FR-positive Tumor Research Articles

Synthesis and Preclinical Evaluation of [18F]AlF-NOTA-Asp2-PEG2-Folate as a Novel Folate-Receptor-Targeted Tracer for PET Imaging.

Recently, the folate receptor (FR) has become an exciting target for the diagnosis of FR-positive malignancies. Nevertheless, suboptimal invivo pharmacokinetic properties, particularly high uptake in the renal and hepatobiliary systems, are important limiting factors for the clinical translation of most FR-based radiotracers. In this study, we developed a novel 18F-labeled FR-targeted positron emission tomography (PET) tracer [18F]AlF-NOTA-Asp2-PEG2-Folate modified with a hydrophilic linker (-Asp2-PEG2) to optimize its pharmacokinetic properties and conducted a comprehensive preclinical assessment. The [18F]AlF-NOTA-Asp2-PEG2-Folate was manually synthesized within 30 min with a non-decay-corrected radiochemical yield of 16.3 ± 2.0% (n = 5). Among KB cells, [18F]AlF-NOTA-Asp2-PEG2-Folate exhibited high specificity and affinity for FR. PET/CT imaging and biodistribution experiments in KB tumor-bearing mice showed decent tumor uptake (1.7 ± 0.3% ID/g) and significantly decreased uptake in kidneys and liver (22.2 ± 2.1 and 0.3 ± 0.1% ID/g at 60 min p.i., respectively) of [18F]AlF-NOTA-Asp2-PEG2-Folate, compared to the known tracer [18F]AlF-NOTA-Folate (78.6 ± 5.1 and 5.3 ± 0.5 % ID/g at 90 min p.i., respectively). The favorable properties of [18F]AlF-NOTA-Asp2-PEG2-Folate, including its efficient synthesis, decent tumor uptake, relatively low renal uptake, and rapid clearance from most normal organs, portray it as a promising PET tracer for FR-positive tumors.

Post-insertion technique to introduce targeting moieties in milk exosomes for targeted drug delivery

BackgroundRecently, increased attention has been given on exosomes as ideal nanocarriers of drugs owing to their intrinsic properties that facilitate the transport of biomolecular cargos. However, large-scale exosome production remains a major challenge in the clinical application of exosome-based drug delivery systems. Considering its biocompatibility and stability, bovine milk is a suitable natural source for large-scale and stable exosome production. Because the active-targeting ability of drug carriers is essential to maximize therapeutic efficacy and minimize side effects, precise membrane functionalization strategies are required to enable tissue-specific delivery of milk exosomes with difficulty in post-isolation modification.MethodsIn this study, the membrane functionalization of a milk exosome platform modified using a simple post-insertion method was examined comprehensively. Exosomes were engineered from bovine milk (mExo) with surface-tunable modifications for the delivery of tumor-targeting doxorubicin (Dox). The surface modification of mExo was achieved through the hydrophobic insertion of folate (FA)-conjugated lipids.ResultsWe have confirmed the stable integration of functionalized PE-lipid chains into the mExo membrane through an optimized post-insertion technique, thereby effectively enhancing the surface functionality of mExo. Indeed, the results revealed that FA-modified mExo (mExo-FA) improved cellular uptake in cancer cells via FA receptor (FR)-mediated endocytosis. The designed mExo-FA selectively delivered Dox to FR-positive tumor cells and triggered notable tumor cell death, as confirmed by in vitro and in vivo analyses.ConclusionsThis simple and easy method for post-isolation modification of the exosomal surface may be used to develop milk-exosome-based drug delivery systems.Graphical

Platinum-Based Nanocomposite Pt@BSA as an Efficient Electrochemical Biosensing Interface for Rapid and Ultrasensitive Determination of Folate Receptor-Positive Tumor Cells.

