pH-sensitive Peptide Research Articles

Microenvironment Responsive Hydrogel Exerting Inhibition of Cascade Immune Activation and Elimination of Synovial Fibroblasts for Rheumatoid Arthritis Therapy

Rheumatoid arthritis (RA) is a progressive autoimmune disease and drug therapy has been restricted due to poor therapeutic efficacy and adverse effects. In RA synovium, dendritic cells present self-antigens to activate cascade immune pathway. Furthermore, downstream macrophages secrete high levels of pro-inflammatory cytokines; Hyperplasia of activated synovial fibroblasts (FLS) is responsible for hypoxic synovium microenvironment, secretion of cytokines/chemokines and erosion of bone/cartilage tissues. Positive feedback loop of inflammation between macrophages and FLS independent of antigen-presentation is constructed. Herein, an injectable pH-sensitive peptide hydrogel encapsulating siRNA/Methotrexate-polyethyleneimine (siMP, including sip65MP, sip38MP, siCD86MP) and Bismuthene nanosheet/Methotrexate-polyethyleneimine (BiMP) is successfully developed. Among them, siCD86MP reduces protein level of co-stimulatory molecule CD86 while sip65MP and sip38MP separately inhibit NF-κB and MAPK-p38 pathways of macrophages and FLS to suppress secretion of cytokines and MMPs. Meanwhile, reduction in anti-apoptotic property of FLS induced by inhibition of NF-κB pathway has a synergistic effect with photodynamic therapy (PDT) and photothermal therapy (PTT) mediated by BiMP for FLS elimination, effectively ameliorating hypoxic synovium microenvironment. After being injected into synovium, hydrogel responds to acidic microenvironment and serves as a reservoir for sustained drug release and inherent retention capacity of which enables cationic nanoparticles to bypass tissue barrier for precise synovium targeting. This brand-new drug delivery system combines modulating cascade immune pathway from beginning to end by RNAi and eliminating FLS for improving synovium microenvironment by phototherapy together, providing a robust strategy for clinical RA treatment.

Enhanced Adsorption Response of pH-Sensitive Peptides: The Role of Membrane Acidity.

pH-sensitive peptides bind and traverse lipid membranes in response to changes in pH. They can be used to target tumors and other acidic tissues. We investigate the influence of acidic lipids on the pH-driven adsorption of recently synthesized peptides. Using a statistical-thermodynamic theory that takes into account the acid-base chemistry of peptides and lipids, we find that the presence of acidic lipids amplifies changes in peptide surface concentration when transitioning from high to low pH. We study cyclic and linear peptides, containing tryptophan, glutamic acid, and arginine residues, examining their behavior in both neutral and acidic membranes. Membrane binding consistently results from the shallow insertion of tryptophan residues with hydrophilic residues facing the aqueous solution. Regardless of the pH, the peptide's geometry predominantly determines the orientation and distribution of residues. Notably, we find that not only the extent of adsorption is pH-sensitive but also the underlying adsorption mechanism: it is barrier-free at low pH but hindered by a large free energy barrier at high pH. Hence, under more acidic conditions, pH-sensitive peptides show facilitated adsorption both kinetically and thermodynamically.

A novel approach to pH-Responsive targeted cancer Therapy: Inhibition of FaDu cancer cell proliferation with a pH low insertion Peptide-Conjugated DGAT1 inhibitor

Targeting enzymes involved in lipid metabolism is increasingly recognized as a promising anticancer strategy. Efficient inhibition of diacylglycerol O-transferase 1 (DGAT1) can block fatty acid (FA) storage. This, in turn, triggers an increase in free polyunsaturated FA concentration, leading to peroxidation and ferroptosis. In this study, we report the development of a pH-sensitive peptide (pHLIP)-drug conjugate designed to selectively deliver DGAT1 inhibitors to cancer cells nested within the acidic microenvironment of tumors. We utilized two previously established pHLIP sequences for coupling with drugs. The study of DGAT1 conjugates in large unilamellar vesicles (LUVs) of different compositions did not reveal enhanced pH-dependent insertion compared to POPC LUVs. However, using in vitro 3D tumor spheroids, significant antiproliferative effects were observed upon exposure to pHLIP-T863 (DGAT1 inhibitor) conjugates, surpassing the inhibitory activity of T863 alone. In conclusion, our study provides the first evidence that pHLIP-based conjugates with DGAT1 inhibitors have the potential to specifically target the acidic compartment of tumors. Moreover, it sheds light on the limitations of LUV models in capturing the pH-dependency of such conjugates.

