Folate Receptor Expression Research Articles

Theranostic pH-sensitive nanoparticles for highly efficient targeted delivery of doxorubicin for breast tumor treatment.

A multifunctional theranostic nanoplatform integrated with environmental responses has been developed rapidly over the past few years as a novel treatment strategy for several solid tumors. We synthesized pH-sensitive poly(β-thiopropionate) nanoparticles with a supermagnetic core and folic acid (FA) conjugation (FA-doxorubicin-iron oxide nanoparticles [FA-DOX@ IONPs]) to deliver an antineoplastic drug, DOX, for the treatment of folate receptor (FR)-overexpressed breast cancer. In addition to an imaging function, the nanoparticles can release their payloads in response to an environment of pH 5, such as the acidic environment found in tumors. After chemical (1H nuclear magnetic resonance) and physical (morphology and super-magnetic) characterization, FA-DOX@IONPs were shown to demonstrate pH-dependent drug release profiles. Western blotting analysis revealed the expression of FRs in three breast cancer cell lines, MCF-7, BT549, and MD-MBA-231. The cell counting kit-8 assay and transmission electron microscopy showed that FA-DOX@IONPs had the strongest cytotoxicity against breast cancer cells, compared with free DOX and non-FR targeted nanoparticles (DOX@IONPs), and caused cellular apoptosis. The FA-DOX@IONP-mediated cellular uptake and intracellular internalization were clarified by fluorescence microscopy. FA-DOX@IONPs plus magnetic field treatment suppressed in vivo tumor growth in mice to a greater extent than either treatment alone; furthermore, the nanoparticles exerted no toxicity against healthy organs. Magnetic resonance imaging was successfully applied to monitor the nanoparticle accumulation. Our results suggest that theranostic pH-sensitive nanoparticles with dual targeting could enhance the available therapies for cancer.

Aconitic acid derived carbon dots: Conjugated interaction for the detection of folic acid and fluorescence targeted imaging of folate receptor overexpressed cancer cells

Herein, we utilized aconitic acid (AA) as the precursor for preparing carbon dots (AA-CDs) by a facile hydrothermal reaction, and the resulting AA-CDs exhibited bright blue fluorescence with an absolute quantum yield of 56.5% in aqueous solution. Preliminary experiments showed that the prepared AA-CDs without further surface passivation or modification could selectively interact with folic acid (FA), resulting in significant fluorescence quenching. Based on this, a sensitive method for FA analysis was developed with a detection limit of 40 nM (S/N = 3), which was then successfully applied for the detection of FA in food and pharmaceutical samples with the average recoveries of 95.0–105.3%. Furthermore, a weak fluorescent nanoprobe (FA-AA-CDs) was fabricated based on the conjugated interaction of FA and AA-CDs, and its feasibility was evaluated as a fluorescence turn-on nanoprobe for targeted imaging of cancer cells using Hela, SMMC-7721, and A549 cells as models that expressed different levels of folate receptors (FRs) on the cell surface. The results exhibited that the fluorescence intensity of these cells was in accordance with their FRs expression levels, testifying its potential of FA-AA-CDs for targeted imaging of cancer cells. Subsequently, the internalization mechanism of FA-AA-CDs into cells was elucidated via receptor-mediated endocytosis using control experiments.

Dual-mode fluorescence biosensor platform based on T-shaped duplex structure for detection of microRNA and folate receptor

In this work, a microfluidic paper-based fluorescent sensor device was constructed for quantitative detection of microRNA (miRNA) and determination of the expression of folate receptor (FR) on the cell surface. It is worth pointing out that simultaneous detection of two biomarkers in cancer was achieved through dynamic self-assembly of T-shaped duplex structure. T-shaped duplex structure can respond to specific triggers and convert to an output due to the conformation changes, it is meaningful to fabricate T-shaped duplex structure into powerful biosensors that can monitor chemical stimuli. Moreover, lamellar MnO2 nanosheets were synthesized, which exhibited excellent quenching ability via adsorbed fluorophore-labeled ssDNA, but the adsorption of the dsDNA was significantly lower than ssDNA because of the shielding of nucleobase pairs by the phosphate groups. This newly designed biosensor provided a sensitive detection of miR-21 from 0.01 to 5.0 fM with a relatively low detection limit of 0.0033 fM. Besides, the present sensing system has a detection of FR in the range from 2.0 to 30.0 ng/mL with the detection limit of 0.667 ng/mL. As biological markers for disease diagnosis, the detection of FR and miRNA will provide a new approach and therapeutic tool for early clinical diagnosis.

