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.

Abstract Activation of the stimulator of interferon genes (STING) pathway causes the release of factors that trigger the immune response in the tumor microenvironment (TME). Transient strong activation leads to immune activation and tumor suppression, while prolonged weak activation leads to immuno-suppression promoting tumor development. Reprogramming of M2-type tumor-associated macrophages (M2-TAMs) toward an M1 phenotype, suppression of cancer-associated fibroblasts (CAFs) and myeloid-derived suppressor cells (mMDSCs), as well as activation of dendritic cells (DCs) to train T-cells are advantageous, while the activation of STING pathway in T-cells is leads to their apoptosis. Thus, targeted delivery of STINGa to specific cells with TME to induce strong transient activation may be a key to therapy. We have developed a delivery approach that uses one or two pH Low Insertion Peptides (pHLIPs) that collaborate in the targeted intracellular delivery of a STINGa. STINGa were conjugated with pHLIPs via S-S cleavable self-immolating linkers. Biophysical studies were performed to ensure proper interactions of pHLIP-STINGa agents with membrane lipid bilayers. pHLIPs extend the lifetime of STINGa in the blood and target them to acidic cancer and stromal cells (CAFs), as well as M2-TAMs, mMDSCs and DCs within TME. The targeting results in selective cytokine activation in tumors with no systemic activation, which triggers the eradication of small (100 mm3) and large (400-700 mm3) CT26 tumors in mice after one or two doses of pHLIP-STINGa. The tumor stroma was destroyed, intratumoral hemorrhage developed, and the level of acidity within the TME was reduced. No tumors developed in mice re-challenged by an additional injection of cancer cells in a 90-day experiment. Thus, targeted delivery of STINGa to cancer cells, tumor stroma and TAMs induces activation of signaling, potentially resulting in the recruitment and infiltration of T- and NK-cells, which gain access to the tumor core. The cytotoxic activity of T- and NK-cells is not impaired by the acidic environment and immune memory is developed. The pHLIP technology may allow transformation of immuno-activating agents into more potent therapeutics, since pHLIP can target and deliver these agents to cancer cells, tumor stroma and myeloid cells. Citation Format: Michael DuPont, Anna Moshnikova, Marissa Iraca, Craig Klumpp, Hannah Visca, Dana Allababidi, Phoebe Pelzer, Donald Engelman, Oleg Andreev, Yana Reshetnyak. Eradication of tumors and development of immunity from pHLIP-targeted intracellular delivery of STINGa [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6758.

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. Since the TME exhibits complex cellular crosstalk interactions, simultaneous targeting and delivery to different cell types leads to a significant synergistic effect for many agents. pHLIPs can also be positioned on the surfaces of various nanoparticles (NPs) for the targeted intracellular delivery of encapsulated payloads. The pHLIP technology is currently advancing in pre-clinical and clinical applications for tumor imaging and treatment.

Abstract Targeted antigen delivery allows activation of the immune system to kill tumor cells. Among the immunogenic epitopes widely investigated is α-Gal, since humans exhibit specific anti-Gal reactivity. However, the repertoire of immunogenic epitopes could be significantly enhanced if therapeutic efficacy did not rely on the presence of natural antibodies, but instead relied on the production of anti-epitope endogenous antibodies induced by immunization against the selected epitope. Therefore, we designed and tested several HA-pHLIP agents, where the HA peptide (YPYDVPDYA) from the influenza virus was selected as an exogenous highly immunogenic epitope, since it is absent in the human genome, and a pH Low Insertion Peptide (pHLIP) was selected as the tumor targeting agent. HA peptides were attached to the N-termini of pHLIPs either directly or via PEG polymers. To enhance the overall binding affinity of HA-pHLIP to anti-HA antibodies, double headed HA-pHLIP agents were introduced, where two HA epitopes were linked to a single pHLIP peptide via PEG12 polymers. The pHLIP sequence was modified to ensure proper pKs of insertion into cell membranes. HA epitopes were targeted to cancer cells in mouse tumors, and remained exposed to the extracelullar space within tumors for about 24 hours. Double-headed HA-pHLIP treatments of imunized mice (a high titer of anti-HA antibodies was developed in mice after immunization with KLH-HA), bearing 4T1 triple negative or B16F10 MHC-I negative melanoma tumors resulted in a 55% reduction of tumor growth. Further reduction of tumor growth was impossible to achive, since all of the anti-HA antibodies in the blood were captured after 3 injections of HA-pHLIP and additional boosts would be required to restore the level of free anti-HA antibodies to induce a more significant therapeutic efficacy. Citation Format: Michael DuPont, Hannah Visca, Anna Moshnikova, Donald Engelman, Yana Reshetnyak, Oleg Andreev. Antigen-pHLIP for tumor treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6759.

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.

Fig. S1: pHLIP-αKu80(γ) binds to the mouse Ku80 sequence. Fig. S2: Validation of tumor vs. stromal cell sorting in EMT6-GFP cells. Fig. S3: Partial suppression of Ku80 expression induces radiosensitivity in cultured cancer cells. Fig. S4: pHLIP-αKu80(γ) causes tumor radiosensitization. Fig. S5: pHLIP-αKu80(γ) causes in vitro radiosensitization. Fig. S6: pHLIP-αKu80(γ) does not induce electrolyte abnormalities. Fig. S7: pHLIP-αKu80(γ) does not cause bone marrow suppression.

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.

