Detecting Functional and Accessible Folate Receptor Expression in Cancer and Polycystic Kidneys.

Folate-based small molecule drug conjugates (SMDCs) are currently under development and have shown promising preclinical and clinical results against various cancers and polycystic kidney disease. Two requisites for response to a folate-based SMDC are (i) folate receptor alpha (FRα) protein is expressed in the diseased tissues, and (ii) FRα in those tissues is accessible and functionally competent to bind systemically administered SMDCs. Here we report on the development of a small molecule reporter conjugate (SMRC), called EC2220, which is composed of a folate ligand for FRα binding, a multilysine containing linker that can cross-link to FRα in the presence of formaldehyde fixation, and a small hapten (fluorescein) used for immunohistochemical detection. Data show that EC2220 produces a far greater IHC signal in FRα-positive tissues over that produced with EC17, a folate-fluorescein SMRC that is released from the formaldehyde-denatured FRα protein. Furthermore, the extent of the EC2220 IHC signal was proportional to the level of FRα expression. This EC2220-based assay was qualified both in vitro and in vivo using normal tissue, cancer tissue, and polycystic kidneys. Overall, EC2220 is a sensitive and effective reagent for evaluating functional and accessible receptor expression in vitro and in vivo.

Folate receptor-targeted aminopterin therapy is highly effective and specific in experimental models of autoimmune uveitis and autoimmune encephalomyelitis.

EC0746 is a rationally designed anti-inflammatory drug conjugate consisting of a modified folic acid-based ligand linked to a γ-hydrazide analog of aminopterin. In this report, EC0746's effectiveness was evaluated against experimental retinal S-antigen (PDSAg) induced autoimmune uveitis (EAU) and myelin-basic-protein induced autoimmune encephalomyelitis (EAE). In both models, functional FR-ß was detected on activated macrophages in local (retinal or central-nervous-system, respectively) and systemic (peritoneal cavity) sites of inflammation. In myelin-rich regions of EAE rats, an increased uptake of (99m)Tc-EC20 (etarfolatide; a FR-specific radioimaging agent) was also observed. EC0746 treatment at disease onset suppressed the clinical severity of both EAU and EAE, and it strongly attenuated progressive histopathological changes in the affected organs. In all parameters assessed, EC0746 activity was completely blocked by a benign folate competitor, suggesting that these therapeutic outcomes were specifically FR-ß mediated. EC0746 may emerge as a useful macrophage-modulating agent for treating inflammatory episodes of organ-specific autoimmunity.

Folate receptor-ß in activated macrophages: ligand binding and receptor recycling kinetics.

Activated macrophages overexpress a receptor for the vitamin folic acid termed the folate receptor ß (FR-ß). Because conjugation of folate to low molecular weight drugs, genes, liposomes, nanoparticles, and imaging agents has minor effects on FR binding, the vitamin can be exploited to target both therapeutic and imaging agents to activated macrophages without promoting their uptake by other healthy cells. In this paper, we characterize the binding, internalization, and recycling kinetics of FR-ß on activated macrophages in inflamed tissues of rats with adjuvant-induced arthritis. Our results demonstrate that saturation of macrophage FR is achieved at injection doses of ∼150-300 nmol/kg, with more rapidly perfused tissues saturating at lower doses than inflamed appendages. After binding, FR-ß internalizes and recycles back to the cell surface every ∼10-20 min, providing empty receptors for additional folate conjugate uptake. Because the half-life of low molecular weight folate conjugates in the vasculature is usually <1 h, these data suggest that targeting of folate conjugates to activated macrophages in vivo can be maximized by frequent dosing at conjugate concentrations that barely saturate FR (∼150 nmol/kg), thereby minimizing nonspecific binding to receptor-negative tissues and maximizing the probability that unoccupied cell surface receptors will be exposed to folate-drug conjugate.

A universal dual mechanism immunotherapy for the treatment of influenza virus infections.

Seasonal influenza epidemics lead to 3-5 million severe infections and 290,000-650,000 annual global deaths. With deaths from the 1918 influenza pandemic estimated at >50,000,000 and future pandemics anticipated, the need for a potent influenza treatment is critical. In this study, we design and synthesize a bifunctional small molecule by conjugating the neuraminidase inhibitor, zanamivir, with the highly immunogenic hapten, dinitrophenyl (DNP), which specifically targets the surface of free virus and viral-infected cells. We show that this leads to simultaneous inhibition of virus release, and immune-mediated elimination of both free virus and virus-infected cells. Intranasal or intraperitoneal administration of a single dose of drug to mice infected with 100x MLD50 virus is shown to eradicate advanced infections from representative strains of both influenza A and B viruses. Since treatments of severe infections remain effective up to three days post lethal inoculation, our approach may successfully treat infections refractory to current therapies.

