High Affinity Folate Receptor Research Articles

Critical Periods in the Neurodevelopment of Autism

Autism Spectrum Disorder (ASD) is a developmental disability that can create significant behavioral and communication challenges. The prevalence of ASD among children at 8 years of age is over 2%, and the prevalence is similar across ethnic groups and countries. Studies have shown that the majority of ASD children make an autoantibody to the high-affinity folate receptor in response to a dietary component. This Folate Receptor Antibody (FRA) blocks transport of folate across the Blood-Brain Barrier (BBB), resulting in a Cerebral Folate Deficiency (CFD). Parents of autistic children also have FRA at substantially higher rates than the general public, which may play a critical role during neurodevelopmental critical periods in the fetus. In clinical trials, ASD children with the FRA had improvement in their communication when placed on a daily supplement of folate in its reduced form, which can enter the brain via a low-affinity transport. We reason that supplementing folate earlier in development, including in utero development, may be most effective in reducing the severity of ASD symptoms by facilitating typical passage through critical neurodevelopmental periods.

Abstract 1494: 5-methyltetrahydrofolate (5-MTHF) as a superior ligand for delivery of small RNAs for cancer therapy

Abstract MicroRNAs (miRNAs) are small noncoding RNAs that regulate a wide array of genes. Because miRNA downregulate translation proteins associated with cancer progression, miRNA-based therapeutics have great potential as anticancer drugs. Nonetheless, the therapeutic potential of miRNA replacement is limited due to lack of safe and efficient delivery vehicles, and inability to target miRNAs to the intended cells/tissues. Thus, identifying molecules that are able to deliver therapeutic miRNA is of critical need. This could be achieved by targeting receptors that are overexpressed on target tumor cells. For example, the folate receptor (FR) is overexpressed on various tumor types including breast, lung, ovarian and brain tumors. Our laboratory has previously developed a unique strategy that directly links a miRNA mimic to the high affinity FR ligand, folate (FolamiRs). In the present work, to further improve the potency of FolamiRs, we introduce the reduced folate metabolite, 5-methyltetrahydrofolate (5-MTHF) that maintains nanomolar affinity for the FR at pH 7, but disengages from the FR at a significantly higher pH than that of folate, which is important when including an endosomal escape agent that typically disrupts endosomal acidification. We synthesize and conjugate 5-MTHF ligand to the potent tumor suppressor miRNA-34a (5-MTHF-miR-34a). We show that 5-MTHF-miR-34a uptake is specific to the cells overexpressing FR and represses target gene expression. Our data supports the use of 5-MTHF in combination with the endosomal escape agent, nigericin, to achieve robust cytosolic localization of therapeutically relevant miRNAs such as miR-34a. This work provides the foundation for future studies using 5-MTHF- conjugated therapeutic agents for targeting additional FR expressing cells, including myeloid derived suppressor cells (MDSCs) and macrophages. Collectively these studies will pave the way for developing 5-MTHF-based small RNA conjugates for treating FR-expressing cancers. The details of these intriguing results will be discussed in the poster. Citation Format: Sudarsan R. Kasireddy, Ahmed M. Abdelaal, Philip S. Low, Andrea L. Kasinski. 5-methyltetrahydrofolate (5-MTHF) as a superior ligand for delivery of small RNAs for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1494.

Folate receptor α is associated with poor clinicopathological perspectives in breast carcinoma

