Pteridines 2015; 26(1): 1–12

Review

Marie Bartouskova, Bohuslav Melichar and Beatrice Mohelnikova-Duchonova* receptor: a potential target in ovarian

Abstract: is the most frequent cause of Introduction gynecological cancer-related death. Unfortunately, many patients are diagnosed at an advanced stage and have a Folic acid antagonists have been used in the treatment poor prognosis. The standard treatment for advanced dis- of cancer for more than six decades, since 1948 [1]. Folic ease involves maximal cytoreductive surgery and chemo- acid is a vitamin required for cell metabolism, DNA syn- therapy based on platinum compounds and . thesis, and repair [2]. Two cellular folate uptake pathways Patients presenting at an advanced stage have a higher have been characterized. The first is mediated by trans- risk of recurrence. The development of drug resistance membrane-reduced folate carriers, which represent the currently represents a major obstacle in the systematic predominant pathway for folate uptake by normal human treatment and, therefore, the discovery of new anticancer cells. Whereas in epithelial cancer cells, membrane pro- agents and approaches should improve the poor progno- teins called folate receptors are often highly expressed and sis of these patients. Folate receptor α is overexpressed in mediate the folate uptake by tumors. Epithelial ovarian epithelial ovarian cancer (EOC), but has limited expres- carcinoma (EOC) has the highest expression of these pro- sion in nonmalignant human tissues. The degree of folate teins, making the folate receptor an attractive candidate receptor expression corresponds with the stage and grade for and targeted drug delivery therapy in of the disease. Because of this, folate receptor α seems several types of , specifically in EOC. to be a potential therapeutic target for the treatment of EOC had an age-standardised rates (ASRs) 13.1 per ­ovarian cancer. Currently, several approaches have been 100,000 inhabitants (2012) [3], with the highest mortality studied to target this protein in ovarian cancer treatment. rate of all gynecologic tumors. Prognostic factors in EOC This review summarizes current knowledge about the are stage, grade, age, histological subtype, performance potential usage of folate receptors as prognostic and pre- status, volume of ascites, extent of residual disease fol- dictive biomarkers as well as their role in the management lowing debulking surgery, and findings at second-look and targeted therapy of ovarian cancer. laparotomy [4]. Parameters of the host response, e.g., the presence of tumor-infiltrating lymphocytes [5] or the Keywords: biomarkers; folate receptor; ovarian cancer; production of neopterin, a biomarker of immune system targeted therapy. activation [6], have also been identified as independ- ent prognostic factors. More research into the prognostic DOI 10.1515/pterid-2014-0013 factors is clearly needed as prognostic factors can help to Received October 28, 2014; accepted November 25, 2014; previously identify patients with poor prognosis and streamline the published online February 6, 2015 development of new approaches and drugs. Currently, the standard treatment for advanced ovarian carcinoma is surgical tumor debulking followed by plati- num and -based combination . However, despite the potentially high response rate to the initial chemotherapy ranging approximately between 60% *Corresponding author: Beatrice Mohelnikova-Duchonova, and 80%, the majority of patients will ultimately experience Department of Oncology, Palacky University Medical School and recurrence [7], and only about 44% survive 5 years after the Teaching Hospital, Olomouc, IP Pavlova 6, 775 25 Olomouc, Czech diagnosis. The development of drug resistance, either de Republic, E-mail: [email protected] Marie Bartouskova and Bohuslav Melichar: Department of novo or induced resistance, significantly limits the long-term Oncology, Palacky University Medical School and Teaching Hospital, efficacy of systemic chemotherapy [8]. For platinum therapy Olomouc, Czech Republic resistant recurrent EOC , liposomal , 2 Bartouskova et al.: Folate receptors in ovarian cancer , , , , [20]. Moderate expression has been found in the brain, and/or other drugs may be used [9]. Therapeutic approaches breast, bladder, and pancreatic cancers [20]. In the above- that target the host response to neoplasia have so far been mentioned quantitative radioligand-binding study, more limited to the experimental setting [10, 11]. than 50% of pancreatic cancer samples were positive for Despite the fact that anti-folic acid chemotherapy has FR expression, but the study using immunohistochemistry been used for more than six decades, a new area for the demonstrated only 13% of pancreatic cancer samples as exploration of anti-folate therapy has been opened