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.

Comparison of folate-conjugated rapamycin versus unconjugated rapamycin in an orthologous mouse model of polycystic kidney disease.

Autosomal-dominant polycystic kidney disease (ADPKD) is a very common genetic disease leading to renal failure. Numerous aberrantly regulated signaling pathways have been identified as promising molecular drug targets for ADPKD therapy. In rodent models, many small-molecule drugs against such targets have proven effective in reducing renal cyst growth. For example, mammalian target of rapamycin (mTOR) inhibition with rapamycin greatly ameliorates renal cystic disease in several rodent models. However, clinical trials with mTOR inhibitors were disappointing largely due to the intolerable extrarenal side effects during long-term treatment with these drugs. Most other potential drug targets in ADPKD are also widely expressed in extrarenal tissues, which makes it likely that untargeted therapies with small-molecule inhibitors against such targets will lead to systemic adverse effects during the necessary long-term treatment of years and decades in ADPKD patients. To overcome this problem, we previously demonstrated that folate-conjugated rapamycin (FC-rapa) targets polycystic kidneys due to the high expression of the folate receptor (FRα) and that treatment of a nonortholgous PKD mouse model leads to inhibition of renal cyst growth. Here we show, in a head-to-head comparison with unconjugated rapamycin, that FCrapa inhibits renal cyst growth, mTOR activation, cell cycling, and fibrosis in an orthologous Pkd1 mouse model. Both unconjugated rapamycin and FC-rapa are similarly effective on polycystic kidneys in this model. However, FC-rapa lacks the extrarenal effects of unconjugated rapamycin, in particular immunosuppressive effects. We conclude that folate-conjugation is a promising avenue for increasing the tissue specificity of small-molecule compounds to facilitate very long-term treatment in ADPKD.

Antiinflammatory Activity of a Novel Folic Acid Targeted Conjugate of the mTOR Inhibitor Everolimus.

Folate receptor (FR)-ß has been identified as a promising target for antimacrophage and antiinflammatory therapies. In the present study, we investigated EC0565, a folic acid-derivative of everolimus, as a FR-specific inhibitor of the mammalian target of rapamycin (mTOR). Because of its amphiphilic nature, EC0565 was first evaluated for water solubility, critical micelle formation, stability in culture and FR-binding specificity. Using FR-expressing macrophages, the effect of EC0565 on mTOR signaling and cellular proliferation was studied. The pharmacokinetics, metabolism and bioavailability of EC0565 were studied in normal rats. The in vivo activity of EC0565 was assessed in rats with adjuvant arthritis, a "macrophage-rich" model with close resemblance to rheumatoid arthritis. EC0565 forms micellar aggregates in physiological buffers and demonstrates good water solubility as well as strong multivalent FR-binding capacity. EC0565 inhibited mTOR signaling in rat macrophages at nanomolar concentrations and induced G0/G1 cell cycle arrest in serum-starved RAW264.7 cells. Subcutaneously administered EC0565 in rats displayed good bioavailability and a relatively long half-life (~12 h). When given at 250 nmol/kg, EC0565 selectively inhibited proliferating cell nuclear antigen expression in thioglycollate-stimulated rat peritoneal cells. With limited dosing regimens, the antiarthritic activity of EC0565 was found superior to that of etanercept, everolimus and a nontargeted everolimus analog. The in vivo activity of EC0565 was also comparable to that of a folate-targeted aminopterin. Folate-targeted mTOR inhibition may be an effective way of suppressing activated macrophages in sites of inflammation, especially in nutrient-deprived conditions, such as in the arthritic joints. Further investigation and improvement upon the physical and biochemical properties of EC0565 are warranted.

Reducing undesirable hepatic clearance of a tumor-targeted vinca alkaloid via novel saccharopeptidic modifications.

During a phase I trial of EC145 (a folate-targeted vinca alkaloid conjugate), constipation was identified as the dose-limiting toxicity, probably from a nonfolate receptor-related liver clearance process capable of releasing unconjugated vinca alkaloid from EC145 and shuttling it to the bile. Here, we report on the selective placement of novel carbohydrate segments (1-amino-1-deoxy-glucitolyl-γ-glutamate) spaced in-between the folate and vinca alkaloid moieties of EC145, which yielded a new agent (EC0489) that is equipotent but less toxic than EC145. Whereas both compounds could cure tumor-bearing mice reproducibly, EC0489 differed from EC145 with i) a shorter elimination half-life, ii) approximately 70% decrease in bile clearance, iii) a 4-fold increase in urinary excretion, and iv) improved tolerability in rodents. This combination of improvements justified the clinical evaluation of EC0489 where currently administered dose levels have exceeded the maximal tolerated dose of EC145 by approximately 70%, thereby reflecting the translational benefits to this new approach.

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.

Impact of high and low folate diets on tissue folate receptor levels and antitumor responses toward folate-drug conjugates.

