Phase I/II Clinical Trial of the Targeted Chemotherapeutic Drug, Folate-Tubulysin, in Dogs with Naturally-Occurring Invasive Urothelial Carcinoma

www.oncotarget.com Oncotarget, 2018, Vol. 9, (No. 97), pp: 37042-37053 Research Paper Phase I/II clinical trial of the targeted chemotherapeutic drug, folate-tubulysin, in dogs with naturally-occurring invasive urothelial carcinoma Nicholas M. Szigetvari1, Deepika Dhawan1, José A. Ramos-Vara2, Christopher P. Leamon3, Patrick J. Klein3, A. Audrey Ruple2, Hock Gan Heng1, Michael R. Pugh3, Satish Rao3, Iontcho R. Vlahov3, Pierre L. Deshuillers2, Philip S. Low4,5, Lindsey M. Fourez1, Ashleigh M. Cournoyer1 and Deborah W. Knapp1,5 1Department of Veterinary Clinical Sciences, Purdue University, West Lafayette, IN, USA 2Department of Comparative Pathobiology, Purdue University, West Lafayette, IN, USA 3Endocyte Inc., West Lafayette, IN, USA 4Department of Chemistry, Purdue University, West Lafayette, IN, USA 5Purdue University Center for Cancer Research, West Lafayette, IN, USA Correspondence to: Deborah W. Knapp, email: [email protected] Keywords: animal model; bladder cancer; canine, clinical trial; targeted chemotherapy Received: August 02, 2018 Accepted: November 26, 2018 Published: December 11, 2018 Copyright: Szigetvari et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Purpose: The purpose was to determine the safety and antitumor activity of a folate-tubulysin conjugate (EC0531) in a relevant preclinical animal model, dogs with naturally-occurring invasive urothelial carcinoma (iUC). Canine iUC is an aggressive cancer with high folate receptor (FR) expression similar to that in certain forms of human cancer. Experimental Design: A 3+3 dose escalation study of EC0531 (starting dose 0.2 mg/kg given intravenously at two-week intervals) was performed in dogs with iUC expressing high levels of FRs (>50% positive tumor cells). Pharmacokinetic (PK) analysis was performed, and the maximum tolerated dose (MTD) was determined. The dose cohort at the MTD was expanded to determine antitumor activity. Results: The MTD of EC0531 was 0.26 mg/kg every two weeks, with grade 3-4 neutropenia and gastrointestinal toxicity observed at higher doses. Treatment at the MTD was well tolerated. Clinical benefit was found in 20 of 28 dogs (71%), including three dogs with partial remission and 17 dogs with stable disease. Plasma EC0531 concentrations in the dogs far exceeded those required to inhibit proliferation of FR- expressing cell in vitro. Unlike human neutrophils, canine neutrophils were found to express FRs, which contributes to the neutropenia at higher doses of EC0531 in dogs. Conclusion: EC0531 was well tolerated and had good antitumor activity in dogs with iUC. It is likely that humans will tolerate higher, potentially more effective doses of folate-tubulysin without myelotoxicity because of the absence of FRs on human neutrophils. The results clearly justify the evaluation of folate-tubulysin in human clinical trials. INTRODUCTION exposure and toxicity to normal tissues. The resulting preferential accumulation of drug in the tumor can Targeted chemotherapy allows the selective delivery increase drug activity with lower systemic exposure, i.e. of a cytotoxic payload to tumor cells while limiting the increasing the therapeutic index. In this way, targeted www.oncotarget.com 37042 Oncotarget delivery of cytotoxic drugs allows reconsideration of tubulysin A had superior potency against colon cancer compounds previously deemed clinically irrelevant due to cells with high P-glycoprotein expression (HCT-15 cells), very low therapeutic index. compared to drugs that are P-glycoprotein substrates One promising, emerging, druggable target for [11]. The concentration of tubulysin A needed to inhibit cancer therapy is folate receptor alpha (FRα), which is cell proliferation by 50% (IC50) of HCT-15 cells was a high-affinity receptor for folate (vitamin B9) [1, 2]. 200 and 2,000 times less than the IC50 of vinblastine Folate is needed by all cells for DNA synthesis [1, 2]. and paclitaxel, respectively [11]. Furthermore, growth Most mammalian cells utilize reduced folate (5-methyl- inhibition was observed in 33% of cell lines tested in tetrahydrofolate), which is transported intracellularly via a standardized cancer cell line panel, the U.S. National the reduced folate carrier [1–3]. In contrast, non-reduced Cancer Institute 60 tumor cell lines for anticancer drug folate is utilized by tumor cells, and typically transported screen. These results are better than those for 95% of via high-affinity folate receptors (FRs), mainly FRα in the other compounds tested in the same manner by the solid tumors [4]. Several human cancer types, such as National Cancer Institute [11]. Despite this