with positively staining [21]. This discrepancy could be explained the advent of targeted therapy and with the ­development by the antibody specificity, thin slicing of samples, where of new targeted approaches in medical oncology. More­ antigen retrieval is compromised, or by the expression over, folate receptors may be not only the target for of isoforms other than α-isoforms, as in the immunohis- anticancer therapy, but also a potential predictive and tochemical study, anti-FRα antibody has been used. Low prognostic biomarker and a target for tumor imaging. FR expression has been reported in the liver, colorectal, This review summarizes published data concerning prostate, lymphoma, and head and neck carcinomas [20]. the folate receptors, their potential usage as prognostic Despite the fact that among all other human tissue types and predictive biomarker as well as their role in the detec- ovarian malignancies have the highest FR expression, the tion and targeted therapy of EOC, and seeks to define level of expression is highly dependent on tumor histol- future research directions. ogy. Almost 100% of nonmucinous primary and metastatic ovarian tumors were found to have high FR expression, whereas mucinous tumors express very low or nondetect- able FR levels [20]. Moreover, it seems that the metastatic Folate receptor – structure, tumors generally express higher levels of FR compared ­expression, and function to the primary tumors [20, 21]. FR expression seems to provide a growth advantage to the tumor by greater folate Folate receptors (FRs) are glycosylphosphatidylinositol- uptake resulting in rapid cellular growth and division [14]. anchored membrane proteins of 38–40 kDa that bind folic Consequently, FR protein expression has been found to acid or folate-drug conjugates with high affinity [12]. FRs be associated with tumor progression, dedifferentiation are encoded by a multigene family FOLR (namely, FOLR1, (grade), decreased survival, and platinum resistance [22– FOLR2, and FOLR3), which is localized to chromosome 24]. Toffoli et al. formulated a hypothesis that FRα might 11q13.3–q14.1 [13]. FRs are membrane proteins that play an increase the repair of DNA damage caused by platinum via important role mainly in the cellular accumulation of increased folate cellular uptake [23]. Patients with subopti- and . Folate (pteroyl-l-glutamic acid, vitamin B9) mal debulking surgery had significantly higher tissue FRα is an essential vitamin that is important for DNA replication expression levels in contrast with patients with optimal and cell division. Three types of folate receptors (FRα, FRβ, surgery. Moreover,­ a higher expression of FRα was an inde- and FRγ) have been distinguished [14]. Each of folate recep- pendent pro­gnostic factor associated with inferior disease- tors has tissue specific distribution and different folate- free survival and overall survival of patients [25]. binding potential [15]. FRα binds folic acid with high affinity There is evidence that FR might be involved in malig- and transports folate by receptor-mediated endocytosis [14]. nant transformation, tumor progression, and treatment FR has been discovered as a tumor marker of EOC cell outcomes. In addition to the modulation of treatment out- line in 1991 [16], and FR expression has been reported in comes of standard platinum-based chemotherapy, FR is 90% of EOC cases. Subsequently, different levels of FR currently studied as a promising potential target for the tar- expression were observed in various human tumors and geted therapy. Moreover, because FR has been confirmed to normal tissues. In contrast to cancer cells, most of the non- be a tumor-associated antigen in ovarian cancer, it may be malignant tissues express low to negligible levels of FR, used for selective targeted delivery of drugs to tumor cells. with the exception of the kidney and the lung. FR is limited to the apical membrane of the proximal tubule cells of human kidney and lung pneumocytes [17, 18]. However, in lung tissue, FR seems not to be able to capture folates Folate receptor as a target for from the circulation as folate-drug conjugates fail to accu- ­anticancer therapy mulate in the lungs of patients [19]. Besides EOC, a number of other cancers have the high levels of FR, namely, renal The FR represents a potential target for targeted therapy cell carcinomas, lung cancer, and endometrial carcinoma in some human cancers [26]. As the above-mentioned Bartouskova et al.: Folate receptors in ovarian cancer 3 studies have shown a significant association of FR expres- Clinical data sion in ovarian cancer cells with biological aggressiveness and tumor phenotype, it is clear that FR plays an impor- In a phase I study, a total of 25 pretreated patients with tant role in tumor development and progression. The role