A targeted tubulysin B hydrazide conjugate for the treatment of LHRH

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A targeted tubulysin B hydrazide conjugate for the treatment of LHRH receptor-positive cancers. jyoti roy, Miranda Kaake, Madduri Srinivasarao, and Philip S. Low Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00164 • Publication Date (Web): 31 May 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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Bioconjugate Chemistry

A targeted tubulysin B hydrazide conjugate for the treatment of LHRH receptor-positive cancers.

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Jyoti Roy1,2, Miranda Kaake2, Madduri Srinivasarao1,2, and Philip S Low1,2,*

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1

Purdue Institute for Drug Discovery, Purdue University, West Lafayette IN 47907 USA

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2

Department of Chemistry, Purdue University, West Lafayette IN 47907 USA

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*Author to whom all correspondence should be addressed:

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Philip S Low

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Email: [email protected]

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Phone: (765)494-5283

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Fax: (765)496-2677

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Department of Chemistry

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Purdue University

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720 Clinic Dr.

14

West Lafayette IN 47907

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The authors declare no potential conflicts of interest.

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract:

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The targeted delivery of chemotherapeutic agents to receptors that are over-expressed on cancer

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cells has become an attractive strategy to concentrate drugs in cancer cells while avoiding uptake

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by healthy cells. Luteinizing hormone-releasing hormone receptor (LHRH-R) has attracted

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considerable interest for this application, since LHRH-R is upregulated in ~86% of prostate

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cancers, ~80% of endometrial cancers, ~80% of ovarian cancers, and ~50% of breast cancers, but

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virtually absent from normal tissues. Although LHRH and related peptides have been used to

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deliver cytotoxic drugs to LHRH-R over-expressing cancer cells, they have suffered from off-

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target delivery of the therapeutic agents to the liver and kidneys. To reduce such unwanted

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uptake by peptide scavenger receptors in the liver and kidneys, we have explored the use of a

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nonpeptidic LHRH-R antagonist (NBI42902) to construct an LHRH-R targeted tubulysin

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conjugate (BOEPL-L3-TubBH). In vitro studies with BOEPL-L3-TubBH demonstrate that the

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conjugate can kill LHRH-R expressing triple-negative breast cancer cells (MDA-MB-231 cells)

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with low nanomolar IC50. Related studies on tumor-bearing mice further reveal that the same

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conjugate can eradicate MDA-MB-231 solid tumors without any measurable side-effects,

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yielding mice that gain weight during therapy and show no evidence of tumor recurrence for at

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least 5 weeks after termination of treatment. That these complete responses are LHRH-R targeted

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was then established by showing that identical treatment of receptor-negative (SKOV3) tumors

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yields no anti-tumor response. Overall, these data provide a proof-of-concept that LHRH-R

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specific targeting of an extremely toxic drug like tubulysin B can treat LHRH-R positive tumors

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without causing significant toxicity to healthy cells.

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Introduction:

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Because nontargeted chemotherapeutic agents exhibit little cell type specificity, they distribute

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indiscriminately into most cells of the body, often causing toxicity to cancer cells and healthy

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cells alike.1,2 One strategy to limit adverse side effects of nontargeted drugs is to tether the

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cytotoxic agent to a targeting ligand that can carry the therapeutic cargo selectively to malignant

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cells, thereby avoiding most uptake by healthy cells. To achieve such tumor-specific targeting, it

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is usually necessary to identify a receptor that is over-expressed on the cancer cells, but largely

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absent from normal cells. Construction of a ligand that can deliver an attached drug directly to

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this receptor should then enable selective accumulation of the cytotoxic drug within the

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malignant tissue.

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One receptor known to be over-expressed in many cancers is the G-protein coupled receptor,

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luteinizing hormone-releasing hormone receptor (also known as gonadotropin-releasing hormone

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receptor).3,4 Luteinizing hormone-releasing hormone receptor (LHRH-R) is upregulated on many

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cancers of the breast (50%),5-8 prostate (86%),8-12 endometrium (80%),12,14-17 and ovary (80%)12-

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15,17

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contrast, the same receptor is essentially absent from healthy tissues except for the pituitary

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gland and reproductive organs, where it naturally functions to induce the synthesis and secretion

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of such gonadotropins as luteinizing hormone (LH) and follicle-stimulating hormone (FSH).