compelling ovarian carcinoma, renal carcinoma, non-small cell lung information, the clinical effectiveness of tubulysins has cancer, colorectal cancer, and metastatic brain cancer, not been well established due to the very low therapeutic have 10- to 100-fold higher expression of FRs relative to index. normal tissues [2, 4]. The high expression of FRs can be Administration of free tubulysin creates exploited for the delivery of cytotoxic drugs conjugated to unacceptable toxicity in mice, even at sub-therapeutic folate [2–5]. Thus, folate-targeted drugs are expected to dosages [12]. Yet, conjugation of tubulysin, specifically largely spare normal tissues by preferentially binding to tubulysin B, to folate creates a drug tolerated at doses tumor cells of select cancer types. 20 times higher than doses of unconjugated tubulysin, Several conjugates of folate and cytotoxic drugs and a drug which has induced complete regression of or folate and immune-modulating drugs have been cancer in mouse xenograft models [12–14]. The insertion developed in an attempt to exploit this disparity in FR of a specific molecular spacer, 1-amino-1-deoxy- expression [2, 3, 6]. One of the more extensively studied glucitolyl-γ-glutamate, in folate-tubulysin conjugates was is a desacetylvinblastine monohydrazide (DAVLBH) especially effective in increasing the therapeutic range folate conjugate (vintafolide, EC145) [2, 3, 5]. In a tumor of the resulting compound (EC0531, Endocyte, Inc., xenograft mouse model, administration of free DAVLBH West Lafayette, IN) [14]. There is considerable interest created unacceptable toxicity before effective anti-tumor in further preclinical (pre-human) evaluation of folate- activity could be observed [7]. Yet, conjugation of the tubulysin conjugates, especially in animal cancer models drug to folate was well tolerated, and led to complete that are relevant to human cancer in regards to tumor remission of multi-drug resistant tumors in mice [2, 3, 7]. heterogeneity, metastatic potential, tumor-host immune Subsequently, a phase I trial demonstrated an acceptable interactions, and inherent or acquired drug resistance. safety profile of vintafolide in people receiving this drug Dogs with specific forms of naturally-occurring for an assortment of solid tumors [8]. Varying degrees cancer, can serve as these highly relevant, in vivo models of clinical effectiveness were observed in phase II and of human cancer in which the outcome of new compounds III trials, with the greatest benefit noted in patients with in dogs is expected to mimic that in humans [15–21]. tumors with the highest FR expression and tumors lacking There are multiple advantages of studying pet dogs with P-glycoprotein expression [2, 3, 5, 9]. These findings specific forms of naturally-occurring cancer as models were encouraging for future targeting of FRs, but also for those same cancers in humans. These similarities indicated the need for additional, potentially more active include tumor histopathology, heterogeneity, genetic and drug conjugates. epigenetic alterations, biological behavior, frequent distant Tubulysins A, B, C, and D are potent anti-mitotic metastases, and response to therapy [17–24]. Likewise, compounds isolated from a mycobacterium, Archangium similarities in FR expression have been reported between gephyra, found in a German soil sample over 40 years dogs and humans [16, 24]. Normal canine tissues have ago [10]. The mechanism of action is similar to that of relatively low FR expression compared to certain canine the vinca alkaloids and dolastatins, causing instability tumor types, just as normal human tissues have relatively of microtubule formation and subsequent cell cycle low FR expression compared to tumor tissues [24]. In one arrest in late G2/M phase [10]. However, tubulysins of the more extensively studied forms of canine cancer, have several potential advantages over other anti-mitotic invasive urothelial carcinoma (iUC), FR over-expression compounds. Unlike vinca alkaloids, tubulysins do not was noted in 76% of iUC cases, and folate uptake in the appear to be a substrate for CYP metabolism, thus cancer was confirmed by scintigraphy [24]. In this same there is less concern for drug-to-drug interactions [11]. study, 78% of human iUC cases had increased expression Tubulysins are also poor substrates for the P-glycoprotein of FRs when detected using the same antibody as in the drug efflux pump, thus retaining efficacy against multi- canine tissues, although the expression level was lower drug resistant (MDR) tumor phenotypes [11]. In vitro, when detected by other antibodies [24]. Canine FRα www.oncotarget.com 37043 Oncotarget protein has >80% predicted homology with the

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