Folate Receptor-expressing Cancer Cells Research Articles

Elucidating a Thermoresponsive Multimodal Photo-Chemotherapeutic Nanodelivery Vehicle to Overcome the Barriers of Doxorubicin Therapy.

In an attempt to circumvent the major pitfalls associated with conventional chemotherapy including drug resistance and off-target toxicity, we have adopted a strategy to simultaneously target both mitochondrial DNA (Mt-DNA) and nuclear DNA (n-DNA) with the aid of a targeted theranostic nanodelivery vehicle (TTNDV). Herein, folic acid-anchored p-sulfo-calix[4]arene (SC4)-capped hollow gold nanoparticles (HGNPs) were meticulously loaded with antineoplastic doxorubicin (Dox) and its mitochondrion-targeted analogue, Mt-Dox, in a pretuned ratio (1:100) for sustained thermoresponsive release of cargo. This therapeutic strategy was enabled to eradicate both n-DNA and Mt-DNA leaving no space to develop drug resistance. The SC4-capped HGNPs (HGNPSC4) were experimented for the first time as a photothermal (PTT) agent with 61.6% photothermal conversion efficiency, and they generated tunable localized heat more efficiently than bare HGNPs. Moreover, the cavity of SC4 facilitated the formation of an inclusion complex with folic acid to target the folate receptor expressing cancer cells and imparted enhanced biocompatibility. The as-synthesized TTNDV was demonstrated to be an ideal substrate for surface-enhanced Raman scattering (SERS) to monitor the molecular-level therapeutic progression in cells and a spheroidal model. A significant reduction in the tumor mass with a marked survival benefit was achieved in syngraft murine models through this synergistic photo-chemotherapy. Collectively, this multifunctional nanoplatform offers a robust approach to treat cancer without any scope of generating Dox resistance and off-target toxicity.

Folic acid conjugates of a bleomycin mimic for selective targeting of folate receptor positive cancer cells

A major challenge in the application of cytotoxic anti-cancer drugs is their general lack of selectivity, which often leads to systematic toxicity due to their inability to discriminate between malignant and healthy cells. A particularly promising target for selective targeting are the folate receptors (FR) that are often over-expressed on cancer cells. Here, we report on a conjugate of the pentadentate nitrogen ligand N4Py to folic acid, via a cleavable disulphide linker, which shows selective cytotoxicity against folate receptor expressing cancer cells.

Quantitative bioimage analytics enables measurement of targeted cellular stress response induced by celastrol-loaded nanoparticles

The cellular stress response, which provides protection against proteotoxic stresses, is characterized by the activation of heat shock factor 1 and the formation of nuclear stress bodies (nSBs). In this study, we developed a computerized method to quantify the formation and size distribution of nSBs, as stress response induction is of interest in cancer research, neurodegenerative diseases, and in other pathophysiological processes. We employed an advanced bioimaging and analytics workflow to enable quantitative detailed subcellular analysis of cell populations even down to single-cell level. This type of detailed analysis requires automated single cell analysis to allow for detection of both size and distribution of nSBs. For specific induction of nSB we used mesoporous silica nanoparticles (MSNs) loaded with celastrol, a plant-derived triterpene with the ability to activate the stress response. To enable specific targeting, we employed folic acid functionalized nanoparticles, which yields targeting to folate receptor expressing cancer cells. In this way, we could assess the ability to quantitatively detect directed and spatio-temporal nSB induction using 2D and 3D confocal imaging. Our results demonstrate successful implementation of an imaging and analytics workflow based on a freely available, general-purpose software platform, BioImageXD, also compatible with other imaging modalities due to full 3D/4D and high-throughput batch processing support. The developed quantitative imaging analytics workflow opens possibilities for detailed stress response examination in cell populations, with significant potential in the analysis of targeted drug delivery systems related to cell stress and other cytoprotective cellular processes.

Pluronic F127-Folate Coated Super Paramagenic Iron Oxide Nanoparticles as Contrast Agent for Cancer Diagnosis in Magnetic Resonance Imaging.