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Because these gonadotropins, in turn, promote the production of steroid sex hormones, it is not

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surprising that estrogen- and androgen-dependent cancers often depend on LHRH-R signaling

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for proliferation and survival.3,4

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LHRH peptide and its analogs have been extensively used as targeting ligands to deliver a range

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of cytotoxic drugs to tumors expressing the LHRH receptor. Several such LHRH conjugates are

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in the early stages of development, whereas others have already advanced to human clinical

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trials. AEZS-108 (AN-152), for example, is an LHRH-targeted doxorubicin conjugate that is

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currently in phase III clinical trials for the treatment of endometrial cancer and in phase II

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clinical trials for the therapy of ovarian cancer.6,14,21 EP-100, in contrast, consists of the LHRH

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hormone linked to a toxic membrane-disrupting peptide that is currently in phase II clinical trials

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for the treatment of advanced LHRH-R positive tumors.22 LHRH-targeted drugs undergoing

and also on some non-hormone dependent cancers of the pancreas,18,19 skin,20 and brain.21 In



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preclinical development include the anti-androgen, CBDIV17, encapsulated in LHRH-

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derivatized micelles,23 and multiple LHRH peptides/analogs linked to the cytotoxic drugs

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cisplatin,24 disorazol,25 ERK inhibitors, PI3K/ERK inhibitors5, or gemcitabine26. Finally,

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researchers have also developed and tested LHRH-targeted paclitaxel-loaded microbubbles for

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ultrasound-stimulated drug release within a tumor mass.27

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Although the above LHRH-targeted therapeutic conjugates have shown promise in treating

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LHRH-R expressing tumors, such peptide-derived drugs often display limitations that can

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adversely impact their tumor specificities. First, because the natural targeting ligand is rapidly

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metabolized in vivo, its premature degradation before arrival in the tumor mass can compromise

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tumor specificity. Second, due to elevated expression of peptide scavenger receptors in the liver,

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kidneys, and other organs, capture and retention of the peptide-targeted conjugates can often

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occur in healthy organs, causing organ damage even in the absence of a targeted receptor.28,29 In

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order to avoid these unwanted side effects, we have undertaken to design a non-peptidic,

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proteolytically stable, small molecule LHRH-R targeting ligand that can deliver an attached

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therapeutic payload selectively to LHRH-R over-expressing cancers without depositing

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measurable drug in the liver or kidneys, etc. We demonstrate below that conjugation of this

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ligand to the microtubule inhibitor, tubulysin B hydrazide, creates an LHRH-R targeted drug that

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can eradicate LHRH-R expressing cancer cells both in vitro and in vivo.

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Results:

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Design and Synthesis of LHRH-R conjugates. To develop LHRH-R targeted tubulysin conjugate

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we searched the literature for a high-affinity LHRH-R antagonist that would also inhibit LHRH

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signaling. Although the LHRH antagonist, NBI42902, met these criteria and exhibited a high

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affinity (Kd= 0.19 nM; Ki= 0.56 nM) for LHRH-R,30 the non-peptidic antagonist could not be

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used directly for drug targeting because it lacked a site for facile conjugation of a therapeutic

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cargo (Fig. 1).

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NBI42902 that could be readily modified without loss of antagonist activity.30 This search

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revealed that the ether functionality on the fluorinated aromatic ring (See Supplementary Fig. S1)

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could be substituted without significant impact on LHRH-R binding affinity,30 and for ease of

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derivatization, we elected to replace this aliphatic ether with an aliphatic carboxylic acid (see

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BOEPL, Fig. 1). The modified ligand, which we named breast, ovarian, endometrial, and

To introduce such a ligation site into NBI42902, we looked for sites on


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prostate cancer ligand (BOEPL), was used for the synthesis of all conjugates reported in this

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article. Finally, to enhance the solubility of these conjugates and to reduce their nonspecific

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uptake by receptor negative cells, the ligand was conjugated to its payload via a hydrophilic

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linker, as shown in Fig. 1.