platinum-resistant epithelial ovarian cancer received far- of FRα in carcinogenesis may be to regulate folate uptake letuzumab administered intravenously on days 1, 8, 15, from serum [27] or generate regulatory signals for tumor and 22 in a 5-week cycle. The maximal tolerable dose of growth [28], and therefore, FRα overexpression phe- farletuzumab of 400 mg/m2 has been established. There notype is associated with ovarian prolifera- was no evidence of serious or severe drug-related adverse tion [29]. The high expression of FRα in ovarian cancer, events of grade 3/4, and there were no anaphylactic reac- together with the effect on tumor biology, predisposes tions. The treatment-related grade 1/2 adverse events were FRα for a potential target for the ovarian cancer treatment. hypersensitivity reactions (15 patients, 60%), fatigue Moreover, FRα could suppress the cytotoxic drug-induced (12 patients, 48%), and diarrhea (four patients, 16%). apoptosis through the caspase pathway by regulating the The most common hypersensitivity reactions, including apoptosis-related molecule Bcl-2 and Bax [25], and there- pyrexia (eight patients, 32%) and chills (five patients, fore, the combination of FRα target therapy and chemo- 20%), were easily controlled with antipyretics and/or anti- therapy has been currently intensively studied. histamines. Rare samples exhibited human anti-human antibodies (HAHA). Farletuzumab was not associated with clinically significant changes in cardiac, pulmonary, Farletuzumab or renal function. There were no objective responses. Nine patients demonstrated a stable disease, and 15 patients Farletuzumab (MORAb-003) is a 145-kDa humanized had a progression of disease based on RECIST criteria [32]. monoclonal antibody against FRα produced in Chinese In conclusion, farletuzumab in the phase I study has been hamster ovary cells [30]. The therapeutic potential of far- proven to be safe and well tolerated. letuzumab in EOC has been shown both in in vitro and Another open-label phase I clinical trial that enrolled in vivo studies (Table 1). a total of 15 platinum-sensitive ovarian cancer patients in first or second relapse received weekly 2.5 mg/kg far- letuzumab intravenously in combination with six cycles Preclinical data of (AUC5-6)/pegylated liposomal doxorubicin (30 mg/m2). We still await for the full report from this trial, Preclinical in vitro studies have shown that farletuzumab avaliable from: http://www.clinicaltrial.gov/ct2/show/ inhibits FRα-dependent cell growth [31]. Moreover, far- study/NCT01004380?term=farletuzumab&rank=1. letuzumab has been shown to mediate immune antitumor The phase II study was performed in platinum-­ activity via the antibody-dependent fixation of complement sensitive ovarian cancer patients in first relapse in order to and cell lysis induction (complement-dependent cytotox- determine the efficacy of farletuzumab as a single agent or icity), as well as via antibody opsonization of tumor cells in combination with platinum and . Patients with followed by the recruitment of immune killer cells (anti- nonsymptomatic relapse (elevated CA125 only) received body-dependent cell-mediated cytotoxicity). In addition, weekly farletuzumab in monotherapy until progression, farletuzumab has reduced tumor growth through inhibi- whereas patients with symptomatic relapse of the disease tion of FRα-mediated lyn kinase phosphorylation [31]. or those who progressed on the single agent farletu- In vivo, nude mice SKOV-3 ovarian tumor xenografts zumab had a combination of six cycles of chemotherapy were treated with murine LK 26 antibody, which is the pre- ­consisting of carboplatin (AUC 5-6) and taxane (­paclitaxel cursor of farletuzumab. A significant reduction of tumor 175 mg/m2 or 75 mg/m2) with farletuzumab growth was demonstrated in 4 weeks [31]. In accord- (37.5 mg/m2, 67.5 mg/m2, or 100 mg/m2). The primary end- ance with these results, farletuzumab in combination point was normalization of CA125. with docetaxel has showed synergistic activity in murine In the arm with the single agent farletuzumab, 28 xenografts bearing FRα-expressing ovarian tumors [29]. patients were enrolled and 25 patients completed at least In relation to side effects, farletuzumab exhibited a low 9 weeks of single agent treatment. The concentration toxicity in nonhuman primates because of the absence of CA125 decreased in two patients and was stable in 18 of FR expression in normal tissues [31]. These encourag- patients. Twenty-one patients were treated with the single ing preclinical data led to initiation of clinical trials with agent farletuzumab; 21 patients crossed over after progres- farletuzumab. sion to combination therapy. Thus, a total of 47 patients 4 Bartouskova et al.: Folate receptors in ovarian cancer [32] Reference Reference [33] Reference [34]

