Folate Binding Protein Research Articles

Interim analysis of a phase I/IIA trial assessing E39+GM-CSF, a folate binding protein vaccine, to prevent recurrence in ovarian and endometrial cancer patients

Interim analysis of a phase I/IIA trial assessing E39+GM-CSF, a folate binding protein vaccine, to prevent recurrence in ovarian and endometrial cancer patients

Abstract P6-10-04: Determining the optimal vaccination strategy using a combination of the folate binding protein (FBP) peptide vaccine (E39+GM-CSF) and an attenuated version (E39') to maximize the immunologic response in breast cancer patients

Abstract BACKGROUND FBP is overexpressed in 20-50% of breast(B) cancers(Ca) and roughly 90% of endometrial(E) and ovarian (Ov) Ca. E39 (FBP191-199, EIWTHSYKV)+GM-CSF is an HLA-A2 restricted FBP peptide vaccine, which has been shown to generate significant in vivo immunologic response(IR) in a phase I/IIa trial in E Ca and Ov Ca patients (pts). There is a risk of inducing immunologic tolerance after multiple inoculations with a highly immunogenic vaccine. Thus, we are investigating a novel vaccination series using combinations of E39 and E39' (EIWTFSTKV, an attenuated version of E39) in a phase Ib, randomized, single-center trial. We are assessing short and long-term IR. Here, we present the initial IR analysis to the primary vaccination series (PVS) within B Ca pts. METHODS HLA-A2 positive B or Ov Ca pts were enrolled after completion of standard of care therapy and randomized into three arms: EE (6 inoculations of E39); EE'(3 inoculations of E39, then 3 of E39'); or E'E(3 of E39', then 3 of E39). Theoretically, due to lower FBP expression and less aggressive chemotherapy regimens, B Ca pts are more antigen naïve and have a less suppressed immune system. Thus, only B Ca pts were included in this analysis. The PVS includes 6 inoculations total (R1-R6), one every 3-4 weeks, and containing 250mcg GM-CSF+500mcg peptide in the first 5 pts per arm and 1000mcg of peptide in second 5 pts. To assess the in vivo IR, local reaction(LR) was measured 48 hours after each inoculation (R1-R6), and delayed type hypersensitivity(DTH) was measured pre-PVS (R0), 1, and 6-months post-PVS (RC1, RC6). Ex vivo IR was measured via dextramer assay for E39-specific CD8+ T-cells at R0, RC1, and RC6. Statistical analyses were completed using appropriate tests. RESULTS Thirty-five B Ca pts were enrolled, with 27 completing the PVS (EE n=10, EE' n=8, E'E n=9). No clinicopathologic differences between groups or significant toxicities > grade 2 were appreciated. LR increased from R1 to R6 in all groups (ΔEE= 24.80mm, p=0.14; ΔEE'=38.13mm, p=0.07; ΔE'E=8.05mm, p=0.38), the greatest increase approaching statistical significance in the EE' arm. The only arm with a statistically significant increase for in vivo DTH from R0-RC1-RC6 was in the EE' arm (ΔEE=-6.17mm, p=0.27; ΔEE'= 44.58mm, p<0.05; ΔE'E=-1.42, p=0.37). Ex vivo analysis of IR revealed no significant difference between groups at R0(p=0.45) or RC6(p=0.72), nor within groups over time (EE p=0.32, EE' p=0.47, E'E p=0.30). CONCLUSION In this phase Ib trial analyzing the IR of B Ca pts receiving a different vaccination strategy, both peptides were noted to be safe and immunogenic. While no difference was seen in E39-specific CD8+ T cells between groups, the in vivo response was enhanced with the use of E39' after E39; this may indicate expansion of more effective clonal populations of CD8+ T cells with this strategy. These results may be specific to B Ca pts who are relatively antigen-naïve with relatively intact immune systems. Further analysis of these pts as this trial continues will determine the optimal vaccination strategy capable of stimulating and maintaining an IR to prevent B Ca recurrence. Citation Format: Jackson DO, Qiao N, Peace KM, Hale DF, Vreeland TJ, Greene JM, Berry JS, Trappey AF, Clifton GT, Ibrahim N, Toms A, Peoples GE, Mittendorf EA. Determining the optimal vaccination strategy using a combination of the folate binding protein (FBP) peptide vaccine (E39+GM-CSF) and an attenuated version (E39') to maximize the immunologic response in breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-10-04.