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122 123

Figure 1. Chemical structures. Structure of LHRH-R targeting ligand NBI42902 and its

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modified version BOEPL, free tubulysin B hydrazide (TubBH), LHRH-R targeting ligand

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conjugated to peptidoglycan linker (BOEPL-L3), LHRH-R targeted tubulysing B hydrazide

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conjugate (BOEPL-L3-TubBH), LHRH-R targeting ligand conjugated to PEG linker (BOEPL-

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L2), and LHRH-R targeted rhodamine conjugate (BOEPL-L2-Rhodamine).

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For the studies below, two different conjugates had to be synthesized: i) a BOEPL-rhodamine

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conjugate for use in evaluating cell binding and internalization, and ii) a BOEPL-tubulysin B

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hydrazide conjugate for analysis of tumor-specific cytotoxicity. The rhodamine conjugate was

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prepared by attaching the dye to the BOEPL ligand via a non-cleavable hydrophilic linker to

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assure that the location of the conjugate could be accurately tracked by following the

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rhodamine’s fluorescence. In contrast, the tubulysin B hydrazide had to be synthesized by

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tethering the drug to the ligand via a self-immolative disulfide linker (Fig. 1) to allow for facile

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release of the unmodified microtubule inhibitor upon entry of the conjugate into a reducing

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endosome within the target cell. Moreover, because the tubulysin conjugate of BOEPL was 5 ACS Paragon Plus Environment


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much more hydrophobic than the rhodamine conjugate of the same ligand, the hydrophilic

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linkers tethering BOEPL to its two payloads had to be independently optimized for each payload.

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Binding and Internalization of rhodamine dye conjugate: Binding and uptake of the LHRH-R

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targeted rhodamine conjugate was evaluated by incubating MDA-MB231 or SKOV3 cancer cells

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for 1h with increasing concentrations of BOEPL-L2-rhodamine either in the presence or absence

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of excess competing for BOEPL-L2. After washing away excess conjugate, cells were

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immediately examined by confocal microscopy to determine the location of any cell-associated

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fluorescence. As shown in Fig. 2, rhodamine conjugate was internalized primarily within the

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intracellular compartments of the MDA-MB231 cells, with little fluorescence remaining on the

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cell surfaces. Total absence of intracellular and cell surface fluorescence in samples treated with

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a 100-fold excess of non-fluorescent BOEPL-L2 demonstrated that this cell-associated

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fluorescence was LHRH receptor mediated. This conclusion was further substantiated by that

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absence of any BOEPL-L2-rhodamine binding or internalization in LHRH receptor negative

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SKOV3 cells (Fig. 2). Importantly, the nearly quantitative intracellular localization of the

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targeted rhodamine conjugate following only 1 hour of incubation suggests that receptor-

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mediated endocytosis of BOEPL-L2-rhodamine is rapid in MDA-MB231 cells. Such rapid

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internalization can be beneficial to drug delivery, since it entraps the payload inside the target

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cell.

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Figure 2. Confocal Imaging. In vitro binding of BOEPL-L2-Rhodamine conjugate to MDA-MB-

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231 (LHRH-R positive) and SKVO3 (LHRH-R negative) cells either in the presence or absence

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of excess of BOEPL-L2 (n=2). 6 ACS Paragon Plus Environment

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In vitro cytotoxicity of tubulysin B hydrazide conjugate: To explore the killing efficiency of the

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BOEPL-L3-tubulysin conjugate, MDA-MB231 and SKOV3 cancer cells were incubated with

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various concentrations of BOEPL-L3-TubBH, either in the presence or absence of 100-fold

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excess BOEPL-L3 to compete for conjugate binding. As shown in Fig. 3, both MDA-MB231

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and SKOV3 cells were very sensitive to free tubulysin B hydrazide, yielding IC50 values of 67

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pM and 5 pM, respectively. In contrast, only the LHRH-R positive MDA-MB231 cells were

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killed by receptor-targeted tubulysin B hydrazide (IC50 ~ 26 nM) and this cytotoxicity could be

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blocked by addition of 100-fold excess BOEPL-L3. As anticipated, receptor negative SKOV3

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cells were largely insensitive to BOEPL-L3-TubBH, suggesting that uptake of the targeted

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cytotoxic conjugate requires binding and endocytosis by the LHRH receptor. The fact that

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SKOV3 cells were readily killed by free tubulysin hydrazide but not by its targeted conjugate

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further reveals that the hydrophilic conjugate is too polar and/or large to diffuse passively into

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receptor-negative cells. This latter property is critical to the prevention of off-target toxicity

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since the self-immolative linker is designed to release free tubulysin hydrazide immediately upon

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entry into a reducing environment such as the interior of virtually all mammalian cells.