SD (9) SD PD (15) (RECIST) Results Results (RECIST) Results CR (3) PR (30) (9) SD PD (2) Results Prematurely Prematurely terminated Failed to meet the to meet Failed endpoint primary

0 events Adverse Adverse events grade 3/4 grade events Adverse Febrile neutropenia, thrombocytopenia, thrombocytopenia, neutropenia, Febrile large diarrhea, pain, abdominal subileus, obstruction, intestinal hyperglycemia and urosepsis, Arm C No Arm C placebo+CBDCA+taxane Arm C

2 2 MTD Dose, mg/m Dose, 100 400 mg/m Arm B Placebo 2.5 mg/kg+ Placebo paclitaxel Farletuzumab 2.5 mg/kg+ Farletuzumab CBDCA+taxane

Previous chemotherapy chemotherapy Previous regimens Arm B combination therapy therapy Arm B combination Farletuzumab+chemotherapy 47 5 (median) Arm A Farletuzumab 2.5 mg/kg+ Farletuzumab paclitaxel Farletuzumab 1.25 mg/kg+ Farletuzumab CBDCA+taxane

Number of of Number patients Arm A Farletuzumab 28 25 Number of of Number patients 417 1100

-dependent cell growth and mediated tumor cytotoxicity through complement-dependent cytotoxicity and antibody-dependent cytotoxicity [31]. cytotoxicity antibody-dependent and cytotoxicity complement-dependent through cytotoxicity tumor mediated and growth cell FR α -dependent inhibited Farletuzumab [31]. xenografts tumor ovarian SKOV-3 in nude mice growth tumor reduced farletuzumab of Precursor [31]. primates in non-human toxicity low exhibited Farletuzumab [29]. model xenograft in murine activity synergistic demonstrated docetaxel with in combination Farletuzumab Primary endpoint Primary endpoint CA125, Normalized response overall (ORR) rate Safety, MTD Safety, Primary endpoint PFS, OS PFS

progression disease; CBDCA, carboplatin; NA,available. CBDCA, not progression disease; carboplatin; sensitive relapse resistant EOC resistant resistant EOC resistant sensitive EOC In vivo data Clinical I Phase Overview of trials with farletuzumab. with trials 1 Overview of Table data Preclinical In vitro Phase II Phase  First platinum- Platinum  Platinum Phase III Phase  Platinum- Platinum  Platinum MTD, maximum tolerated dose; EOC, epithelial ovarian carcinoma; PFS, progression-free survival; OS, overall survival; CR, complete response; PR, partial response; SD, stable disease; disease; stable SD, response; response; PR, partial CR, complete survival; overall OS, survival; PFS, progression-free carcinoma; ovarian epithelial dose; EOC, tolerated maximum MTD, PD, ­ Bartouskova et al.: Folate receptors in ovarian cancer 5 were treated with combination therapy. CA125 normalized Unfortunately, the phase III randomized trials in both in 38 of these patients after six cycles of combined therapy, platinum-sensitive and platinum-resistant EOC failed to and treatment continued only with farletuzumab in confirm the promising results of the in vitro and in vivo mono­therapy until progression. According to the RECIST studies. Nevertheless, the phase II trials demonstrated criteria, complete response was observed in 7% (3), partial the safety of farletuzumab. The question remains whether response in 68% (30), stable disease in 21% (9), and 5% (2) the design of the randomized trials took into account the of patients had progression. Farletuzumab alone as well proper selection of patients that could profit from anti-FR as in combination with chemotherapy was well tolerated. therapy. It would be interesting to divide patients to sub- The most common adverse events were febrile neutrope- groups according to FR expression status. From phase II nia, thrombocytopenia, abdominal pain, diarrhea, large trials, it seems to be possible that the subgroup of patients intestinal obstruction, subileus, urosepsis, and hyper- who may have benefit from anti-FR therapy exists, but the glycemia [33]. Phase II indicated that the combination of biomarkers that would predict treatment response are cur- farletuzumab with carboplatin and taxane might enhance rently unknown. the response rate and duration of response in platinum- sensitive EOC patients in first relapse and served as a basis for subsequent trials. Based on promising results of early phase I and Folate receptor and targeted drug phase II clinical