Genome-wide characterization of folate transporter proteins of eukaryotic pathogens

Background:Medically important pathogens are responsible for the death of millions every year. For many of these pathogens, there are limited options for therapy and resistance to commonly used drugs is fast emerging. The availability of genome sequences of many eukaryotic protozoa is providing important data for understanding parasite biology and identifying new drug and vaccine targets. The folate synthesis and salvage pathway are important for eukaryote pathogen survival and organismal biology and may present new targets for drug discovery.Methods:We applied a combination of bioinformatics methods to examine the genomes of pathogens in the EupathDB for genes encoding homologues of proteins that mediate folate salvage in a bid to identify and assign putative functions. We also performed phylogenetic comparisons of identified proteins. .Results:We identified 234 proteins to be involve in folate transport in 63 strains, 28 pathogen species and 12 phyla, 60% of which were identified for the first time. Many of the genomes examined contained genes encoding transporters such as folate-binding protein YgfZ, folate/pteridine transporter, folate/biopterin transporter, reduced folate carrier family protein, folate/methotrexate transporter FT1. The mitochondrion is the predicted location of the majority of the proteins, with 15% possessing signal peptides. Phylogeny computation shows the similarity of the proteins identified.Conclusion:These findings offer new possibilities for potential drug development targeting folate-salvage proteins in eukaryotic pathogens.

Folate binding protein: therapeutic natural nanotechnology for folic acid, methotrexate, and leucovorin.

Blood serum proteins play a critical role in the transport, biodistribution, and efficacy of systemically-delivered therapeutics. Here, we have investigated the concentration- and ligand-dependent aggregation of folate binding protein (FBP), focusing in particular on folic acid, an important vitamin and targeting agent; methotrexate, an antifolate drug used to treat cancer and rheumatoid arthritis; and leucovorin which is used to decrease methotrexate toxicity. We employed atomic force microscopy to characterize, on a particle-by-particle basis, the volumes of the FBP nanoparticles that form upon ligand binding. We measured the distribution of FBP nanoparticle volumes as a function of ligand concentration over physiologically- and therapeutically-relevant ranges. At physiologically-relevant concentrations, significant differences in particle volume distributions exist that we hypothesize are consistent with different trafficking mechanisms for folic acid and methotrexate. In addition, we hypothesize leucovorin is trafficked and delivered like folic acid at therapeutically-relevant concentrations. We propose that changes in dosing procedures could improve the delivery and therapeutic index for methotrexate and other folic acid-targeted drug conjugates and imaging agents. Specifically, we suggest pre-binding the drugs to FBP may provide a better formulation for drug delivery of methotrexate for both cancer and rheumatoid arthritis. This would be analogous to pre-binding paclitaxel to albumin, which is already used in the clinic.

Folate-Binding Protein Self-Aggregation Drives Agglomeration of Folic Acid Targeted Iron Oxide Nanoparticles.

Folate-conjugated nanomaterials have been widely investigated for drug and imaging-agent delivery. In this work, two folic acid (FA) conjugated iron oxide particles (IOP), a ∼40 nm diameter FA-IOP and a ∼450 nm diameter FA-IOP(FA-SeraMag), were synthesized. Both particles aggregated in the presence of serum folate-binding protein (FBP) at physiological concentration and buffer conditions. Mixing 0.01% w/w FA-conjugated iron oxide particles with FBP-induced agglomeration generated an average hydrodynamic particle diameter of 3800 ± 1100 nm for ∼40 nm FA-IOP and 4030 ± 1100 nm for FA-SeraMag as measured by dynamic light scattering (DLS). The presence of excess human serum albumin (HSA) (600 μM) did not prevent agglomeration of the ∼40 nm FA-IOP; however, it did inhibit agglomeration of FA-SeraMag. Atomic force microscopy measurement provided additional insight into particle morphology with the detection of individual particles in the agglomerate. This behavior is an example of a triggered cascade. A protein structural change is induced by FA binding, and the structural change favors aggregation of the ∼4 nm diameter FBPs on the particle surface; this further triggers the agglomeration of both the ∼40 and ∼450 nm diameter IOPs.