Synovium microenvironment-responsive injectable hydrogel inducing modulation of macrophages and elimination of synovial fibroblasts for enhanced treatment of rheumatoid arthritis

Rheumatoid arthritis (RA) is a progressive autoimmune disease accompanied by joint swelling, cartilage erosion and bone damage. Drug therapy for RA has been restricted due to poor therapeutic effect, recurrence and adverse effects. Macrophages and synovial fibroblasts both play important roles in the pathology of RA. Macrophages secrete large amount of pro-inflammatory cytokines, while synovial fibroblasts are tightly correlated with hypoxia synovium microenvironment, cytokine release, recruitment of pro-inflammatory cells, bone and cartilage erosion. Therefore, in this timely research, an injectable and pH-sensitive peptide hydrogel loading methotrexate (MTX) and bismuthene nanosheet/polyethyleneimine (BiNS/PEI) has been developed to reduce the activity of macrophages and eliminate over-proliferated synovial fibroblasts simultaneously. MTX can reduce the cytokine secretion of macrophages/anti-apoptosis property of synovial fibroblasts and BiNS/PEI can eliminate synovial fibroblasts via photodynamic therapy (PDT) and photothermal therapy (PTT) routes. The hydrogel was injected into the acidic inflammatory synovium for precise targeting and served as a drug reservoir for pH responsive and sustained drug release, while improving the bioavailability and reducing the toxicity of MTX. Excellent therapeutic efficacy has been achieved in both in vivo and in vitro studies, and this unique drug delivery system provides a new and robust strategy to eliminate synovial fibroblasts and modulate immune system for RA treatment in clinical.Graphic

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.

PH sensitive antimicrobial peptide with targeted activity in a mildly acidic microenvironment

Antimicrobial peptides (AMPs) are short peptides that can interact with the cellular membrane, which can actively inhibit the growth of diverse microorganisms. In recent years, AMPs have become a promising antimicrobial agent, however, the lack of selectivity and unwanted cytotoxicity of AMPs limit their development as therapeutic drugs. In this study, we employed a pH-sensitive histidine-glutamic acid (“HE”) peptide pro-sequence to modify an AMP, model amphipathic peptide (MAP), resulting in a pH-sensitive AMP termed HE-MAP. At bacterial infection sites, due to the inflammatory response and pathogen-host cell interactions, there is typically a mildly acidic pH. This microenvironment provides a unique recognition site for the selective distribution of HE-MAP. Under neutral pH, the HE pro-sequence can mask the cationic charge of MAP, therefore HE-MAP only exhibits its toxic function only under mildly acidic pH where the cationic charge is unmasked. The results from this study demonstrated that HE-MAP could specifically inhibit the growth of Escherichia coli (E. coli) and Acinetobacter baumannii (A. baumannii) under pH 6–6.5 and exhibited low cytotoxicity toward mammalian cells and erythrocytes at normal physiological pH 7.4. These results indicate that HE-MAP is potentially a new antimicrobial agent with pH sensitive selectivity.