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Figure 3. Potency of TubBH and BOEPL-L3-TubBH. In vitro cytotoxicity assay showing dose

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response activity of TubBH (black triangle) and LHRH-R targeted tubulysin B hydrazide

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conjugate (BOEPL-L3-TubBH) either in the presence (grey circle) or absence (black square) of



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excess of BOEPL-L3 in LHRH-R positive MDA-MB-231 (A) and LHRH-R negative SKOV3

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(B) cells (n=3).

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In vivo efficacy of tubulysin B hydrazide conjugate: To investigate the efficacy of BOEPL-L3-

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TubBH conjugate in nu/nu athymic nude mice bearing LHRH-R positive tumors (MDA-

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MB231), mice were treated 3x/week for 3 weeks with either saline control, targeted BOEPL-L3-

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TubBH, or targeted BOEPL-L3-TubBH in the presence of 100-fold excess of free BOEPL-L3.

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As shown in Fig. 4, MD-MB231 tumor bearing mice that received targeted tubulysin B

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hydrazide showed complete elimination of their tumors, whereas mice treated with saline

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displayed rapid tumor growth. Similarly, mice co-injected with excess competing ligand

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exhibited no suppression of tumor growth compared to the control group (Fig. 4A). These

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strongly argue that the efficacy of the LHRH receptor-targeted tubulysin B hydrazide is receptor

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mediated. However, to unequivocally establish that the anti-tumor efficacy of the targeted

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conjugate (BOEPL-L3-TubBH) is LHRH-R mediated, mice bearing LHRH-R negative tumors

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(SKOV3, ovarian cancer) were divided into analogous treatment groups and monitored similarly.

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As expected, no reduction in tumor volume was observed in either the targeted or control groups,

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despite the fact that SKOV3 tumors are very tubulysin B hydrazide sensitive (Fig. 4C); i.e.

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demonstrating that the toxicity seen in MDA-MB-231 tumors was not due to nonspecific

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systemic release of free tubulysin B hydrazide. Finally, to explore the effect of the targeted

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tubulysin B hydrazide conjugate on systemic toxicity, body weights of mice were monitored over

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the course of the study. As seen in Fig. 4B & 4D, none of the mice bearing either MDA-MB-231

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or SKVO3 tumors experienced significant weight loss, suggesting that the targeted conjugate is

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not toxic to either receptor positive or negative cancers.

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After completion of the treatment, mice in the MDA-MB231 targeted group were monitored with

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no further therapy for an additional five weeks. During this time the tumor volume and weight

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were monitored three time a week (Fig. 5). None of the mice showed any indication of tumor

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growth or weight loss. These data suggest that the LHRH-R targeted tubulysin B hydrazide

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conjugate (BOEPL-L3-TubBH) constitutes a promising candidate for treatment of cancers that

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over-express LHRH receptors.


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Figure 4. Therapeutic efficacy of BOEPL-L3-TubBH. LHRH-R positive (MDA-MB-231) and

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negative (SKOV3) cancer cells were s.c implanted in to nu/nu athymic mice and treatment was

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initiated once the tumor volume reached ~100 mm3. Mice were randomized into several groups

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with 5 mice in each group. Mice in control group received saline, whereas mice in the

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competition and treatment group received 2 µmol/kg of BOEPL-L3-TubBH either in the

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presence or absence of 100-fold excess of BOEPL-L3 respectively for 3 times per week for 3

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weeks. All the test agents were administered intravenously through tail vein. A and C represent

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tumor volume (mm3), and B & D represent animal body weight (g).

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Figure 5. Therapeutic efficacy of BOEPL-L3-TubBH against subcutaneous LHRH-R positive

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MDA-MB231 tumor growth. Mice were treated with the LHRH-R targeted tubulysin B

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hydrazide conjugate (BOEPL-L3-TubBH) for thrice a week for 5 weeks (n=5). After completion

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of the study, tumor volume (A; mm3) and animal body weight (B; g) were continuously



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monitored for 75 days post tumor implantation. The dotted vertical line specifies the last day of

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dosing.