Preclinical evaluation of EC145, a folate-vinca alkaloid conjugate.

We recently developed a new group of folate-conjugated Vinca alkaloids, one of which, EC145, emerged as a candidate for clinical development. Brief treatment of nude mice bearing approximately 100 mm(3) folate receptor-positive human xenografts led to complete response (CR) in 5/5 mice and cures (i.e., remission without a relapse for >90 days post-tumor implantation) in 4/5 mice. Multiple CRs and cures were also noted when EC145 was used to treat mice initially bearing tumors as large as 750 mm(3). Likewise, complete cures (5/5) resulted following the treatment of an aggressive folate receptor-positive J6456 lymphoma model. The activity of EC145 was not accompanied by noticeable weight loss or major organ tissue degeneration. Furthermore, no significant antitumor activity (0/5 CR) was observed in EC145-treated animals that were co-dosed with an excess of a benign folate ligand, thus demonstrating the target-specific activity of EC145. The enhanced therapeutic index due to folate conjugation was also evidenced by the fact that the unconjugated drug (desacetylvinblastine monohydrazide) was found to be completely inactive when administered at nontoxic dose levels and only marginally active when given at highly toxic dose levels. Subsequent dose regimen studies confirmed that EC145 given on a more frequent, qdx5 schedule resulted in the most effective antitumor response as compared with an equivalent total dose given on thrice- or single-injection-per-week schedule. Taken together, these studies show that EC145 has significant antiproliferative activity and tolerability, thus lending support to an ongoing phase 1 trial for the treatment of advanced malignancies.

Preclinical Evaluation of Bispecific Adaptor Molecule Controlled Folate Receptor CAR-T Cell Therapy With Special Focus on Pediatric Malignancies.

Chimeric antigen receptor (CAR)-T cell therapy has transformed pediatric oncology by producing high remission rates and potent effects in CD19+ B-cell malignancies. This scenario is ideal as CD19 expression is homogeneous and human blood provides a favorable environment for CAR-T cells to thrive and destroy cancer cells (along with normal B cells). Yet, CAR-T cell therapies for solid tumors remain challenged by fewer tumor targets and poor CAR-T cell performances in a hostile tumor microenvironment. For acute myeloid leukemia and childhood solid tumors such as osteosarcoma, the primary treatment is systemic chemotherapy that often falls short of expectation especially for relapsed and refractory conditions. We aim to develop a CAR-T adaptor molecule (CAM)-based therapy that uses a bispecific small-molecule ligand EC17, fluorescein isothiocyanate (FITC) conjugated with folic acid, to redirect FITC-specific CAR-T cells against folate receptor (FR)-positive tumors. As previously confirmed in rodents as well as in human clinical studies, EC17 penetrates solid tumors within minutes and is retained due to high affinity for the FR, whereas unbound EC17 rapidly clears from the blood and from receptor-negative tissues. When combined with a rationally designed CAR construct, EC17 CAM was shown to trigger CAR-modified T cell activation and cytolytic activity with a low FR threshold against tumor targets. However, maximal cytolytic potential correlated with (i) functional FR levels (in a semi-log fashion), (ii) the amount of effector cells present, and (iii) tumors' natural sensitivity to T cell mediated killing. In tumor-bearing mice, administration of EC17 CAM was the key to drive CAR-T cell activation, proliferation, and persistence against FR+ pediatric hematologic and solid tumors. In our modeling systems, cytokine release syndrome (CRS) was induced under specific conditions, but the risk of severe CRS could be easily mitigated or prevented by applying intermittent dosing and/or dose-titration strategies for the EC17 CAM. Our approach offers the flexibility of antigen control, prevents T cell exhaustion, and provides additional safety mechanisms including rapid reversal of severe CRS with intravenous sodium fluorescein. In this paper, we summarize the translational aspects of our technology in support of clinical development.

Preclinical pharmacokinetics, tissue distribution, and antitumor activity of a folate-hapten conjugate-targeted immunotherapy in hapten-immunized mice.