Acid-etched layered double hydroxides armed with dual “Doorkeepers” for Immunomodulatory-NIR III photodynamic therapy of hepatocellular carcinoma

Immune checkpoint blockade (ICB) therapy for hepatocellular carcinoma (HCC) is limited due to its immunosuppressive tumor microenvironment (TME). Proinflammatory pyroptosis as a new schedule can induce immunogenic cell death (ICD), thereby reversing immunosuppressive TME and improving ICB efficiency. Herein, we report an acid-etched layered double hydroxides nanosheets (E-LDH)-based smart nanoplatforms armed with H2O2/pH dual “doorkeepers” (denoted as CTEP) targeting HCC to realize membrane-anchored photodynamic therapy (PDT) and evoke potent pyroptosis to effectively improve anti-tumor immunity. The hybrid Hepa1-6 cancer cell membrane (CM) and thylakoid (TK) membrane is cloaked on the surface of pH-(low)-insertion peptide (pHLIP) modified E-LDH to obtain CTEP. Once CTEP nanoplatforms arrive at tumor regions, the dual “doorkeepers” will orderly open to perform PDT precisely. Given the fact that TK membranes have the catalase mimetic function, CTEP is able to respond to high concentrations of H2O2 in TME and rupture the hybrid CM-TK membrane to expose pH-sensitive peptide pHLIP. Membrane-localized PDT can induce gasdermin-D (GSDMD) mediated pyroptosis, effectively stimulating tumor immunogenicity. Pyroptotic cell death releases damage-associated molecular patterns (DAMPs) and consequently robust IaCB ability. CTEP-evoked pyroptosis together with anti-PD-L1 blockade therapy activates adaptive immunity in HCC, successfully inhibiting the progression of primary and distant HCC tumors with mitigation of the side effects. This research has demonstrated that CTEP with excellent biocompatibility and homologous targeting ability can act as a potent pyroptosis inducer to improve immune efficacy. The combination of PDT and immunotherapy via a photo-pyroptosis pathway can be a smart strategy for HCC treatment.

PH Sensitive Peptide Hydrogel Encapsulating Anti-angiogenesis Drug Conbercept and Chemotherapy Drug DOX for the Combined Treatment of Retinoblastoma

Retinoblastoma(RB) is a malignant tumor originating from retina. Radiotherapy, chemotherapy, photodynamic therapy, cryotherapy and surgery are commonly used in clinical RB treatment, but the overall efficacy is poor and often...

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 .

Abstract 2715: Utilizing a pH sensitive peptide (pHLIP) for tumor targeted delivery of an immunogenic peptide motif

Abstract Chimeric antigen receptor (CAR) T cell and immune-cell therapies are revolutionizing how patients are treated in the clinic by targeting tumor-specific antigens. Specifically, advances in antigen targeting of CD20 and CD19 with CAR T cell therapy has increased overall survival of this patient population and has influenced to expand T cell repertoire to novel antigen targets. However, limitations to these cell-based therapies are the lack of development against solid tumor antigens, partly due to the 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. This project aims to artificially deliver an antigen to tumor cells by harnessing a universal property of tumors: acidic microenvironment. The pH low insertion peptide (pHLIP) is a technology that targets solid tumors based on the low pH produced by the Warburg effect. pHLIP-conjugates have undergone extensive development for drug delivery, with one notable candidate CBX12 entering phase I clinical trials in 2021. Our system uses a pHLIP-conjugated SIINFEKL peptide (derived from ovalbumin) to serve as our model antigen system, with SIINFEKL-targeting T cells (OT1) that can be used as a tool to investigate antigen delivery, recognition, and functionality. We have observed through immunofluorescence that B16 F10 mouse melanoma cells treated in vitro with the 1uM pHLIP-SIINFEKL construct under acidic conditions can recruit OT1 cells and induce a T cell synapse. Preliminary in vivo experiments in C57BL/6-Tg OT1 mice bearing B16 F10 flank tumors showed that treatment with pHLIP-SIINFEKL by intravenous administration led to a suppression of tumor growth compared to controls, with approximately 50% smaller fold change in tumor volume. Cumulatively, these initial results suggest that pHLIP-SIINFEKL can be delivered to tumors to recruit OT1 cells to engage in antigen recognition and lead to efficacy in tumor growth delay in vivo. This technology has the capability to 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 tumor targeted delivery of an immunogenic peptide motif [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 2715.