Developing a cytosensing strategy based on electrochemical approaches has attracted wide interest due to the low cost, rapid response, and simple instrumentation. In this work, an electrochemical cytosensor employing the Pt@BSA nanocomposite as the biosensing substrate along with the covalent cross-linking of targeting molecules folic acid (FA) was constructed for highly sensitive determination of folate receptor (FR)-positive tumor cells. The prepared Pt@BSA nanocomposite revealed excellent biocompatibility for cell adhesion and proliferation, which was confirmed by cell viability evaluation using thiazolyl blue tetrazolium bromide (MTT) colorimetric methods. Due to the satisfactory electrical conductivity originating from Pt@BSA and the high binding affinity of FA to FR on the cell surface, an ultrasensitive and specific cytosensing device was designed for rapid and quantitative determination of HeLa cells (a model system) by differential pulse voltammetry (DPV) tests. This proposed cytosensor resulted in a wide HeLa cell determination range of 2.8 × 101-2.8 × 106 cells mL-1 with a low DPV detection limit of 9 cells mL-1. The developed cytosensing approach exhibited highly specific recognition of FR-positive tumor cells, excellent inter-assay reproducibility with a relative standard deviation (RSD) of 4.7%, acceptable intra-assay precision, and favorable storage stability, expanding the application of electrochemical measurement technology in the biomedical field of early detection and diagnosis of cancers.

Enhanced Anti-Tumor Effect of Folate-Targeted FA-AMA-hyd-DOX Conjugate in a Xenograft Model of Human Breast Cancer

Folate-aminocaproic acid-doxorubicin (FA-AMA-hyd-DOX) was firstly synthesized by our group. It was indicated that FA-AMA-hyd-DOX was pH-responsive, and had strong cytotoxicity on a folate receptor overexpressing cell line (KB cells) in vitro. The aim of our study was to further explore the potential use of FA-AMA-hyd-DOX as a new therapeutic drug for breast cancer. The cellular uptake and the antiproliferative activity of the FA-AMA-hyd-DOX in MDA-MB-231 cells were measured. Compared with DOX, FA-AMA-hyd-DOX exhibited higher targeting ability and cytotoxicity to FR-positive tumor cells. Subsequently, the tissue distribution of FA-AMA-hyd-DOX was studied, and the result confirmed that DOX modified by FA can effectively increase the selectivity of drugs in vivo. After determining the maximum tolerated dose (MTD) of FA-AMA-hyd-DOX in MDA-MB-231 tumor-bearing nude mice, the antitumor effects and the in vivo safety of FA-AMA-hyd-DOX were systematically evaluated. The data showed that FA-AMA-hyd-DOX could effectively increase the dose of DOX tolerated by tumor-bearing nude mice and significantly inhibit MDA-MB-231 tumor growth in vivo. Furthermore, FA-AMA-hyd-DOX treatment resulted in almost no obvious damage to the mice. All the positive data suggest that FA-targeted FA-AMA-hyd-DOX is a promising tumor-targeted compound for breast cancer therapy.

Core–Shell Structured NaYF4:Yb,Er Nanoparticles with Excellent Upconversion Luminescent for Targeted Drug Delivery

A core–shell-structured composite by combining mesoporous silica with upconversion luminescence (UCL) property had considerable application potentials in the fields of drug delivery, disease diagnosis, and therapy. In this paper, a monodisperse, core–shell-structured NaYF4:Yb,Er@nSiO2@mSiO2 nanoparticle (UCNP@MSNs-FA) with excellent UCL property was successfully fabricated by a facile two-step sol–gel strategy and by modifying the surface with folic acid (FA) to strengthen its tumor-targeting performance. The properties of the composite were studied extensively, which indicated that UCNP@MSNs-FA possessed good dispersion, typical core–shell-structured with high specific surface area (132.775 m2/g), and excellent UCL property that improve cell imaging and drug delivery. The viability of L929 cells and hemolysis assay demonstrated the good biocompatibility of the composite. By using doxorubicin hydrochloride (DOX) as a model drug, the drug loading content and encapsulation efficiency of UCNP@MSNs-FA could reach as high as 11.2% and 37.3%, respectively, which proved the effectiveness to load anticancer drugs. In addition, the DOX-UCNP@MSNs-FA system exhibited sustained drug release and strong pH-dependent performance, in which the drug release would be accelerated at the slightly acidic microenvironment in the tumor. Moreover, experimental results indicated that DOX-UCNP@MSNs-FA exhibited specific cytotoxicity to KB cells but weakened toxicity to FR-negative cells. Therefore, the as-prepared UCNP@MSNs-FA could be used potential for simultaneous targeted anti-cancer drug delivery and cell imaging and enhance the therapeutic efficacy against FR-positive tumor cells.