BackgroundBreast carcinoma is the commonest malignancy in females. Folate is required for the biosynthesis of nucleotide bases, amino acids, and other cellular methylation reactions in proteins and phospholipids. The high affinity folate receptor alpha (FRα) has been shown to be expressed in several kinds of human cancers. MethodsIn this descriptive-analytic study, sections from formalin-fixed paraffin-embedded tissue blocks of 50 cases of invasive ductal breast carcinoma (IDC) as well as 15 cases of non-neoplastic breast specimens were immunohistochemically stained with FRα antibody. Histopathological evaluation for various clinicopathological parameters was done and was correlated with FRα expression. ResultsPositive FRα expression was more frequently detected in IDC (64%) compared to non-neoplastic breast specimens (20%). In IDC, Positive FRα expression was significantly associated with high tumor grade (p = 0.007), large tumor size (p < 0.001), high lymph node stage (p = 0.004), presence of angiolymphatic emboli (p = 0.001), presence of perineural invasion (p = 0.001). Significant association between FRα positivity and negative hormone receptors (estrogen and progesterone) (p < 0.001) and triple negative cases (p = 0.0021). ConclusionOur work demonstrates that FRα is over expressed in IDC compared to non-neoplastic breast tissue. Folate receptor α expression was associated with poor clinicopathological perspective. This work suggests that FRα may be an independent prognostic factor and supports the possibility of using FRα-targeted therapies of breast carcinoma. However, our work requires validation on larger cohort with correlation with survival data of patients.

Maternally Contributed Folate Receptor 1 Is Expressed in Ovarian Follicles and Contributes to Preimplantation Development.

Folates have been shown to play a crucial role for proper development of the embryo as folate deficiency has been associated with reduced developmental capacity such as increased risk of fetal neural tube defects and spontanous abortion. Transcripts encoding the reduced folate carrier RFC1 (SLC19A1 protein) and the high-affinity folate receptor FOLR1 are expressed in oocytes and preimplantation embryos, respectively. In this study, we observed maternally contributed FOLR1 protein during mouse and human ovarian follicle development, and 2-cell mouse embryos. In mice, FOLR1 was highly enriched in oocytes from primary, secondary and tertiary follicles, and in the surrounding granulosa cells. Interestingly, during human follicle development, we noted a high and specific presence of FOLR1 in oocytes from primary and intermediate follicles, but not in the granulosa cells. The distribution of FOLR1 in follicles was noted as membrane-enriched but also seen in the cytoplasm in oocytes and granulosa cells. In 2-cell embryos, FOLR1-eGFP fusion protein was detected as cytoplasmic and membrane-associated dense structures, resembling the distribution pattern observed in ovarian follicle development. Knock-down of Folr1 mRNA function was accomplished by microinjection of short interference (si)RNA targeting Folr1, into mouse pronuclear zygotes. This revealed a reduced capacity of Folr1 siRNA-treated embryos to develop to blastocyst compared to the siRNA-scrambled control group, indicating that maternally contributed protein and zygotic transcripts sustain embryonic development combined. In summary, maternally contributed FOLR1 protein appears to maintain ovarian functions, and contribute to preimplantation development combined with embryonically synthesized FOLR1.

Abstract 262: Combination therapy of folate-targeted chemotherapeutics with anti-PD-1 antibody against folate receptor-positive tumors in immunocompetent murine models

Abstract The PD-1/PD-L1 (programmed cell death protein 1; programmed death-ligand 1) pathway plays a key role in immunological tolerance and autoimmunity. PD-1 is primarily expressed on activated T and B cells while its receptor, PD-L1, is expressed in a variety of cell types including resting lymphocytes, DCs, and macrophages. Recently, multiple human solid tumor types (melanoma, NSCLC, RCC, ovarian, and colorectal cancer) have been found to overexpress PD-L1 within its tumor microenvironment. More importantly, PD-1/PD-L1 blocking agents (pembrolizumab, nivolumab, lambrolizumab, MPDL3280A) have been found active as a single agent or in combination with chemotherapy and some have produced durable clinical responses. On the other hand, human epithelial tumors are also known to overexpress the high-affinity folate receptor (FR)-alpha that is capable of bringing folate-linked drugs into the cell cytosol via endocytosis. Using flow cytometry, we assessed PD-L1 expression in various FR-positive syngeneic mouse tumor models (M109, Renca, L1210A, 4T1-Cl2, ID8-Cl15). PD-L1 expression was found to vary significantly among different tumor types and could be found not only on CD45- malignant cells but also on tumor stromal cells such as F4/80+CD11b+ tumor-associated macrophages. Using a rat anti-mouse PD-1 antibody (clone RMP1-14), we studied in-vivo efficacy of a folate-targeted chemotherapy in combination with PD-1/PD-L1 blockage in PD-L1-expressing tumor models. In syngeneic models with high PD-L1 expression and tumor-infiltrating lymphocytes, a synergistic and curable anti-tumor effect was observed and the animals developed tumor-protective immunity against rechallenge. Thus, our preliminary results suggest that folate-targeted chemotherapeutics may also be enhanced by PD-1/PD-L1 blockage by means of overcoming tumor-mediated immune suppression Citation Format: Yingjuan June Lu, Leroy W. Wheeler, Vicky Cross, Elaine Westrick, Alex Lloyd, Christopher P. Leamon. Combination therapy of folate-targeted chemotherapeutics with anti-PD-1 antibody against folate receptor-positive tumors in immunocompetent murine models. [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 262.

