pH Low Insertion Peptide Research Articles

Tumor-specific antigen delivery for T-cell therapy via a pH-sensitive peptide conjugate.

Identifying an optimal antigen for targeted cancer therapy is challenging as the antigen landscape on cancerous tissues mimics that of healthy tissues, with few unique tumor-specific antigens identified in individual patients. pH low insertion peptides (pHLIPs) act as a unique delivery platform that can specifically target the acidic microenvironment of tumors, sparing healthy tissue in the process. We developed a pHLIP-peptide conjugate to deliver the SIINFEKL peptide, an immunogenic fragment of ovalbumin, to tumor cells in vivo. When processed intracellularly, SIINFEKL is presented for immune recognition through the major histocompatibility complex (MHC) class I pathway. We observed selective delivery of pHLIP-SIINFEKL both in vitro and in vivo using fluorescently labeled constructs. In vitro, treatment of melanoma tumor cells with pHLIP-SIINFEKL resulted in recognition by SIINFEKL-specific T cells (OT1), leading to T cell activation and effector function. Mechanistically, we show that this recognition by OT1 cells was abrogated by siRNA/shRNA knockdown of multiple components within the MHC class I pathway in the target tumor cells, indicating that an intact antigen processing pathway in the cancer cells is necessary to mediate the effect of pHLIP-directed SIINFEKL delivery. In vivo, pHLIP-SIINFEKL treatment of tumor-bearing mice resulted in recruitment of OT1 T cells and suppression of tumor growth in two syngeneic tumor models in immunocompetent mice, with no effect when mutating either the pHLIP or SIINFEKL components of the conjugate. These results suggest that pHLIP-mediated peptide delivery can be used to deliver novel artificial antigens that can be targeted by cell-based therapies.

Engraftment of a Surrogate Antigen onto Tumor Cell Surface via pHLIP Peptide to Universally Target CAR-T Cell Therapy to Solid Tumors

CAR-T cells and monoclonal antibodies (mAbs) are immunotherapeutics that have shown efficacies against certain malignancies. However, their broad application is hindered by the scarcity of tumor-associated antigens on tumor cell surfaces. Previous investigations unveiled the unique capacity of pH-low insertion peptide (pHLIP) to anchor to plasma membranes under acidic conditions. Considering that an acidic tumor microenvironment is a hallmark of solid tumors, we engineered a novel peptide, Myc-pHLIP, by tethering a surrogate epitope tag, the c-Myc-tag, to pHLIP. We evaluated the efficiency of Myc-pHLIP in inserting the artificial c-Myc-tag onto the plasma membrane of malignant cells and determined if this engraftment could convert it into a therapeutic target for CAR-T cells or mAbs. Our in vitro experiments demonstrated that incubating Myc-pHLIP with tumor cells in acidic media triggered significant killing by either Myc-targeted CAR-T cells (Myc-CAR-T), or by an anti-Myc mAb in the presence of NK cells. In vivo studies demonstrated substantial antitumor effects with sequential administration of Myc-pHLIP followed by either Myc-CAR-T or Myc-mAb. These findings establish that Myc-pHLIP has the potential to act as a universal surrogate tumor antigen capable of directing CAR-T cells or mAbs to treat any solid tumors by concurrently targeting both malignant and stromal cells.

Selective Recruitment of Antibodies to Cancer Cells and Immune Cell-mediated Killing via In Situ Click Chemistry.

Many current cancer immunotherapies function by redirecting immune system components to recognize cancer biomarkers and initiate a cytotoxic attack. The lack of a universal tumor biomarker limits the therapeutic potential of these approaches. However, one feature characteristic of nearly all solid tumors is extracellular acidity. This inherent acidity provides the basis for targeted drug delivery via the pH-low insertion peptide (pHLIP), which selectively accumulates in tumors in vivo due to a pH-dependent membrane insertion propensity. Previously, we established that we could selectively decorate cancer cells with antigen-pHLIP conjugates to facilitate antibody recruitment and subsequent killing by engineered effector cells via antibody-dependent cellular cytotoxicity (ADCC). Here, we present a novel strategy for opsonizing antibodies on target cell surfaces using click chemistry. We utilize pHLIP to facilitate selective tetrazine - trans-cyclooctene ligation of human IgGs to the cancer cell surface and induce ADCC. We demonstrate that our approach activates the primary ADCC signaling pathway via CD16a (FcγRIIIa) receptors on effector cells and induces the killing of cancer cell targets by engineered NK cells.