Enhanced and Selective Antiproliferative Activity of Methotrexate-Functionalized-Nanocapsules to Human Breast Cancer Cells (MCF-7).

Methotrexate is a folic acid antagonist and its incorporation into nanoformulations is a promising strategy to increase the drug antiproliferative effect on human breast cancer cells by overexpressing folate receptors. To evaluate the efficiency and selectivity of nanoformulations containing methotrexate and its diethyl ester derivative, using two mechanisms of drug incorporation (encapsulation and surface functionalization) in the in vitro cellular uptake and antiproliferative activity in non-tumoral immortalized human keratinocytes (HaCaT) and in human breast carcinoma cells (MCF-7). Methotrexate and its diethyl ester derivative were incorporated into multiwall lipid-core nanocapsules with hydrodynamic diameters lower than 160 nm and higher drug incorporation efficiency. The nanoformulations were applied to semiconfluent HaCaT or MCF-7 cells. After 24 h, the nanocapsules were internalized into HaCaT and MCF-7 cells; however, no significant difference was observed between the nanoformulations in HaCaT (low expression of folate receptors), while they showed significantly higher cellular uptakes than the blank-nanoformulation in MCF-7, which was the highest uptakes observed for the drug functionalized-nanocapsules. No antiproliferative activity was observed in HaCaT culture, whereas drug-containing nanoformulations showed antiproliferative activity against MCF-7 cells. The effect was higher for drug-surface functionalized nanocapsules. In conclusion, methotrexate-functionalized-nanocapsules showed enhanced and selective antiproliferative activity to human breast cancer cells (MCF-7) being promising products for further in vivo pre-clinical evaluations.

Hyaluronic Acid-Based Optical Probe for the Diagnosis of Human Osteoarthritic Cartilage.

Osteoarthritis is typically caused by cartilage injury, followed by localized inflammatory responses and tissue deterioration. Early treatment of osteoarthritis is often impossible due to the lack of diagnostic options. Recent studies have supported that different imaging probes can be used for arthritis detection in mice. However, none of these diagnostic tools have been tested on human articular cartilage. To fill this gap, an optical imaging probe was developed to target activated macrophages and the accumulation of imaging probes on tissue was used to assess the severity of human osteoarthritis.Methods: The probe was fabricated using hyaluronic acid (HA) particles conjugated with near-infrared dye and folic acid (FA). The ability of the FA-HA probes to detect activated macrophages and quantify cartilage injury was evaluated using a cell culture model in vitro and human osteoarthritic cartilage explants ex vivo.Results: Our cell study results supported that the FA-HA probes are cell compatible (up to 0.5mg/mL) and can detect activated macrophages in 30 minutes. Using human articular cartilage, we verified the existence of activated macrophages on osteoarthritic cartilage with highly up-regulated expression of folate receptors (~13 folds by comparison with healthy control). In addition, we found that FA-HA probes had higher binding amounts (~3 folds) to osteoarthritic tissue than healthy ones. Histological analyses confirmed that there was a strong linear relationship (R=0.933) between the fluorescent intensity of tissue-associated probe and the extent of folate receptors on osteoarthritic cartilage. Finally, the co-localization of the imaging probe, folate receptors and cartilage degeneration on the tissue sections indicated the extraordinary accuracy and efficiency of this osteoarthritis diagnostic probe.Conclusions: Our results support the probe as an effective diagnostic tool to detect the area and severity of osteoarthritic human articular cartilage.

Large Stokes-shifted NIR-emission from nanospace-induced aggregation of perylenemonoimide-doped polymer nanoparticles: imaging of folate receptor expression.

The development of a water-soluble, perylenemonoimide (PMI) dye-doped polymer nanoparticle (PNP) with NIR emission for live-cell imaging is demonstrated. The large Stokes-shifted NIR emission is due to confined nanospace-induced aggregation offered by the polymer matrix. Later, folic acid functionalised PNP (PNP-FA) is successfully employed to differentiate folate receptor positive and negative cancer cells.