High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity.

Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here, we isolated a high affinity (HA) folate receptor beta (FRβ)-specific scFv (2.48nM KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T-cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ+ AML in vitro and in vivo compared to a low affinity (LA) FRβ CAR (54.3nM KD). Using the HA-FRβ IgG, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34+ hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T-cells lysed mature CD14+ monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T-cells retained effective anti-tumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T-cells is highly effective against AML and reduces the risk for long-term myeloid toxicity.

Fc-Small Molecule Antibody Mimetics.

Antibody therapeutics are a promising drug class due to their high specificity and favorable pharmacokinetics. While there are many methods for the development of antibodies specific to disease associated antigens, selecting antibodies against functional epitopes with high specificity and affinity can be difficult for certain epitopes. We describe a generalizable method for synthesizing antibody mimetics by site specifically conjugating small molecules (with high affinity and specificity to disease associated antigens) to an Fc fragment to develop drugs with the benefits of an antibody. As a proof of concept, an E269pAcPhe Fc antibody Fc fragment was produced and subsequently site-specifically labeled with a linker-modified folic acid compound to generate an Fc-folic acid antibody-mimetic. This was chosen as the model system because the high-affinity folate receptor FR-α is highly expressed in a number of cancer types including breast and ovarian cancer. The specificity of the Fc-folic acid conjugate was assessed via flowcytometry with the folate-receptor positive breast cancer cell line MDA-MB-231 by measuring Fc-folic acid binding in both the absence and presence of an excess of folic acid. Fc-small molecule conjugates could be developed into a unique class of antibody-like therapeutics.

Expression of high affinity folate receptor in breast cancer brain metastasis.

High affinity folate receptor (HFR) can be overexpressed in breast cancer and is associated with poor prognosis, however the expression in breast cancer brain metastases (BCBM) is unknown. The aim of this study was to analyze the rate of HFR expression in BCBM and its role in the prognosis of this high-risk cohort. We analyzed 19 brain metastasis (BM) and 13 primary tumors (PT) from a total of 25 patients. HFR status was assessed by immunohistochemistry. Median follow-up was 4.2 years (range 0.6-18.5). HFR was positive in 4/19 BM (21.1%) and in 1/13 PT (7.7%). Positive samples had low H-scores (range 1-50). 56% of patients had apocrine differentiation. OS was similar between patients with positive HFR (median OS 48 months) and negative HFR (median OS 69 months) (P = 0.25); and between patients with apocrine differentiation (median OS 63 months) and those without apocrine differentiation (median OS 69 months) (P = 0.49). To the best of our knowledge, this is the first analysis of HFR expression in BCBM. While previous studies associated the presence of HFR with worse prognosis; in our cohort HFR was positive in only 21.1% of BM with low levels of positivity. Neither HFR nor apocrine features had impact in OS.