Pilot study of CBX-12 pharmacodynamics in patients with advanced solid tumors.

TPS3187 Background: Topoisomerase 1 (TOP1) inhibitors effectively kill a diverse range of tumor cells but have limited clinical utility due to severe off-target toxicities. Targeted therapeutic strategies have been developed to circumvent these limitations, although there remains an unmet need for antigen-agnostic tumor targeting. CBX-12 is a peptide conjugate that uses a novel pH-low insertion peptide (pHLIP) linked to TOP1 inhibitor exatecan to selectively target the low-pH tumor microenvironment and deliver the payload intracellularly. Recently published results of the first-in-human study of CBX-12 (NCT04902872) demonstrated single-agent activity in 4 patients, including a complete response in a patient with ovarian cancer. The predominant reported toxicity was myelosuppression with the most common grade 3/4 treatment-related adverse event being neutropenia in 28.6% of patients. Here we report our new pharmacodynamic (PD) biomarker-driven pilot study of CBX-12 in patients with advanced solid tumors. The primary objective of this study is to assess the effects of CBX-12 on biomarkers of DNA damage response (DDR) in biopsy specimen to establish the degree and duration of CBX-12 target engagement. The secondary objectives are to assess the effects of CBX-12 on TOP1 molecular response; determine any association between tumor TOP1 levels, DDR modulation, and plasma exatecan levels; evaluate the effect of CBX-12 on CD8+ T cell infiltration and activation in the tumor microenvironment; assess the objective response rate using RECIST v 1.1; and assess safety and tolerability of CBX-12. The planned PD endpoints aim to confirm and further enrich knowledge of the mechanisms underlying response or resistance to CBX-12 treatment, and guide future complementary treatment combination strategies. The study results may also help identify future patient populations likely to benefit from CBX-12 treatment. Methods: CBX-12 will be administered intravenously once weekly in 28-day cycles at the recommended phase 2 dose. Mandatory biopsies will be collected at baseline, 24-36 hours post first dose, and on cycle 3 day 1 to assess both short- and long-term effects of CBX-12 on the tumor. Mandatory research blood samples will be collected at specified timepoints for pharmacokinetic, circulating tumor cell, and cell-free DNA analyses. Clinical trial information: NCT05691517 .

Neutron spin echo shows pHLIP is capable of retarding membrane thickness fluctuations

Cell membranes are responsible for a range of biological processes that require interactions between lipids and proteins. While the effects of lipids on proteins are becoming better understood, our knowledge of how protein conformational changes influence membrane dynamics remains rudimentary. Here, we performed experiments and computer simulations to study the dynamic response of a lipid membrane to changes in the conformational state of pH-low insertion peptide (pHLIP), which transitions from a surface-associated (SA) state at neutral or basic pH to a transmembrane (TM) α-helix under acidic conditions. Our results show that TM-pHLIP significantly slows down membrane thickness fluctuations due to an increase in effective membrane viscosity. Our findings suggest a possible membrane regulatory mechanism, where the TM helix affects lipid chain conformations, and subsequently alters membrane fluctuations and viscosity.

Acidity-targeting transition-aided universal chimeric antigen receptor T-cell (ATT-CAR-T) therapy for the treatment of solid tumors

The use of CAR-T cells in treating solid tumors frequently faces significant challenges, mainly due to the heterogeneity of tumor antigens. This study assessed the efficacy of an acidity-targeting transition-aided universal chimeric antigen receptor T (ATT-CAR-T) cell strategy, which is facilitated by an acidity-targeted transition. Specifically, the EGFRvIII peptide was attached to the N-terminus of a pH-low insertion peptide. Triggered by the acidic conditions of the tumor microenvironment, this peptide alters its structure and selectively integrates into the membrane of solid tumor cells. The acidity-targeted transition component effectively relocated the EGFRvIII peptide across various tumor cell membranes; thus, allowing the direct destruction of these cells by EGFRvIII-specific CAR-T cells. This method was efficient even when endogenous antigens were absent. In vivo tests showed marked antigen modification within the acidic tumor microenvironment using this component. Integrating this component with CAR-T cell therapy showed high effectiveness in combating solid tumors. These results highlight the capability of ATT-CAR-T cell therapy to address the challenges presented by tumor heterogeneity and expand the utility of CAR-T cell therapy in the treatment of solid tumors.