trials, randomized, double-blind, pla- delivery in anticancer therapy cebo-controlled phase III studies have been conducted. The final results have so far not been published, and Targeted delivery of anticancer therapy exclusively to only limited preliminary data have been reported. cancer cells is certainly an attractive treatment approach The phase III (NCT00849667) trial aimed to evaluate in medical oncology. This concept brings several advan- the efficacy and safety of the weekly farletuzumab in tages, mainly improvements in efficacy of anticancer combination with carboplatin and taxane in patients therapy and reduction of drug toxicity as high toxicity and with platinum-sensitive ovarian cancer in first relapse. insufficient activity are currently the biggest limitations Patients were randomized to one of three arms, includ- of anticancer therapy [35]. The frequent overexpression of ing placebo plus carboplatin/taxane, farletuzumab at folate receptors in cancer cells may represent a target for 1.25 mg/kg intravenously plus carboplatin/taxane, or tumor-selective drug delivery [36]. In general, two strat- farletuzumab at 2.5 mg/kg intravenously plus carbopla- egies have been shown to be effective for targeted deliv- tin/taxane. The primary endpoints were progression- ery of drugs to FR-positive cancer cells [36]. First is the free survival; the secondary endpoints were safety and coupling of an anticancer drug to monoclonal antibody tolerability of weekly doses 1.25 mg/kg or 2.5 mg/kg of against the receptor. However, in this case, the targeting farletuzumab in combination with chemotherapy (car- ligand is highly immunogenic with a range of side effects boplatin/taxane), overall survival, CA125-defined pro- and with the inability for repeated administrations in the gression-free survival, duration of second compared to case of development of secondary antibodies. The second first remission, and patient quality of life. The median approach is coupling of anticancer drugs to folic acid, a progression-free survival was 9.0 months in the placebo high affinity FR ligand. Currently, there are five folic acid- group, 9.5 months in the 1.25-mg/kg farletuzumab arm, based small-molecule drug conjugates (SMDCs) [26]. and 9.7 months in the 2.5-mg/kg farletuzumab arm. With no statistically significant differences between the arms, the study did not meet its primary endpoints Folate-desacetylvinblastine monohydrazide [34]. A similar phase III study that randomized plat- (EC140; DAVLBH) inum-resistant or refractory-relapsed ovarian cancer patients into two arms (paclitaxel with farletuzumab EC140 was produced by coupling a peptidic analog of the 2.5 mg/kg and paclitaxel with placebo) also failed in the folic acid to DAVLBH via an acylhydrazone bond [37]. primary outcome focused on progression-free survival and overall survival. The study has been prematurely terminated as it did not meet the pre-specified criteria Preclinical data for continuation after interim futility analysis, available from: http://www.clinicaltrial.gov/ct2/show/NCT00738 EC140 is a water-soluble conjugate with a high affinity for 699?term=NCT00738699&rank=1. FR-positive cells. EC140 has been shown to have a specific 6 Bartouskova et al.: Folate receptors in ovarian cancer and dose-responsive activity in vitro [37]. Subsequently, the activity has been described in vivo, in both syngeneic and xenograft models with minimal to moderate toxic- ity [37]. However, immediately after that, it was demon- Unpublished data Unpublished Reference [41] Reference [42] strated that EC145 (vintafolide) was more active and less Reference toxic than EC140. Vintafolide was shown to induce more durable complete responses in tumor-bearing animals [38]. Furthermore, EC145 was shown to be FR specific as it was not active against a FR-negative tumor model [39]. Therefore, subsequent research aiming to evaluate clini- cal use of folate-vinca alkaloids has focused mainly on vintafolide (EC145). Prematurely terminated Prematurely Results PFS 5.0 months (B) vs. PFS 2.7 months (A) Results PR (1) (1) SD Results (RECIST) EOC only (RECIST) EOC Results