Abstract P6-01-15: Expression of high affinity folate receptor in breast cancer brain metastasis

Abstract Background: Folic acid is required by proliferating cells for the synthesis of nucleotide bases among other tasks. The high affinity folate receptor (HFR) is a membrane protein that is upregulated in several cancers of epithelial origin and rarely present in most normal cells. High expression of HFR has been documented in ovarian, breast, kidney, uterine and lung cancers. In breast cancer patients, HFR is overexpressed in 33% of primary tumors (PT) and this is associated with poor prognosis. The HFR expression in breast cancer brain metastases (BCBM) is unknown. The aim of this study was to analyze the incidence of HFR expression in BCBM and its role in the prognosis of this high-risk patient subgroup. Methods: We analyzed a database of 42 patients who underwent craniotomy with resection of breast cancer brain metastasis (BCBM). Of those, we collected 19 brain metastasis (BM) and 13 PT. HFR status was assessed by immunohistochemistry. Log-Rank test analyzed differences in overall survival (OS) between groups. Results: Median age was 51 years (range 24-74). Median follow-up was 4.2 years (range 0.6-18.5). HFR was positive in 4/19 BM (21.1%) and in 1/13 PT (7.7%). Positive samples had low H-scores (range 1-50). Staining with H&amp;E revealed that 11/19 BM (57.9%) and 7/13 PT (53.8%) had apocrine differentiation. Analysis of OS showed no difference between patients with positive HFR (median OS 48 months) and negative HFR (median OS 69 months) (P=0.25). Similarly, there was no difference in OS between patients with apocrine differentiation (median OS 63 months) and those without apocrine differentiation (median OS 69 months) (P=0.49). Conclusions: To the best of our knowledge, this is the first analysis of HFR expression in BCBM. While previous studies associated the presence of HFR with worse prognosis; in this cohort of high-risk patients, HFR was positive in only 21.1% of BM with low levels of positivity. Around half of our patients had apocrine differentiation. Neither the status of HFR nor the presence of apocrine features had impact in OS in our cohort. Citation Format: Jose P Leone, Rohit Bhargava, Adrian Lee, Adam Brufsky. Expression of high affinity folate receptor in breast cancer brain metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-01-15.

Abstract 1677: Preclinical activity of Vintafolide/MK-8109 monotherapy and in combination with standard of care therapy in triple-negative breast cancer models

Abstract Vintafolide (also known as EC145 or MK-8109), is a small molecule drug conjugate for the treatment of cancers expressing high affinity folate receptor (FR). Vintafolide consists of the anti-mitotic vinca alkaloid desacetylvinblastine monohydrazide (DAVLBH) chemically linked to folic acid. Binding of vintafolide to FR located on the cell surface delivers the chemotherapy payload directly to the tumor cell. Clinical investigations of vintafolide are underway in ovarian and lung cancer, indications with a high prevalence of FR expression. Triple negative breast cancer (TNBC) may represent an additional indication for vintafolide due to the prevalence of FR expression (∼30%), sensitivity to vinca alkaloids, and unmet medical need. Vintafolide and/or DAVLBH were evaluated in preclinical models of TNBC as monotherapies and in combination with taxanes (paclitaxel or docetaxel). Taxanes represent a commonly used standard of care therapy for TNBC. A panel of TNBC cell lines was generally sensitive to DAVLBH, with IC50s ranging from 4-67 nM. In the majority of cell lines tested, DAVLBH in combination with paclitaxel provided combination benefit and induced more cell death than either single agent. Vintafolide and DAVLBH were further evaluated in vivo in the FR-high MDA-MB-231 and FR-low CAL51 TNBC xenograft models. Vintafolide was dosed at its MTD of 9.6 mg/kg three times per week (TIW), and at 1.5 mg/kg TIW. DAVLBH was dosed at its MTD of 0.77 mg/kg TIW. Mice were dosed for 3 weeks followed by a 6 week follow-up. Both vintafolide doses resulted in marked MDA-MB-231 tumor regressions of 56-78% at the end of therapy (Day 21) and 75% cures (6 of 8 mice) over the 6 week follow-up period. In the CAL51 model, vintafolide produced 8% and 76% tumor growth inhibition (TGI), respectively, at the 1.5 and 9.6 mg/kg doses, and no cures. DAVLBH was less efficacious compared to vintafolide, giving 96% TGI in the MDA-MB-231 model and 46% TGI in the CAL51 model. In mechanism of action studies in the MDA-MB-231 xenograft model, a dose of vintafolide that gave tumor regressions/cures was associated with increased phospho-histone H3 staining, indicative of a mitotic block of tumor cells, and consistent with the mechanism of action of vinca alkaloids. In combination therapy experiments in vivo, docetaxel (20 mg/kg weekly) monotherapy achieved near tumor stasis (98% TGI) of MDA-MB-231 tumors over 3 weeks of therapy, but all tumors re-grew upon cessation of treatment. Since vintafolide monotherapy at 1.5 mg/kg TIW gave significant regressions and cures, a combination benefit with docetaxel could not be determined. However, DAVLBH and docetaxel combination therapy delayed tumor re-growth over single agents upon follow-up, suggesting benefit of a vinca alkaloid / taxane combination. Taken together, these preclinical data support further investigation of vintafolide monotherapy and in combination with taxane therapy in TNBC. Citation Format: Brian B. Haines, Jennifer O'Neil, Marlene C. Hinton, Christopher Ware, Tammie C. Yeh, Tianxiao Sun, Kristen L. Picard, Theresa Zhang, Emmett V. Schmidt, Isabelle Dussault. Preclinical activity of Vintafolide/MK-8109 monotherapy and in combination with standard of care therapy in triple-negative breast cancer models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1677. doi:10.1158/1538-7445.AM2014-1677