Antigen-independent tumor targeting by CBX-12 (alphalex™-exatecan) induces long-term antitumor immunity.

Aims: To determine whether antigen-independent targeting of the TOP1 inhibitor exatecan to tumor with a pH-sensitive peptide (CBX-12) produces superior synergy with immunotherapy compared with unconjugated exatecan. Materials & methods: In vitro and ex vivo functional assays were performed via FACS and ELISA assays. In vivo efficacy was evaluated in the syngeneic CT26 model. Results: CBX-12 combined with anti-PD-1 or anti-CTLA4 results in delayed tumor growth and complete response, with cured animals displaying long-term antitumor immunity. CBX-12 stimulates expression of MHC1 and PD-L1 and is an inducer of immunogenic cell death, producing long-term immune recognition of tumor cells and resultant antitumor immunity. Conclusion: The authors' data provide the rationale for exploring immunotherapy combinations with CBX-12 in clinical trials.

Cyanine Dye Coupling Mediates Self-assembly of a pH Sensitive Peptide into Novel 3D Architectures.

Synthetic multichromophore systems are of great importance in artificial light harvesting devices, organic optoelectronics, tumor imaging and therapy. Here, we introduce a promising strategy for the construction of self-assembled peptide templated dye stacks based on coupling of a de novo designed pH sensitive peptide with a cyanine dye Cy5 at its N-terminus. Microscopic techniques, in particular cryogenic TEM (cryo-TEM) and cryo-electron tomography technique (cryo-ET), reveal two types of highly ordered three-dimensional assembly structures on the micrometer scale. Unbranched compact layered rods are observed at pH 7.4 and two-dimensional membrane-like assemblies at pH 3.4, both species displaying spectral features of H-aggregates. Molecular dynamics simulations reveal that the coupling of Cy5 moieties promotes the formation of both ultrastructures, whereas the protonation states of acidic and basic amino acid side chains dictates their ultimate three-dimensional organization.

Cyanine Dye Coupling Mediates Self‐assembly of a pH Sensitive Peptide into Novel 3D Architectures

AbstractSynthetic multichromophore systems are of great importance in artificial light harvesting devices, organic optoelectronics, tumor imaging and therapy. Here, we introduce a promising strategy for the construction of self‐assembled peptide templated dye stacks based on coupling of a de novo designed pH sensitive peptide with a cyanine dye Cy5 at its N‐terminus. Microscopic techniques, in particular cryogenic TEM (cryo‐TEM) and cryo‐electron tomography technique (cryo‐ET), reveal two types of highly ordered three‐dimensional assembly structures on the micrometer scale. Unbranched compact layered rods are observed at pH 7.4 and two‐dimensional membrane‐like assemblies at pH 3.4, both species displaying spectral features of H‐aggregates. Molecular dynamics simulations reveal that the coupling of Cy5 moieties promotes the formation of both ultrastructures, whereas the protonation states of acidic and basic amino acid side chains dictates their ultimate three‐dimensional organization.

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

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

Targeting cancer cells in acidosis with conjugates between the carnitine palmitoyltransferase 1 inhibitor etomoxir and pH (low) insertion peptides

Targeting enzymes involved in tumor metabolism is a promising way to tackle cancer progression. The inhibition of carnitine palmitoyltransferase 1 (CPT1) by etomoxir (Eto) efficiently slows down the growth of various cancers. Unfortunately, the clinical use of this drug was abandoned because of hepatotoxic effects. We report the development of pH-sensitive peptide (pHLIP)-drug conjugate to deliver Eto selectively to cancer cells exposed to acidic microenvironmental conditions. A newly designed sequence for the pHLIP peptide, named pHLIPd, was compared with a previously published reference pHLIP peptide, named pHLIPr. We showed that the conjugate between pHLIPd and Eto has a better pH-dependent insertion and structuration than the pHLIPr-based conjugate inside POPC vesicles. We observed antiproliferative effects when applied on acid-adapted cancer cells, reaching a larger inhibitory activity than Eto alone. In conclusion, this study brings the first evidence that pHLIP-based conjugates with a CPT1 inhibitor has the potential to specifically target the tumor acidic compartment and exert anticancer effects while sparing healthy tissues.