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Discussion:

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All LHRH receptor targeted drug conjugates described to date employ an LHRH peptide or one

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of its analogs as the targeting ligand. However, because such peptides are rapidly digested or

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captured by scavenger receptors in the liver and kidneys, concerns can logically be raised that

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cytotoxic conjugates of an LHRH peptide might cause damage to one or both organs28-29. Indeed,

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when normal rats were injected intravenously with

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the injected dose was observed two hours later in the kidneys (>5% ID/g), liver (>2% ID/g),

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stomach (>6% ID/g), and intestines (>4 % ID/g), suggesting significant uptake of LHRH is

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indeed observed in healthy tissues.31, 32 Although a small fraction of this retention could be due

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to slow clearance of the decapeptide from the liver and kidneys, when linked to a cytotoxic drug

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for cancer therapy, the unwanted accumulation of drug in these normal organs could cause

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unnecessary toxicity. This concern is especially worrisome when tubulysin is used as the

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warhead, since tubulysins in their nontargeted forms have proven to be undosable in vivo due to

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severe toxicities to malignant and healthy cells alike.33 Our strategy for avoiding such off-target

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toxicities was to conjugate tubulysin to a modified version of the nonpeptidic LHRH-R

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antagonist, NBI42902 (K(i) ~0.56 nM).30 However, due to lack of an appropriate functional

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group, derivatization of NBI42902 proved to be difficult, but could be overcome by replacing the

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ether group on NBI42902 with a carboxyl group. Because the ether formed part of the molecule

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that does not interact with the receptor, the selectivity and specificity of the resulting conjugate

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for LHRH-R was not remarkably different from the parent antagonist. Moreover, our ability to

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target tubulysin B hydrazide to LHRHR positive tumors without causing weight loss argues

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strongly that the conjugate was primarily concentrated in the tumor masses. In fact, when we

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prepared and tested a conjugate of the same targeting ligand linked to a

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conjugate revealed little or no uptake in the liver or GI tract and only temporary uptake in the

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kidneys (unpublished data).

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While the absence of overt toxicity constitutes an essential property of a viable cancer drug

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candidate, lack of toxicity alone is insufficient to justify development of a therapeutic lead. In the

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paragraphs above, we have documented that the tubulysin B conjugate of the NBI42902

99m

Tc-labeled LHRH, a high percentage of


99m

Tc chelate, the

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derivative (BOEPL-L3-TubBH) can completely eradicate LHRH-R positive human tumor

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xenografts in athymic mice. Thus, whereas the other reported LHRH-R targeted therapies have

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only succeeded in slowing tumor growth,6,21-25 BOEPL-L3-Tubulysin was found to yield

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complete responses that persisted for at least 5 weeks following termination of treatment (i.e. the

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length of the monitoring period). Taken together with cytotoxicity data on i) LHRH receptor

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negative tumors (SKOV3), and ii) LRHR positive tumors that were blocked with excess ligand

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(BOEPL-L3), our results demonstrate that the therapeutic efficacy of BOEPL-L3-TubBH is

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indeed receptor-mediated. While the tumor specificity of such a targeted drug should almost

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always improve the toxicity profile of the drug, it should also be remembered that the receptor

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targeting strategy will simultaneously impose a limitation on the amount of drug that can be

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delivered, since this amount will invariably be determined by the number of receptors per cancer

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cell and the rate of receptor recycling into intracellular compartments and back to the cell

262

surface.34,35

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Because virtually all naturally occurring tumors are heterogeneous, monotherapies seldom yield

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sustained responses in humans, prompting oncologists to search for synergistic combination

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therapies to treat most cancers. Fortunately, multiple established treatment options already exist

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for cancers that overexpress LHRH-R, yielding many FDA-approved drugs that might be mixed

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with BOEPL-L3-tubulysin and examined for synergy in the treatment of prostate8-12

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endometrial,12,14-17 ovarian,2,15-17 and breast5-8 cancers. Thus, because LHRH stimulation of

269

LHRH receptor is required for sex steroids production and since these same hormones can drive

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progression of breast, ovarian, endometrial and prostate cancers, especially during early stages of

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the respective diseases, conjugation of an LHRHR antagonist (i.e. NBI42902) to a highly

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cytotoxic drug might not only inhibit production of tumor-sustaining sex steroids, but also kill

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malignant cells that over-express the LHRH receptor. It will obviously be important in future

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studies to explore the impact of BOEPL-L3-TubBH therapy on androgen and estrogen

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production and to determine whether the BOEPL-L3-TubH conjugate might suppress

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reproductive tumor growth by multiple mechanisms of action. It will also be important to

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evaluate the pharmacokinetics and pharmacodynamics of the conjugate and any metabolites to

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determine whether it might qualify for further development.