Abstract B007: Improved disease-free survival in endometrial and ovarian cancer patients with low folate binding protein expression after treatment with the E39 peptide vaccine in a Phase I/IIa trial

Abstract Introduction: One of the most encouraging examples of targeted therapy for cancer is trastuzumab, but its success is dependent on levels of expression of its target, HER2. We have found that HER2 expression levels also have a significant impact on the efficacy of HER2-directed peptide vaccines. Analogous to HER2 in breast cancer, Folate Binding Protein (FBP) is over-expressed on ovarian and endometrial cancer cells (up to 80 - 90-fold higher) and increased FBP expression is associated with aggressive disease. As a result, multiple FBP-directed therapies are being developed. We are investigating E39 + GM-CSF, which is an HLA-A2-restricted FBP-derived peptide vaccine used to prevent recurrence in disease-free endometrial and ovarian cancer patients (pts) after standard of care (SOC), multi-modality therapy. We have shown that E39 is safe, effectively generates E39-specific immune responses, and may improve DFS when optimally dosed in a phase I/IIa trial.1 Little is known about the effects of FBP expression levels on FBP-directed therapies, including our E39 vaccine. Purpose: Here, we report clinical outcomes of patients based on FBP expression levels from a phase I/IIa trial of the E39+GM-CSF vaccine given for the prevention of recurrence in disease-free endometrial and ovarian cancer patients. Methods: Disease-free, HLA-A2+ pts were vaccinated (VG), while HLA-A2- pts were followed as untreated controls (CG). The VG received 6 monthly inoculations of E39+GM-CSF, including either 100, 500, or 1000mcg of peptide and 250mcg of GM-CSF. FBP expression testing was performed by immunohistochemistry and the results were graded 0-4+ based on the percentage of positively staining cells. Patient's tumors were then categorized as low expression (FBPlo) if scored 0-1+ or high expression (FBPhi) if 2-4+. The pts were monitored for evidence of clinical recurrence through the SOC follow-up by their treating oncology team. Demographics, FBP expression and disease-free survival (DFS) were analyzed using appropriate statistical tests. Results: Thirty-eight enrolled pts underwent FBP expression testing (CG n = 20; VG n = 18). There were no clinicopathologic differences between groups (p≥0.1). Nineteen pts were found to be FBPlo (CG, n = 11; VG, n = 8) and 19 were FBPhi (CG, n = 9; VG, n = 10). Median follow up was 16.3 months. There was no significant difference in overall DFS between the CG and the VG (34.6% vs. 34.6%, p = 0.208). In FBPlo pts, there was improved DFS in the VG vs. CG (85.7% vs. 17.5%, p = 0.01) while there was no such difference in FBPhi pts (VG:13.9% vs. CG:44.4%, p = 0.83). Though groups were small, there was a dose-dependent effect on DFS; pts receiving 1000mcg (n = 4) had improved DFS compared to the <1000mcg pts (n = 4) and the CG (n = 11; 100% vs. 66.7% vs.17.5%, respectively, p = 0.03). Comparing FBPlo and FBPhi pts in the VG, the FBPlo pts had improved DFS (85.7% vs. 15.6%, p = 0.052). In the CG, FBPlo pts did worse (17.5% vs. 44.4% in FBPhi, p = 0.371). Conclusion: This phase I/IIa trial has previously demonstrated that E39 is well-tolerated, elicits a strong, dose-dependent in vivo immune response and may improve DFS when properly dosed. This focused analysis based on FBP expression level revealed a DFS benefit in FBPlo, but not FBPhi, endometrial and ovarian cancer pts treated with E39. This may be due to immunotolerance from significantly higher endogenous exposure to the FBP antigen. This is concordant with findings in our trials of HER2-directed peptide vaccines in breast cancer pts. These findings warrant further study as they may have important implications regarding the target population for future E39 peptide vaccine trials. 1. Jackson DO, et al. Interim analysis of a phase I/IIa trial assessing E39+GM-CSF, a folate binding protein vaccine, to prevent recurrence in ovarian and endometrial cancer patients. In Press. Citation Format: Kaitlin M. Peace, Diane F. Hale, Timothy J. Vreeland, Doreen O. Jackson, Julia M. Greene, John S. Berry, IV, Alfred F. Trappey, Garth S. Herbert, Guy T. Clifton, Mark O. Hardin, Kathleen M. Darcy, Chad A. Hamilton, G. Larry Maxwell, George E. Peoples. Improved disease-free survival in endometrial and ovarian cancer patients with low folate binding protein expression after treatment with the E39 peptide vaccine in a Phase I/IIa trial [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B007.