PH-Sensitive Peptide Hydrogels as a Combination Drug Delivery System for Cancer Treatment.

Conventional antitumor chemotherapeutics generally have shortcomings in terms of dissolubility, selectivity and drug action time, and it has been difficult to achieve high antitumor efficacy with single-drug therapy. At present, combination therapy with two or more drugs is widely used in the treatment of cancer, but a shortcoming is that the drugs do not reach the target at the same time, resulting in a reduction in efficacy. Therefore, it is necessary to design a carrier that can release two drugs at the same site. We designed an injectable pH-responsive OE peptide hydrogel as a carrier material for the antitumor drugs gemcitabine (GEM) and paclitaxel (PTX) that can release drugs at the tumor site simultaneously to achieve the antitumor effect. After determining the optimal gelation concentration of the OE polypeptide, we conducted an in vitro release study to prove its pH sensitivity. The release of PTX from the OE hydrogel in the medium at pH 5.8 and pH 7.4 was 96.90% and 38.98% in 7 days. The release of GEM from the OE hydrogel in media with pH of 5.8 and 7.4 was 99.99% and 99.63% in 3 days. Transmission electron microscopy (TEM) and circular dichroism (CD) experiments were used to observe the microstructure of the peptides. The circular dichroism of OE showed a single negative peak shape when under neutral conditions, indicating a β-folded structure, while under acidic conditions, it presented characteristics of a random coil. Rheological experiments were used to investigate the mechanical strength of this peptide hydrogel. Furthermore, the treatment effect of the drug-loaded peptide hydrogel was demonstrated through in vitro and in vivo experiments. The results show that the peptide hydrogels have different structures at different pH values and are highly sensitive to pH. They can reach the tumor site by injection and are induced by the tumor microenvironment to release antitumor drugs slowly and continuously. This biologically functional material has a promising future in drug delivery for combination drugs.

Improving Cancer Detection and Treatment by pH‐Sensitive Peptide Nanoparticle Drug Delivery Platform: Pharmacokinetics, Toxicity, and Immunogenicity Profile

Cancer Detection In article number 2100081, I-Wei Chen, Rong Zhou, and co-workers demonstrate that a peptide-based nanodelivery platform made of pH-sensitive peptides (blue, turning red when protonated in low pH) and decorated by PEG (yellow) can be used to formulate poorly water-soluble small molecules to improve cancer diagnosis and treatment. The pH-sensitive nanoparticles, stable in blood, self-dissolve in a mildly acidic extracellular space of the tumor, thereby releasing encapsulated molecular drug cargos (silver spheres) near tumor cells.

TfR-T12 short peptide and pH sensitive cell transmembrane peptide modified nano-composite micelles for glioma treatment via remodeling tumor microenvironment

Two kinds of amphiphilic block copolymers of TfR-T12-PEG-PLGA and TATH7-PEG-PLGA were synthesized to self-assembly nano-composite micelles for encapsulating paclitaxel and imiquimod synchronously. TfR-T12 peptide modified nano-composite micelles can pass through BBB in a TfR-mediated way to achieve targeted delivery of chemotherapeutic drugs, and pH sensitive TATH7 peptide modified nano-composite micelles enhanced uptake efficiency more significantly under pH 5.5 medium than pH 7.4 medium. The results of pharmacodynamic evaluation in vivo showed that the nano-composite micelles had achieved good anti-tumor effect in subcutaneous and normotopia glioma models, and effectively prolonged the life cycle of tumor-bearing mice. The nano-composite micelles regulated the immunosuppression phenomenon of tumor microenvironment significantly, and promoted the M1 polarization of TAMs, then enhanced the proliferation and activation of CD8+ T cells in tumor microenvironment. It comes to conclusion that the nano-composite micelle achieves the purpose of effective treatment of glioma by chemotherapy combined with immunotherapy.