Tumoral acidosis promotes adipose tissue depletion by fostering adipocyte lipolysis

ObjectiveTumour progression drives profound alterations in host metabolism, such as adipose tissue depletion, an early event of cancer cachexia. As fatty acid consumption by cancer cells increases upon acidosis of the tumour microenvironment, we reasoned that fatty acids derived from distant adipose lipolysis may sustain tumour fatty acid craving, leading to the adipose tissue loss observed in cancer cachexia. MethodsTo evaluate the pro-lipolytic capacities of acid-exposed cancer cells, primary mouse adipocytes from subcutaneous and visceral adipose tissue were exposed to pH-matched conditioned medium from human and murine acid-exposed cancer cells (pH 6.5), compared to naive cancer cells (pH 7.4). To further address the role of tumoral acidosis on adipose tissue loss, a pH-low insertion peptide was injected into tumour-bearing mice, and tumoral acidosis was neutralised with a sodium bicarbonate buffer. Prolipolytic mediators were identified by transcriptomic approaches and validated on murine and human adipocytes. ResultsHere, we reveal that acid-exposed cancer cells promote lipolysis from subcutaneous and visceral adipocytes and that dampening acidosis in vivo inhibits adipose tissue depletion. We further found a set of well-known prolipolytic factors enhanced upon acidosis adaptation and unravelled a role for β-glucuronidase (GUSB) as a promising new actor in adipocyte lipolysis. ConclusionsTumoral acidosis promotes the mobilization of fatty acids derived from adipocytes via the release of soluble factors by cancer cells. Our work paves the way for therapeutic approaches aimed at tackling cachexia by targeting the tumour acidic compartment.

Abstract 4163: Improving the dynamic range of pH low insertion peptides for targeted imaging of pancreatic tumors

Abstract Objective: Current intraoperative imaging modalities such as near-infrared fluorescent imaging (NIR) or ultrasound are limited due to operator experience and depth, and targeted imaging of pancreatic tumors would be beneficial. However, specific pancreatic tumor biomarkers available for targeting in a novel imaging protocol are lacking. As such, we focus on developing a novel targeted imaging paradigm utilizing multispectral optoacoustic imaging (MSOT) to target the acidic microenvironment of pancreatic tumors. Peptides know as pH low insertion peptides (pHLIPs) when bound to dye allow for contrasted imaging selectively based on pH using MSOT. We have identified the need for a pHLIP with improved dynamic range to improve imaging accuracy. Here, we synthesize the novel pHLIP V7FS for comparison to V3 with aims of improving the dynamic range of imaging with MSOT in pancreatic cancer. Methods: Using microwave chemistry, V7FS and V3 were synthesized with >90% purity. Peptides were conjugated to HiLyte 750 dye, and peptide-dye conjugation confirmed via spectroscopy. Intraoperative measurements of human pancreatic tumor and surrounding uninvolved tissue were obtained using a microsensor. Near-infrared fluorescent imaging was used to measure probe uptake in S2VP10 and S2013 pancreatic cancer cells lines grown in pH specific media (7.4, 6.8, 6.6) in replicates. MSOT was then used to image probes in phantoms mimicking tissue and then in mice with pancreatic tumors from orthotopically implanted S2VP10. Statistical analysis was performed using ANOVA. Results: Compared to surrounding uninvolved pancreatic tissue, human pancreatic tumor was more acidic with pH 6.2-6.7 vs 7.2-7.3 (p<0.01). Signal at pH 7.4, 6.8 and 6.6 was higher for V7FS-750 compared to V3-750 (p=0.0105). Signal for V7FS-750 was centered around pH 6.6 and signal for V3-750 centered around pH 6.8. Interaction between probe and pH did not reach significance (p=0.745). Peak uptake in orthotopic murine models was observed at 3 hours following intravenous injection. Conclusions: V7FS-750 was shown to have higher signal accumulation compared to V3-750 favoring a more acidic environment (pH 6.6 vs pH 6.8). This novel pHLIP was demonstrated to be viable for use in an imaging paradigm utilizing MSOT in an orthotopic murine model. Continued improvement in the dynamic range of these probes is promising for the use of pHLIPS in imaging of pancreatic cancer with MSOT. Citation Format: Ryan Bynum, Emma Sanderson, Barish H. Edil, Ajay Jain, Lacey R. McNally. Improving the dynamic range of pH low insertion peptides for targeted imaging of pancreatic tumors [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 4163.