Abstract CT073: Comparing an attenuated booster (E39’) vs E39 booster to potentiate the clinical benefit of the folate binding protein (FBP)-derived vaccine (E39 + GM-CSF) in a phase I/IIa trial to prevent recurrence in endometrial (EC) and ovarian cancer (OC) patients

Abstract Background: We have completed treatment in a phase I/IIa trial utilizing E39 (GALE-301, FBP 191-199, EIWTHSYKV), an HLA-A2 restricted, FBP-derived peptide + GM-CSF vaccine to prevent recurrence in EC and OC patients (pts). This vaccine has been shown to be safe and immunogenic, with promising early clinical results. Booster inoculations have improved disease-free survival (DFS) in our previous peptide vaccine trials, but repeated boosting can theoretically lead to overstimulation and loss of vaccine-induced T cells. To avoid this, we have developed an attenuated version of E39, E39’ (GALE-302, EIWTFSTKV), which has been shown to expand FBP-specific cytolytic T cells in vitro and in vivo. Therefore to assess this strategy, E39-vaccinated pts from the phase I/IIa trial were randomized to receive a booster series of either E39’ or E39 + GM-CSF and compared for safety, immunologic response, and DFS. Methods: HLA-A2+ pts were vaccinated (VG), and HLA-A2- pts were followed as controls (CG). Six monthly intradermal inoculations of E39 + 250mcg GM-CSF were administered to the VG to complete the primary vaccine series (PVS). Patients were then randomized to receive 2 booster inoculations of 500mcg of E39’ or E39 + 250mcg GM-CSF at 6 (B1) and 12 (B2) months post-PVS. Local reactions (LR) were recorded 48-72 hours after each booster. Demographic, safety, immunologic, and DFS data were collected and evaluated with the appropriate statistical tests. Results: A total of 51 pts were enrolled; 29 in VG and 22 in CG. 17 pts continued on to the booster series and were randomized. For B1, 9 received E39’ and 8 received E39; B2 included 7 pts in each group. There were no significant clinicopathologic differences between groups. No difference in toxicities were seen with no grade 3 or 4 toxicities in either group. The average LR for the E39’ vs E39 groups were 79.7+14.0 mm vs 82.1+8.3 mm, respectively for B1 (p = 0.45) and 74.1+11.5 mm vs 78+11.2 mm, respectively for B2 (p = 0.41). Clinically, the recurrence rate was 22.2% in the E39’ boost group vs 25% for E39. The estimated 2-year DFS for B1 pts for E39’, E39 and the CG were 66.7%, 58.3%, and 36.0%, respectively; and for B2 pts were 66.7%, 66.7%, and 36.0%. Comparing just the boosted groups, for B1 the hazard ratio (HR) for E39’ vs E39 = 0.71 (95% CI: 0.1 - 5.13), and for B2 the HR for E39’ vs E39 = 0.82 (95% CI: 0.05 - 13.24). Conclusion: The use of an attenuated peptide (E39’) booster was safe and as immunogenic as the wildtype peptide (E39) in this randomized trial of optimal boosting strategies. More importantly, there appears to be a potential clinical advantage to the attenuated booster in this small trial. These results must be corroborated by further evaluation of this attenuated peptide vaccine and boosting strategy in a larger clinical trial. Citation Format: Doreen O. Jackson, Timothy J. Vreeland, Diane F. Hale, Garth S. Herbert, Julia M. Greene, Erika J. Schneble, John S. Berry, Alfred F. Trappey, Guy T. Clifton, John C. Elkas, Chad Hamilton, Kathleen M. Darcy, George L. Maxwell, George E. Peoples. Comparing an attenuated booster (E39’) vs E39 booster to potentiate the clinical benefit of the folate binding protein (FBP)-derived vaccine (E39 + GM-CSF) in a phase I/IIa trial to prevent recurrence in endometrial (EC) and ovarian cancer (OC) patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT073.

The primary analysis of a phase I/IIa dose finding trial of a folate binding protein vaccine, E39 + GM-CSF in ovarian and endometrial cancer patients to prevent recurrence.

The primary analysis of a phase I/IIa dose finding trial of a folate binding protein vaccine, E39 + GM-CSF in ovarian and endometrial cancer patients to prevent recurrence.