Herein, we present a detailed analysis on the effects of feeding laboratory mice both high and low folic acid (folate)-containing diets as related to associated changes in serum and red blood cell (RBC) folate levels, tissue-derived folate receptor levels, and the ability of folate-drug conjugates to bind and effectuate activity against folate receptor (FR)-positive tumor xenografts. Our data show that serum and RBC folate concentrations sharply drop immediately after mice are switched to low folate diets; however, both parameters reach steady-state, "human-like" levels after 6 weeks. Interestingly, tissue-related folate binding capacities were also lowered during the dietary modulation period, whereas the net uptake of a radiolabeled folate conjugate was simultaneously increased 2.6- and 5-fold in FR-positive kidney and tumor tissue, respectively. Finally, the performances of several clinically and preclinically relevant folate-drug conjugates were evaluated against tumors in mice that were fed high or low folate diets. Except when administered at a dose level 6-fold less than that required to saturate endogenous FRs, no significant loss of antitumor activity was observed. From these findings, we conclude that lowering the dietary intake of folates in mice has little impact on the biological activity of repetitively dosed folate-targeted agents but that low folate diet regimens will reduce serum and RBC folate levels down to levels that more closely approximate the normal human ranges.

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.

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.

Targeting folate receptors to treat invasive urinary bladder cancer.

Folate receptors (FR) may be of use for targeted delivery of cytotoxic drugs in invasive urothelial carcinoma (iUC), for which improved therapy is needed. FR expression and function in iUC were explored and the antitumor activity and toxicity of a folate-targeted vinblastine conjugate were evaluated in dogs with naturally occurring iUC, an excellent model for human iUC. FR immunohistochemistry was carried out on iUC and normal human and dog bladder tissues together with nuclear scintigraphy in dogs to monitor iUC folate uptake. Dose escalation of a folate-targeted vinblastine compound, EC0905, was conducted in dogs with biopsy-confirmed, FR-positive iUC. FRs were detected by immunohistochemistry (PU17) in most primary iUC and many nodal and lung metastases from dogs, and scintigraphy confirmed folate uptake in both primary and metastatic lesions. The maximum tolerated dose of EC0905 in dogs was 0.25 mg/kg IV weekly, with neutropenia at higher doses. Tumor responses included partial remission (≥ 50% reduction in tumor volume) in five dogs and stable disease (<50% change in tumor volume) in four dogs. Immunoreactivity to PU17 was similar in humans (78% of primary iUC, 80% of nodal metastases). Less immunoreactivity to mab343 (22% of cases) occurred. FR-ß was noted in 21% of human iUC cases. Our findings suggest folate-targeted therapy holds considerable promise for treating iUC, where FR-ß may be important in addition to FR-α.

In vivo structural activity and optimization studies of folate-tubulysin conjugates.

Herein we report on the potencies of 4 related folate-conjugated tubulysins constructed with either tubulysin B hydrazide (EC0305), tubulysin A hydrazide (EC0510), the N,O-acetal derivative of natural tubulysins (EC0317) or a tubulysin B ester (EC0302). Our results confirmed that EC0305 is the most favorable conjugate of the group due to its potent antitumor activity [100% cures at 1 micromol/kg, three times a week (TIW) for 2 weeks] and its favorably low toxicity profile. In contrast, the natural tubulysin B drug proved to be inactive against a human nasopharyngeal tumor model when administered at doses near to or greater than the maximum tolerated dose (MTD). When tested against more chemoresistant folate receptor expressing M109 and 4T1-cl2 tumors, EC0305 displayed superior antitumor activity over a previously disclosed folate conjugate of desacetylvinblastine monohydrazide (EC145). These studies demonstrate that EC0305 has significant antiproliferative activity against FR expressing tumors, including those which are generally more chemoresistant, and that EC0305 should be considered for development as a candidate for the treatment of advanced FR-expressing human cancers.

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.

Comparative preclinical activity of the folate-targeted Vinca alkaloid conjugates EC140 and EC145.

EC140 is a water soluble folate conjugate of desacetylvinblastine monohydrazide (DAVLBH), which is constructed with an endosome-cleavable acyl hydrazone bond. This agent has proven to be active and specific against well established, subcutaneous folate receptor (FR)-positive tumors in multiple animal models. Recent structure-activity and optimization studies have yielded a disulfide bond-containing counterpart to EC140, herein referred to as EC145. This new conjugate was found to retain high affinity for FR-positive cells, and it produced specific, dose-responsive activity in vitro. Comparative in vivo efficacy tests confirmed that, like EC140, EC145 displays activity against both syngeneic and xenograft tumor models. However, EC145 was found to be more active and better tolerated than EC140; hence, more durable complete responses were consistently observed in EC145-treated tumor-bearing animals. Furthermore, EC145 was not found to be active against a FR-negative tumor model. Additional preclinical studies are therefore warranted to better understand EC145's breadth of activity against FR-positive tumors.

Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay.

The folate receptor (FR) is a valuable therapeutic target that is highly expressed on a variety of cancers. The current development of folate-targeted cancer therapies has created the need for quantitating functional FRs in clinical specimens. In this article, we report on the creation of a highly sensitive radioactive binding method for quantitatively measuring FR expression in frozen tissue homogenates. Expression was positive in approximately 89% of human ovarian carcinomas but was negligible in both mucinous ovarian carcinomas and normal ovary. Expression was also significant in carcinomas of the kidney, endometrium, lung, breast, bladder, and pancreas. Normal tissues from humans and six different laboratory species were also analyzed; surprisingly, some interspecies variability in FR expression (especially in kidney, spleen, and lung tissue) was found. Interestingly, normal human lung tissue displayed high expression levels, whereas expression in normal lung of the other species was negligible. However, considering that folate-drug conjugates fail to accumulate in the lungs of patients, the consequence of this finding was not considered to be of clinical concern. Overall, this new methodology is reliable for determining functional FR expression levels in tissues, and it could possibly be a useful clinical test to determine patient candidacy for FR-targeted therapeutics.

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.