Abstract 6758: Eradication of tumors and development of immunity from pHLIP-targeted intracellular delivery of STINGa

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.

Abstract 6759: Antigen-pHLIP for tumor 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.

Abstract 3193: Utilizing a pH sensitive peptide (pHLIP) for antigen-directed T cell therapy

Abstract Many targeted therapies, such as antibody drug conjugates (ADCs) and chimeric antigen receptor (CAR) T cell therapies, rely on the identification of an optimal antigen as the cell marker for targeting cancer cells. However, there is a paucity of antigen candidates that are sufficiently specific to epithelial cancers. Therefore, technologies that can mark tumor cells for immune recognition could prove beneficial in the treatment of these solid cancers. pH low insertion peptides (pHLIPs) act as a unique delivery platform that can specifically target the acidic microenvironment of tumors, sparing healthy tissue in the process. We developed a pHLIP-peptide conjugate (pHLIP-SIINFEKL) to deliver the SIINFEKL peptide, an immunogenic fragment of ovalbumin, to investigate antigen delivery and recognition to tumors in vivo. When processed intracellularly, SIINFEKL is presented for immune recognition through the major histocompatibility complex (MHC) class I pathway. We observed selective delivery of pHLIP-SIINFEKL both in vitro and in vivo using fluorescently labeled constructs. In vitro, treatment of melanoma tumor cells with pHLIP-SIINFEKL resulted in recognition by SIINFEKL-specific T cells (OT1), leading to T cell activation and effector function. Mechanistically, we show that this recognition by OT1 cells was abrogated by siRNA knockdown of the β2 microglobulin (β2m) component of MHC class I in the target tumor cells, indicating that an intact antigen processing pathway in the cancer cells is necessary to mediate the effect of pHLIP-directed SIINFEKL delivery. In vivo, pHLIP-SIINFEKL treatment of tumor-bearing mice resulted in recruitment of OT1 T cells and suppression of tumor growth in two syngeneic tumor models in immunocompetent mice, with no effect when mutating either the pHLIP or SIINFEKL components of the conjugate. These results suggest that pHLIP-mediated peptide delivery can provide a novel approach to decorate solid tumors with engineered antigens for enhanced immune recognition and anti-tumor efficacy. Citation Format: Annali M. Yurkevicz, Yanfeng Liu, Peter M. Glazer. Utilizing a pH sensitive peptide (pHLIP) for antigen-directed T cell therapy [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 3193.

Abstract 731: pHLIP targeted intracellular delivery of calicheamicin

Abstract Calicheamicin is a potent, cell-cycle independent enediyne antibiotic that binds and cleaves DNA. Toxicity has led to its approved use in a targeted form as antibody-drug conjugates (ADC) for the treatment of liquid tumors. Mylotarg, one of the first such ADCs, was voluntarily withdrawn from the market due to safety concerns. However, Mylotarg was recently reapproved for acute myeloid leukemia based on new data that demonstrated efficacy with an acceptable safety profile. Besponsa, a related ADC, was approved for acute lymphocytic leukemia in 2017. In both agents, Mylotag and Besponsa, calicheamicin is conjugated to antibodies via an acid-labile hydrazone linker. 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) via a disulfide bond to generate a linkerless cytoplasmic release. The cytoplasmic reduction of the disulfide releases the calicheamicin, which leads to its activation, DNA binding, and strand scission. pHLIP is a tumor-targeting agent now in clinical trials with imaging and therapeutic payloads. 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 mice, treatment led to the growth inhibition of immuno-suppressive CT26 tumors with no signs of toxicity. Biodistribution studies confirmed tumor targeting with no accumulation of the agent in organs and tissues. The fluorescent version of the agent was observed within the tumor mass and tumor-stroma interface, where CD206+ M2 tumor-associated macrophages (M2-TAMs) reside. Treatment of tumors led to the depletion of M2-TAMs within the tumor core. Thus, pHLIP-calicheamicin could be developed into an effective therapeutic for the treatment of solid tumors with abundant immuno-suppressive M2-TAMs. Citation Format: Michael DuPont, Craig Klumpp, Marissa Iraca, Dana Allababidi, Hannah Visca, Donald Engelman, Oleg Andreev, Anna Moshnikova, Yana Reshetnyak. pHLIP targeted intracellular delivery of calicheamicin [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 731.