Folate content of cultivated and wild traditional leafy vegetables found in Nigeria

Folate is an important micronutrient in the prevention of neural tube defects and cardiovascular disease. Given its importance, it is necessary to establish accurately the folate content of food that could contribute significantly to dietary intake. The aim of the study was to quantify the folate content in raw and cooked traditional leafy and non‐leafy vegetables because there is a dearth of information on the folate content leafy vegetables found in Nigeria. The vegetables included: Corchorus olitorius, Adansonia digitata, Abelmoscus esculentus (fruit), Abelmoscus esculentus (leaves), Amaranthus hybridus, Launaea taraxacifolia, Solanum macrocarpon, and Crassocephalum crepidioides. They were further cooked in boiling water, until assessed as cooked according to Nigerian eating habits.Folate is found in foods mainly as polyglutamate. The analysis of folate in the raw and cooked vegetable samples followed the process of extraction, deconjugation (using chicken pancreas deconjugase), derivatisation (a series of chemical reactions to convert all folate present in the deconjugated sample extracts, to 5‐methyltetrahydrofolic acid (THF‐5CH3) monosodium glutamate and / or diglutamate), purification by affinity chromatography with Folate Binding Protein and quantification by HPLC equipped with fluorimetric detection.Folate content in the raw samples ranged from 183.4 μg/100g (FW) in Corchorus olitorius to 21.5 μg/100g (FW) in Solanum macrocarpon leaves; and in the boiled samples from 48.6 μg/100g (FW) in Launaea taraxacifolia to 8.5 μg/100g (FW) in Solanum macrocarpon. Loss of folate in the boiled vegetables varied from 46.6% in Launaea taraxacifolia to 88.4% in Adansonia digitata.Traditional green leafy vegetables are good sources of folate. However cooking of the vegetables caused a considerable decrease in the folate content of the vegetables. Considering that most leafy vegetables are consumed in their cooked form in Nigeria, preparation methods of traditional leafy vegetables that would allow for optimal retention of folate are necessary.Support or Funding InformationThis work was supported by an Agropolis Foundation grant for the internship of SIE and FWMD at Institut National de la Recherch Agronomique (INRA), Avignon, France. AWARD Fellowship provided the platform and opportunity of a 6‐month research attachment/ Advanced science training in INRA to conduct this research; University of Ibadan granted the permission to participate in the Advanced Science training during which this study was carried out. Labex Agro also supported this work. Uncultivated/wild and cultivated traditional green leafy vegetables studied Scientific name Family English name Local/common name Status Amaranthus hybridus L. Amaranthaceae Pig weed Efo tete/ green Cultivated Abelmoscus manihot (L.) Medikus (leaves and tender shoots) Malvaceae Okro leaves Ilasa Adansonia digitatad Malvaceae Baobab leaves Luru / kuka Uncultivated Corchorus olitorius L. Malvaceae Jute mallow Ewedu Cultivated Crassocephalum crepidioides (Benth.) S. Moore Asteraceae Ebolo Uncultivated Launaea taraxacifolia (Wild.) Amin, ex C. Jeffrey Asteraceae African lettuce/wild lettuce Yanrin Uncultivated Solanum macrocarpon L. Solanaceae Efo igbagba Cultivated/wild Folate content of vegetables: RESULTS – Folate content of traditional green leafy vegetables Vegetable Condition Moisture Folic acid Loss Local/common name Scientific name % μg/100g % Ewedu Corchorus olitorius Fresh 82.59 183.36±12.9 74.2 Boiled 95.13 47.31 ±3.7 Luru / kuka Adansonia digitata Dried ‐ 149.64±13.9 88.4 Boiled 90.11 17.36±2.1 Okro/ ila Abelmoscus esculentus fruit Fresh 87.09 106.00±15.6 66.3 Boiled 95.77 35.69±8.49 Ilasa Abelmoscus manihot Fresh 77.05 46.75±9.49 n/a Efo tete/ green Amaranthus hybridus Fresh 84.34 62.15±13.7 48.2 Boiled 91.58 32.19±4.9 Yanrin Launaea taraxacifolia Fresh 88.12 91.13±11.2 46.6 Boiled 92.16 48.63±8.4 Efo igbagba Solanum macrocarpon Fresh 84.82 21.54±3.1 60.6 Boiled 89.55 8.48±1.2 Ebolo Crassocephalum crepidioides Fresh 89.07 70.98±4.9 75.8 Boiled 94.1 17.18±1.7