Vintafolide (EC145)

Vintafolide (EC145) is a folate-vinca alkaloid (folate- desacetylvinblastine monohydrazide) conjugate with a -destabilizing effect targeting FR-express- ing cells [40] and a disulfide bond-containing analog No Arm C Abdominal pain, leukopenia, leukopenia, pain, Abdominal peripheral neutropenia, sensory neuropathy Adverse events Adverse Constipation, fatigue Constipation, EC140 [38]. The therapeutic potential of vintafolide in 3/4 events Adverse EOC has been shown both in in vitro and in vivo studies (Table 2). Placebo+PLD Arm B 2.5 Dose, mg Dose, 2.5 mg MTD Preclinical data

In vitro, vintafolide displayed a strong synergistic activity against nasopharyngeal KB cell line when combined with doxorubicin. In vivo, all vintafolide drug combinations, namely, pegylated liposomal doxorubicin, , car- boplatin, paclitaxel, docetaxel, topotecan, and produced far greater antitumor effect compared to single agents alone, without significantly increasing overall tox- Vintafolide 2.5 mg+PLD Vintafolide Arm A PLD+vintafolide 100 Arm B 8 icity [43]. Moreover, these results were not observed with regimens chemotherapy Previous combinations of EC140 or [43]. In vivo, nude mice with nasopharyngeal carcinoma KB cells or J6456 murine lymphoma cell xenografts were treated by vintafolide. The treatment of FR-positive nude mice bearing human xenografts led to complete response 640 Number of patients of Number 49 PLD alone Arm A 32 Number of patients of Number in five out of five mice, and four out of five mice have been cured (i.e., remission without a relapse for > 90 days post-tumor implantation). Vintafolide treatment was not accompanied by noticeable weight loss or major organ tissue degeneration. Furthermore, the enhanced thera- PFS Primary endpoint PFS Primary endpoint Safety, MTD Safety, peutic index due to folate conjugation was also indicated [41]. doxorubicin with when combined KB cells nasopharyngeal against activity synergistic strong displayed Vintafolide [41]. to the single agents alone compared effect antitumor greater far produced chemotherapy plus Vintafolide [42]. toxicity tolerable with xenografts 5 mice to CR in 5 out led treatment Vintafolide Primary endpoint by the fact that the unconjugated drug (desacetylvin- blastine monohydrazide) was found to be completely inactive when administered at nontoxic dose levels and only marginally active when given at highly toxic dose resistant EOC resistant resistant EOC resistant tumors  Platinum- PD, progression disease; disease; stable SD, response; response; PR, partial CR, complete survival; PFS, progression-free carcinoma; ovarian epithelial dose; EOC, tolerated maximum MTD, doxorubicin. liposomal PLD, pegylated Proceed study Proceed Phase III Phase Precedent study Precedent  Platinum- Phase II Phase Advanced  Advanced solid Overview of trials with vintafolide. with trials 2 Overview of Table Preclinical data Preclinical In vitro levels [40]. Taken together, vintafolide had significant In vivo data Clinical I Phase Bartouskova et al.: Folate receptors in ovarian cancer 7 antiproliferative activity, tolerable toxicity, and seemed to the FR-targeting investigational diagnostic agent EC20 be a potent, folate-targeted anticancer agent for phase I during the screening period. However, the study has clinical trials. been suspended, and the results have so far not been published. Nevertheless, vintafolide is also tested in other types of human cancer. A multicenter trial of Clinical data vintafolide in advanced FR-positive adenocarcinoma of the lung brought also inconclusive results [44]. The Phase I study has been conducted in cohorts of three to patients with FR-positive lesions had a trend of better six patients with a wide range of refractory solid tumors overall survival (47.2 weeks vs. 14.9 weeks) compared to determine the maximum-tolerated dose of vintafolide to patients with at least one, but not all, FR-positive administered as a bolus intravenous injection or 1-h infu- target lesions, although the statistical significance was sion on days 1, 3, 5 and days 15, 17, and 19 of each 28-day not reached (p = 0.101) [44]. To investigate the clinical cycle with dose escalation. The maximal tolerated dose benefit, a randomized phase II trial evaluating the activ- was 2.5 mg regardless of the type of administration. ity of vintafolide plus docetaxel vs. docetaxel alone in The dose-limiting toxicity was constipation. The other highly FR positive lung tumors has been initiated. It is reported adverse events were nausea, fatigue, and vom- evident from the data that vintafolide has been shown iting, mostly grade 2 toxicities [42]. Vintafolide has been to be a promising candidate for the treatment of ovarian proven to have an acceptable safety profile in patients cancer, and the ongoing trials will address the clinical with advanced cancer. use of this drug. The phase II PRECEDENT trial compared vintafolide (2.5 mg intravenously three times per week during weeks 1 and 3) combined with pegylated liposomal doxorubicin Folate-maytansinoid conjugate (EC131) (PLD, 50 mg/m2 intravenously once every 28 days) with PLD monotherapy in a total of 149 pretreated patients Folate-maytansinoid conjugate (EC131) is a new folate with platinum-resistant EOC. The primary aim was to receptor targeted drug-conjugate. Maytansinoid is a strong compare progression-free survival. Median progression- microtubule-inhibiting agent. Folate-maytansinoid conju- free survival of 5.0 months for vintafolide plus PLD was gate was prepared by covalently attaching the folic acid to significantly longer