Abstract 462: Developing the miR-888 cluster as a therapeutic target for prostate cancer

Abstract Prostate Cancer (PCa) is a serious health crisis in the United States. 20% of men diagnosed with PCa will progress to fast-growing, advanced disease. There are no curative treatment options for PCa that has spread to distant sites and the 5-year survival rate for metastatic disease remains 30%. Recognizing the need for better PCa therapeutics, we are developing novel anti-microRNA delivery reagents targeted against the noncoding RNA miR-888 cluster to block prostate tumor progression. Our lab identified these oncomiRs in a biomarker discovery screen as elevated in human metastatic PCa cell lines and prostatic fluids from patients with high-grade PCa. We noted that over-expression of miR-888 cluster members (miR-888, miR-891a) promoted prostate cell growth and induced invasiveness in vitro. miR-888 and miR-891a also accelerated xenograft tumor growth in mice. Treatment of aggressive PCa cells in culture with synthetic antisense inhibitors against individual cluster members reversed these growth and invasion phenotypes, highlighting their clinical potential. We are testing pH Low Insertion Peptide (pHLIP)-Peptide Nucleic Acid (PNA)-based antimiR conjugates against miR-888 and miR-891a. The delivery vehicle pHLIP is a 36-amino acid peptide that at low pH (< pH 6.5) adopts an alpha-helical conformational change and facilitates the insertion of its C-terminus across lipid bilayers to transport conjugated anti-miRNA cargo into cells. When administered systemically, pHLIP effectively targets solid tumors with acidic microenvironments. We found that these reagents are readily internalized by human PCa cells cultured at pH 6.0 and in prostate xenograft tumors when delivered systemically in mice. Treatment significantly repressed miR-888 and miR-891a and acted to slow prostate cell growth in vitro. We are currently moving these studies into mouse prostate models. Furthermore, we are investigating the downstream signaling pathways for the miR-888 cluster. Our work indicates that the miR-888 cluster network coordinates the repression of the Tissue Inhibitors of Metalloproteinase (TIMP) and SMAD4 anti-metastatic pathways to drive extracellular matrix degradation and prostate cell invasion, and as a consequence, induces matrix metalloproteinase (MMP) activity. Expression profiling arrays and luciferase reporter assays indicated that multiple members of the miR-888 cluster co-suppress TIMPs in the prostate and similarly elevate multiple MMPs as well as cadherins, fibronectin and heparinase, and co-modulated several immune response genes. We are currently studying the consequence of various CRISPR-mediated miR-888 cluster deletions to understand how this non-coding RNA cluster functionally overlaps to promote aggressive PCa. This work will establish new clinical tools for aggressive PCa and optimize patient treatment management. Citation Format: Katherine Routon, Trevor Fachko, Vishal Kasina, Raman Bahal, Aurora Esquela Kerscher. Developing the miR-888 cluster as a therapeutic target for prostate cancer [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 462.

Abstract 4160: V7 targeted liposomes detect pancreatic adenocarcinoma via multispectral optoacoustic tomography