Folate bio-fortification of yoghurt and fermented milk: a review

Folate, an essential vitamin, plays an important role in human life for the synthesis of nucleotides, vitamins and some amino acid. This vitamin cannot be synthesized by humans and must be obtained exogenously to prevent folate deficiency, neural tube defects (NTDs) and other related diseases. Folic acid, the chemically synthesized form of folate, is used for fortification and supplementation, but it can cause adverse effects such as vitamin B12 deficiency in high intake. Therefore, novel methods to increase concentrations of naturally occurring folate in foods have grabbed the interest of researchers. The application of bacterial cultures in food fermentation is a novel strategy to increase “natural” folate levels. Dairy products, especially yoghurt, are an appropriate choice for bio-fortification of folate as they contain folate-binding protein which improves folate bioavailability. This study will review the folate production by probiotic bacteria in fermented milk based on various strains and culture conditions.

Substrate-Triggered Exosite Binding: Synergistic Dendrimer/Folic Acid Action for Achieving Specific, Tight-Binding to Folate Binding Protein.

Polymer-ligand conjugates are designed to bind proteins for applications as drugs, imaging agents, and transport scaffolds. In this work, we demonstrate a folic acid (FA)-triggered exosite binding of a generation five poly(amidoamine) (G5 PAMAM) dendrimer scaffold to bovine folate binding protein (bFBP). The protein exosite is a secondary binding site on the protein surface, separate from the FA binding pocket, to which the dendrimer binds. Exosite binding is required to achieve the greatly enhanced binding constants and protein structural change observed in this study. The G5Ac-COG-FA1.0 conjugate bound tightly to bFBP, was not displaced by a 28-fold excess of FA, and quenched roughly 80% of the initial fluorescence. Two-step binding kinetics were measured using the intrinsic fluorescence of the FBP tryptophan residues to give a KD in the low nanomolar range for formation of the initial G5Ac-COG-FA1.0/FBP* complex, and a slow conversion to the tight complex formed between the dendrimer and the FBP exosite. The extent of quenching was sensitive to the choice of FA-dendrimer linker chemistry. Direct amide conjugation of FA to G5-PAMAM resulted in roughly 50% fluorescence quenching of the FBP. The G5Ac-COG-FA, which has a longer linker containing a 1,2,3-triazole ring, exhibited an ∼80% fluorescence quenching. The binding of the G5Ac-COG-FA1.0 conjugate was compared to poly(ethylene glycol) (PEG) conjugates of FA (PEGn-FA). PEG2k-FA had a binding strength similar to that of FA, whereas other PEG conjugates with higher molecular weight showed weaker binding. However, no PEG conjugates gave an increased degree of total fluorescence quenching.

Folate receptor-targeted multimodal polymersomes for delivery of quantum dots and doxorubicin to breast adenocarcinoma: In vitro and in vivo evaluation

In this study, we report the design and delivery of tumor-targeted, quantum dot (QD) and doxorubicin (DOX)-encapsulated PEG-PLGA nanopolymersomes (NPs) for the imaging and chemotherapy of breast cancer. To achieve active cancer targeting, QD and DOX-encapsulated NPs were conjugated with folate for folate-binding protein receptor-guided delivery, which overexpressed in many cancer cells. Hydrophobic DOX and hydrophilic MSA-capped QD were encapsulated in the bilayer and core of the PEG-PLGA nanopolymersomes, respectively.The data show that the formulated NPs sustained DOX release for a period of 12 days. Fluorescence microscopy and MTT assay demonstrated that the developed folate-targeted DOX-QD NPs had higher cytotoxicity than non-targeted NPs and the free form of the drug; moreover, they preferentially accumulated in 4T1 and MCF-7 cells in vitro.In vivo experiments including whole organ tissue–homogenate analysis and organ fluorescence microscopy imaging of BALB/c mice bearing 4T1 breast adenocarcinoma showed that the folate receptor-targeted QD encapsulated NPs accumulate at tumor sites 6h following intravenous injection. Acute toxicity studies of the prepared targeted QD-loaded NPs showed no evidence of long-term harmful histopathological and physiological effects on the treated animals. The in vivo tumor inhibitory effect of folic acid (FA)-QD-DOX NPs demonstrated an augmented therapeutic efficacy of targeted formulation over the non-targeted and free drug. The data obtained illustrate a high potential of the prepared targeted theranostic nanoplatform in the treatment and imaging of breast cancer. This study may open new directions for preparation of QD-based theranostic polymersomes for clinical application.