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Experimental Procedure:



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Materials: H-Cys(Trt)-2-Cl-Trt resin and protected amino acids were purchased from Chem-

281

Impex

282

hexafluorophosphate

283

dimethylformamaide (DMF), piperidine, isopropryl alcohol (IPA), dichloromethane (DCM),

284

dmithelysulfoxide (DMSO) and most other chemicals were purchased from Sigma Aldrich.

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Tubulysin B hydrazide and its activated derivative was a kind gift from Endocyte Inc. (West

286

Lafayette, IN). RPMI media and fetal bovine serum (FBS) were purchased from GIBCO (Grand

287

Island, NY). Glutamine, penicillin-streptomycin and trypsin were procured from BD Biosciences

288

(San Jose, CA).

289

Conjugate Synthesis:

290

Synthesis of BOEPL-L2: The LHRH-R antagonist (BOEPL) was synthesized per the previously

291

published procedure30 except a carboxylic acid derivative was used in the last step instead of the

292

ether shown Supplemental Fig. 1. This modification was introduced to enable its subsequent

293

coupling to a linker. The desired linker was prepared by standard solid phase peptide synthesis as

294

shown in supplementary Fig. S1, after which BOEPL was coupled via its carboxyl to the linker.

295

The product, BOEPL-L2 (Fig. 1) was then cleaved from the resin using a hydrolysis solution

296

comprised of TFA:water:triisopropylsilane:ethanedithiol (95%: 2.5%: 2.5%: 2.5%) and the crude

297

BOEPL-L2 conjugate was purified by reverse phase HPLC [A=2 Mm ammonium acetate buffer

298

(pH 5.0), B= acetonitrile, solvent gradient 0% B to 80% B in 35 min] to afford the desired

299

product at 70% yield. LRMS-LC/MS (m/z): [M+H]+ calcd for C47H61F2N5O12S, 958.08; found

300

959.

301

Synthesis of BOEPL-L2-Rhodamine: To synthesize the rhodamine conjugate of BOEPL-L2,

302

purified BOEPL-L2 and rhodamine maleimide (1 eq) were dissolved in anhydrous DMSO

303

containing DIPEA (2 eq). The reaction mixture was stirred under argon atmosphere at room

304

temperature (See supplementary Fig. S1) during which the reaction’s progress was monitored by

305

analytical LRMS-LCMS. Following completion of the reaction (~1h) crude product was purified

306

by preparative RP-HPLC [A= 2mM ammonium acetate buffer (pH 7.0), B=acetonitrile, solvent

307

gradient 0% B to 50% B in 35 min] to yield 90% of the desired product. LRMS-LC/MS (m/z):

308

[M+H]+ calcd for, C75H84F2N8O17S, 1439.59; found 1440.

Intl.

(Chicago,

IL).

2-(1H-7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyl

methanaminium

(HATU),

diisopropylethylamine


uronium

(DIPEA),

N,N’

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309

Synthesis of BOEPL-L3: BOEPL-L3 was prepared by the standard solid phase peptide synthesis

310

described in supplementary Fig. S2, after which the final product was cleaved from the resin as

311

described above. Crude BOEPL-L3 was purified by RP-HPLC [A=2 Mm ammonium acetate

312

buffer (pH 5.0), B= acetonitrile, solvent gradient 0% B to 80% B in 35 min] to yield 80% of the

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desired product. LRMS-LC/MS (m/z): [M+H]+ calcd for C67H94F2N10O23S, 1477.59; found 1478.

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Synthesis of BOEPL-L3-TubBH: Tubulysin B hydrazide was synthesized as described below.