Contrast agents have been widely used in medicine to enhance contrast in magnetic resonance imaging (MRI). Among them, super paramagnetic iron oxide nanoparticles (SPION) have been reported to have low risk in clinical use. In our study, F127-Folate coated SPION was fabricated in order to efficiently target tumors and provide imaging contrast in MRI. SPION alone have an average core size of 15 nm. After stabilizing with Pluronic F127, the nanoparticles reached a hydrodynamic size of 180 nm and dispersed well in various kinds of media. The F127-Folate coated SPION were shown to specifically target folate receptor expressing cancer cells by flow cytometry analysis, confocal laser scanning microscope, as well as in vitro MRI. Furthermore, in vivo MRI images have shown the enhanced negative contrast from the F127-Folate coated SPION in tumor-bearing mice. In conclusion, our F127-Folate coated SPION have shown great potential as a contrast agent in MRI, as well as in the combination with drug delivery for cancer therapy.

Abstract 3571: Use of a universal CAR T cell plus bispecific adapters to eradicate antigenically heterogeneous tumors

Abstract Chimeric antigen receptor (CAR) T cell therapies have attracted considerable attention because of their abilities to focus the killing power of T cells specifically onto tumor antigen expressing cancer cells. Despite impressive successes in the clinic, CAR T cell technologies still suffer from an inability to kill cancer cells that mutate to avoid expression of the CAR T cell recognized antigen. While current CAR T cell technologies would require the engineering of a new CAR to recognize a second tumor-specific antigen, we have instead developed a universal CAR that can recognize any tumor antigen. In this modification of standard CAR T cell technology, we fuse an scFv that binds fluorescein to the exoplasmic domain of a T cell receptor that contains the usual 4-1BB and CD3 zeta activation domains within its cytoplasmic domain. We then promote engagement of the resulting CAR T cell with the desired cancer cell by administering a bispecific adapter molecule that contains fluorescein linked to a tumor-specific ligand via a short spacer. Upon addition of this adapter, simultaneous binding of the fluorescein to the CAR and the tumor-specific ligand to the cancer cell mediates multivalent binding of the CAR T cell to the cancer cell. With the simultaneous administration of a cocktail of different tumor-specific adaptors, each comprised of a fluorescein linked to a distinct tumor-specific ligand, multiple orthogonal cancer cells within a heterogeneous solid or liquid tumor can be simultaneously eliminated by a single CAR T cell. To test the ability of our proposed universal CAR T cell to eradicate solid tumors with multiple orthogonal tumor antigens, we first demonstrate the capacity of our anti-fluorescein CAR T cell to kill a variety of cultured human cancer cells in vitro upon addition of the optimal adapter. In this effort, we show that the same anti-fluorescein CAR T cell can kill: i) multiple folate receptor-expressing cancer cells upon addition of folate-fluorescein, ii) PSMA-expressing cancer cells upon addition of a PSMA ligand-fluorescein conjugate, iii) carbonic anhydrase IX-expressing cancer cells upon addition of a CA9 ligand-fluorescein conjugate, and iv) neurokinin-1 receptor expressing cancer cells upon addition of a NK1R ligand-fluorescein conjugate. We then demonstrate that this same universal killing potency is also effective in vivo by transfecting separate MDA-MB-231 breast cancer cell cultures with one of the above tumor antigens and then implanting the MDA-MB-231 cells expressing the orthogonal tumor antigens in NSG mice and showing that our universal CAR T cell can eradicate the resulting tumors upon administration of the appropriate bispecific adapter. Taken together these results establish that a single anti-fluorescein CAR T cell can be exploited to eliminate antigenically heterogeneous tumors upon addition of the correct cocktail of ligand-fluorescein conjugates. Citation Format: Yong Gu Lee, Isaac Marks, Madduri Srinivasarao, Ananda Kumar Kanduluru, Sakkarapalayam M. Mahalingam, Haiyan Chu, Philip S. Low. Use of a universal CAR T cell plus bispecific adapters to eradicate antigenically heterogeneous tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3571.