Effect of novel peptide vaccines on secondary recurrences and survivorship.

e274 Background: According to the NCI Office of Cancer Survivorship, in 2014, it is estimated there are 14.5 million cancer survivors in the US. By 2024, it is projected there will be 19 million cancer survivors. Peptide-based vaccines are being developed to prevent recurrence in cancer patients following standard of care and increase survivorship by inducing immune responses using antigenic epitopes derived from tumor associated antigens. Two peptide vaccines, nelipepimut-S, a HLA-A2/A3-restricted immunogenic peptide derived from the HER2 protein and E39, a HLA-A2-restricted, folate binding protein (FBP) derived peptide, are being developed to prevent breast cancer and gynecological cancers recurrence, respectively. Methods: Early phase dose finding clinical trials results for both agents have been previously reported by Mittendorf et al. (2014) and Greene et al. (2015). Both agents are concomitantly administered with GM-CSF in the adjuvant setting. Toxicity was monitored and recurrences were documented. Disease free survival (DFS) was analyzed by Kaplan-Myers curves and groups were compared by log-rank test. Results: In the nelipepimut-S Phase 1/2 trial, 97 patients were enrolled in the node positive group. The reported 3-year DFS was 100% in the optimally dosed (1000 mcg vaccine + 250 mcg GM-CSF) vaccinated group vs. 77.8% in the control (p = 0.035, n = 45). In the E39 Phase 1/2 trial, with 9.8 months median follow-up, among optimally dosed (1000 mcg), there is only one recurrence (6.7% vs. 5I% CG, p = 0.01). The 2-year DFS estimate is 19.2% (CG) vs. 85.7% (1000 mcg) (p = 0.09). Nelipepimut-S and E39 were well tolerated. Conclusions: Preliminary results from both Phase 1/2 trials indicate these peptide vaccines derived from the HER2 protein and FBP may be administered chronically, thereby preventing recurrence and increase survivorship in patients with breast, ovarian, and endometrial cancers. Nelipepimut-S is currently being investigated in three ongoing adjuvant clinical trials – 1) single agent in a registrational Phase 3, PRESENT, under a FDA-approved SPA; 2) combination with trastuzumab in IHC 1+/2+ and IHC 3+. Early results of E39 are encouraging and a Phase 2b is being planned in gynecological malignancies. Clinical trial information: NCT00841399, NCT00584789.

Folate-based single cell screening using surface enhanced Raman microimaging

Recent progress in nanotechnology and its application to biomedical settings have generated great advantages in dealing with early cancer diagnosis. The identification of the specific properties of cancer cells, such as the expression of particular plasma membrane molecular receptors, has become crucial in revealing the presence and in assessing the stage of development of the disease. Here we report a single cell screening approach based on Surface Enhanced Raman Scattering (SERS) microimaging. We fabricated a SERS-labelled nanovector based on the biofunctionalization of gold nanoparticles with folic acid. After treating the cells with the nanovector, we were able to distinguish three different cell populations from different cell lines (cancer HeLa and PC-3, and normal HaCaT lines), suitably chosen for their different expressions of folate binding proteins. The nanovector, indeed, binds much more efficiently on cancer cell lines than on normal ones, resulting in a higher SERS signal measured on cancer cells. These results pave the way for applications in single cell diagnostics and, potentially, in theranostics.

Modular Integration of Upconverting Nanocrystal–Dendrimer Composites for Folate Receptor‐Specific NIR Imaging and Light‐Triggered Drug Release

Upconversion nanocrystals (UCNs) display near-infrared (NIR)-responsive photoluminescent properties for NIR imaging and drug delivery. The development of effective strategies for UCN integration with other complementary nanostructures for targeting and drug conjugation is highly desirable. This study reports on a core/shell-based theranostic system designed by UCN integration with a folate (FA)-conjugated dendrimer for tumor targeting and with photocaged doxorubicin as a cytotoxic agent. Two types of UCNs (NaYF4:Yb/Er (or Yb/Tm); diameter = ≈50 to 54 nm) are described, each displaying distinct emission properties upon NIR (980 nm) excitation. The UCNs are surface modified through covalent attachment of photocaged doxorubicin (ONB-Dox) and a multivalent FA-conjugated polyamidoamine (PAMAM) dendrimer G5(FA)6 to prepare UCN@(ONB-Dox)(G5FA). Surface plasmon resonance experiments performed with G5(FA)6 dendrimer alone show nanomolar binding avidity (KD = 5.9 × 10(-9) M) to the folate binding protein. This dendrimer binding corresponds with selective binding and uptake of UCN@(ONB-Dox)(G5FA) by FAR-positive KB carcinoma cells in vitro. Furthermore, UCN@(ONB-Dox)(G5FA) treatment of FAR(+) KB cells inhibits cell growth in a light dependent manner. These results validate the utility of modularly integrated UCN-dendrimer nanocomposites for cell type specific NIR imaging and light-controlled drug release, thus serving as a new theranostic system.

