Melissa Nelson

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.

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. A series of vinca alkaloid-containing SMDCs were synthesized and purified by HPLC and LCMS. The SMDCs were screened in vitro against folate receptor (FR)-positive cells, and anti-tumor activity was tested against well-established subcutaneous FR-positive tumor xenografts. The cytotoxic and anti-tumor activity was directly compared to that produced by vintafolide. Among all the folate vinca alkaloid SMDCs tested, DAVLBH-containing SMDCs were active, while those constructed with vincristine, vindesine, or vinorelbine analogues failed to produce meaningful biological activity. Within the DAVLBH series, having a bioreleasable, self-immolative linker system was found to be critical for activity since multiple analogues constructed with thioether-based linkers all failed to produce meaningful activity both in vitro and in vivo. Substitutions of some or all of the natural amino acids within vintafolides hydrophilic spacer module did not significantly change the in vitro or in vivo potency of the SMDCs. Vintafolide remains one of the most potent folate-vinca alkaloid SMDCs produced to date, and continued clinical development is warranted.

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. Experimental Design: FR-expressing KB, M109, IGROV, and L1210 cells were first exposed to graded concentrations of vintafolide, either alone or in combination with doxorubicin (active ingredient in PLD), and isobologram plots and combination index values generated. The vintafolide combinations were also studied in mice bearing various FR-expressing tumors. Results: Vintafolide displayed strong synergistic activity against KB cells when combined with doxorubicin, and no less-than-additive effects resulted when tested against M109, IGROV, and L1210 cells. In contrast, when either desacetylvinblastine hydrazide (DAVLBH; the vinca alkaloid moiety in vintafolide) or vindesine (the vinca alkaloid most structurally similar to DAVLBH) were tested in combination with doxorubicin, less-than-additive antitumor effects were observed. In vivo, all vintafolide drug combinations produced far greater antitumor effect (complete responses and cures) compared with the single agents alone, without significant increase in overall toxicity. Importantly, these benefits were not observed with combinations of PLD and DAVLBH or vindesine. Conclusions: On the basis of these encouraging preclinical results, clinical studies to evaluate vintafolide drug combination therapies are now under way. Clin Cancer Res; 20(8); 2104–14. ©2014 AACR.

Abstract 832: Pre-clinical development of EC1456: A potent Folate targeted Tubulysin SMDC

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Folate targeted small molecule drug conjugates (SMDC) have shown promising results in early stage clinical trials with Vintafolide now being evaluated in a phase 3 trial in FR-positive ovarian cancer patients. We have now developed a new group of folate conjugated Tubulysins, of which EC1456 has emerged as a lead candidate for clinical development. Treatment of nude mice bearing folate receptor (FR) positive human xenografts with EC1456 led to complete remissions (CRs) or cures in 100% of the mice at various doses and schedules. The observed activity was not accompanied by any noticeable weight loss or major organ tissue degeneration. In contrast, no significant anti-tumor activity (0 % CRs) was observed in EC1456-treated animals that were co-dosed with an excess of a benign folate ligand, thus demonstrating EC1456s target-specific activity. The enhanced therapeutic index due to folate conjugation was also evidenced by the fact that the un-targeted free drug (tubulysin B or its hydrazide form) was found to be completely inactive even when administered at highly toxic dose levels. Furthermore, when challenged with larger tumors, EC1456 again displayed remarkable anti-tumor activity with 100% cures in tumors up to 750 mm3. Complete cures were also observed in other FR positive models such as MDA-MB-231 TNBC and M109 lung tumor models. In support of our clinical strategy to treat drug resistant FR-positive cancer patients we have also shown that EC1456 was highly active against FR expressing paclitaxel and cisplatin-resistant cell lines. Taken together, these studies demonstrated that EC1456 has significant anti-tumor growth activity and tolerability, thus lending support to the ongoing Phase 1 clinical evaluation of EC1456 for advanced malignancies. Citation Format: Joseph A. Reddy, Alicia Bloomfield, Melissa Nelson, Ryan Dorton, Marilynn Vetzel, Christopher P. Leamon. Pre-clinical development of EC1456: A potent Folate targeted Tubulysin SMDC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 832. doi:10.1158/1538-7445.AM2014-832