Abstract Purpose: Pancreatic ductal adenocarcinoma (PDA) remains one of the most aggressive malignancies with a 5-year survival rate of 9-10%. Insufficient early detection coupled with limitations in operating surgery renders PDA as one of the most challenging tumors to treat. Accurate localization of tumor margins can tremendously improve surgical treatment outcomes in patients. Recently, nanotechnology has emerged as a promising platform for theranostic applications in the field of drug discovery, although the clinical success is limited by their tumor-specific uptake. Hypoxia due to aberrant vascularization is a hallmark of PDA and can be exploited for active targeting. pH-low insertion peptide variant 7 (V7) is used as hypoxia and acidosis-dependent biomarker for selective targeting of pancreatic cancer tissue. This work is focused on using IR 780 contrast agent loaded, V7 peptide conjugated liposomes for the active targeting of PDA towards enhanced surgical outcomes. Methods: Liposomes encapsulating IR780 dye were prepared using thin film hydration technique. V7 peptide was conjugated to liposomes using an SMCC linker and liposomes were characterized for their size, polydispersity and surface potential. In-vitro uptake of IR780-V7-liposome by S2VP10 at pH 7.4, 6.8, and 6.6 was evaluated through NIR fluorescence imaging using an Odyssey Infrared System. Athymic mice were orthotopically implanted with S2VP10 cells. After tumors were 3 mm, V7-liposomes and untargeted liposomes (0.01 OD) were intravenously injected and biodistribution and tumor specific uptake was accessed using multispectral optoacoustic imaging (MSOT). Results: Dynamic light scattering (DLS) revealed the size of liposomes to be 109 nm with a polydispersity index of 0.198 and zeta potential of +35.8 mV. Surface conjugation of V7 peptide was confirmed using the zeta potential measurement. NIR fluorescence imaging in S2VP10 cells established enhanced tumor specific uptake of V7 conjugated liposomes than passively targeted liposomes (p<0.05). Biodistribution of V7-liposomes demonstrated tumor specific accumulation (PDAC tumor 23.7 a.u, liver 3.6 a.u., and kidney 1.1 a.u.) while untargeted liposomes had the greatest uptake in liver (PDAC tumor 2.1 a.u., liver 6.3 a.u., and kidney 0.7 a.u.) p<0.05. Conclusion: pH-specific actively targeted liposomes can be used to specifically target PDA with minimal off-target binding effects. V7-IR780-liposome formulation can be used to efficiently deliver contrast agent for imaging tumor margins and to expand the potential of MSOT towards clinical applications. Citation Format: Happy Agarwal, Phillip Chuong, William E. Grizzle, Barish H. Edil, Ajay Jain, Lacey R. McNally. V7 targeted liposomes detect pancreatic adenocarcinoma via multispectral optoacoustic tomography [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 4160.

Biosynthesized tumor acidity and MMP dual-responsive plant toxin gelonin for robust cancer therapy.

Among all kinds of anticancer agents, small molecule drugs produce an unsatisfactory therapeutic effect due to the lack of selectivity, notorious drug resistance and side effects. Therefore, researchers have begun to pay extensive attention to macromolecular drugs with high efficacy and specificity. As a plant toxin, gelonin exerts potent antitumor activity via inhibiting intracellular protein synthesis. However, gelonin lacks a translocation domain, and thus its poor cellular uptake leads to low outcomes of antitumor response. Here, tumor acidity and matrix metalloproteinase (MMP) dual-responsive functional gelonin (Trx-PVGLIG-pHLIP-gelonin, TPpG), composed of a thioredoxin (Trx) tag, a pH low insertion peptide (pHLIP), an MMP-responsive motif PVGLIG hexapeptide and gelonin, was innovatively proposed and biologically synthesized by a gene recombination technique. TPpG exhibited good thermal and serum stability, showed MMP responsiveness and could enter tumor cells under weakly acidic conditions, especially for MMP2-overexpressing HT1080 cells. Compared to low MMP2-expressing MCF-7 cells, TPpG displayed enhanced in vitro antitumor efficacy to HT1080 cells at pH 6.5 as determined by different methods. Likewise, TPpG was much more effective in triggering cell apoptosis and inhibiting protein synthesis in HT1080 cells than in MCF-7 cells. Intriguingly, with enhanced stability and pH/MMP dual responsiveness, TPpG notably inhibited subcutaneous HT1080 xenograft growth in mice and no noticeable off-target side effect was observed. This ingeniously designed strategy aims at providing new perspectives for the development of a smart platform that can intelligently respond to a tumor microenvironment for efficient protein delivery.

Targeting acidic pre-metastatic niche in lungs by pH low insertion peptide and its utility for anti-metastatic therapy.

