A Novel Method of Magnetic Nanoparticles Functionalized with Anti-Folate Receptor Antibody and Methotrexate for Antibody Mediated Targeted Drug Delivery.

Therapeutic effects of anticancer medicines can be improved by targeting the specific receptors on cancer cells. Folate receptor (FR) targeting with antibody (Ab) is an effective tool to deliver anticancer drugs to the cancer cell. In this research project, a novel formulation of targeting drug delivery was designed, and its anticancer effects were analyzed. Folic acid-conjugated magnetic nanoparticles (MNPs) were used for the purification of folate receptors through a novel magnetic affinity purification method. Antibodies against the folate receptors and methotrexate (MTX) were developed and characterized with enzyme-linked immunosorbent assay and Western blot. Targeting nanomedicines (MNP-MTX-FR Ab) were synthesized by engineering the MNP with methotrexate and anti-folate receptor antibody (anti-FR Ab). The cytotoxicity of nanomedicines on HeLa cells was analyzed by calculating the % age cell viability. A fluorescent study was performed with HeLa cells and tumor tissue sections to analyze the binding efficacy and intracellular tracking of synthesized nanomedicines. MNP-MTX-FR Ab demonstrated good cytotoxicity along all the nanocomposites, which confirms that the antibody-coated medicine possesses the potential affinity to destroy cancer cells in the targeted drug delivery process. Immunohistochemical approaches and fluorescent study further confirmed their uptake by FRs on the tumor cells' surface in antibody-mediated endocytosis. The current approach is a useful addition to targeted drug delivery for better management of cancer therapy along with immunotherapy in the future.

Phase Ib trial of folate binding protein (FBP)-derived peptide vaccines, E39 and an attenuated version, E39': An analysis of safety and immune response.

In this randomized phase Ib trial, we tested combining the E39 peptide vaccine with a vaccine created from E39', an attenuated version of E39. Patients with breast or ovarian cancer, who were disease-free after standard of care therapy, were enrolled and randomized to one of three arms. Arm EE received six E39 inoculations; arm EE' received three E39 inoculations followed by three E39'; and arm E'E received three E39' inoculations, followed by three E39. Within each arm, the first five patients received 500 µg of peptide and the remainder received 1000 µg. Patients were followed for toxicity, and immune responses were measured. This initial analysis after completion of the primary vaccination series has confirmed the safety of both vaccines. Immune analyses suggest incorporating the attenuated version of the peptide improves immune responses and that sequencing of E39 followed by E39' might produce the optimal immune response. TRIAL REGISTRATION: NCT02019524.

High prevalence of serum folate receptor autoantibodies in children with autism spectrum disorders.

BACKGROUND: Supplementation of folic acid by pregnant mothers is thought to lower the risk of autism spectrum disorders (ASDs) in the offspring. Folic acid is taken up by cells via receptors with high affinity for folate and reduced folic acid derivatives. However, this is blocked by the presence of folate receptor autoantibodies (FRAA). Cerebral FRAA have been detected with high frequency in children with ASDs, suggesting the existence of a link between folic acid uptake and disease aetiology. METHODS: We investigated the frequency of FRAA in serum samples from 40 children with ASDs and 42 gender- and age-matched children with typical development (TD). Serum FRAA concentrations were measured by enzyme-linked immunosorbent assay. RESULTS: We found a significant difference in the frequency of serum FRAA in the two study cohorts. Serum FRAA were present in 77.5% (31/40) of children with ASDs compared with 54.8% (23/42) of TD children (p = 0.03746, Fischer's exact test). Thus, serum FRAA are more prevalent in children with ASDs than in TD children. CONCLUSIONS: Our data suggest that children with ASDs may have defects in folic acid absorption that play a role in the onset of ASDs.

Folate receptor expression on murine and human adipose tissue macrophages.

OBJECTIVE AND DESIGN: Adipose tissue macrophages (ATMs) have been implicated in a number of obesity-related diseases. Because the activated macrophages associated with many types of autoimmune and inflammatory diseases express a folate receptor (FR) that can be exploited for FR-targeted drug delivery, we examined the visceral adipose tissue of obese mice and humans to determine whether ATMs also express FR that are accessible by folate conjugates. MATERIAL OR SUBJECTS: C57BL/6 or FATSO mice fed on either a low- or high-fat diet were used in murine studies. Human adipose tissue were obtained from healthy volunteers during adipose reduction surgery. METHODS: Visceral adipose tissue was collected from both obese mice and humans, collagenase digested, and stained with folate-Oregon Green and antibodies for macrophage markers including F4/80, mannose receptor (CD206), CD11b, and CD11c. Cells were then examined for expression of the above markers by flow cytometry. Furthermore, the ability of folate conjugates to target the FR-expressing ATMs in obese mice was evaluated in vivo. RESULTS: A subset of the ATMs harvested from obese mice were found to express FR. Subpopulations of ATMs also simultaneously express both pro- and anti-inflammatory markers, and FR is expressed on both subsets. We then demonstrate that FR-expressing ATMs can be targeted with folate-linked fluorescent dyes in vivo. CONCLUSIONS: FR are expressed on multiple subsets of ATMs and these subsets can be targeted with folate-linked drugs, allowing for the possible development of FR-targeted therapies for obesity-related inflammatory diseases.

