Pre-clinical studies of EC2629, a highly potent folate- receptor-targeted DNA crosslinking agent.

Folate receptor (FR)-targeted small molecule drug conjugates (SMDCs) have shown promising results in early stage clinical trials with microtubule destabilizing agents, such as vintafolide and EC1456. In our effort to develop FR-targeted SMDCs with varying mechanisms of action, we synthesized EC2629, a folate conjugate of a DNA crosslinking agent based on a novel DNA-alkylating moiety. This agent was found to be extremely potent with an in vitro IC50 ~ 100× lower than folate SMDCs constructed with various microtubule inhibitors.

Prostate-Specific Membrane Antigen-Specific Antitumor Activity of a Self-Immolative Tubulysin Conjugate.

Prostate-specific membrane antigen (PSMA) is a biomarker that is overexpressed on prostate cancer, and it is also present on the neovasculature within many non-prostate solid tumors. Herein, we report on the construction and biological testing of novel tubulysin B-containing therapeutic agents for the treatment of PSMA-expressing cancer. One of these compounds, EC1169, emerged as a lead candidate for preclinical development and phase 1 clinical testing.

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.

Pre-clinical evaluation of EC1456, a folate-tubulysin anti-cancer therapeutic.

EC1456 is a folate-tubulysin conjugate constructed with an all-D enantiomeric spacer/linker configuration. When tested against folate receptor (FR)-positive cells, EC1456 demonstrated dose-responsive activity with an approximate 1000-fold level of specificity. Treatment of nude mice bearing FR-positive human xenografts (as large as 800 mm) with non-toxic doses of EC1456 led to cures in 100% of the mice.

Latent Warheads for Targeted Cancer Therapy: Design and Synthesis of pro-Pyrrolobenzodiazepines and Conjugates.

Pyrrolobenzodiazepines (PBDs) and their dimers (bis-PBDs) have emerged as some of the most potent chemotherapeutic compounds, and are currently under development as novel payloads in antibody-drug conjugates (ADCs). However, when used as stand-alone therapeutics or as warheads for small molecule drug conjugates (SMDCs), dose-limiting toxicities are often observed. As an elegant solution to this inherent problem, we designed diazepine-ring-opened conjugated prodrugs lacking the imine moiety.

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.

High Levels of Expression of P-glycoprotein/Multidrug Resistance Protein Result in Resistance to Vintafolide.

Targeting surface receptors overexpressed on cancer cells is one way to specifically treat cancer versus normal cells. Vintafolide (EC145), which consists of folate linked to a cytotoxic small molecule, desacetylvinblastine hydrazide (DAVLBH), takes advantage of the overexpression of folate receptor (FR) on cancer cells. Once bound to FR, vintafolide enters the cell by endocytosis, and the reducing environment of the endosome cleaves the linker, releasing DAVLBH to destabilize microtubules.

Folate-vinca alkaloid conjugates for cancer therapy: a structure-activity relationship.

Vintafolide is a potent folate-targeted vinca alkaloid small molecule drug conjugate (SMDC) that has shown promising results in multiple clinical oncology studies. Structurally, vintafolide consists of 4 essential modules: (1) folic acid, (2) a hydrophilic peptide spacer, (3) a disulfide-containing, self-immolative linker, and (4) the cytotoxic drug, desacetylvinblastine hydrazide (DAVLBH). Here, we report a structure-activity study evaluating the biological impact of (i) substituting DAVLBH within the vintafolide molecule with other vinca alkaloid analogues such as vincristine, vindesine, vinflunine, or vinorelbine; (ii) substituting the naturally (S)-configured Asp-Arg-Asp-Asp-Cys peptide with alternative hydrophilic spacers of varied composition; and (iii) varying the composition of the linker module.

Rational combination therapy of vintafolide (EC145) with commonly used chemotherapeutic drugs.

Purpose: When evaluated in patients with ovarian and other cancer, vintafolide (EC145), a potent folate-targeted vinca alkaloid conjugate, displayed a toxicity profile that seemed to be nonoverlapping with many standard-of-care cancer therapeutics. It was, therefore, hypothesized that combining vintafolide with certain approved anticancer drugs may afford greater therapeutic efficacy compared with single-agent therapy. To explore this concept, vintafolide was evaluated in combination with pegylated liposomal doxorubicin (PLD; DOXIL), cisplatin, carboplatin, paclitaxel, docetaxel, topotecan, and irinotecan against folate receptor (FR)-positive models.

Acid mediated formation of an N-acyliminium ion from tubulysins: a new methodology for the synthesis of natural tubulysins and their analogs.

Tubuylsins are extremely potent cytotoxic agents which inhibit tubulin polymerization and lead to cell cycle arrest and apoptosis. Tubulysins have been isolated from fermentation mixtures and have been chemically synthesized; however, these efforts have been hampered by poor yields and arduous purifications. In contrast, treatment of a mixture of natural tubulysins A, B, C, G, and I, obtained from a fermentation batch with trifluoroacetic acid results in the formation of a single N-acyliminium ion.

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.

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).

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.

Preclinical antitumor activity of a novel folate-targeted dual drug conjugate.

We have designed a new type of tumor-targeted agent by tethering two different drug molecules, with distinct biological mechanisms of action, to the same ligand. This compound, named EC0225, represents the "first in class" multidrug, folate receptor (FR)-targeted agent to be disclosed. It was constructed with a single folate molecule, extended by a hydrophilic peptide-based spacer, which was in turn attached to mitomycin and Vinca alkaloid units via two separate disulfide-containing linkers.

Folate receptor-specific antitumor activity of EC131, a folate-maytansinoid conjugate.

EC131, a new folate receptor (FR)-targeted drug conjugate, was prepared by covalently attaching the vitamin folic acid (FA) to a potent microtubule-inhibiting agent, maytansinoid DM1, via an intramolecular disulfide bond. When tested on cells in culture, EC131 was found to retain high affinity for FR-positive cells and to provide FR-specific cytotoxicity with an IC(50) in the low nanomolar range. The activity of EC131 was completely blocked in the presence of an excess of free FA, and no activity was detected against FR-negative cells.

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.

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.

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.

Synthesis and biological evaluation of EC140: a novel folate-targeted vinca alkaloid conjugate.

A novel folate conjugate of desacetylvinblastine monohydrazide (DAVLBH), herein referred to as EC140, was designed and evaluated for biological activity against folate receptor (FR)-positive cells and tumors. EC140 was produced by coupling a peptidic analogue of the vitamin folic acid to DAVLBH via an acylhydrazone bond. This water-soluble conjugate was found to retain high affinity for FR-positive cells, and it produced specific, dose-responsive activity in vitro.

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.

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.

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.