NK Cell-Mediated Antitumor Effects of a Folate-Conjugated Immunoglobulin Are Enhanced by Cytokines.

Optimally effective antitumor therapies would not only activate immune effector cells but also engage them at the tumor. Folate conjugated to immunoglobulin (F-IgG) could direct innate immune cells with Fc receptors to folate receptor-expressing cancer cells. F-IgG bound to human KB and HeLa cells, as well as murine L1210JF, a folate receptor (FR)-overexpressing cancer cell line, as determined by flow cytometry. Recognition of F-IgG by natural killer (NK) cell Fc receptors led to phosphorylation of the ERK transcription factor and increased NK cell expression of CD69. Lysis of KB tumor cells by NK cells increased by about 5-fold after treatment with F-IgG, an effect synergistically enhanced by treatment with IL2, IL12, IL15, or IL21 (P< 0.001). F-IgG also enhanced the lysis of chronic lymphocytic leukemia cells by autologous NK cells. NK cells significantly increased production of IFNγ, MIP-1α, and RANTES in response to F-IgG-coated KB target cells in the presence of the NK cell-activating cytokine IL12, and these coculture supernatants induced significant T-cell chemotaxis (P< 0.001). F-IgG-coated targets also stimulated FcR-mediated monocyte effector functions. Studies in a murine leukemia model confirmed the intratumoral localization and antitumor activity of F-IgG, as well as enhancement of its effects by IL12 (P =0.05). The antitumor effect of this combination was dependent on NK cells and led to decreased tumor cell proliferation in vivo Thus, F-IgG can induce an immune response against FR-positive tumor cells that is mediated by NK cells and can be augmented by cytokine therapy.

Imaging-guided high-efficient photoacoustic tumor therapy with targeting gold nanorods

Photoacoustic therapy using the large photoacoustic effect of agents for selectively killing cancer cells is demonstrated. Herein, a highly efficient photoacoustic treatment using gold nanorods (AuNRs) and its antitumor effect are reported. Folic acid conjugated AuNRs are designed to specifically target folate receptor-expressing cancer cells. Following photoacoustic treatment, most of the cancer cells with intracellular AuNRs die within 20s. Compared with single-walled carbon nanotubes and indocyanine green containing nanoparticles, AuNRs can produce much stronger shock waves by absorbing the optical energy and thus induced the more efficient cell death at equal molar concentrations. In addition, the laser-induced shock waves can be detected for photoacoustic imaging. Our in vivo experiments demonstrated that the AuNR-mediated photoacoustic treatment resulted in efficient tumor suppression in mice. Thus, both efficient cancer cell diagnostics and selective photoacoustic treatment can be realized with a single-particle formulation. From the Clinical EditorNanotechnology has enabled the development of many novel methods for the treatment of cancer. One of these is photoacoustic therapy. In this article, the authors demonstrated the efficacy of Folic acid conjugated gold nanorods in killing cancer cells after photoacoustic treatment. The findings should provide impetus for future clinical studies.

Radiosensitization effect of folate-conjugated gold nanoparticles on HeLa cancer cells under orthovoltage superficial radiotherapy techniques