Folic acid (pteroylglutamic acid) represents a useful ligand for targeted cancer therapies because it binds to a common epithelial tumor antigen known as the folate receptor. We previously devised an immunotherapy strategy that uses a bispecific ligand, a folate-hapten (FITC) conjugate, to redirect endogenously induced anti-FITC antibodies to folate receptor-positive tumor cells following parenteral administration. Here, we present results from preclinical pharmacokinetic and tissue biodistribution studies using a radioactive folate-FITC conjugate and results from dose optimization studies done in tumor-bearing animals. Folate-FITC was found to be rapidly eliminated in non-immunized mice; however, in immunized hosts, folate-FITC was shown to form immune complexes with FITC-specific antibodies, the consequence of which was a approximately 173-fold increase in drug exposure (i.e., area under the curve). Using a newly developed ELISA assay, the extent of circulating anti-FITC antibodies occupied by parenterally given folate-FITC was determined to be proportional to the given dose. Furthermore, high doses of folate-FITC were found to promote the cosaturation of tumor cell surface folate receptors and circulating FITC-specific antibodies, blocking the immune recognition of tumor cells and thereby reducing antitumor activity. Nonetheless, by extending the duration of treatment and administering subsaturating doses of folate-FITC, enhanced antitumor response was observed in mice bearing established folate receptor-positive M109 tumors. Overall, results from the present study may help to guide clinicians through on-going clinical investigations of folate-targeted immunotherapy.

Enhancing the therapeutic range of a targeted small-molecule tubulysin conjugate for folate receptor-based cancer therapy.

PURPOSE: EC0305 represents a folate-tubulysin B construct capable of specifically eradicating folate receptor (FR)-positive subcutaneous tumors from mice (Leamon et al., Cancer Res 68:9839-9844, 8). Herein we report on the use of multiple polar carbohydrate segments (e.g. 1-amino-1-deoxy-glucitolyl-γ-glutamate) placed in-between the folate and tubulysin B moieties of EC0305 creating a new conjugate, herein referred to as EC0531, with more desirable biological properties. METHODS: The synthesis of EC0531 and its tritium-labeled counterpart are described. EC0531's affinity for FR binding and specific cytotoxic activity was assessed using standard in vitro assays. Human tumor xenografts were used to directly compare EC0305 and EC0531's antitumor activity. Finally, bile duct cannulated, female Sprague-Dawley rats were used to compare hepatobiliary clearance of these two targeted chemotherapeutic agents. RESULTS: EC0531 tightly binds to the FR with an affinity about half that of folic acid. It was found to specifically inhibit the growth of FR+ cells (IC50 of ~2 nM) in a dose-dependent manner. Using 3H-labeled compounds, more than a 12-fold higher amount of tubulysin was measured in a FR + human tumor xenograft compared to the unconjugated drug, a finding that explains, in part, why EC0531 displays curative activity, whereas the unconjugated tubulysin agent is essentially inactive. EC0531 was found to produce greater FR-specific anti-tumor activity at lower dose levels than EC0305; furthermore, EC0531's maximum tolerated dose level was significantly higher than that of EC0305, likely because EC0531's saccharopeptidic-based spacer allows for ~sixfold reduction in hepatic clearance. CONCLUSIONS: These data provide additional evidence that the therapeutic range of targeted small-molecule drug conjugates can be favorably increased using molecular spacers constructed with 1-amino-1-deoxy-glucitolyl-γ-glutamate residues.

Preclinical evaluation of (99m)Tc-EC20 for imaging folate receptor-positive tumors.

UNLABELLED: Our group previously reported on the synthesis and characterization of a novel (99m)Tc-based folate-peptide chelator called EC20. This agent was found to bind folate receptor (FR)-positive cells and tissues with high affinity and was deemed useful for radiodiagnostic applications. In this study, we investigated the effect of D-amino acid substitution within EC20 on its tissue biodistribution. Expanded in vivo studies were also performed with unmodified (99m)Tc-EC20 to determine the effect of tumor FR expression, tumor size, tumor location, route of dose administration, and rodent diet on the agent's tissue biodistribution pattern. METHODS: EC20 and EC53, the all-D-isomer of EC20, were synthesized and radiolabeled with (99m)Tc. The relative affinity of EC53 to the FR with respect to EC20 was then determined in cultured tumor cells. The ability of (99m)Tc-EC20 and (99m)Tc-EC53 to target tumors in vivo was examined using BALB/c mice with subcutaneously inoculated M109 or 4T1 cells, yielding 0.1- to 0.5-g tumors in 20 d. RESULTS: The D-amino acid substitutions of EC20 were found to reduce the uptake of the agent into tumor and major organs. Subsequent studies using the original (99m)Tc-EC20 agent confirmed that its net tumor uptake was specific and proportional to FR expression levels in tumor cells as well as linear with respect to the overall tumor size. Further, (99m)Tc-EC20 uptake was found to be independent of both solid tumor location (intraperitoneal vs. subcutaneous) and the route of administration (intraperitoneal vs. intravenous). Interestingly, leucovorin supplementation of a commonly used folate-deficient laboratory chow had no effect on the agent's overall tissue biodistribution pattern. But, tumor-to-nontumor ratios could be increased up to 2.7-fold when 1 equivalent of free folic acid was coinjected with (99m)Tc-EC20. CONCLUSION: Taken together, these results confirm that (99m)Tc-EC20 has the potential to be a clinically useful noninvasive radiodiagnostic agent for detecting the locus of FR-positive cancers.