compared to median progression-free maytansinoid DM1 with use as an intramolecular disulfide survival of only 2.7 months for PLD monotherapy. The bond [45]. best efficacy was observed in patients with lesions 100% positive for FR with median progression-free survival of 5.5 months compared with 1.5 months for PLD alone Preclinical data (p = 0.013). FR negative patients had no benefit from the addition of vintafolide [41]. Overall survival was evaluated In vitro, EC131 has been highly cytotoxic toward FR-posi- as a secondary endpoint. There were no statistically sig- tive nasopharyngeal carcinoma KB cells. nificant differences between the two arms in overall sur- In vivo, the antitumor activity of EC131 was explored vival. In general, the drug combination was well tolerated, against BALB/c mice bearing subcutaneously injected FR- although higher frequency of neutropenia, leukopenia, positive lung adenocarcinoma cells (M109), which express abdominal pain, and peripheral neuropathy was observed similar numbers of folate receptors as human ovarian car- in the vintafolide plus PLD arm. On the other hand, this cinoma [20]. Tumors in the untreated animals attained arm had a higher total median cumulative PLD dose per a size of approximately 1500 mm3 by day 42, whereas patient and a higher frequency of safety evaluations [41]. tumors in the EC131-treated group of animals attained the Thus, vintafolide plus PLD has been the first combination same volume by day 45. The treatment led to complete demonstrating an improvement over standard therapy, response in one out of five mice and partial response in with marginally increased toxicity, in a randomized trial three out of five mice. The treated group and the untreated in patients with platinum-resistant EOC. group differed significantly on day 18 post-tumor implan- A phase III study (PROCEED) started in 2010 as a tation. EC131 treatment was not observed to lead to weight double-blind trial evaluating PLD in combination with loss or organ tissue degeneration. The antitumor activity placebo or PLD with vintafolide. The trial planned to of EC131 was reduced by application of folic acid leading enroll approximately 500 FR-positive patients, and all to no response to EC131 treatment in a group of mice with participants were scheduled to undergo imaging with a diet containing folic acid. Thus, the antitumor activity 8 Bartouskova et al.: Folate receptors in ovarian cancer was proven to be dependent on the binding of EC131 to in median overall survival time in comparison with the tumor-associated FR [45]. Furthermore, EC131 also showed untreated control group (p = 0.006). No noticeable toxicity marked antitumor activity in FR-positive human KB tumor was found in the context of weight loss or physical change xenografts. The EC131 treatment led to complete response in behavior. The EC72 treatment led to the complete in 4/5 of the mice and partial response in 1/5 of the mice. response in one out of four mice and partial response in No signs of drug toxicity were detected during and after three of four mice. Mitomycin C-treated animals did not the therapy. Taken together, these results indicate that survive significantly longer than the controls, presum- EC131 has a significant activity in xenograft models and ably due to mitomycin C-related toxicity [47]. The anti- seemed to be a potent folate-targeted anticancer agent [45] tumor activity of EC72 has been shown also in xenograft waiting for further validation studies and clinical trials models using xenografts with human KB carcinoma cells. (Table 3). The survival of EC72-treated mice was doubled compared to untreated controls. There was no evidence of toxicity [47]. In conclusion, both tumor models proved the anti- Folate-mitomycin C conjugate (EC72) tumor activity of EC72 in a dose-dependent manner and the lack of serious toxicity. EC72 seems to be a promising EC72 is a folate drug conjugate, which was prepared by candidate for further validation studies and future clini- covalently attaching folate to the potent DNA crosslinker cal trials (Table 3). mitomycin C, a cytotoxic drug with a high antitumor and antibiotic activity [46]. Imaging methods Preclinical data In light of the development of FR-targeted anticancer In vitro, EC72 exhibits dose-dependent activity against therapy, the need for quantification of functional FRs has a panel of FR-positive cell lines (KB, ID8, and M109 cell occurred in clinical cancer samples. Several methodical lines). Similarly to EC131, the activity of EC72 is also approaches have been reported to analyze the FR-positive blocked by the presence of excess free folic acid and does cells, namely, polymerase chain reaction detecting mRNA not affect normal tissues with low levels of FR [46]. expression levels [48], immunohistochemistry, and radio- In vivo therapy experiments were performed in both ligand binding assay detecting protein expression levels syngeneic and xenograft models. In a syngeneic model, [20–25]. Quantitative radioligand binding assay has an BALB/c mice bearing subcutaneously injected FR-posi- advantage of sensitively measuring only functional recep- tive lung adenocarcinoma cells (M109) have been treated tors which is very important for FR-targeted therapies. by intraperitoneal injection of EC72 or mitomycin C start- On the other hand, immunohistochemistry can avoid ing 4 days after implantation of M109 tumor cells. The specificity concerns regarding nonmalignant cells as acti- EC72-treated animals had a significant 185% increase vated macrophages that may contribute to radioligand