Dysregulated extracellular pH, the universal feature of tumor, works as an evolutional force to drive dissemination of tumor cells. It is well-established that tumor acidity is associated with tumor growth and metastasis. However, the pH of pre-metastatic niche remains unclear. We hypothesized that primary tumor cells remotely prime acidity in secondary organ to achieve metastatic colonization. Herein, we demonstrated that the pH responsive probe pH Low Insertion Peptide (pHLIP) was notably accumulated in pre-metastatic lungs of 4T1.2 breast tumor-bearing mice. The pHLIP-targeted lungs showed high amounts of lactate and overexpressed glycolysis-related proteins. Pharmacological inhibition of glycolysis suppressed the lung acidification induced by 4T1.2 cancer cell culture supernatant and delayed subsequent metastatic burden of disseminated tumor cells. In the acidic lungs, pHLIP was primarily localized in alveolar type 2 cells which strongly expressed glycolysis-related proteins. 4T1.2-derived extracellular vesicles expressed some of the glycolysis-related proteins, and their administration increased pHLIP accumulation and glycolytic enhancement in lungs. pHLIP-conjugated dexamethasone effectively attenuated lung metastatic burden by disrupting pro-inflammatory response in the acidic lungs. From these results, targeting the metastasis-supporting microenvironment by pHLIP technology creates possibility to identify pre-metastatic organ and prevent metastatic recurrence.

D-to-E and T19V Variants of the pH-Low Insertion Peptide and Their Doxorubicin Conjugates Interact with Membrane at Higher pH Ranges Than WT.

To improve targeted cargo delivery to cancer cells, pH-Low Insertion Peptide (pHLIP) variants were developed to interact with the membrane at pH values higher than those of the WT. The Asp-to-Glu variants aim to increase side chain pKa without disturbing the sequence of protonations that underpin membrane insertion. The Thr19 variants represent efforts to perturb the critical Pro20 residue. To study the effect of cargo on pHLIP insertion, doxorubicin (Dox), a fluorescent antineoplastic drug, was conjugated to selected variants near the inserting C-terminus. Variants and conjugates were characterized on a POPC membrane using Trp and Dox fluorescence methods to define the entire pH range of insertion (pHinitial-pHfinal). Compared to WT with a pHi-pHf range of 6.7-5.6, D25E-D31E-D33E, D14E-D25E-D31E-D33E, and T19V-D25E variants demonstrated higher pHi-pHf ranges of 7.3-6.1, 7.3-6.3, and 8.2-5.4, respectively. The addition of Dox expanded the pHi-pHf range, mainly by shifting pHi to higher pH values (e.g., WT pHLIP-Dox has a pHi-pHf range of 7.7-5.2). Despite the low Hill coefficient observed for the conjugates, D14E-D25E-D31E-D33E pHLIP-Dox completed insertion by a pHf of 5.7. However, the Dox cargo remained in the hydrophobic membrane interior after pHLIP insertion, which may impede drug release. Finally, a logistic function can describe pHLIP insertion as a peripheral-to-TM (start-to-finish) two-state transition; wherever possible, we discuss data deviating from such sigmoidal fitting in support of the idea that pH-specific intermediate states distinct from the initial peripheral state and the final TM state exist at intervening pH values.

Tumor-Targeted Delivery of IL-2 by Fusing with a pH Low Insertion Peptide for Antitumor Immunotherapy.

As a pleiotropic cytokine, interleukin-2 (IL-2) can effectively regulate lymphocyte proliferation, survival, and active antitumor immune responses in tumor microenvironments. Although the ability of IL-2 to boost immune responses was reported in cancer patients, its short circulating half-life and high toxicity hinder its broad and continual clinical application. Herein, we developed a novel tumor target agent by fusing pH low insertion peptides (pHLIP) with IL-2, forming the fusion protein pHLIP-IL2. Based on the low pH insertion property of pHLIP, the pHLIP-IL2 fusion protein could be selectively delivered to the acidic tumor microenvironments and then promote the proliferation of killer immune cells to elicit tumor regression. We found that pHLIP-IL2 fusion proteins can be significantly enriched in tumor tissues and can effectively reduce tumor size in diverse tumor models, including breast cancer and melanoma, without apparent adverse effects. These data suggest that the pHLIP-IL2 fusion protein may be a promising solution for the continual and extensive application of IL-2, and pHLIP-IL2 is a potential and valuable therapeutic drug for cancer patients with antitumor immunotherapy.