Strategy to prevent drug-related hypersensitivity in folate-targeted hapten immunotherapy of cancer.

Cancer vaccine/immunotherapy rarely involves systemic administration of an immunogenic compound to an actively immunized host. We have developed such a strategy that utilizes folate to deliver antigenic haptens [e.g., fluorescein (FITC) and dinitrophenyl] to folate receptor-positive tumors in a hapten-pre-vaccinated host. Here, we investigated the safety of this novel approach and developed strategies to prevent drug-related hypersensitivity. Using FITC as the model hapten, we identified a potential source of allergic species in folate-FITC preparations by LC-MS/MS. In mice and guinea pigs, we tested the significance of this impurity by passive cutaneous anaphylaxis and active systemic anaphylaxis assays. We studied the effect of immunogen (e.g., KLH-FITC) dose and derived a desensitization regimen that was further evaluated in a murine tumor model. Administration of folate-FITC with low multi-haptenated contaminants (e.g. bis-FITC) resulted in hypersensitivity in underimmunized animals. However, this drug-related hypersensitivity may be independently prevented by (1) increasing the immunogen dose and/or (2) desensitizing animals with folate-FITC during vaccination. In addition, such manipulation in vivo did not appear to negatively alter the effectiveness of immunotherapy. This study provided confidence on the safety of folate-hapten-targeted cancer immunotherapy in an actively immunized host.

Synthesis and biological evaluation of EC72: a new folate-targeted chemotherapeutic.

A novel folate conjugate of mitomycin C, herein referred to as EC72, was designed and evaluated for biological activity against FR-positive cells and tumors. EC72 was produced by coupling folic acid-gamma-cysteine to 7-N-modified MMC via a disulfide bond. This water soluble conjugate was found to retain high affinity for FR-positive cells, and it produced dose responsive activity in vitro against a panel of folate receptor (FR)-positive cell lines. EC72's activity was considered to be targeted and specific for the FR since (i) excess folic acid blocked biological activity, and (ii) FR-negative cell lines were unresponsive to this drug. Initial in vivo tests confirmed EC72's activity in both syngeneic and xenograft models, and this activity occurred in the apparent absence of gross or pathological toxicity. These results are significant, since daily dosing of EC72 for more than 30 consecutive days yielded no evidence of myelosuppression or toxicity to major organs, including the FR-positive kidneys. The latter observation supports published data, indicating that the apically oriented kidney proximal tubule FRs function to salvage folates prior to their excretion and to return these molecules back into systemic circulation. Overall, EC72's performance in vitro and in vivo warrants further preclinical study before this novel targeted chemotherapeutic is considered for clinical investigation.

Synthesis and biological evaluation of EC20: a new folate-derived, (99m)Tc-based radiopharmaceutical.

A new peptide derivative of folic acid was designed to efficiently coordinate (99m)Tc. This new chelate, referred to as EC20, was found to bind cultured folate receptor (FR)-positive tumor cells in both a time- and concentration-dependent manner with very high affinity (K(D) approximately 3 nM). Using an in vitro relative affinity assay, EC20 was also found to effectively compete with (3)H-folic acid for cell binding when presented either alone or as a formulated metal chelate. Following intravenous injection into Balb/c mice, (99m)Tc-EC20 was rapidly removed from circulation (plasma t(1/2) approximately 4 min) and excreted into the urine in a nonmetabolized form. Data from gamma scintigraphic and quantitative biodistribution studies performed in M109 tumor-bearing Balb/c mice confirmed that (99m)Tc-EC20 predominantly accumulates in FR-positive tumor and kidney tissues. These results suggest that (99m)Tc-EC20 may be clinically useful as a noninvasive radiodiagnostic imaging agent for the detection of FR-positive human cancers.

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

OBJECTIVE: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.