Enhancing tumor response to targeted chemotherapy through up-regulation of folate receptor α expression induced by dexamethasone and valproic acid

Several folate-drug conjugates are currently undergoing clinical trials for application in oncology. However, the efficacy of folate-targeted therapy strongly depends on the folate receptor (FR) abundance at the surface of cancer cells. Recently, it has been postulated that up-regulation of FRα by means of chemo-sensitizing agents could enhance the anticancer activity of FR-drug conjugates. In this study, we demonstrate in vitro that a combination of dexamethasone (Dexa) and valproic acid (VPA) increases FRα expression selectively at the surface of FR-overexpressing cancer cells. The same stimulation was observed in vivo in KB-tumor xenografts when mice are treated with this combined treatment. This effect is reversible since treatment interruption induces the return of FR expression at basal level. When incubated with Dexa and VPA, the β-galactosidase-responsive folate-monomethyl auristatin E (MMAE) conjugate, called MGAF, exhibits higher cytotoxic activity on several FR-positive human cancer cell lines, compared to its administration as a single agent. This improved toxicity results from the enhanced concentration of MMAE released within cancer cells after internalization and subsequent enzymatic activation of MGAF. Higher deposition of MMAE is also observed in vivo after up-regulation of FR expression level in tumor xenografts, induced by the prior administration of the Dexa/VPA combination. In this model, MGAF/Dexa/VPA combined therapy results in an 81% inhibition of tumor growth compared to the control group, while MGAF used in monotherapy is inefficient. Since Dexa and VPA are currently used in humans, this finding could be of great interest for further development of folate-drug conjugates, in particular for those that are presently under clinical investigation.

In vivo MR imaging of folate-receptor expression with the folate-specific nanospheres in a C6 glioblastoma model

Purpose: To assess the ability of magnetic albumin nanospheres conjugated with folate (FA-MAN) to provide FR-specific enhancement of C6 glioblastoma on magnetic resonance (MR) images.Procedures: Active targeting effect of magnetic albumin nanospheres conjugated with folate (FA-MAN) was evaluated based on MR images and histopathological analysis. MR imaging of subcutaneously transplanted C6 glioblastomas was performed after intravenous injection of FA-MAN, non-targeted (magnetic albumin nanospheres, MAN) and FA-inhibited (magnetic albumin nanospheres conjugated with folate plus folate, FA-MAN + FA) agents at designated time points. The T2 relaxation times in tumors were compared among different treatment groups and were correlated with histopathological findings. Prussian blue staining and in vivo toxicology assay were also performed simultaneously.Results: Upon MR imaging in vivo, T2 relaxation time of the tumor sites in the group administrated with FA-MAN (T2 is 49 ms, 46 ms and 45 ms at 24 h, 48 h and 72 h, respectively) has statistical difference compared to those in the groups of MAN (T2 is 56 ms, 56 ms and 61 ms at 24 h, 48 h and 72 h, respectively) and FA-MAN + FA nanospheres (T2 is 56 ms, 57 ms and 56 ms at 24 h, 48 h and 72 h, respectively). Prussian blue-stained results demonstrated that more iron particles accumulated in the tumors of the targeted group than those of the other groups. Toxicology assay showed that no noticeable body weight losses were observed after monitoring 31 days, and the results of routine blood parameters, liver and kidney function biomarkers also demonstrated that the nanoshperes did not influence the respectively physiological index. Besides, no obvious pathological injuries on the major organs were examined.Conclusion: Folate-conjugated magnetic albumin nanospheres were more effective in targeting C6 glioblastoma in vivo.

Nanoparticle targeted folate receptor 1-enhanced photodynamic therapy for lung cancer

ObjectiveDespite modest improvements, the prognosis of lung cancer patients has still remained poor and new treatment are urgently needed. Photodynamic therapy (PDT), the use of light-activated compounds (photosensitizers) is a treatment option but its use has been restricted to central airway lesions. Here, we report the use of novel porphyrin-lipid nanoparticles (porphysomes) targeted to folate receptor 1 (FOLR1) to enhance the efficacy and specificity of PDT that may translate into a minimally-invasive intervention for peripheral lung cancer and metastatic lymph nodes of advanced lung cancer. Materials and methodsThe frequency of FOLR1 expression in primary lung cancer and metastatic lymph nodes was first analyzed by human tissue samples from surgery and endobronchial ultrasonography-guided transbronchial needle aspiration (EBUS-TBNA). Confocal fluorescence microscopy was then used to confirm the cellular uptake and fluorescence activation in lung cancer cells, and the photocytotoxicity was evaluated using a cell viability assay. In vivo fluorescence activation and quantification of uptake were investigated in mouse lung orthotopic tumor models, followed by the evaluation of in vivo PDT efficacy. ResultsFOLR1 was highly expressed in metastatic lymph node samples from patients with advanced lung cancer and was mainly expressed in lung adenocarcinomas in primary lung cancer. Expression of FOLR1 in lung cancer cell lines corresponded with the intracellular uptake of folate-porphysomes in vitro. When irradiated with a 671nm laser at a dose of 10J/cm2, folate-porphysomes showed marked therapeutic efficacy compared with untargeted porphysomes (28% vs. 83% and 24% vs. 99% cell viability in A549 and SBC5 lung cancer cells, respectively). Systemically-administered folate-porphysomes accumulated in lung tumors with significantly enhanced disease-to-normal tissue contrast. Folate-porphysomes mediated PDT successfully inhibited tumor cell proliferation and activated tumor cell apoptosis. ConclusionFolate-porphysome based PDT shows promise in selectively ablating lung cancer based on FOLR1 expression in these preclinical models.