Abstract A38: Nanoreformulation of platinum-based drugs to address disparate cancer treatment outcomes.

Abstract Modest responses and systemic toxicities during treatment of advanced cancer limits cancer drug efficacy, especially in African Americans. We determined the utility of cisplatin-loaded biodegradable XPclad nanoparticles coated with folate-conjugated polycaprolactone-polyethylene glycol. XPclad nanoparticles under in vitro conditions exhibited sustained release of the encapsulated cisplatin. Prostate cancer (PCa) cell lines (PC3, LNCaP, PTEN-CaP2, etc.), but not normal prostatic epithelial cells (PrEC and RWPE-1), expressed high levels of folate receptor α (FRα/FR1). Similar to cancer cells, Treg cells but not T helper cells express high affinity folate receptor (FRδ/FR4). Treatment using 20 μM of cisplatin solution resulted in &amp;gt; 90% cell death of PCa cells, PrEC, and RWPE-1 cells as well as purified CD4+CD25HiFoxP3+ T cells (Tregs) and CD4+CD25LoFoxP3- T cells (T helper). However, XPclad nanoparticles, containing cisplatin (equivalent to a 200 nM dose), were taken up and induced the same level of apoptosis in PCa and Treg cells as cisplatin solution, but did not significantly affect PrEC or RWPE-1 cells or T helper cells. Finally, we show that PC3 tumor-bearing mice that received either cisplatin solution (8 mg/kg/week) or cisplatin-encapsulated folate-coated XPclad nanoparticles (equivalent cisplatin dose = 0.08 mg/kg/week) resulted in significant tumor regression. Mice receiving cisplatin solution had significantly higher kidney and liver toxicities than naïve or XPclad nanoparticle-treated mice. Our studies show XPclad nanoparticles enhance tumor therapy efficacy with significantly lower to no toxicity and suggest that FR-targeted nanotherapy modulates the destruction of both tumor cells and Treg cells for increased efficacy. GRANT SUPPORT: G12 RR03034, U54 CA118638 and P60MD00525 Citation Format: James W. Lillard, S. Singh, R. Singh. Nanoreformulation of platinum-based drugs to address disparate cancer treatment outcomes. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr A38.

Abstract 5704: Curcumin-folate conjugate for increased bioavailability and targeted delivery of curcumin to cancer cells