Synthesis of NaYF4:Yb,Er upconversion nanoparticle-based optomagnetic multifunctional composite for drug delivery system

Optomagnetic multifunctional composite has attracted much attention in recent years because of its promising application prospect in bioimaging, analysis, detection, disease diagnosis, and targeted drug delivery. To explore a dual-targeted therapy for cancer, a novel class of optomagnetic multifunctional composite (UCNP-Fe3O4@MSNs-FA) was successfully synthesized by using upconversion nanoparticles (UCNPs) as nucleus, embedding Fe3O4 nanoparticles into the SiO2 coating layer, and modifying the surface with folic acid (FA) to strengthen its tumor targeting performance. The properties of the composite were extensively studied. The obtained composite possesses excellent upconversion fluorescence, good dispersion, high specific surface area (229.347 m2/g), and saturation magnetization value (10.9 A·m2/g). Its drug loading content and encapsulation efficiency can reach as high as 14.2% and 47.3%, respectively, using doxorubicin hydrochloride (DOX) as model drug. The DOX-UCNP-Fe3O4@MSNs-FA system shows excellent sustained drug release and strong pH-dependent performance, in which the drug release would be accelerated at the slightly acidic microenvironment in the tumor; thus, the system can realize the targeted treatment of cancers. The viability of L929 cells demonstrates the good biocompatibility of the composite. Furthermore, DOX-UCNP-Fe3O4@MSNs-FA exhibits specific cytotoxicity to folate receptor (FR) positive tumor cells, whereas DOX has weak toxicity to FR-negative cells. Therefore, the as-prepared UCNP-Fe3O4@MSNs-FA can potentially be used as an anti-cancer targeted drug delivery system and enhance the therapeutic efficacy against FR-positive tumor cells.

Pre-clinical studies of EC2629, a highly potent folate- receptor-targeted DNA crosslinking agent

Folate receptor (FR)-targeted small molecule drug conjugates (SMDCs) have shown promising results in early stage clinical trials with microtubule destabilizing agents, such as vintafolide and EC1456. In our effort to develop FR-targeted SMDCs with varying mechanisms of action, we synthesized EC2629, a folate conjugate of a DNA crosslinking agent based on a novel DNA-alkylating moiety. This agent was found to be extremely potent with an in vitro IC50 ~ 100× lower than folate SMDCs constructed with various microtubule inhibitors. EC2629 treatment of nude mice bearing FR-positive KB human xenografts led to cures in 100% of the test animals with very low dose levels (300 nmol/kg) following a convenient once a week schedule. The observed activity was not accompanied by any noticeable weight loss (up to 20 weeks post end of dosing). Complete responses were also observed against FR-positive paclitaxel (KB-PR) and cisplatin (KB-CR) resistant models. When evaluated against FR-positive patient derived xenograft (PDX) models of ovarian (ST070), endometrial (ST040) and triple negative breast cancers (ST502, ST738), EC2629 showed significantly greater anti-tumor activity compared to their corresponding standard of care treatments. Taken together, these studies thus demonstrated that EC2629, with its distinct DNA reacting mechanism, may be useful in treating FR-positive tumors, including those that are classified as drug resistant.

Development of Folate Receptor-Targeted PET Radiopharmaceuticals for Tumor Imaging-A Bench-to-Bedside Journey.

The folate receptor-α (FR-α) is overexpressed in many epithelial cancers, including ovary, uterus, kidneys, breast, lung, colon and prostate carcinomas, but shows limited expression in normal tissues such as kidneys, salivary glands, choroid plexus and placenta. FR-α has therefore emerged as a promising target for the delivery of therapeutic and imaging agents to FR-positive tumors. A series of folate-based PET (positron emission tomography) radiopharmaceuticals have been developed for the selective targeting of FR-positive malignancies. This review provides an overview on the research progress made so far regarding the design, radiosynthesis and the utility of the folate-derived PET radioconjugates for targeting FR-positive tumors. For the most part, results from folate radioconjugates labeled with fluorine-18 (t1/2 = 109.8 min) and gallium-68 (t1/2 = 67.7 min) have been presented but folates labeled with “exotic” and new PET radionuclides such as copper-64 (t1/2 = 12.7 h), terbium-152 (t1/2 = 17.5 h), scandium-44 (t1/2 = 3.97 h), cobalt-55 (t1/2 = 17.5 h) and zirconium-89 (t1/2 = 78.4 h) are also discussed. For tumor imaging, none of the reported PET radiolabeled folates reported to date has made the complete bench-to-bedside journey except [18F]AzaFol, which made it to patients with metastatic ovarian and lung cancers in a multicenter first-in-human trial. In the near future, however, we expect more clinical trials with folate-based PET radiopharmaceuticals given the increasing clinical interest in imaging and the treatment of FR-related malignancies.