Improving folate (vitamin B9) stability in biofortified rice through metabolic engineering.

Biofortification of staple crops could help to alleviate micronutrient deficiencies in humans. We show that folates in stored rice grains are unstable, which reduces the potential benefits of folate biofortification. We obtain folate concentrations that are up to 150 fold higher than those of wild-type rice by complexing folate to folate-binding proteins to improve folate stability, thereby enabling long-term storage of biofortified high-folate rice grains.

2764 Preliminary results of the phase I/IIa dose finding trial of a folate binding protein vaccine (E39+GM-CSF) in ovarian and endometrial cancer patients to prevent recurrence

2764 Preliminary results of the phase I/IIa dose finding trial of a folate binding protein vaccine (E39+GM-CSF) in ovarian and endometrial cancer patients to prevent recurrence

Characterization of Folic Acid and Poly(amidoamine) Dendrimer Interactions with Folate Binding Protein: A Force-Pulling Study.

Atomic force microscopy force-pulling experiments have been used to measure the binding forces between folic acid (FA) conjugated poly(amidoamine) (PAMAM) dendrimers and folate binding protein (FBP). The generation 5 (G5) PAMAM conjugates contained an average of 2.7, 4.7, and 7.2 FA per dendrimer. The most probable rupture force was measured to be 83, 201, and 189 pN for G5-FA2.7, G5-FA4.7, and G5-FA7.2, respectively. Folic acid blocking experiments for G5-FA7.2 reduced the frequency of successful binding events and increased the magnitude of the average rupture force to 274 pN. The force data are interpreted as arising from a network of van der Waals and electrostatic interactions that form between FBP and G5 PAMAM dendrimer, resulting in a binding strength far greater than that expected for an interaction between FA and FBP alone.

Preliminary results of the phase I/IIa dose finding trial of a folate binding protein vaccine (E39+GM-CSF) in ovarian and endometrial cancer patients to prevent recurrence.

5576Background: Folate binding protein (FBP) is an immunogenic protein over-expressed in serous endometrial (EC) and ovarian cancer (OC). We’ve conducted a phase I/IIa trial using E39 (GALE-301, FB...

The role of Glycine N‐methyltransferase (GNMT) in cellular 1‐carbon metabolism

GNMT is a folate binding protein commonly diminished in human hepatoma. We have demonstrated that GNMT assists cellular methyl group homeostasis in vitro. In the present study we postulated that GNMT assists cellular 1‐carbon metabolism and helps maintain genome integrity by promoting nucleotide biosynthesis. To test the hypothesis, GNMT was over‐expressed in GNMT‐null cell lines cultured in conditions of folate abundance or restriction. The partitioning of folate dependent 1‐carbon groups was investigated using stable isotopic tracers and GC/MS. DNA damage was assessed as uracil content in cell models, and in Gnmt wildtype (Gnmt(+/+)), heterozygote (Gnmt(+/‐)) and knockout (Gnmt(‐/‐)) mice under folate deplete, replete, or supplementation conditions. We discovered that GNMT‐ cells utilized more exogenous formate in purine synthesis, suggesting that GNMT may promote endogenous formate generation. Furthermore, compared to GNMT‐, GNMT+ cells had increased percentage of the thymidine specie derived from the mitochondria as formate, supporting our hypothesis. In conclusion, GMMT assists cellular 1‐carbon metabolism in numerous ways. GNMT helps methyl group homeostasis, assists formate generation; supports methylene‐folate dependent pyrimidine synthesis and formylfolate dependent purine syntheses; and minimizes uracil incorporation into DNA in folate depletion. Loss of GNMT impairs nucleotide biosynthesis. Over‐expression of GNMT improves DNA integrity by reducing uracil misincorporation in DNA both in vitro and in vivo. The present study gives new insights into the role of GNMT on cellular 1‐carbon metablic kinetic.