Abstract Specific types of high risk Human Papillomavirus (HR-HPV) types, particularly the HPV type 16 and HPV 18 are known to cause cervical cancer. The expression of two viral oncogenes E6 and E7 responsible for tumorigenic transformation, is mainly dependent on specific host-cell transcription factor, Activator Protein 1 (AP-1) which acts as a signaling epicenter for cervical cancer. Although recently two prophylactic HPV vaccines have been developed, there is no therapeutic molecule available for the treatment of cervical lesions. We demonstrate that Curcumin (Diferuloylmethane), a yellow pigment, used in traditional medicines and present in the dietary spice turmeric (Curcuma longa), can selectively down regulate HPV 18 transcription as well as AP-1 binding activity. Curcumin can also reverse the expression dynamics of c-fos and fra-1 in tumorigenic HeLa cells by mimicking their expression pattern in normal cells. But the bioavailability of curcumin is extremely poor due to its hydrophobicity, rapid metabolism and lack of specificity in targeting cancer cells. Therefore, a folic acid-curcumin conjugate for mutation has been developed by attaching one molecule of curcumin with two molecules of folic acid (Cur-2FA) to make curcumin hydrophilic, enhance its bioavailability and to target only cancer cells which specifically express high level of folate receptors. The effect of both native curcumin and Cur-2FA on HPV and AP-1 has been analyzed in HPV positive cervical cancer and HPV negative breast cancer cell lines, by band shift assay, confocal microscopy, flow cytometry and western blotting including their cytotoxic potential and targeted cellular uptake of curcumin. In comparison to native curcumin, curcumin-2FA has been found to be atleast two times more effective in inducing down regulation of HPV transcription, AP-1 activity and expression of its components particularly c-fos and fra-1. Curcumin-2FA was found to be more potent than curcumin in inducing apoptosis, inhibits tumor cell proliferation and Cur-2FA conjugate was specifically taken up by cancer cells. In mice, curcumin-2FA was more bioavailable and had a longer half-life than curcumin. We demonstrate that this nontoxic low molecular weight curcumin-2Folic acid conjugate formulation specifically targets cancer cells which overexpress high affinity folate receptor and enhanced cellular uptake facilitating increased bioavailability and targeted delivery to cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5704. doi:1538-7445.AM2012-5704

A phase I pharmacokinetic and safety analysis of epothilone folate (BMS-753493): A first-in-human clinical experience of a folate receptor-targeted chemotherapeutic agent dosed once daily on days 1, 2, 3, and 4 of a 21-day cycle.

e13005 Background: The high-affinity folate receptor alpha is selectively overexpressed in some epithelial tumors, including ovarian, endometrial, renal, and breast, and in a few nonepithelial neop...

Phase I pharmacokinetic and safety analysis of epothilone folate (BMS-753493): First-in-human clinical experience of a folate receptor-targeted chemotherapeutic agent administered on days 1, 4, 8, and 11 of a 21-day cycle.

2607 Background: The high-affinity folate receptor alpha is selectively overexpressed in some epithelial tumors, including ovarian, endometrial, renal, and breast carcinomas, and in a few nonepithelial neoplasms. BMS-753493, a folate conjugate of epothilone analog BMS-748285, was designed to target folate receptor-expressing tumor cells. Methods: BMS-753493 was investigated in two parallel first-in-human phase I/II studies in subjects with advanced solid cancers. These studies differed only in dosing schedule. Both studies dosed 4 times every 21 days and followed a 3+3 open-label dose escalation scheme during phase I. CA190-001, discussed here, dosed once daily on days 1, 4, 8, and 11, while CA190-002 (companion abstract) dosed once daily on days 1-4. Results: Phase I escalated 5 cohorts to 33 mg. At 33 mg, three dose-limiting toxicities (DLTs) were observed, one each in 3 of 4 patients treated. The first DLT consisted of grade 2 ALT elevation resulting in omission of two doses. The second DLT consisted of grade 3 diarrhea. The third DLT consisted of grade 3 esophagitis. The 26 mg cohort was expanded to confirm the maximum tolerated dose, and one DLT (skipping day 4 and 11 doses due to Grade 2 AST elevation) was observed among 7 subjects treated as of December 2009. The most common related AEs were fatigue, diarrhea, nausea, vomiting, anorexia, and ALT/AST elevations. Although no complete or partial responses occurred, target lesion shrinkage up to 18% from baseline was observed. Plasma exposures (Cmax and AUC) of BMS-753493 and BMS-748285 on Day 1 and Day 4 increased in a dose-related manner in the dose range of 5 to 33 mg. All data are preliminary. Conclusions: At the doses and schedules tested, BMS-753493 demonstrated at best moderate activity with manageable toxicity. Pharmacokinetic data: BMS-753493, day 4 Dose (mg) N Cmax (ng/mL)GeoMean (CV%) AUC(0-T) (ng*h/mL) GeoMean (CV%) 5 3 583 (23) 232.3 (14) 10 4 1,343 (32) 642.4 (38) 17 6 2,427 (30) 1,409.3 (32) 26 3 3,892 (27) 2,467.5 (46) 33 3 3,914 (23) 2,919.9 (4) Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Bristol-Myers Squibb Bristol-Myers Squibb Bristol-Myers Squibb Bristol-Myers Squibb Bristol-Myers Squibb