A novel dual-modality imaging agent targeting folate receptor of tumor for molecular imaging and fluorescence-guided surgery.

Folate receptor (FR) is an ideal target for cancer imaging because it is frequently overexpressed in major types of human tumor, whereas its expression in normal organs is highly limited. Combining nuclear and fluorescence-imaging techniques provides a novel approach for cancer imaging and monitoring the surgery. The objective of this study was to report the synthesis and characteristics of a dual-modality imaging agent, Tc-99m Folate-Gly-His-Glu-Gly-Glu-Cys-Gly-Lys(-5-carboxy-X-rhodamine)-NH2 (Folate-ECG-ROX), and verify its feasibility as both molecular imaging agent and intra-operative guidance. Folate-ECG-ROX was synthesized using Fmoc solid-phase peptide synthesis. Radiolabeling of Folate-ECG-ROX with Tc-99m was done using ligand exchange via tartrate. Binding affinity and in vitro cellular uptake studies were performed. Gamma camera imaging, biodistribution and ex vivo imaging studies were performed using KB and HT-1080 tumor-bearing murine models. Tumor tissue slides were prepared and analyzed with immunohistochemistry staining and confocal microscopy. Surgical removal of tumor nodules in murine models with peritoneal carcinomatosis was performed under the fluorescence-imaging system. After radiolabeling procedures with Tc-99m, Tc-99m Folate-ECG-ROX complexes were prepared in high yield (> 97%). The binding affinity value (Kd) of Tc-99m Folate-ECG-ROX for KB cells was estimated to be 6.9 ± 0.9nM. In gamma camera imaging, tumor to normal muscle uptake ratio of Tc-99m Folate-ECG-ROX increased with time (3.4 ± 0.4, 4.4 ± 0.7, and 6.6 ± 0.8 at 1, 2, and 3h, respectively). In biodistribution study, %IA/g for KB tumor was 2.50 ± 0.80 and 4.08 ± 1.16 at 1 and 3h, respectively. Confocal microscopy with immunohistochemistry staining detected strong Tc-99m Folate-ECG-ROX fluorescence within KB tumor tissue which is correlating with the fluorescent activity of anti-FR antibody. Under real-time optical imaging, the removal of visible nodules was successfully performed. In vivo and in vitro studies revealed substantial and specific uptake of Tc-99m Folate-ECG-ROX in FR-positive tumors. Thus, Tc-99m Folate-ECG-ROX could provide both pre-operative molecular imaging and fluorescence image-guidance for tumor.

Voltammetric and biological studies of folate-targeted non-lamellar lipid mesophases

Folate-targeted lipid nanostructures are promising strategies for the development of biocompatible drug delivery systems. The objective of this study was to evaluate the efficacy of drug delivery to cancer cells by folate-targeted lipid mesophases, cubosomes (CUB) and hexosomes (HEX), loaded with doxorubicin (DOX). Three cancer-derived cell lines (KB, HeLa, T98G) exhibiting different expressional levels of folate receptor protein (FR) were used. DOX-loaded folate-targeted CUB and HEX dispersions were characterized via small angle X-ray scattering and dynamic light scattering to assess their physicochemical properties. DOX release characteristics were evaluated by electrochemical methods and demonstrated structure-dependent release capabilities. A slow release rate was observed for hexosomes, while cubosomes offered more rapid drug transport. Analysis of the release kinetics revealed that the total amount of DOX released from cubosomes is linearly dependent on the square root of time, implying that the release process follows the Higuchi diffusion model. Assessment of drug uptake performed on cancer-derived cell lines demonstrated that DOX accumulation in cancer cell depends not only on the release capability of the applied mesophase, but also, on the level of folate receptor protein present in the cancer cells. FR-functionalized CUB and HEX enabled faster drug delivery to cancer cells as a result of receptor-ligand interactions. In addition, doxorubicin encapsulated into FR-cubosomes demonstrated significantly improved anti-tumor activity promoting the necrosis of tumor cells, while DOX-loaded FA-hexosomes acted via induction of the apoptotic state. Overall, our data indicates that folate-modified formulations are promising drug delivery systems and can be considered as potential therapeutic tools in the targeted therapy of FR-positive tumors.