Due to the high atomic number of gold nanoparticles (GNPs), they are known as new radiosensitizer agents for enhancing the efficiency of superficial radiotherapy techniques by increasing the dose absorbed in tumor cells wherein they can be accumulated selectively. The aim of this study was to compare the effect of various common low energy levels of orthovoltage x-rays and megavoltage γ-rays (Co-60) on enhancing the therapeutic efficiency of HeLa cancer cells in the presence of conjugated folate and non-conjugated (pegylated) GNPs. To achieve this, GNPs with an average diameter of 52 nm were synthesized and conjugated to folic acid molecules. Pegylated GNPs with an average diameter of 47 nm were also synthesized and used as non-conjugated folate GNPs. Cytotoxicity assay of the synthesized folate-conjugated and pegylated GNPs was performed using different levels of nanoparticle concentration incubated with HeLa cells for 24 h. The radiosensitizing effect of both the conjugated and pegylated GNPs on the cells at a concentration of 50 µM was compared using MTT as well as clonogenic assays after exposing them to 2 Gy ionizing radiation produced by an orthovoltage x-ray machine at four different kVps and γ-rays of a Co-60 unit. Significant differences were noted among various irradiated groups with and without the folate conjugation, with an average dose enhancement factor (DEF) of 1.64 ± 0.05 and 1.35 ± 0.05 for the folate-conjugated and pegylated GNPs, respectively. The maximum DEF was obtained with the 180 kVp x-ray beam for both of the GNPs. Folate-conjugated GNPs can significantly enhance the cell killing potential of orthovoltage x-ray energies (especially at 180 kVp) in folate receptor-expressing cancer cells, such as HeLa, in superficial radiotherapytechniques.

A Phase I Study of Folate Immune Therapy (EC90 Vaccine Administered With GPI-0100 Adjuvant Followed by EC17) in Patients With Renal Cell Carcinoma

This is the first phase I, open-label study to assess the safety, pharmacokinetics, and antitumor activity of a novel immunotherapeutic regimen known as Folate Immune (EC90 vaccine administered with GPI-0100 adjuvant followed by EC17, a folate-targeted hapten immunotherapy that targets folate receptor expressing cancer cells), which is designed to convert poorly immunogenic tumors to highly immunogenic tumors in patients with metastatic renal cell carcinoma. Three to 6 patients were enrolled in each cohort. In the vaccination phase, patients were given once weekly vaccinations of 0.2 mg of EC90 plus 3.0 mg of GPI-0100 for 3-5 weeks. In the treatment phase, patients were treated with 0.031, 0.092, or 0.276 mg/kg of EC17, 5 d/wk, for weeks 3, 4, or 6. Forty-one patients were enrolled in the study of which 33 patients received ≥1 treatment of EC17. Two dose-limiting toxicities were observed including grade 4 anaphylaxis and grade 3 pancreatitis. During the vaccination phase, mild to moderate injection site reactions were the most frequently reported adverse events. During the treatment phase, transient hypersensitivity reactions were the most common adverse event. Partial response was noted in 4% (1/28) of patients, and stable disease was noted in 54% (15/28) of patients after cycle 1 and was maintained in the majority of patients entering the extension phase of the study. EC90 vaccine with GPI-0100 adjuvant followed by EC17 is safe and well tolerated. The recommended regimen for further studies is 4 weekly vaccinations with 0.2 mg of EC90 plus 3.0 mg GPI-0100 followed by treatment with 0.3 mg of EC17.

Efficient delivery of gold nanoparticles by dual receptor targeting.

Targeted delivery of inorganic nanoparticles, particularly of gold, may find wide utility in a number of biomedical applications due to their unique physicochemical and optoelectronic properties. [1-6] However, efficient delivery of a threshold amount of such particles is required for successful biomedical applications including imaging, detection/diagnosis and therapy. [7, 8] Efficient delivery of gold nanoparticles may be achieved by simultaneous targeting of multiple receptors on the cancer cells. [9] Hence, selection of appropriate targets is crucial for successful application of multiple/dual receptor targeted drug delivery system. Herein, we demonstrate the fabrication and characterization of gold nanoconjugates containing EGFR (epidermal receptor growth factor) and FR (folate receptor) antibodies on a single gold core, as a proof of principle study, to enhance the loading of gold nanoparticles (AuNPs) to EGFR and FR expressing cancer cells. We further demonstrate that the dual receptor targeted system (DRTS) is more efficient in delivering AuNPs to EGFR and FR expressing cancer cells than their corresponding single receptor targeting systems (SRTS). Such selective delivery of GNPs could be utilized in numerous biomedical applications such as detection, diagnosis and therapy. The folate receptor α (FRα) and epidermal growth factor receptor (EGFR) is known to be overexpressed in a number of malignancies including ovarian cancer. [10] Farletuzumab, a monoclonal antibody to FRα is in Phase III clinical trials both alone or in combination with platinum/taxane chemotherapy for the patients experiencing recurrence. [11] Several monoclonal antibodies directed against EGFR (trastuzumab, cetuximab, pertuzumab, and panitumumab) and small molecule tyrosine kinase inhibitors (erlotinib and gefitinib) have been investigated and are currently in different phases of clinical trials in ovarian cancer. [12] Thus, FR and EGFR represent important targets for tumor-specific delivery of anticancer drugs. Herein, we demonstrate, as a proof of principle study, fabrication and characterization of a dual receptor targeted system (DRTS) and demonstrate that it is much more efficient in delivering AuNPs to EGFR and FR expressing ovarian cancer cells Skov3-ip and OVCAR-5 than their corresponding single receptor targeting systems (SRTS). Such selective delivery of AuNPs has the potential to be used in numerous biomedical applications including detection, diagnosis and therapeutics.