Folate regulation of axonal regeneration in the rodent central nervous system through DNA methylation

The folate pathway plays a crucial role in the regeneration and repair of the adult CNS after injury. Here, we have shown in rodents that such repair occurs at least in part through DNA methylation. In animals with combined spinal cord and sciatic nerve injury, folate-mediated CNS axon regeneration was found to depend on injury-related induction of the high-affinity folate receptor 1 (Folr1). The activity of folate was dependent on its activation by the enzyme dihydrofolate reductase (Dhfr) and a functional methylation cycle. The effect of folate on the regeneration of afferent spinal neurons was biphasic and dose dependent and correlated closely over its dose range with global and gene-specific DNA methylation and with expression of both the folate receptor Folr1 and the de novo DNA methyltransferases. These data implicate an epigenetic mechanism in CNS repair. Folic acid and possibly other nontoxic dietary methyl donors may therefore be useful in clinical interventions to promote brain and spinal cord healing. If indeed the benefit of folate is mediated by epigenetic mechanisms that promote endogenous axonal regeneration, this provides possible avenues for new pharmacologic approaches to treating CNS injuries.

High‐affinity folate receptor in cardiac neural crest migration: A gene knockdown model using siRNA

Folate supplementation reduces the incidence of congenital heart defects, but the nature of this protective mechanism remains unclear. Immunolabeling demonstrated that the neural tube and neural crest (NC) cells were rich in the high-affinity folate receptor FOLR1and during the early stages of development FOLR1 was found principally in these cells. Suppression of Folr1 expression in the nascent cardiac NC by site-directed short-interfering RNA (siRNA) altered cardiac NC cell mitosis and subsequent migration patterns leading to abnormal development of the pharyngeal arch arteries (PAA) and outflow tract. qPCR analysis demonstrated that the siRNA treatment significantly reduced Folr1 24 hr after treatment. These treatments also significantly reduced mitosis in the neural tube, but adjacent, nontreated areas were unaffected. In summary, a brief reduction in the expression of Folr1 during a critical stage of NC development had long-term consequences for the development of the PAA and outflow tract.

Folate Targeting Enables Durable and Specific Antitumor Responses from a Therapeutically Null Tubulysin B Analogue

The membrane-bound high-affinity folate receptor (FR) is highly expressed on a wide range of primary and metastatic human cancers, such as those originating in ovary, lung, breast, endometrium, kidney, and brain. Because folate-linked conjugates bind to and become internalized within FR-expressing cells (similar to that of free folic acid), we explored the possibility of using the folate ligand to target a potent, semisynthetic analogue of the microtubule inhibitor tubulysin B to FR-enriched tumors. When tested in vitro, a novel folate conjugate, herein referred to as EC0305, was found to specifically inhibit the growth of a panel of FR-positive cell lines (IC50 range, 1-10 nmol/L) in a dose-dependent manner, whereas cells lacking FR expression were unaffected. The potency of EC0305 was also confirmed against a human KB xenograft-nu/nu mouse cancer model. Here, a brief three times per week, 2-week regimen yielded remarkable antitumor activity (100% tumor-free animals) without causing significant weight loss or major organ tissue degeneration. In contrast, antitumor activity was completely abolished in EC0305-treated animals that were co-dosed with an excess of a nontoxic folate-containing analogue, thereby confirming that the antitumor effect of this agent was mediated by FRs. The advantage provided by folate conjugation was further proved by the untargeted free drug, which was found to be completely inactive at both tolerable and highly toxic dose levels. Collectively, these results show that this potent antiproliferative tubulysin compound can be specifically delivered to FR-positive tumors to provide substantial therapeutic benefit using well-tolerable dosing regimens.