Mechanistic Target of Rapamycin Is a Novel Molecular Mechanism Linking Folate Availability and Cell Function

Folate deficiency has been linked to a wide range of disorders, including cancer, neural tube defects, and fetal growth restriction. Folate regulates cellular function mediated by its involvement in the synthesis of nucleotides, which are needed for DNA synthesis, and its function as a methyl donor, which is critical for DNA methylation. Here we review current data showing that folate sensing by mechanistic target of rapamycin (mTOR) constitutes a novel and distinct pathway by which folate modulates cell functions such as nutrient transport, protein synthesis, and mitochondrial respiration. The mTOR signaling pathway responds to growth factors and changes in nutrient availability to control cell growth, proliferation, and metabolism. mTOR exists in 2 complexes, mTOR complex (mTORC) 1 and mTORC2, which have distinct upstream regulators and downstream targets. Folate deficiency in pregnant mice caused a marked inhibition of mTORC1 and mTORC2 signaling in multiple maternal and fetal tissues, downregulation of placental amino acid transporters, and fetal growth restriction. In addition, folate deficiency in primary human trophoblast (PHT) cells resulted in inhibition of mTORC1 and mTORC2 signaling and decreased the activity of key amino acid transporters. Folate sensing by mTOR in PHT cells is independent of the accumulation of homocysteine and requires the proton-coupled folate transporter (PCFT; solute carrier 46A1). Furthermore, mTORC1 and mTORC2 regulate trophoblast folate uptake by modulating the cell surface expression of folate receptor α and the reduced folate carrier. These findings, which provide a novel link between folate availability and cell function, growth, and proliferation, may have broad biological significance given the critical role of folate in normal cell function and the multiple diseases that have been associated with decreased or excessive folate availability. Low maternal folate concentrations are linked to restricted fetal growth, and we propose that the underlying mechanisms involve trophoblast mTOR folate sensing resulting in inhibition of mTORC1 and mTORC2 and downregulation of placental amino acid transporters.

Abstract 4017: Development and application of an immunohistochemistry-based assay for evaluating functional and accessible folate receptor expression in vivo

Abstract With the development of molecularly-targeted therapeutics, it is critical to have a reliable method for predicting response to therapy of patients with target-expressing diseases. Folate receptor (FR) is overexpressed in a number of disease states while it is low in most healthy individuals with the exception of the kidney. FR-targeted small molecule drug conjugates (SMDCs) are currently under development and have shown promising pre-clinical and clinical results. Two premises that a patient would respond to the folate-drug conjugates are: 1). FR protein expressed in diseased tissues is functionally competent for folate binding and 2). functional FR is accessible to intravenously-infused SMDCs. Although anti-FR antibody-based immunohistochemical (IHC) assays are being developed as companion diagnostic strategies for antibody-drug conjugate (ADC) therapies, it fails to address the functionality and accessibility of FR in an in vivo context. To circumvent these limitations, we have developed an FR-targeted, crosslinkable small molecule reporter conjugate (SMRC), FRRC, which contains three modules: 1. a high-affinity folate ligand that binds with FR on diseased cells; 2. a small hapten, FITC, which can be detected by anti-FITC antibodies; and 3. an amino acid spacer in between these two modules that can crosslink FRRC to FR in-situ during formalin fixation. After intravenous (i.v.) injection, FRRC would dock to the accessible and functional receptors in vivo. By processing biopsied tissues and performing anti-FITC IHC staining, we can evaluate the cellular localization and relative abundance of functional FR in heterogeneous tissues. To evaluate our design, we synthesized a compound we refer to as FRRC and examined its properties both in vitro and in vivo. By testing FRRC in cell lines, we found that it can bind to FR and is detected in formalin-fixed cells by anti-FITC immunostaining. To validate this assay in vivo, FRRC was injected via the tail vein into mice bearing FR-positive KB tumor xenografts and its specific accumulation and kinetics in tumor and other tissues were evaluated by performing anti-FITC IHC staining on formalin-fixed paraffin embedded tissues. Our results were shown to be in agreement with previous folate-based functional FR imaging and bio-distribution studies. In contrast, EC17, which lacks the crosslinkable spacer module, showed significantly reduced binding in in vitro and in vivo assays. Owing to its modular design, additional SMRCs with different ligands and small haptens (rhodamine, DNP) have also been designed to assess the functional binding of other receptor and membrane-expressed proteins. In conclusion, our assay is an effective tool for evaluating functional and accessible receptor expression in vitro and in vivo, and has the potential to be useful in patient or disease selection for our SMDC therapeutics. Citation Format: Haiyan Chu, Jonathan M. Shillingford, Nikki Parker, Melissa Nelson, Jeremy F. Vaughn, Albert Felten, Yingjuan Lu, Joseph A. Reddy, Iontcho R. Vlahov, Christopher P. Leamon. Development and application of an immunohistochemistry-based assay for evaluating functional and accessible folate receptor expression in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4017. doi:10.1158/1538-7445.AM2017-4017