Folic acid-nanoscale gadolinium-porphyrin metal-organic frameworks: fluorescence and magnetic resonance dual-modality imaging and photodynamic therapy in hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) is the most common primary liver cancer and severely threatens human health. Since the prognosis of advanced HCC remains poor, there is an urgent need to develop new therapeutic approaches. Porphyrin metal-organic frameworks are a class of porous organic-inorganic hybrid functional materials with good biocompatibility.MethodsGadolinium-porphyrin metal-organic frameworks were used as a skeleton for folic acid (FA) conjugation to synthesize a novel type of nanoparticle, denoted as folic acid-nanoscale gadolinium-porphyrin metal-organic frameworks (FA-NPMOFs). The FA-NPMOFs were characterized using transmission electron microscopy, Fourier transform infrared spectroscopy and thermogravimetric-differential thermal analysis. The biotoxicity and imaging capability of the FA-NPMOFs were determined using HepG2 cells and embryonic and larval zebrafish. The delivery and photodynamic therapeutic effect of FA-NPMOFs were explored in transgenic zebrafish with doxycycline-induced HCC.ResultsFA-NPMOFs were spherical in structure with good dispersion and water solubility. They showed low biotoxicity, emitted bright red fluorescence, and exhibited an excellent magnetic resonance imaging capability, both in vitro and in vivo. Meanwhile, the FA-NPMOFs exhibited a strong affinity for folate receptor (FR)-expressing cells and were delivered to the tumor site in a targeted manner. Moreover, HCC tumor cells were eliminated following laser irradiation.ConclusionFA-NPMOFs can be used for dual-modality imaging and photodynamic therapy in HCC and show promise for use as a carrier in new therapies for HCC and other FR-positive tumors.

Targeting and sensing of some cancer cells using folate bioreceptor functionalized nitrogen-doped graphene quantum dots

In recent years, study of folate receptor (FR) expression related to targeting, drug delivery and counting of tumoral cells have been followed. In this work, a fast and simple strategy was reported to determine the FR expressed cancer cells based on the selective bonding of the folic acid/folate (FA) to the FR-positive tumor cells. The folate decorated Nitrogen-doped graphene quantum dots (N-GQDs) were utilized as selective targeting of the MKN 45 cells. Fluorescent microscopy imaging investigations revealed that the produced FA conjugated N-GQDs could specifically attach to the target FR-positive tumor cells. Due to the fluorescence emission of N-GQDs, the developed cytosensor is free from attaching any fluorescent ligand i.e. Rhodamine B to capture the florescence microscopy images and also flow cytometry analysis. The fabricated cytosensor possesses a dynamic range from 100 to 7.0 × 104 cell·mL−1 with high selectivity. Furthermore, the cytosensor also could visualized the MCF 7 and HT 29 cells where the dynamic ranges were 100 to 1.0 × 104 and 500 to 4.0 × 104 cells·mL−1, respectively. In vitro toxicity tests has shown low toxicity of the synthesized N-GQDs where the minimum viability is 68%. The proposed FA-N-GQDs based cytosensor provides a novel platform for detection of MKN 45, HT 29 and MCF 7 cancer cell lines which could be used in multi-channel cancer diagnosis biodevice.

Combining Albumin-Binding Properties and Interaction with Pemetrexed to Improve the Tissue Distribution of Radiofolates.

