mm3) CT26 tumors in mice after a single dose of pHLIP-STINGa. The tumor stroma was destroyed (the number of CAFs was reduced by 98%), intratumoral hemorrhage developed, and the level of acidity within the TME was reduced. Further, no tumors developed in 20 out of 25 tumor-free mice re-challenged by an additional injection of cancer cells. The therapeutic effect on CT26 tumors was insignificant in nude mice, lacking T-cells. Thus, targeted delivery of STINGa to tumor stroma and TAMs induces activation of signaling, potentially resulting in the recruitment and infiltration of T-cells, which gain access to the tumor core. The cytotoxic activity of T-cells is not impaired by an acidic environment and immune memory is developed.

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.

PET Imaging of Acidic Tumor Environment With 89Zr-labeled pHLIP Probes.

Acidosis of the tumor microenvironment is a hallmark of tumor progression and has emerged as an essential biomarker for cancer diagnosis, prognosis, and evaluation of treatment response. A tool for quantitatively visualizing the acidic tumor environment could significantly advance our understanding of the behavior of aggressive tumors, improving patient management and outcomes. 89Zr-labeled pH-low insertion peptides (pHLIP) are a class of radiopharmaceutical imaging probes for the in vivo analysis of acidic tumor microenvironments via positron emission tomography (PET). Their unique structure allows them to sense and target acidic cancer cells. In contrast to traditional molecular imaging agents, pHLIP's mechanism of action is pH-dependent and does not rely on the presence of tumor-specific molecular markers. In this study, one promising acidity-imaging PET probe ([89Zr]Zr-DFO-Cys-Var3) was identified as a candidate for clinical translation.

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. Fluorescence microscopy, whole-body and organ imaging, immunohistochemistry, and FACS analysis were employed. RESULTS: We find that cultured, activated macrophages readily internalize pHLIPs. The uptake is higher in glycolytic macrophages activated by LPS and INF-γ compared to macrophages activated by IL-4/IL-13. Fluorescent pHLIPs target LPS-induced lung inflammation in mice. In addition to marking cancer cells within the tumor microenvironment, fluorescent pHLIPs target CD45+, CD11b+, F4/80+, and CD206+ tumor-associated macrophages with no significant targeting of other immune cells. Also, fluorescent pHLIPs target CD206-positive cells found in the inguinal lymph nodes of animals inoculated with breast cancer cells in mammary fat pads. CONCLUSIONS: pHLIP peptides sense low cell surface pH, which triggers their insertion into the cell membrane. Unlike cancerous cells, activated macrophages do not retain inserted pHLIPs on their surfaces, instead their highly active membrane recycling moves the pHLIPs into endosomes. Targeting activated macrophages in diseased tissues may enable clinical visualization and therapeutic opportunities.

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.].

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. There is a high unmet need for a more universal tumor targeting technology to broaden the application of cytotoxic payloads. Acidification of the extracellular milieu arises from metabolic adaptions associated with the Warburg effect in cancer. Here we report the development of a pH-sensitive peptide-drug conjugate to deliver the topoisomerase inhibitor, exatecan, selectively to tumors in an antigen-independent manner. Using this approach, we demonstrate potent in vivo cytotoxicity, complete suppression of tumor growth across multiple human tumor models, and synergistic interactions with a PARP inhibitor. These data highlight the identification of a peptide-topoisomerase inhibitor conjugate for cancer therapy that provides a high therapeutic index, and is applicable to all types of human solid tumors in an antigen-independent manner.