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. Vintafolide has shown efficacy and improved tolerability compared with DAVLBH in FR-positive preclinical models. As the first FR-targeting drug to reach the clinic, vintafolide has achieved favorable responses in phase II clinical trials in FR-positive ovarian and lung cancer. However, some FR-positive patients in these clinical trials do not respond to vintafolide. We sought to identify potential biomarkers of resistance to aid in the future development of this and other FR-targeting drugs. Here, we confirm that high P-glycoprotein (P-gp) expression was the strongest predictor of resistance to DAVLBH in a panel of 359 cancer cell lines. Furthermore, targeted delivery of DAVLBH via the FR, as in vintafolide, fails to overcome P-gp–mediated efflux of DAVLBH in both in vitro and in vivo preclinical models. Therefore, we suggest that patients whose tumors express high levels of P-gp be excluded from future clinical trials for vintafolide as well as other FR-targeted therapeutics bearing a P-gp substrate. Mol Cancer Ther; 15(8); 1998–2008. ©2016 AACR.

Abstract 2145: PSMA-specific anti-tumor activity of the targeted-tubulysin conjugate, EC1169.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Prostate-specific membrane antigen (PSMA) is a biomarker that is overexpressed on prostate cancer, and its also present on the neovasculature within many non-prostate solid tumors. We previously reported on EC0652, a 99mTc-labeled small molecule that specifically binds to PSMA-positive tumors and is currently being clinically evaluated for diagnostic imaging applications. Herein, we report on the construction and biological testing of novel tubulysin B-containing, companion therapeutic agents for the treatment of PSMA-expressing cancer. One of these compounds, EC1169, recently emerged as a lead candidate for preclinical development. This water-soluble conjugate was shown to have high affinity for PSMA-positive cells. When tested in vitro, EC1169 was found to inhibit the growth of PSMA-positive cells, but it displayed no activity against PSMA-negative cells. Brief treatment of nude mice bearing PSMA-positive LNCaP human xenografts with EC1169 led to complete remissions in 5/7 mice, and cures (i.e. tumor free >90 days post implantation) in 2/7 mice. Furthermore, this activity occurred in the absence of weight loss or major organ tissue degeneration. In contrast, the non-targeted tubulysin B drug proved to be inactive against the LNCaP tumor model when administered at doses near to or greater than the maximum tolerated level. PSMA-negative KB tumors did not appreciably respond to EC1169 therapy, thereby confirming this compounds targeted specificity for PSMA-expressing cells. Finally, treatment of LNCaP-bearing mice with docetaxel (the most active chemotherapeutic agent approved for prostate cancer), was found to produce only modest anti-tumor activity (2/4 partial responses, 1/4 cures), and this outcome was also associated with severe weight loss (18%). Taken together, these results strongly indicate that PSMA-positive tumors may be more effectively treated using highly potent, PSMA-targeted small-molecule drug conjugates using regimens that do not cause unwanted toxicity. Citation Format: Joseph A. Reddy, Alicia Bloomfield, Ryan Dorton, Melissa Nelson, Marilynn Vetzel, Spencer Hahn, Iontcho Vlahov, Christopher Leamon. PSMA-specific anti-tumor activity of the targeted-tubulysin conjugate, EC1169. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2145. doi:10.1158/1538-7445.AM2013-2145

Abstract 3622: Predicting response to EC145 therapy using the folate receptor-specific radiodiagnostic imaging agent, 99mTc-EC20

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL EC145 is a potent folate-targeted Vinca alkaloid conjugate that is currently being evaluated in a global phase 3 trial for the treatment of platinum-resistant ovarian cancer. By design, response to EC145 should be dependent on folate receptor (FR) expression. Therefore, characterizing the presence and functionality of the FR in real time is critical for the selection of patients for whom FR-targeted therapy may be beneficial. A promising method thats currently being co-developed with EC145 is to image patients prior to therapy using 99mTc-EC20, which is a small molecular weight folate-targeted companion imaging agent. Similar to EC145, 99mTc-EC20 binds to the FR with high affinity (Kd ∼ 3 nM) in a manner that is both saturable and independent of the cell type. 99mTc-EC20s binding specificity has been confirmed using validated preclinical models where it was found that i) binding of 99mTc-EC20 to cells or accumulation within solid tumors is dependent on FR expression (but independent of isoform), and ii) excess folic acid can competitively block 99mTc-EC20s cell/tissue binding. Furthermore, EC145 was also found to displace pre-bound 99mTc-EC20 from FR-positive tumors and kidneys in mice, strongly suggesting that EC145 and 99mTc-EC20 distribute similarly in vivo. Taken together, these findings indicate that the FR mediates tissue uptake of 99mTc-EC20. Since 99mTc-EC20 provides a non-invasive, real-time assessment of functionally active as well as anatomically-accessible FRs, this radiodiagnostic agent may predict what tissues will accumulate and potentially respond to folate-targeted drugs, such as EC145. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3622. doi:1538-7445.AM2012-3622

