Comparative preclinical activity of the folate-targeted Vinca alkaloid conjugates EC140 and EC145 Christopher P. Leamon * , Joseph A. Reddy, Iontcho R. Vlahov, Elaine Westrick, Nikki Parker, Jeffrey S. Nicoson and Marilynn Vetzel Endocyte, Inc., 3000 Kent Ave., West Lafayette, IN EC140 is a water soluble folate conjugate of desacetylvinblastine monohydrazide (DAVLBH), which is constructed with an endo- some-cleavable acyl hydrazone bond. This agent has proven to be active and specific against well established, subcutaneous folate re- ceptor (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 xeno- graft 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. ' 2007 Wiley-Liss, Inc. Key words: folate receptor; targeting; endocytosis; chemotherapy; Vinca alkaloid The toxicity of an oncolytic drug should theoretically be reduced if it were specifically targeted to the cancer cells or to some related, but specific cellular defect. This alternative chemo- therapeutic approach has successfully been exemplified in recent years by the clinical successes of tumor-targeted/tumor-specific agents, such as Herceptin and Gleevec. 1 But, other drug targeting techniques are also being explored, such as that which exploits the receptor-mediated uptake pathway for folic acid. Vitamin B9, or folic acid, is a ligand capable of targeting cova- lently attached bioactive agents quite specifically, and with very high affinity, to folate receptor (FR)-positive cells. 2 The FR, which is a well known tumor associated protein, can actively internalize bound folates and folate-drug conjugates via the natu- ral process of endocytosis. 2,3 This receptor is present at very high levels in most ovarian and other gynecological cancers, as well as at high to moderate levels in brain, lung, kidney and breast carci- nomas. 4–12 But, its presence on normal tissues appears to either be (i) limited in quantity, (ii) inconsequential for targeted drug appli- cations   or (iii) simply inaccessible to blood-borne folates. 6,11–17 Because of this unique expression/accessibility pattern, the con- cept of ‘folate-drug targeting’ has been effectively pursued as an alternative method for treating FR-positive cancer. Indeed, folate- targeted delivery of drug payloads as diverse as small radiophar- maceutical agents to large DNA-containing formulations have successfully been exemplified both at the preclinical and clinical levels (see Low and Antony 18 for a comprehensive overview of this technology). There are only a few examples of folate-targeted chemotherapy applications, and these investigations were either limited to in vitro evaluations, or were shown to yield low to moderate levels of antitumor activity in animal models. 15,17,19 Our laboratory recently published on the synthesis and in vitro/in vivo activity of a novel folate-desacetylvinblastine monohydrazide (DAVLBH) conjugate, called EC140. 14 In contrast to other folate-drug conju- gates, this microtubule depolymerizing agent was shown to pro- duce marked activity against well established subcutaneous (s.c.) FR-positive tumors. In our latest efforts, linker optimization stud- ies have yielded a disulfide bond-containing counterpart to EC140, herein referred to as EC145. As shown later, comparative in vivo efficacy tests confirmed that EC145 is more active and bet- ter tolerated than EC140, which are a couple of early reasons why EC145 was ultimately chosen for clinical development. Material and methods Materials Pteroic acid (Pte) and N 10 -trifluoroacetylpteroic acid were pre- pared according to Xu et al. 20 Peptide synthesis reagents were pur- chased from NovaBiochem (La Jolla, CA) and Bachem (San Car- los, CA). Folate-free RPMI media (FFRPMI) and PBS were obtained from Gibco, Grand Island, NY. 3 H-thymidine was pur- chased from Moravek Biochemicals, Brea, CA. Vinblastine sulfate and all other common reagents were purchased from Sigma (St. Louis, MO) or other major suppliers. Test articles DAVLBH, EC119 à , EC140 and EC145 were produced by Endocyte (West Lafayette, IN). Their syntheses, purifications and analytical characterizations have been described in detail else- where. 14,21 Cell culture Three types of cells were used for the presented studies. KB cells are FR-positive human, nasopharyngeal cells (originally obtained from ATCC). Madison 109 (M109) are FR-positive lung adenocarcinoma cells that are syngeneic to Balb/c mice (gift from Alberto Gabizon). M109’s are typically passed in vivo and only kept in vitro for no more than 2 passages. 4T1 is a FR-negative breast carcinoma cell line that is also syngeneic to the Balb/c mouse (gift from former Rhone Poulenc Rorer). When in vitro, cells were maintained in FFRPMI containing 10% heat-inactivated fetal calf serum (HIFCS) at 37°C in a 5% CO 2 /95% air-humidified Grant sponsor: Indiana 21st Century Fund; Grant number: 511040805. *Correspondence to: Endocyte, Inc., 3000 Kent Ave., West Lafayette, IN 47906, USA Fax: 765-463-9271. E-mail: chrisleamon@endocyte.com Received 18 January 2007; Accepted after revision 23 March 2007 DOI 10.1002/ijc.22853 Published online 5 June 2007 in Wiley InterScience (www.interscience. wiley.com).   For example, kidney proximal tubules are normal, FR-positive cells, which transfer folate-drug conjugates back into the bloodstream through a mechanism that does not produce renal toxicity (see Refs. 13–15). Abbreviations: CR, complete response; DAVLBH, desacetylvinblastine monohydrazide; DPM, disintegrations per minute; FFRPMI, folate-free RPMI media; FR, folate receptor; HIFCS, heat-inactivated fetal calf serum; LCK, log cell kill; MTD, maximum tolerated dose; PR, partial response; Pte, pteroic acid; s.c., subcutaneous; T/C, treated over control. à EC119 consists of Pteroic acid-gGlu-Asp-Arg-Asp-Asp-Cys, which is the folate–peptide moiety found within both the EC140 and EC145 struc- tures. Int. J. Cancer: 121, 1585–1592 (2007) ' 2007 Wiley-Liss, Inc. Publication of the International Union Against Cancer