[Establishment and evaluation of enzyme-linked immunosorbent assay for measuring human autoantibody IgG to folate receptor].

OBJECTIVE: To establish and evaluate a newly established method of enzyme-linked immunosorbent assay (ELISA) for measuring human autoantibody to folate receptor (FR). METHODS: Folate receptor was extracted and purified from healthy woman placenta tissues. The protein was coated on 96-well plates. Goat monoclonal antibody was used as detecting antibody to set up the indirect ELISA procedure. The sensitivity, precision and linearity of the method were evaluated. Further, the method was compared with the ELISA method with commercialized bovine folate binding protein (FBP) by determining autoantibody levels in 24 individuals. RESULTS: The measuring range of the standard curve was from 6.25 × 10⁻4 to 8 × 10⁻² (the IgG concentration of pooled plasma from healthy donors was defined as 1). The lowest detectable level was 3.13 × 10⁻4. The intra- and inter-assay coefficients of variations were 2.74%-8.07% and 4.16%-8.23%, respectively. Linearity test results were considered within acceptable limits. The data from FBP-ELISA and FR-ELISA were highly correlated (r=0.954, P<0.001); The value from FR-ELISA was higher by 14% than that from FBP-ELISA. CONCLUSION: The ELISA method for measuring human autoantibody IgG to folate receptor was successfully established using human FR as coating protein. The method is sensitive and repeatable and can be used in large-scale population study.

Neural tube defects, folate, and immune modulation.

Periconceptional supplementation with folic acid has led to a significant worldwide reduction in the incidence of neural tube defects (NTDs). However, despite increasing awareness of the benefits of folic acid supplementation and the implementation of food fortification programs in many countries, NTDs continue to be a leading cause of perinatal morbidity and mortality worldwide. Furthermore, there exists a significant subgroup of women who appear to be resistant to the protective effects of folic acid supplementation. The following review addresses emerging clinical and experimental evidence for a role of the immune system in the etiopathogenesis of NTDs, with the aim of developing novel preventative strategies to further reduce the incidence of NTD-affected pregnancies. In particular, recent studies demonstrating novel roles and interactions between innate immune factors such as the complement cascade, neurulation, and folate metabolism are explored.

Lack of association between folate receptor autoantibodies and conotruncal congenital heart defects.

Conotruncal cardiac defects are partially prevented by maternal folic acid supplementation. However, the biochemical mechanism is unknown. Maternal autoantibodies to folate receptors, previously associated with increased risk for neural tube defects, also may account for this effect. This study aimed to examine the titers of folate receptor-blocking autoantibodies in mothers of children with conotruncal congenital heart defects and to compare them with those in the general population. Serum samples were obtained from 22 women whose pregnancies were complicated by conotruncal congenital heart malformations. Groups of samples were analyzed for autoantibodies against [(3)H] folic acid-labeled folate receptors, quantitative amounts of immunoglobulin G (IgG) and IgM autoantibodies to the folate receptor, and for ability to block-bind folic acid to receptors. No elevated levels of antibodies binding to [(3)H] folic acid-labeled folate receptors were found. No difference was found in antifolate receptor alpha-IgG or IgM median levels between cases (261 vs. 240 µg/mL) and control subjects (773 vs. 924 µg/mL). There was no increased blocking of folic acid binding between cases [0.69 ng/mL; 95 % confidence interval (CI), 0.006-0.01] and control subjects (0.69 ng/mL; 95 % CI, 0.003-0.013). Although epidemiologic evidence suggests that periconceptual folic acid may prevent many conotruncal congenital heart defects, the current study suggests that this effect is unlikely to be explained by the presence of maternal autoantibodies to folate receptor. These data suggest that a strategy of screening women for such autoantibodies will not identify a high-risk group of women to target for supplemental folic acid to prevent congenital heart defects.

Folate-targeted immunotherapies: Passive and active strategies for cancer.

The glycoprotein FRα is a membrane-attached transport protein that is shielded from the immune system in healthy cells. However, it is upregulated in various malignancies, involved in cancer development and is also immunogenic. Furthermore, FRα is a tumor-associated antigen endowed with unique properties, thus rendering it a suitable target for immunotherapeutic development in cancer. Various anti- FRα immunotherapeutic strategies are thus currently being developed and clinically assessed for the treatment of various solid tumors. These approaches include passive anti-FRα immunotherapies, such as monoclonal antibodies, or active immunotherapies, such as CART, folate haptens and vaccines. In this review, we will explore the advances in the field of FRα-based immune therapies and discuss both their successes and shortcomings in the clinical setting.