Controlled release of targeted anti-leukemia drugs azacitidine and decitabine monitored using surface-enhanced Raman scattering (SERS) spectroscopy

A new targeted drug delivery system with controlled release of anti-cancer drugs, azacitidine and decitabine, was investigated to enhance the efficacy of cancer treatment and reduce the effects of high drug toxicity to healthy tissues. The proposed drug nanocarriers are based on gold nanoparticles (AuNPs) modified with mercaptobenzoic acid (MBA) linker to enable the immobilization of azacitidine (AZA) and decitabine (DAC) on AuNPs in the form of AuNP@MBA/AZA,DAC entities. The cancer cell recognition was accomplished by covalently binding folic acid (FA) ligands to para-aminothiophenol (PATP) in the mixed SAM shell on gold nanoparticle nanocarriers, AuNP@MBA,PATP. The FA ligand was used due to the strong expression of folic acid receptors (FR) in the membrane of cancer cells. This enables the functionalized carriers to target only cancer cells owing to the efficient FA-FR binding property. The amide bonds between the linkers and azacitidine/decitabine are pH sensitive and undergo acid hydrolysis in a low pH environment of the cytosol in cancer cells. Using the solutions of different pH, the release of azacitidine/decitabine was monitored by surface-enhanced Raman scattering spectroscopy (SERS) measurements of the MBA Raman modes at 1586 cm-1 and 1074 cm-1 . At pH 7.4, the release of the drug was found to be negligible, while at pH 4.0 and 5.5 a continuous drug release was observed over 3 hours. The utilization of SERS monitoring for the drug release was based on the strong Raman signals which are generated by the MBA linker when it is bound to a plasmonic AuNP. During the immobilization of azacitidine/decitabine on AuNP carriers, the SERS signals are strongly reduced due to the shielding by drug molecules but they increase sharply upon the drug release confirming the amide bond breakage and successful drug delivery.

Folate receptor expression in bladder cancer and its correlation with tumor behaviors and clinical outcome

BackgroundFolate receptor (FR) has been recognized as having the capacity to become a biological marker for early diagnosis or prognostication of bladder cancer in previous studies. The aim of this study was to evaluate the expression of FR in bladder cancer, and its potential relevance to clinicopathological characteristics and patient survival. Materials and methodsSurgical specimens of cancer tissue were obtained from 78 patients with bladder cancer. The relative expression levels of FR in the bladder cancer tissue were measured by immunohistochemistry stain and graded according to stain intensity. Thereafter, the correlation with clinicopathological parameters and patient survival was analyzed. ResultsThe staining intensity and positivity rate of FR were significantly higher in low grade tumor tissues than in high grade tissues (P = 0.0035 and 0.003). Nevertheless, in the univariate Cox proportional hval. There was no correlation of FR expression or intensity with patient survival was seen (P > 0.01). ConclusionIn addition to tumor grade and stage, the expression of FR in bladder cancer is related to cellular differentiation. However, no correlation with patient survival was seen in this limited study.

Relationship between CD4 Regulatory T Cells and Anergy In Vivo.