Abstract 2686: Anti-tumor effects of a folate-immunoglobulin conjugate are enhanced by cytokine treatment in vitro and in vivo

Abstract Folate conjugation is a means of selectively targeting therapeutics to folate receptor (FR)-expressing cancer cells. A novel folate-bound immunoglobulin (F-IgG) was tested for its ability to target natural killer (NK) cells to folate-receptor expressing cancer cells in the presence or absence of NK-activating cytokines. FR expression by the KB and HeLa cell lines was confirmed by immunoblot analysis (IB) and flow cytometry. Binding of F-IgG to NK cell Fc receptors led to increased phosphorylation of the epidermal growth factor related kinase (ERK) as measured by IB. Lysis of FR+ KB tumor cells by NK cells was increased 8-fold following treatment with F-IgG as compared to C-IgG (p&amp;lt;0.0001 across E:T ratios from 6.25:1 to 50:1). NK cell lysis of F-IgG- coated KB target cells was significantly enhanced following treatment of NK cells with IL-2, IL-12, IL-15 and IL-21 (all at 10ng/mL). NK cell production of IFN-γ, RANTES and MIP-1α was significantly enhanced by IL-12 in response to F-IgG-coated KB target cells as compared to control-treated cells (p&amp;lt;0.005; IFN-γ-2100 vs. 1000pg/mL, RANTES-800 vs. 150pg/mL, MIP-1α 1800 vs. 500pg/mL, respectively). Studies using the L1210JF murine leukemia model confirmed the anti-tumor activity of F-IgG and the ability of NK-activating cytokines to significantly enhance its effects. NK cell depletion in tumor-bearing mice demonstrated that the anti-tumor effects of IL-12 and F-IgG are dependent on NK cells. These studies indicate that F-IgG induces an immune response by NK cells against FR-positive cancer cell lines and that cytokine treatment has a synergistic effect on this response both in vitro and in vivo. Thus F-IgG has a potential to be used as a therapeutic antibody for the treatment of FR-positive cancers in combination with immune modulatory cytokines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2686. doi:10.1158/1538-7445.AM2011-2686

Multimodal Bacteriochlorophyll Theranostic Agent

The complimentary ability of different noninvasive imaging technologies with therapeutic modalities can be used in tandem providing high-resolution and highly sensitive imaging of events at the molecular and cellular level providing a means for image-guided therapy. There is increasing interest in using porphyrin-based photosensitizers as theranostics to take advantages of their near-infrared fluorescent properties for imaging and their strong singlet oxygen generation abilities for photodynamic therapy. Here we report a targeted multimodal bacteriochlorophyll theranostic probe. This probe consists of a bacteriochlorophyll derivative, a pharmacokinetics modification peptide linker and folate for targeted delivery to folate receptor expressing cancer cells. We demonstrate its multimodal theranostic capability, its folate receptor targeting ability and its utility for both NIR fluorescence imaging and photodynamic therapy purposes both in vitro and in vivo.