Folic-acid-based radioconjugates have been developed for nuclear imaging of folate receptor (FR)-positive tumors; however, high renal uptake was unfavorable in view of a therapeutic application. Previously, it was shown that pre-injection of pemetrexed (PMX) increased the tumor-to-kidney ratio of radiofolates several-fold. In this study, PMX was combined with the currently best performing radiofolate ([177Lu]cm13), which is outfitted with an albumin-binding entity. Biodistribution studies were carried out in mice bearing KB or IGROV-1 tumor xenografts, both FR-positive tumor types. SPECT/CT was performed with control mice injected with [177Lu]folate only and with mice that received PMX in addition. Control mice showed high uptake of radioactivity in KB and IGROV-1 tumor xenografts, but retention in the kidneys was also high, resulting in tumor-to-kidney ratios of ~0.85 (4 h p.i.) and ~0.60 (24 h p.i.) or ~1.17 (4 h p.i.) and ~1.11 (24 h p.i.) respectively. Pre-injection of PMX improved the tumor-to-kidney ratio to values of ~1.13 (4 h p.i.) and ~0.92 (24 h p.i.) or ~1.79 (4 h p.i.) and ~1.59 (24 h p.i.), respectively, due to reduced uptake in the kidneys. It was found that a second injection of PMX—3 h or 7 h after administration of the radiofolate—improved the tumor-to-kidney ratio further to ~1.03 and ~0.99 or ~1.78 and ~1.62 at 24 h p.i. in KB and IGROV-1 tumor-bearing mice, respectively. SPECT/CT scans readily visualized the tumor xenografts, whereas accumulation of radioactivity in the kidneys was reduced in mice that received PMX. In this study, it was shown that PMX had a positive impact in terms of reducing the kidney uptake of albumin-binding radiofolates; hence, the administration of PMX resulted in ~1.3–1.7-fold higher tumor-to-kidney ratios. This is, however, a rather moderate effect in comparison to the previously shown effect of PMX on conventional radiofolates (without albumin binder), which led to 5–6-fold increased tumor-to-kidney ratios. An explanation for this result may be the different pharmacokinetic profiles of PMX and long-circulating radiofolates, respectively. Despite the promising potential of this concept, it is believed that a clinical translation would be challenging, particularly when PMX had to be injected more than once.

Pre-clinical evaluation of EC1456, a folate-tubulysin anti-cancer therapeutic

EC1456 is a folate-tubulysin conjugate constructed with an all-D enantiomeric spacer/linker configuration. When tested against folate receptor (FR)-positive cells, EC1456 demonstrated dose-responsive activity with an approximate 1000-fold level of specificity. Treatment of nude mice bearing FR-positive human xenografts (as large as 800 mm3) with non-toxic doses of EC1456 led to cures in 100% of the mice. Combinations of low dose EC1456 with standard of care agents such as platins, taxanes, topotecan and bevacizumab, safely and significantly augmented the growth inhibitory effects of these commonly used agents. When tested against FR-positive human tumor xenograft models having confirmed resistance to a folate-vinca alkaloid (vintafolide), cisplatin or paclitaxel, EC1456 was found to generate partial to curative responses. Taken together, these studies demonstrate that EC1456 has significant anti-proliferative activity against FR-positive tumors, including models which were anticancer drug resistant, thereby justifying a Phase 1 trial of this agent for the treatment of advanced human cancers.

Comparison Study of Two Differently Clicked 18F-Folates-Lipophilicity Plays a Key Role.

Within the last decade, several folate-based radiopharmaceuticals for Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET) have been evaluated; however, there is still a lack of suitable 18F-folates for clinical PET imaging. Herein, we report the synthesis and evaluation of two novel 18F-folates employing strain-promoted and copper-catalyzed click chemistry. Furthermore, the influence of both click-methods on lipophilicity and pharmacokinetics of the 18F-folates was investigated. 18F-Ala-folate and 18F-DBCO-folate were both stable in human serum albumin. In vitro studies proved their high affinity to the folate receptor (FR). The lipophilic character of the strain-promoted clicked 18F-DBCO-folate (logD = 0.6) contributed to a higher non-specific binding in cell internalization studies. In the following in vivo PET imaging studies, FR-positive tumors could not be visualized in a maximum intensity projection images. Compared with 18F-DBCO-folate, 18F-Ala-folate (logD = −1.4), synthesized by the copper-catalyzed click reaction, exhibited reduced lipophilicity, and as a result an improved in vivo performance and a clear-cut visualization of FR-positive tumors. In view of high radiochemical yield, radiochemical purity and favorable pharmacokinetics, 18F-Ala-folate is expected to be a promising candidate for FR-PET imaging.

Reduced 18F-Folate Conjugates as a New Class of PET Tracers for Folate Receptor Imaging.