Selective suppression of effector CD4+ T cell functions is necessary to prevent immune cell-mediated damage to healthy tissues. This appears especially true during pregnancy or in individuals predisposed to autoimmunity. Foxp3+ regulatory T (Treg) cells and induction of anergy, an acquired state of T cell functional unresponsiveness in Foxp3- cells, have both been implicated as mechanisms to suppress dangerous immune responses to tissue-restricted self-Ags. Anergic CD4+ T cells and Treg cells share a number of phenotypic and mechanistic traits-including the expression of CD73 and folate receptor 4, and the epigenetic modification of Treg cell signature genes-and an interesting relationship between these two subsets has recently emerged. In this review, we will compare and contrast these two subsets, as well as explore the role of anergy in the generation of peripheral Treg cells.

Differential expression of folate receptor 1 in medulloblastoma and the correlation with clinicopathological characters and target therapeutic potential.

Medulloblastoma is the most common malignant brain tumor in children. Folate receptor 1 (Folr1) was abundantly expressed in some epithelial malignancies. However the expression profile and the role of clinicopathological significance and therapeutic target potential in medulloblastoma still remain elusive. Currently we detected the expression of Folr1 in medulloblastoma and identified the diagnostic application by evaluating the clinical, pathological and neuroimaging values. Then we developed a target therapeutic compound with Folr1, which exhibited promising efficiency in treatment of medulloblastoma. Folr1 expression was up-regulated in medulloblastoma and positively correlated with percentage of Ki-67 and MMP9 labeling, pathological subtypes, serum Folr1 levels and CSF spreading on MRI. The level of serum Folr1 showed rational sensitivity and specificity in predicting histological subgroups. Strong Folr1 expression was recommended as the independent value regarding the prognosis of patients with medulloblastoma. Folr1 targeted therapy attenuated the tumor growth and metastasis with down-regulation of MMPs proteins and activation of apoptosis. Immunostaining analysis in the xenograft samples showed the decreased Ki-67 and MMP9 index providing the strong evidences that Folr1 targeted application can suppress the proliferation and invasion. Our findings uncovered in Folr1 a predictive candidate and therapeutic target for medulloblastoma.

Mouse IP-10 Gene Delivered by Folate-modified Chitosan Nanoparticles and Dendritic/tumor Cells Fusion Vaccine Effectively Inhibit the Growth of Hepatocellular Carcinoma in Mice.

Dendritic cells (DC) and tumor cell fusion vaccine (DC/tumor cell fusion vaccine) is considered an effective approach in cancer biotherapy. However, its therapeutic effects in early clinical trials have been suboptimal partially due to the immunosuppressive tumor environment. In this study, we used nanoparticles of folate (FA)-modified chitosan, a non-viral vector capable of targeting tumor cells with high expression of FA receptors. FA-chitosan nanoparticles were used as biological carriers for the expression plasmid of the mouse interferon-induced protein-10 (mIP-10) gene, a potent chemoattractant for cytotoxic T cells. The combination of FA-chitosan/mIP-10 and DC/tumor cell fusion vaccine against hepatocellular carcinoma (HCC) effectively inhibited the growth of implanted HCC tumors and prolonged the survival of mice. The combination therapy significantly reduced myeloid-derived suppressor cells (MDSC) in mouse spleen, local tumor, and bone marrow while increasing tumor-specific IFN-γ responses. Furthermore, the combination therapy significantly inhibited tumor cell proliferation while promoting their apoptosis. Taken together, our data illustrate that the mIP-10 enhances the anti-tumor effect of DC/tumor cell fusion vaccine by alleviating the immunosuppressive tumor environment.

Control of targeting ligand display by pH-responsive polymers on gold nanoparticles mediates selective entry into cancer cells.

Selective targeting of cells for intracellular delivery of therapeutics represents a major challenge for pharmaceutical intervention in disease. Here we show pH-triggered receptor-mediated endocytosis of nanoparticles via surface ligand exposure. Gold nanoparticles were decorated with two polymers: a 2 kDa PEG with a terminal folate targeting ligand, and a di-block copolymer including a pH-responsive and a hydrophilic block. At the normal serum pH of 7.4, the pH-responsive block (apparent pKa of 7.1) displayed a hydrophilic extended conformation, shielding the PEG-folate ligands, which inhibited cellular uptake of the nanoparticles. Under pH conditions resembling those of the extracellular matrix around solid tumours (pH 6.5), protonation of the pH-responsive polymer triggered a coil-to-globule polymer chain contraction, exposing folate residues on the PEG chains. In line with this, endocytosis of folate-decorated polymer-coated gold nanoparticles in cancer cells overexpressing folate receptor was significantly increased at pH 6.5, compared with pH 7.4. Thus, the tumour acidic environment and high folate receptor expression were effectively exploited to activate cell binding and endocytosis of these nanoparticles. These data provide proof-of-concept for strategies enabling extracellular pH stimuli to selectively enhance cellular uptake of drug delivery vectors and their associated therapeutic cargo.