Targeting of Porous Hybrid Silica Nanoparticles to Cancer Cells

Mesoporous silica nanoparticles functionalized by surface hyperbranching polymerization of poly(ethylene imine), PEI, were further modified by introducing both fluorescent and targeting moieties, with the aim of specifically targeting cancer cells. Owing to the high abundance of folate receptors in many cancer cells as compared to normal cells, folic acid was used as the targeting ligand. The internalization of the particles in cell lines expressing different levels of folate receptors was studied. Flow cytometry was used to quantify the mean number of nanoparticles internalized per cell. Five times more particles were internalized by cancer cells expressing folate receptors as compared to the normal cells expressing low levels of the receptor. Not only the number of nanoparticles internalized per cell, but also the fraction of cells that had internalized nanoparticles was higher. The total number of particles internalized by the cancer cells was, therefore, about an order of magnitude higher than the total number of particles internalized by normal cells, a difference high enough to be of significant biological importance. In addition, the biospecifically tagged hybrid PEI-silica particles were shown to be noncytotoxic and able to specifically target folate receptor-expressing cancer cells also under coculture conditions.

The construction and in vitro testing of photo-activatable cancer targeting folated anti-CD3 conjugates

The construction and in vitro testing of a photo-activatable anti-tumour immuno-regulatory antibody is described. In this ‘cloaked’ folated anti-CD3 antibody conjugate, the folate portion of the conjugate is free to bind to folate receptor expressing cancer cells, whilst the anti-CD3 activity is effectively rendered inert by a coating of photo-labile 2-nitrobenzyl groups. On irradiation with UV-A light the activity of the anti-CD3 antibody is restored, not only when it is required, but more importantly, only where it is required. The conjugate can then attract killer T-cells to the surface of the tumour cells and kill them. Unirradiated normal tissues, to which the conjugate has been targeted by specific and non-specific binding, remain unharmed. We believe that these ‘photo-switchable’ conjugates could be used to markedly improve the targeting of the immune response to folate receptor (FR) expressing ovarian and breast cancers whilst minimising the side effects in the rest of the body.

Folate targeting of haptens to cancer cell surfaces mediates immunotherapy of syngeneic murine tumors.

A variety of human cancers overexpress a cell surface receptor with high affinity for the vitamin, folic acid ( K(d) approximately 10(-10)M). Covalent attachment of therapeutic agents to folic acid has been shown to allow efficient targeting of the folate-drug conjugates to folate receptor-expressing cancer cells, with little or no uptake by normal tissues except the kidneys. We report here the use of folate's ability to deliver attached molecules specifically to cancer cells to convert poorly immunogenic tumors into highly immunogenic tissue targets. By linking folic acid to a model hapten, we have been able to decorate folate receptor-expressing cancer cell surfaces with >10(6) haptens/cell in vivo. Following marking of such cells with haptens, the cells are observed to become opsonized with autologous anti-hapten antibodies, which is presumed to mediate cell removal via antibody-dependent cellular cytotoxicity (ADCC). Supplemental administration of low levels of ADCC-activating cytokines [e.g. interleukin-2 (IL-2) and interferon-alpha (IFN-alpha)] has been shown to synergize with the folate-targeted immunotherapy. Thus, using M109 syngeneic lung cancer cells injected intraperitoneally into Balb/c mice that were previously immunized against fluorescein, a significant extension of life span is observed following treatment with folate-fluorescein conjugates, and complete cures are observed upon supplementation with moderate levels of IL-2 and IFN-alpha. Because control tumor-bearing mice treated with the same cytokines but with non-targeted fluorescein show no extension of life span, we conclude that tumor-specific opsonization is an essential step in this immunotherapy. Finally, because the anti-fluorescein antibodies are unable to access the folate receptors on the apical membranes of the kidney proximal tubules, no kidney or other normal tissue cytotoxicity is observed. These data suggest that retargeting of haptens to folate receptor-expressing cancers might constitute a method for mobilizing the immune system specifically against poorly immunogenic tumors.