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

Abstract

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 folatelinked 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 (IC 50 range, 1-10 nmol/L) in a dosedependent 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. [Cancer Res 2008;68(23):9839-44]

Development of Novel Quaternary Ammonium Linkers for Antibody–Drug Conjugates

“…Conversion of the C-terminal carboxylate to include a circulation-stable conjugatable group appears more promising. For example, a modest 3-fold reduction in free drug potency on KB cells was observed upon conversion of the carboxylate in tubulysin B to the corresponding hydrazide as part of a folate-targeted conjugate (16). However, with tubulysin 3, conversion to the hydrazide (5) resulted in around a 5-to 20-fold loss of potency (Table 1).…”

“…Derivatization of the C-terminus through conversion of the carboxylic acid to a hydrazide (16) or modification of the tubuphenylalanine ring (17) appears more promising. However, these changes also attenuate potency relative to the unmodified parent tubulysin.…”

Reducing Undesirable Hepatic Clearance of a Tumor-Targeted Vinca Alkaloid via Novel Saccharopeptidic Modifications

“…Cells were seeded in individual 12-well Falcon plates and allowed to form nearly confluent monolayers overnight in folate-deficient RPMI 1640 medium supplemented with 10% fetal bovine serum. Following a detailed published procedure (Leamon et al, 2008b), a 2-h pulse, 70-h chase assay format was used to evaluate the cytotoxic effects of increasing concentrations of EC0489. Viability was assessed by measuring […”

Rational Combination Therapy of Vintafolide (EC145) with Commonly Used Chemotherapeutic Drugs

“…As a result, "folate targeting" has been successfully applied toward the delivery of a wide variety of therapeutic-and imaging-based agents to tumors that express the FR protein (9)(10)(11)(12). Compared with their nontargeted counterparts, folic acid-bearing drugs and delivery systems have repeatedly shown greater cancer cell specificity and selectivity in numerous preclinical studies (13)(14)(15)(16)(17)(18)(19)(20). Hence, this targeting strategy leads to improvements in the safety and efficacy of anticancer agents, resulting in an increased therapeutic advantage.…”