P3.02c-027 Phase I and PK Study of the Folate Receptor-Targeted Small Molecule Drug Conjugate (SMDC) EC1456 in Advanced Cancer: Lung Cancer Subset: Topic: IT

EC1456 is composed of folic acid conjugated through a releasable linker system to a potent microtubule inhibitor, tubulysin B hydrazide. EC1456 targets folate receptor (FR)-expressing cancer cells, which occur in approximately 60% of NSCLC cases and 14% of SCLC. The objectives of the ongoing Phase 1 are to determine the safety, PK, and optimal dosing schedule of EC1456 in advanced cancer pts. FR expression (not required for enrollment) is characterized in all pts using 99mTc-etarfolatide. 63 pts were dosed weekly (QW) or twice-weekly (BIW), for 2 consecutive weeks of a 3-week cycle. 8 NSCLC and 3 SCLC pts received doses ranging from 1.0-12.5 mg/m2. Toxicities are primarily Grade (Gr) 1 and 2. Common adverse events (AE) are GI, fatigue, and metabolic changes. Gr 3 infusion reaction (4.5 mg/m2) and Gr 3 headache (10.0 mg/m2) were seen in the QW cohort. The safety profile in lung cancer pts was similar to the overall population.Tabled 1BIW (N=32)QW (N=31)BIW Lung (N=4)QW Lung (N=7)AllDrug RelatedAllDrug RelatedAllDrug RelatedAllDrug Related≥ 1 AE32 (100%)25 (78%)29 (94%)23 (74%)4 (100%)4 (100%)7 (100%)4 (57%)≥ 1 grade 3 or 4 AE19 (59%)6 (19%)14 (45%)4 (13%)2 (50%)0 (0%)1 (14%)0 (0%)≥ 1 serious AE12 (38%)2 (6%)14 (45%)5 (16%)1 (25%)0 (0%)2 (29%)0 (0%)Serious drug related AEsConstipation (2/63 pts); Abd pain, Anemia, Headache, Infusion reactions, and SVT(1/63 pts each) Open table in a new tab Response and durability of response is demonstrated in the figure: EC1456 is well tolerated, with early indications of efficacy suggested by durable stable disease, and responses in this refractory population. Updated safety, PK, and efficacy data will be presented at the meeting.