Final analysis of a phase I/IIa trial of the folate-binding protein-derived E39 peptide vaccine to prevent recurrence in ovarian and endometrial cancer patients.

BACKGROUND: E39, an HLA-A2-restricted, immunogenic peptide derived from the folate-binding protein (FBP), is overexpressed in multiple malignancies. We conducted a phase I/IIa trial of the E39 + GM-CSF vaccine with booster inoculations of either E39 or E39' (an attenuated version of E39) to prevent recurrences in disease-free endometrial and ovarian cancer patients(pts). Here, we present the final 24-month landmark analysis. PATIENTS AND METHODS: HLA-A2 + patients receiving E39 + GM-CSF were included in the vaccine group (VG), and HLA-A2- pts (or HLA-A2 + patients refusing vaccine) were followed as the control group (CG). VG group received 6 monthly inoculations as the primary vaccine series (PVS) and were randomized to receive either E39 or E39' booster inoculations. Demographic, safety, immunologic, and disease-free survival (DFS) data were collected and evaluated. RESULTS: Fifty-one patients were enrolled; 29 in the VG and 22 in the CG. Fourteen patients received <1000 µg and 15 received 1000 µg of E39. There were no clinicopathologic differences between VG and CG or between dose groups. E39 was well tolerated. At the 24 months landmark, DFS was 55.5% (VG) vs 40.0% (CG), P = 0.339. Patients receiving 1000 µg and boosted patients also showed improved DFS (P < 0.03). DFS was improved in the 1000 µg group after treatment of primary disease (90.0% vs CG:42.9%, P = 0.007), but not in recurrent patients. In low-FBP expressing patients, DFS was 100.0% (1000 µg), 50.0% (<1000 µg), and 25.0% (CG), P = 0.029. CONCLUSIONS: This phase I/IIa trial reveals that E39 + GM-CSF is safe and may be effective in preventing recurrence in high-risk ovarian and endometrial cancer when optimally dosed (1000 µg) to FBP low patients being treated for primary disease.

Biomarker-Based Metabolic Labeling for Redirected and Enhanced Immune Response.

Installation of an antibody-recruiting moiety on the surface of disease-relevant cells can lead to the selective destruction of targets by the immune system. Such an approach can be an alternative strategy to traditional chemotherapeutics in cancer therapy and possibly other diseases. Herein we describe the development of a new strategy to selectively label targets with an antibody-recruiting moiety through its covalent and stable installation, complementing existing methods of employing reversible binding. This is achieved through selective delivery of 1,3,4- O-acetyl- N-azidoacetylmannosamine (Ac3ManNAz) to folate receptor-overexpressing cells using an Ac3ManNAz-folate conjugate via a cleavable linker. As such, Ac3ManNAz is converted to cell surface glycan bearing an azido group, which serves as an anchor to introduce l-rhamnose (Rha), a hapten, via a click reaction with aza-dibenzocyclooctyne (DBCO)-Rha. We tested this method in several cell lines including KB, HEK-293, and MCF7 and were able to demonstrate the following: 1) Rha can be selectively installed to the folate receptor overexpressing cell surface and 2) the Rha installed on the target surface can recruit anti-rhamnose (anti-Rha) antibodies, leading to the destruction of target cells via complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP).

Folate-Modified Chitosan Nanoparticles Coated Interferon-Inducible Protein-10 Gene Enhance Cytotoxic T Lymphocytes' Responses to Hepatocellular Carcinoma.

Adoptive therapy using tumor antigen-specific cytotoxic T lymphocytes (CTLs) is a promising approach for treatment of human cancers. Due to immune suppression in cancer patients, it is difficult for tumor antigen-specific CTLs to arrive at tumor tissues. Interferon-inducible protein-10 (IP-10) is a powerful chemokine that effectively attracts CTLs to tumor tissues and improves their anti-tumor activity. Increase over expression of IP-10 in tumor tissues can efficiently promote efficacy of adoptive therapy. Folate-modified chitosan nanoparticles coating the human IP-10 gene (FA-CS-hIP-10) were therefore developed in this study. The FA-CS-hIP-10 nanoparticles were specifically bound to folate receptors on hepatoma cells and promoted the expression of IP-10, to improve the activity of pMAGE-A1(278-286) specific CTLs. Combination of the FA-CS-hIP-10 and pMAGE-A1(278-286) specific CD8+ CTLs efficiently increased secretion of IFN-γ, inhibited tumor growth and extended survival of nude mice with subcutaneously transplanted human hepatocellular carcinoma. Our results demonstrated that the mechanism behind this novel therapeutic approach involved inhibition of angiogenesis and proliferation, and also promoted apoptosis of tumor cells. Our study provides a potentially novel approach for treatment of human hepatocellular carcinoma by improving the activity of tumor antigen-specific CTLs.