Purpose: pHLIPs (pH-Low Insertion Peptides) target cell surface acidity resulting from the metabolism of cells in a solid tumor. The acidity causes pHLIPs to insert as transmembrane helices, and attached cargoes can be delivered into targeted cells. STING agonists can trigger an immune response when they are inside cells. pHLIP targeted delivery of a STING agonist (STINGa) effectively triggers cytokine secretion to mobilize the immune system to eradicate tumors while avoiding side effects. Experimental Procedures: Constructs consisting of a pHLIP peptide (Var3) linked to a STINGa (diABZI) via a self-immolating linker were synthesized and characterized. Activation of the STING pathway by the agent was confirmed in cells. Small (100 mm3) and large (400-700 mm3) tumors were grown in female Balb/c mice following flank injections of CT26 colorectal cancer cells. Randomized groups of mice were injected i.p. or i.v. with pHLIP-STINGa made with L- or D- amino acid pHLIP peptides, STINGa alone, pHLIP alone or vehicle. Tumor size and body weight were then measured 3 times per week. The durability of the response to the agent was tested by a second injection of CT26 cells after 60 days. In separate experiments, the identities of targeted immune and stromal cells, the production of cytokines, the PK, tumor targeting and biodistribution were assessed. Results and Conclusions: pHLIP extends the lifetime of a STINGa in the blood 6-fold and delivers STINGa to acidic cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), myeloid derived suppressor cells (mMDSCs), and dendritic cells (DCs). The resulting activation of cytokines within the tumor microenvironment (TME) triggers the eradication of small and large CT26 tumors in mice after a single dose of pHLIP-STINGa. The tumor stroma was destroyed, intratumoral hemorrhage developed, and the TME pH increased, which is important for the efficient cytotoxic activity of immune cells. 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. Inhibition of MHC-I negative B16F10 melanoma tumor growth was also observed in Balb/c mice. Thus, targeted delivery of the STINGa to the tumor stroma and TAMs activates signaling, potentially resulting in the recruitment and infiltration of both T-cells and NK-cells, which gain access to the tumor core. The cytotoxic activity of immune cells is enhanced, and immune memory is developed. Citation Format: Donald M. Engelman, Yana K. Reshetnyak, Oleg A. Andreev. Durable eradication of tumors by single injections of a pHLIP-STING agonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1804.

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.

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. This study by Wu et al. reports a cryo-electron microscopy (cryo-EM) structure of the five-protein channel through which aberrant proteins are extracted from the ER, providing insights into how recognition of misfolded proteins is coupled to their transport through a hydrophobic channel that acts to thin the ER membrane, further facilitating their dislocation to the cytosol [1].

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. This study by Wu et al. reports a cryo-electron microscopy (cryo-EM) structure of the five-protein channel through which aberrant proteins are extracted from the ER, providing insights into how recognition of misfolded proteins is coupled to their transport through a hydrophobic channel that acts to thin the ER membrane, further facilitating their dislocation to the cytosol1.

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. 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. 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. 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.

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. Twenty-eight ex-vivo bladder specimens, from patients undergoing robotic assisted laparoscopic radical cystectomy for bladder cancer, were treated via intravesical incubation for 15-60 minutes with pHLIP conjugated to indocyanine green (ICG) or IR-800 near infrared fluorescent (NIRF) dyes at concentrations of 4-8 μM. White light cystoscopy identified 47/58 (81%) and NIRF pHLIP cystoscopy identified 57/58 (98.3%) of malignant lesions of different subtypes and stages selected for histopathological processing. pHLIP NIRF imaging improved diagnosis by 17.3% (p < 0.05). All carcinoma-in-situ cases missed by white light cystoscopy were targeted by pHLIP agents and were diagnosed by NIRF imaging. We also investigated the interactions of pHLIP-amanitin with urothelial cancer cells of different grades. pHLIP-amanitin produced concentration- and pH-dependent inhibition of the proliferation of urothelial cancer cells treated for 2 hrs at concentrations up to 4 μM. A 3-4x enhanced cytotoxicity of pHLIP-amanitin was observed for cells with a 17p loss after 2 hrs of treatment at pH6. Potentially, pHLIP technology may improve the management of urothelial cancers, including imaging of malignant lesions using pHLIP-ICG for diagnosis and surgery, and the use of pHLIP-amanitin for treatment of superficial bladder cancers via intravesical instillation.

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. In doing so, we elucidate a general trend that miR-125b is more pro-apoptotic when its anti-apoptotic targets are overexpressed, whereas it is more anti-apoptotic when its pro-apoptotic targets are overexpressed. We show that it is possible to completely reverse miR-125b's function in apoptosis by modifying the expression levels of its target genes. Furthermore, miR-125b's function may also be altered by the presence of anticancer drugs. These results suggest that the function of a microRNA can vary substantially and is dependent on its target gene expression levels.

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. We generate a simple mathematical model to describe the transmembrane insertion process and then integrate it into a pharmacokinetic model, which predicts the tumor vs. normal tissue biodistribution of the most studied pHLIP, "wild-type pHLIP," over time after a single intravenous injection. From these models, we gain insight into the various mechanisms behind pHLIP tumor targeting and delivery, as well as the various biological parameters that influence it. Furthermore, we analyze how changing the properties of pHLIP can influence the efficacy of tumor targeting and delivery, and we predict the properties for optimal pHLIP phenotypes that have superior tumor targeting and delivery capabilities compared with wild-type pHLIP.