Leukemia-Specific Delivery of Mutant NOTCH1 Targeted Therapy

Several studies strongly support the development of Notch1 inhibitors for targeted cancer therapy. This idea is most compelling for T cell acute lymphoblastic leukemia/lymphoma (T-ALL) where activating mutations of NOTCH1 are present in 55-60% of the cases, and cancer dependency has been well established.We previously demonstrated that a small-molecule inhibitor of SERCA (Sarco/Endoplasmic Reticulum Calcium ATPase), thapsigargin, had NOTCH1 dependent anti-leukemia activity in mouse models of human T-ALL. Interestingly thapsigargin preferentially inhibits the maturation of mutant NOTCH1 compared to wild type NOTCH1 and NOTCH2 receptors. This selectivity provides a therapeutic window not observed before with other Notch modulators, including γ-secretase inhibitors or antibody-based approaches.To avoid the potential non-specific toxicityof thapsigargin secondary to intracellular calcium shifts, we leveraged the addiction of ALL to folic acid (FA), and we tagged folate to a permissive site on an active alcohol derivative of thapsigargin (8-O-debutanoylthapsigargin) via a cleavable ester linkage (JQ-FT). We explored folate conjugation of thapsigargin as an anti-cancer strategy for several reasons: the restricted expression of folate receptors (FR1-4) on cancer cells, the affinity of folic acid for FR binding and the FR endocytic mechanism that allows the internalization of large probes.We determined that folate-conjugates, including JQ-FT, enter T-ALL cells by FR2 binding and endocytosis. Importantly, T-ALL cell lines demonstrated stronger folic acid fluorescence-tagged probe (FL-FITC) labeling compared to human CD3+ T cells, providing support for leukemia-specific targeting. To determine if these effects were FR mediated, we treated T-ALL cells with JQ-FT in the presence or absence of the competitive inhibitor FA (10 and 100 μM). Co-treatment with FA diminished the negative growth effects of JQ-FT on T-ALL cells. This result supports FR-mediated internalization of JQ-FT.Next, we demonstrated that JQ-FT alters the growth of T-ALL cell lines, induces apoptosis and a G1/G0 arrest. Similar to thapsigargin, JQ-FT treatment decreased the levels of the activated form of NOTCH1, ICN1, and trans-membrane NOTCH1, while it increased full-length NOTCH1. Consequently, the NOTCH1 transcriptional targets, MYC, DTX1 and HES1, were repressed as measured by qRT-PCR. As predicted, the addition of free FA to the media rescued the decrement of ICN1.To assess the translational significance of these findings, we studied patient-derived xenograft (PDX) cells. JQ-FT treatment inhibited the viability of NOTCH1-mutated PDX cells in vitro. In addition, JQ-FT treatment resulted in a loss of transmembrane NOTCH1, leading to the depletion of ICN1. In contrast, no effect was observed in PDX T-ALL cells possessing wild-type NOTCH1. Consistent with these results, no transcriptional changes were observed in NOTCH1 target genes in wild type PDX samples, supporting the notion that mutated NOTCH1 receptors are more sensitive to SERCA inhibitors, including JQ-FT treatment, than are wild type NOTCH1 receptors in primary patient T-ALL.To explore the therapeutic efficacy of JQ-FT in vivo, we studied its effects on a syngeneic T-ALL mouse model carrying a NOTCH1 L1601P ΔPEST, a common mutation observed in the human disease. We first established the maximal tolerated dose (MTD) of JQ-FT as 60 mg/kg/day in mice administered by daily intraperitoneal injection. Notably, this MTD is 150-fold improved over our prior established MTD of unconjugated thapsigargin. Following five days of treatment, a decrease in tumor growth was observed, confirmed pathologically by a decrease in the leukemic infiltration in the bone marrow, spleen and liver, and clinically by a reduction in spleen weight. Pharmacodynamic modulation of the Notch pathway was validated by measurement of reduced ICN1 expression in T-ALL cells from JQ-FT-treated animals as compared to vehicle-treated controls.In summary, we report the development of the first-in-class NOTCH1 inhibitor with dual selectivity for leukemia over normal cells and NOTCH1-mutant over wild-type receptors. JQ-FT is recognized by folate receptors on the leukemia cell surface and delivered into cells as a mutant NOTCH1-targeted anti-leukemic agent. In mechanistic and translational models of T-ALL, we demonstrated NOTCH1-specific inhibition in vitro and in vivo. DisclosuresBradner:Novartis Institutes for BioMedical Research: Employment.

Folic acid-conjugated amphiphilic alternating copolymer as a new active tumor targeting drug delivery platform.

Targeted drug delivery using polymeric nanostructures is an emerging cancer research area, engineered for safer, more efficient, and effective use of chemotherapeutic drugs. A pH-responsive, active targeting delivery system was designed using folic acid functionalized amphiphilic alternating copolymer poly(styrene-alt-maleic anhydride) (FA-DABA-SMA) via a biodegradable linker 2,4-diaminobutyric acid (DABA). The polymeric template is pH responsive, forming amphiphilic nanostructures at pH 7, allowing the encapsulation of hydrophobic drugs on its interior. Moreover, the structure is stable only at neutral pH and collapses in the acidic tumor microenvironment, releasing drugs on-site from its core. The delivery vehicle is investigated using human pancreatic PANC-1 cancer cells and RAW-Blue™ mouse macrophage reporter cell line, both of which have overly expression of folic acid receptors. To trace the cellular uptake by both cell lines, curcumin was selected as a dye and drug mimic owing to its fluorescence nature and hydrophobic properties. Fluorescent microscopy of FA-DABA-SMA loaded with curcumin revealed a significant internalization of the dye by human pancreatic PANC-1 cancer cells compared to those with unfunctionalized polymers (SMA). Moreover, the FA-DABA-SMA polymers exhibit rodlike association specific to the cells. Both empty SMA and FA-DABA-SMA show little toxicity to PANC-1 cells as characterized by WST-1 cell proliferation assay. These results clearly indicate that FA-DABA-SMA polymers show potential as an active tumor targeting drug delivery system with the ability to internalize hydrophobic chemotherapeutics after they specifically attach to cancer cells.