Table 3 FR-targeted delivery drugs in in vivo and in vitro studies.

Drug conjugated to folate In vitro In vivo on xenograftsa Clinical trials References

EC72 Mitomycin C Specific and dose-responsive activity Minimal toxicity No [46, 47] on FR-positive cells Doubled OS EC131 Myatansinoid Specific activity on FR-positive cells CR in 4/5 mice No [45] PR in 1/5 mice Minimal toxicity EC140 Desacetylvinblastine Specific and dose-responsive activity CR in 1/5 mice No [37, 38] monohydrazide on FR-positive cells PR in 4/5 mice Minimal to moderate toxicity EC145 Desacetylvinblastine Specific and dose-responsive activity CR in 5/5 mice Phase I–III [38–44] on FR-positive cells. Minimal toxicity More active and less toxic than EC140. aNude mice bearing FR-positive human KB cells. CR, complete response; PR, partial response; OS, overall survival. Bartouskova et al.: Folate receptors in ovarian cancer 9 signaling. Thus, both methods are currently used to estab- vintafolide. In addition to histological verification of FRs lished FR expression status in trials. by immunohistochemistry, SPECT with radiofolates seems As several folic acid-based radiopharmaceuticals tar- to be another approach to verify FR-positive tumors that, geting FR have been developed, some of these agents have in addition, brings the possibility to evaluate the treat- been tested in imaging FR via single-photon emission ment response. computed tomography (SPECT) or via positron emission tomography (PET) [49]. With regard to PET imaging, the first 18F-labeled folate Conclusion has been synthetized for application in 2006 by amide coupling of the prosthetic group 4-[18F]fluorobenzylamine FRα has been shown to be a biomarker of ovarian cancer and native folic acid [50]. Despite the good visualization of as it is overexpressed in EOC, but has limited expression FR-positive tumors, the preparation of the radiopharma- in nonmalignant human tissues. The degree of FR expres- ceutical was complicated and time consuming and pro- sions corresponds with the stage and grade of the disease. vided a low region-selective product. Therefore, another Because of this, FRα seems to be an ideal target for EOC radiofolate was developed using the copper-catalyzed treatment. Moreover, the FR expression levels correlated azide-alkyne cycloaddition (CuAAC; click reaction) result- with the response to platinum-based chemotherapy, and ing in the first 18F-click-labeled folate in high radiochemi- it seems that FR may increase the repair of DNA damage cal yields, but in vivo animal PET imaging revealed an caused by platinum. Together, it is hypothesized that the unfavorable biological distribution profile with a poor addition of anti-FR therapy to conventional chemotherapy signal-to-noise ratio caused by the polarity of the radi- may be synergistic rather than additive. ofolate [51]. Fischer et al. used the efficacy of CuAAC for Currently, two principal approaches have been 18F-radiolabeling in combination with the inevitable polar- studied to use this protein in ovarian cancer treatment. ity of [18F]FDG to enhance pharmacodynamics and signifi- The first is the development of antibodies against FR (far- cantly enhanced tumor-to-background ratio [52]. Another letuzumab) and the second is the use of FR for targeted approach is 18F-fluorination, which led to the development delivery of anticancer drugs selectively to cancer cells of 2′-[18F]fluorofolic acid with promising results in vivo, as (vintafolide/EC145, EC140, EC131, and EC72). Despite the a clear-cut visualization of FR-positive KB tumors, and fact that all these drugs demonstrated antitumor activity healthy tissues have been shown [53, 54]. Recently, several in in vitro and in in vivo models as well as a lack of severe new 18F-folate was developed, being available in very high adverse events, none of them showed so far a benefit for radiochemical yields via a fast and convenient two-step EOC patients in phase III clinical trials. Based on prom- radiosynthesis and showed good in vivo behavior waiting ising results of phase I and phase II, farletuzumab and for testing in clinical trials [49, 55]. vintafolide have been tested in phase III trials recently. Radionuclides showing promising results in preclini- However, both of these trials failed, and the questions cal in vivo SPECT are 111In-diethylenetriamine pentaacetic regarding the efficacy of this approach remain. It is not acid (DTPA)-folates, 99mTc-folates, and 67Ga-folates [56– clear how to design randomized trials to ensure a proper 58]. The first radionuclide tested in clinical trials was selection of patients that could benefit from anti-FR 111In-DTPA-folate [59]. However, 99mTc-based imaging therapy. Several approaches have been tested, includ- agents showed more rapid pharmacokinetics and lower ing immunohistochemistry, radioligand-labeled folates cost [19]. In vivo, 99mTc-etarfolatide evaluation of FRs in PET and SPECT. However, all these methods are semi- correlated to the immunohistochemistry staining results quantitative approaches and have limitations with regard in 61% of 154 patients [60]. The authors suggest that the to the specificity and sensitivity. From the phase II trial, it discrepancy may reflect a different FR expression in the seems to be possible that the subgroup(s) of patients who primary tumor and metastatic lesions. The administra- may have benefit from anti-FR therapy exists, but these tion of 99mTc-etarfolatide was safe as none of the serious subgroups still remain to be identified. Moreover, FR has adverse events were related to the administration of etar- been used for specific drug delivery of anticancer agents folatide. 99mTc-etarfolatide imaging has been tested in two as vinca-alkaloids and mitomycin C. However, in clinical clinical phase II trials with vintafolide to identify the pres- practice, the most effective drugs for EOC are still pacli- ence of FR in recurrent/refractory ovarian or endometrial taxel and platinum compounds. tumors and the correlation of FR expression with response More research is needed to answer the question to FR-targeted therapy [9, 61]. In both studies, etarfolatide regarding FR isoform selectivity and the role of FR iso- has been able to identify patients likely to benefit from forms in ovarian cancer development and progression. 10 Bartouskova et al.: Folate receptors in ovarian cancer

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