Abstract P047: A phase 1/2 trial of CBX-12, an alphalexTM peptide drug conjugate, in patients with advanced or metastatic refractory solid tumors

Abstract Background Tumor-targeted drug delivery technologies are urgently needed to overcome the lack of tumor selectivity, a major drawback of conventional chemotherapy. In addition, the acidic intercellular microenvironment in solid tumors traps weak acid/base chemotherapy agents, preventing necessary intracellular concentrations in tumour cells. An alphalex conjugate, which contains a low-pH insertion peptide, a linker, and a payload, is designed to overcome these limitations. Unlike antibody drug conjugates, these peptide drug conjugates 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, inserts directionally in the cell membrane delivering the linker and payload intracellularly where the linker is cleaved. CBX-12 consists of the pH-sensitive peptide, a self-immolating linker, and the topoisomerase 1 (TOP1) inhibitor exatecan. Methods CBX-12-101 is a first-in-human, open-label, dose-escalation, safety, pharmacokinetics (PK), and biomarker study of CBX-12 in patients with advanced or metastatic refractory solid tumors. CBX-12 is administered as a 1-hour intravenous infusion. Two dosing schedules, daily x 5 every 3 weeks (Part A) and daily x 3 every 3 weeks (Part B), are being evaluated. The starting dose in Part A is 0.25 mg/kg. Single-patient cohorts will be enrolled initially, with dose-escalations up to 100% of the prior dose, until a patient has a ≥ Grade 2 adverse event (AE) considered possibly related to CBX-12 during Cycle 1 (the DLT period), at which time 2 additional patients will be enrolled in that cohort, and a 3 + 3 design will subsequently be utilized. Further dose escalations may be no more than 50% of the prior dose. After at least 2 cohorts have been evaluated in Part A, Part B will open for accrual. Maximum tolerated doses and recommended phase 2 doses will be determined for each dosing schedule. The PK of the CBX-12 conjugate and free exatecan will be determined. The stability of the conjugate in circulation will be evaluated by the ratio of CBX-12 to free exatecan. Pre- and on-treatment tumor biopsies are required. Biomarker and pharmacodynamic evaluations including measuring intratumor exatecan levels, TOP1, gamma H2AX and schlafen-11 are planned. Antitumor activity will be assessed per RECIST v 1.1. Phase 2 expansion cohorts are planned in patients with platinum-resistant ovarian and small cell lung cancer. As of July 2021, patients are enrolling in Part A Cohort 2 at a dose of 0.50 mg/kg. Citation Format: Anthony Tolcher, Joseph Paul Eder, David Sommerhalder, Sophia Gayle, Paul Pearson, Deb Chapman, Arthur P. DeCillis, Anish Thomas, Funda Meric-Bernstam. A phase 1/2 trial of CBX-12, an alphalexTM peptide drug conjugate, in patients with advanced or metastatic refractory solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P047.

Kinetics of pore formation in stearoyl-oleoyl-phosphatidylcholine vesicles by pH sensitive cell penetrating peptide GALA

In order to engineer endosomal escape of drug carrying liposomes into the cytoplasm of target cells, the kinetics of bilayer poration by cell penetrating peptides needs to be well understood. To this end, we have studied pH-dependent pore formation in stearoyl-oleoyl-phosphatidylcholine vesicles as a function of concentration of the peptide GALA. Using laser scanning confocal microscopy, we measured the rate of fluorophore transport from the suspending medium into giant unilamellar vesicles across bilayer pores induced by GALA under acidic pH conditions. We also measured the mean pore size of GALA-induced pores in large unilamellar vesicles by electron microscopy. We fitted a mathematical model of pore formation kinetics to the measured rate of fluorophore transport across the giant vesicle bilayer to estimate the rate of pore formation as a function of GALA concentration. We observed that the number of pores per vesicle and the pore density increased with increasing GALA concentration