Abstract 5359: Antitumor efficacy of EC1456 in patient derived xenograft models of ovarian, endometrial, NSCLC and TNBC

EC1456, a potent folate-targeted tubulysin conjugate, is currently being evaluated in a phase 1 dose escalation study in patients with advanced solid tumors. To assist in upcoming cancer indication selection decisions for advanced trials, we tested EC1456 against eight patient-derived xenograft (PDX) cancer models. Thus, EC1456 was evaluated as a single agent or in combination with standard of care (SOC) treatments against two folate receptor (FR)-positive models each of ovarian, endometrial, non-small cell lung (NSCLC) and triple negative breast (TNBC) tumor models. Nude mice engrafted subcutaneously with PDX tumors received 4 μmol/kg/week EC1456 for two weeks, following a once a week (SIW) or two times a week (BIW) schedules, either alone or in combination with 15 mg/kg, SIW x 2 Paclitaxel (ovarian and endometrial), 1 mg/kg SIW x 2 eribulin mesylate (TNBC) or 15 mg/kg, SIW docetaxel (NSCLC). EC1456 alone demonstrated tumor growth suppression or stasis in most of the tumor models and significantly greater anti-tumor activity (including cures) with no detectable increase in toxicity when tested in combination with the SOC agents. Overall, EC1456 significantly augmented the growth inhibitory effects of SOC treatments against all four PDX cancer indications examined, thus lending support to future clinical development of this novel FR-targeted agent. Citation Format: Joseph A. Reddy, Alicia Bloomfield, Christina Taylor, Katherine Hargett, Melissa Nelson, Christopher Leamon. Antitumor efficacy of EC1456 in patient derived xenograft models of ovarian, endometrial, NSCLC and TNBC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5359. doi:10.1158/1538-7445.AM2015-5359

Abstract 2570: Combination studies of EC145 with approved anti-cancer drugs for ovarian cancer

EC145, a potent folate-targeted Vinca alkaloid conjugate, is currently being evaluated in a phase 2 study of EC145 + DOXIL® compared with DOXIL® alone, in women with platinum-resistant ovarian cancer. Since EC1459s toxicity profile is appearing to be non-overlapping with many of the existing approved drugs for ovarian cancer, we tested the effects of combining EC145 with some of these drugs. Thus, EC145 was evaluated in combination with cisplatin, carboplatin, docetaxel, topotecan or DOXIL® against folate receptor (FR)-positive human tumor xenograft models in vivo. In these studies, EC145 was purposely administered at a non-curative dose level while the non-targeted combination drugs were given at ∼50-75% of their MTD values. When tested as single agents against the FR-positive KB tumor model, partial anti-tumor responses were typically observed. In contrast, significantly greater anti-tumor activity with no detectable increase in toxicity was observed when EC145 was tested in combination. For example, 100% cures resulted when EC145 was combined with cisplatin or docetaxel, 80% cures with carboplatin or DOXIL®, and 40% cures with topotecan. Overall, these data provide support for the evaluation of EC145 in combination with numerous agents with different mechanisms of action for the treatment of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2570. doi:10.1158/1538-7445.AM2011-2570

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 mm3) with non-toxic doses of EC1456 led to cures in 100% of the mice. Combinations of low dose EC1456 with standard of care agents such as platins, taxanes, topotecan and bevacizumab, safely and significantly augmented the growth inhibitory effects of these commonly used agents. When tested against FR-positive human tumor xenograft models having confirmed resistance to a folate-vinca alkaloid (vintafolide), cisplatin or paclitaxel, EC1456 was found to generate partial to curative responses. Taken together, these studies demonstrate that EC1456 has significant anti-proliferative activity against FR-positive tumors, including models which were anticancer drug resistant, thereby justifying a Phase 1 trial of this agent for the treatment of advanced human cancers.