Ambient Light Resistant Shortwave Infrared Fluorescence Imaging for Preclinical Tumor Delineation via the pH Low-Insertion Peptide Conjugated to Indocyanine Green.

Shortwave infrared (900-1,700 nm) fluorescence imaging (SWIRFI) has shown significant advantages over visible (400-650 nm) and near-infrared (700-900 nm) fluorescence imaging (reduced autofluorescence, improved contrast, tissue resolution, and depth sensitivity). However, there is a major lag in the clinical translation of preclinical SWIRFI systems and targeted SWIRFI probes. Methods: We preclinically show that the pH low-insertion peptide conjugated to indocyanine green (pHLIP ICG), currently in clinical trials, is an excellent candidate for cancer-targeted SWIRFI. Results: pHLIP ICG SWIRFI achieved picomolar sensitivity (0.4 nM) with binary and unambiguous tumor screening and resection up to 96 h after injection in an orthotopic breast cancer mouse model. SWIRFI tumor screening and resection had ambient light resistance (possible without gating or filtering) with outstanding signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) values at exposures from 10 to 0.1 ms. These SNR and CNR values were also found for the extended emission of pHLIP ICG invivo (>1,100 nm, 300 ms). Conclusion: SWIRFI sensitivity and ambient light resistance enabled continued tracer clearance tracking with unparalleled SNR and CNR values at video rates for tumor delineation (achieving a tumor-to-muscle ratio above 20). In total, we provide a direct precedent for the democratic translation of an ambient light resistant SWIRFI and pHLIP ICG ecosystem, which can instantly improve tumor resection.

CBX-12-101: A first-in-human study of CBX-12, an alphalex peptide drug conjugate (PDC) in patients (pts) with advanced or metastatic solid tumors.

3087 Background: Tumor-targeted drug delivery technologies are urgently needed to overcome conventional chemotherapy’s lack of tumor selectivity. Alphalex conjugates, which contain a pH-low insertion peptide (pHLIP), a linker and a payload, are designed to overcome this lack of selectivity. Unlike antibody drug conjugates, alphalex PDCs target tumors in an antigen-agnostic manner. At pH ≥7.0, the peptide is unstructured. In the low-pH tumor microenvironment the peptide forms an alpha helix and inserts directionally into the tumor cell membrane delivering the linker and payload intracellularly where the linker is cleaved releasing the payload in the cytosol. CBX-12 consists of the pH-sensitive peptide, a self-immolating linker, and the topoisomerase 1 (TOP1) inhibitor exatecan. Methods: In this Phase 1 trial, cohorts of pts were treated with escalating doses of CBX-12 in a 3 + 3 design on three dosing schedules: daily x 5 every 3 weeks, daily x 3 every 3 weeks or once weekly. The primary objectives are safety, tolerability and to determine the MTD and/or RP2D. Secondary and exploratory objectives include evaluation of plasma PK of CBX-12 and exatecan, anti-tumor activity per RECIST v1.1, and measurement of anti-drug antibodies. Results: As of 26 Jan 2023, 42 pts have been treated on three schedules. The most frequent treatment-related AEs (TRAEs) were anaemia (42.9%), nausea (38.1%), fatigue (33.3%), ANC and WBC decreased (33.3% each), vomiting (26.2%), diarrhoea (23.8%) and thrombocytopenia (19.0%). The most frequent Gr3/4 TRAEs were ANC decreased (28.6%), anemia (23.8%), WBC decrease (16.7%), thrombocytopenia (14.3%). Dose limiting toxicity was observed in 6 pts: myelosuppression (6 pts) febrile neutropenia (2 pts), and sepsis (2 pts). Responses were observed in 2 of 9 pts with ovarian cancer and 2 of 8 with breast cancer. The MTD on the daily x 3 schedule is 45mg/m2. Dose evaluation continues on the weekly schedule. Conclusions: In this FIH study of a pH-targeting alphalex PDC, CBX-12 demonstrated single-agent antitumor activity including 4 responses with the dominant toxicity of myelosuppression. Phase 2 expansion cohorts are planned in pts with platinum-resistant ovarian cancer and hormone-receptor positive, HER2 negative breast cancer. Clinical trial information: NCT04902872 .