5-Methyltetrahydrofolate (5-MTHF), a reduced folate form, is the biologically active folate involved in many different metabolic processes. To date, there are no studies available in the literature on 18F-labeled 6 S- and 6 R-5-MTHF radiotracers for imaging folate receptor (FR)-α-positive tissues. Therefore, the goal of this study was to synthesize four 18F-labeled 5-MTHF derivatives conjugated at either the α- or γ-carboxylic functionality of glutamate and to assess their suitability for FR-targeting. Organic syntheses of the precursors and the four reference compounds, namely, 6 S-α, 6 S-γ, 6 R-α, and 6 R-γ-click-fluoroethyl-5-MTHF, were carried out in low to moderate overall chemical yields. The radiosyntheses of the α- and γ-conjugated 18F-labeled folate derivatives were accomplished in approximately 100 min, low radiochemical yields (1-7% d.c.) and high molar activities (139-245 GBq/μmol). Radiochemically pure tracers were obtained after the addition of a mixture of antioxidants consisting of sodium ascorbate and l-cysteine. In vitro, all four 5-MTHF conjugates showed similar binding affinities to FR-α (IC50 = 17.7-24.0 nM), whereas folic acid showed a significantly higher binding affinity to the FR-α. Cell uptake and internalization experiments with KB cells demonstrated specific uptake and internalization of the radiofolate conjugates. Metabolite studies in mice revealed high in vivo stability of the radiotracers in mice. Biodistribution and positron emission tomography (PET) imaging studies in FR-positive KB tumor-bearing mice demonstrated that the 6 S- and 6 R-5-MTHF conjugates exhibited a different accumulation pattern in various organs including the kidneys and the liver, whereas no significant differences in radioactivity accumulation in the kidneys and the liver were found for both the α- and γ-conjugated diastereoisomers. Despite the considerably lower binding affinities of the 5-MTHF derivatives compared to the corresponding folic acid conjugates similar high KB tumor uptake was observed for all the folate conjugates investigated (8-11% IA/g). Based on these results, we conclude that 18F-labeled 5-MTHF conjugates are a promising new class of radiotracers for targeting FR-positive tumor tissues.

Abstract 3228: Development and characterization of in vitro assays to detect and quantitate tubulysin B hydrazide in biological samples

Abstract EC1456 is a folate-targeted small molecule drug conjugate (SMDC) of tubulysin B hydrazide (TubBH) that is currently in Phase I clinical studies for treatment of patients with folate receptor (FR)-positive tumors. The ability to detect, quantitate, and localize drug in tissues and biological fluids is particularly important for the characterization of its biodistribution and pharmacodynamic properties. To aid in the biological characterization of our targeted TubBH conjugates, our lab had a rabbit polyclonal antibody produced against TubBH for use in various assays. An IgG-purified fraction of antiserum was used to develop competitive ELISA, immunohistochemical, and flow cytometry methods for analysis of biological samples, such as xenograft tumors from animals dosed with drug. Before assay development, a direct ELISA was performed to determine antibody titer and specificity. The TubBH antibody showed excellent binding to immobilized TubBH, whereas a rabbit IgG negative control antibody had minimal binding. Next, a competitive ELISA was developed in which soluble TubBH competed for antibody binding to immobilized antigen. Multiple experimental parameters were optimized, including coating antigen concentration, antibody dilution, and sample matrix extraction method. Using the optimized conditions, the resulting standard curves for TubBH in acetonitrile-extracted KB tumor sample matrix showed good sensitivity and dose-response. It was determined that, in addition to untargeted TubBH, the antibody could also be used to quantitate intact EC1456, as well as a metabolite of the drug lacking the hydrazide. The utility of the antibody was further demonstrated utilizing a PSMA-targeted version of TubBH. Once optimized, the ELISA method was used to analyze FR-positive KB and FR-negative A549 xenograft tumor homogenates from animals dosed with either EC1456 or untargeted TubBH. The levels of TubBH were higher 4h-post EC1456 dose in the FR-positive KB tumors compared to the FR-negative A549 tumors; however, levels were similar after 24h. Moreover, untargeted TubBH resulted in lower tumor concentrations of the drug compared to EC1456 in KB tumors. An immunohistochemical method developed using the antibody showed strong, focal, cytoplasmic staining in FFPE tumor sections of KB tumors from animals dosed with EC1456, while no staining was observed in tumors from undosed animals. Finally, the antibody was successfully used to detect cell surface FR-bound EC1456 on fixed and unfixed KB cells in vitro by flow cytometry. This experiment confirmed that the antibody is able to recognize the conjugate even when it is situated in the FR binding pocket. Collectively, these results show that this antibody against TubBH can be used as a powerful tool for analysis of tumor drug uptake for folate-targeted TubBH in xenograft models and could hold great potential for use in patient sample analysis as well. Citation Format: Nikki L. Parker, Jonathan M. Shillingford, Melissa Nelson, Joseph A. Reddy, Christopher P. Leamon. Development and characterization of in vitro assays to detect and quantitate tubulysin B hydrazide in biological samples [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 3228. doi:10.1158/1538-7445.AM2017-3228