Folate receptor overexpression is associated with poor outcome in breast cancer

The high affinity folate receptor is a membrane-associated glycoprotein that is preferentially expressed in cancers of epithelial origin and rarely expressed in normal cells. We examined its expression pattern in breast cancer, utilizing a tissue microarray containing samples from 63 invasive breast cancers from women with divergent clinical outcomes. Thirty-three women comprised the poor outcome group with a median time to recurrence of 1.9 years. Thirty women, the good outcome group, were free of recurrence for a minimum of 7 years after diagnosis. The intensity of folate receptor staining was strongly correlated with outcome. There were two summary categories of staining intensity: weak (n = 42) or strong (n = 21). In the strong staining group, 17 of 21 women (81%) have recurred and their median survival is 2.4 years. In the weak staining group, 16 of 42 women (38%) have recurred. Their median survival is not estimable. After adjustment for tumor size, nodal status, ER status, adjuvant therapy, histology and tumor grade, strong staining for the folate receptor remained significantly associated with poor outcome, p < 0.001. Our work requires validation in a larger cohort, but supports the possibility of using folate receptor-targeted approaches in the management of breast cancer.

Synthesis and Functional Evaluation of DNA-Assembled Polyamidoamine Dendrimer Clusters for Cancer Cell-Specific Targeting

We sought to produce dendrimers conjugated to different biofunctional moieties (fluorescein [FITC] and folic acid [FA]), and then link them together using complementary DNA oligonucleotides to produce clustered molecules that target cancer cells that overexpress the high-affinity folate receptor. Amine-terminated, generation 5 polyamidoamine (G5 PAMAM) dendrimers are first partially acetylated and then conjugated with FITC or FA, followed by the covalent attachment of complementary, 5'-phosphate-modified 34-base-long oligonucleotides. Hybridization of these oligonucleotide conjugates led to the self-assembly of the FITC- and FA-conjugated dendrimers. In vitro studies of the DNA-linked dendrimer clusters indicated specific binding to KB cells expressing the folate receptor. Confocal microscopy also showed the internalization of the dendrimer cluster. These results demonstrate the ability to design and produce supramolecular arrays of dendrimers using oligonucleotide bridges. This will also allow for further development of DNA-linked dendrimer clusters as imaging agents and therapeutics.

Folate-targeted imaging of activated macrophages in rats with adjuvant-induced arthritis.

To determine whether overexpression of the high-affinity folate receptor (FR) on activated macrophages can be exploited to selectively target imaging agents to sites of inflammation in rats with adjuvant-induced arthritis (AIA). Folic acid was conjugated to a (99m)Tc chelator (the complex termed EC20), and its distribution was visualized using gamma scintigraphy in healthy rats, rats with AIA, and arthritic rats that had been depleted of macrophages. To confirm that uptake was mediated by the FR, excess folic acid competition studies were conducted, and tissue FR levels were quantitated using a radioligand binding assay. Flow cytometry was also used to investigate uptake of folate conjugates into macrophages of both arthritic and healthy rats. EC20 concentrated in the arthritic extremities of diseased rats but not in the extremities of healthy rats. The intensity of images of affected tissues was greatly reduced in the presence of excess competing folic acid. The livers and spleens of arthritic animals also showed enhanced uptake of EC20 and increased levels of FR. Depletion of macrophages from arthritic animals reduced tissue FR content and concomitantly abolished uptake of EC20. In addition, macrophages isolated from livers of rats with AIA exhibited a significantly higher binding capacity for folate conjugates than did macrophages obtained from healthy rats. Although EC20 is currently undergoing clinical evaluation for use in the imaging of ovarian carcinomas, the present results suggest that it may also be useful for assaying the participation of activated macrophages in inflammatory processes such as rheumatoid arthritis.