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

Abstract 4574: Combinatorial strategies of folate receptor-targeted chemotherapy guided by improved understanding of tumor microenvironment and immunomodulation

Abstract Inflammation disorder is a major component of cancer progression and drug resistance. Tumor microenvironment due to its complex nature presents physical and physiological barriers to both chemotherapy and immunotherapy. On the other hand, the folate receptor (FR) family includes two cysteine-rich GPI-anchored membrane glycoproteins (FRα/β) that are capable of bringing folate-linked small-molecule drugs (SMDCs) into the cell cytosol via endocytosis. Consequently, both receptor isoforms have been considered promising therapeutic targets due to frequent overexpression of FRα by cancer cells of epithelial origins and FRβ by tumor-associated macrophages (TAMs). TAMs are strongly immunosuppressive and high TAM frequency has been correlated with poor patient prognosis and treatment outcome. While a folate-targeted chemotherapy can be very powerful for sensitive tumor types, resistance does regularly occur in FR-positive tumors for reasons that are largely unknown. Moreover, there seems to be considerable disconnect between a drug candidate’s in-vitro activity and in-vivo efficacy. Using syngeneic mouse models that represent different tumor immune microenvironments (M109, Renca, 4T1-Cl2), we investigated the immunomodulatory properties of SMDCs and devised combinatorial strategies with standard-of-care (SOC) agents to break the tumor-induced immune tolerance. Our results indicated that a successful treatment goes beyond targeting the cancer cells themselves; instead, it requires a good match between our compound and a “sensitive” tumor microenvironment. We found that within “TAM-rich” solid tumors, an effective SMDC may be one with dual mechanisms of action that affect both FRα-positive tumor cells and FRβ-positive TAMs. Moreover, the process of choosing partner drugs, such as immune checkpoint inhibitors and inhibitors of myeloid-derived suppressor cells, should be guided by the immunomodulatory properties of both SMDCs and SOC agents to overcome the frequently observed chemo- and immuno-resistance. Citation Format: Yingjuan Lu, Leroy W. Wheeler, Vicky A. Cross, Elaine M. Westrick, Alex M. Lloyd, Theresa P. Johnson, Nikki L. Parker, Christopher P. Leamon. Combinatorial strategies of folate receptor-targeted chemotherapy guided by improved understanding of tumor microenvironment and immunomodulation [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 4574. doi:10.1158/1538-7445.AM2017-4574

Enhanced antitumor effect of targeted nanoliposomal bleomycin.

Folate receptor (FR)-mediated drug delivery is a promising approach for active targeting of drugs to the FR-positive tumor cells. Bleomycin (BLM) is an antitumor antibiotic with poor therapeutic activity as a result of its limited diffusion into tumor cells. The aim of this study was to investigate whether FR-targeted PEGylated nanoliposomes (FPNL) can effectively deliver BLM to tumor cells and enhance its in vitro and in vivo efficacy. FPNL and PNL (non-targeted) were prepared by thin film hydration method, and their physiochemical properties, cellular uptake, tissue distribution and tumor inhibitory effects were investigated. In Lewis lung cancer (LLC1) cells, FPNL containing BLM showed 2.38-fold and 3.26-fold higher cytotoxicity compared to PNL-BLM and free BLM, respectively. Moreover, the uptake of FPNL by these cells was increased as compared to the PNL. Furthermore, FPNL showed significantly higher tumor distribution of BLM in the LLC1 cells and more tumor inhibition efficacy compared to free BLM and PNL. Both formulations of nanoliposomes had longer plasma half-life than that of free BLM. Therefore, FPNL may be suitable carriers for targeted drug delivery to FR-positive tumor cells.