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Briefly, BOEPL-L3 was dissolved in argon purged HPLC grade water and adjusted to pH7.0

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using a NaCO3-saturated solution of the same water. To this reaction mixture was added

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disulfide activated tubulysin B hydrazide (1 eq) in THF and the reaction mixture was allowed to

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stir at room temperature under argon atmosphere. The progress of the reaction was monitored

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using analytical LRMS-LCMS, and after its completion (~30 min.) the crude product was

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purified by preparative RP-HPLC [A=2 Mm ammonium acetate buffer (pH 7.0), B= acetonitrile,

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solvent gradient 0% B to 80% B in 35 min] to yield 90% of the desired product. LRMS-LC/MS

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(m/z): [M+H]+ calcd for C112H161F2N17O34S3, 2423.78; found 2424.

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Cell culture and confocal microscopy: LHRH-R positive breast cancer cells (MDA-MB231) and

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LHRH-R negative ovarian cancer cells (SKVO3) were cultured in RPMI 1640 medium

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supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 ˚C in a 95%

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humidified air and 5% CO2 atmosphere. MDA-MB231 and SKVO3 cells were seeded into

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chambered coverglass plates and allowed to grow to confluence over 48 h. Spent medium was

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replaced with 0.5 mL of fresh medium and incubated with 100 nM of the LHRH-R targeted

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rhodamine dye (BOEPL-L2 -rhodamine) either in the presence or absence of 100-fold excess of

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BOEPL-L2. After incubation for 1h, the cells were washed 3x in incubation solution and then

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replaced with 0.5 ml of fresh culture medium. Images were acquired using Olympus confocal

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microscopy.

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The cell culture of MDA-MB231 and SKOV3 cell lines used in this study was initiated by

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thawing frozen vials (2016) from a master stock saved from the original cell lines previously

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purchased from ATCC. All experiments were performed within two or three passages following

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thawing of the cell lines. No mycoplasma test was performed on both the cell lines.

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In vitro determination of cell viability. MDA-MB231 and SKVO3 cells were seeded at a

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concentration of 100,000 cells per well on a 24 well plate and allowed to grow in monolayers. 13 ACS Paragon Plus Environment


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Spent medium was removed and cells were incubated in various concentrations of BOEPL-L3-

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TubBH either in the presence or absence of 100-fold excess of BOEPL-L3 in fetal bovine serum

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free medium. After incubating for 2 h at 37 ˚C, cells were rinsed three times with fresh medium

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and then incubated in 0.5 ml fresh medium for an additional 66 h at 37 ˚C. Spent medium was

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then replaced with 0.5 ml of fresh medium containing 3H-thymidine and incubated for additional

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4 h. Cells were again washed 3x with fresh medium and then incubated with 0.5ml of 2.5 %

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trichloroacetic acid for 10 min at room temperature. After removing the trichoroacetic acid, cells

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were dissolved in 0.25 N NaOH. Cell viability was determined by counting the incorporation of

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3

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IC50 value of the BOEPL-L3-TubBH was derived from a plot of the percent of 3H-thymidine

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incorporation versus log concentration using Graph Pad Prism 4.

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Animal husbandry and therapy: 4-5-week-old Athymic nu/nu female mice were purchased from

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Harlan Laboratories. Mice were housed in a sterile environment on a standard 12-hour light–dark

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cycle and maintained on normal rodent chow. All animal procedures were approved by the

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Purdue Animal Care and Use Committee in accordance with National Institutes of Health

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guidelines. 5-6 weeks old female nu/nu athymic nude mice were subcutaneously injected with

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5.0 × 106 breast cancer cells (MDA-MB231) or ovarian cancer cells SKVO3 into their shoulders.

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Tumors were measured in two perpendicular directions 3 times per week with Vernier calipers,

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and their volumes were calculated as 0.5 x L x W2, where L is the longest axis (in millimeters),

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and W is the axis perpendicular to L (in millimeters). Treatment was initiated once the tumor

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volume reached ∼100 mm3. Dosing solutions were prepared in sterile saline and injected

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intravenously. Each mouse received 2µmol/kg of BOEPL-L3-TubBH either in the presence or

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absence of 100-fold excess of BOEPL-L3 or saline. Mice were administered with the test agents

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3x per week for 3 weeks and weighed as a measure of drug toxicity at each dosing.

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Supporting Information:

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Synthetic schemes and characterization of BOEPL-L2-Rhodamine, BOEPL-L3, and BOEPL-L3-

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TubBH.

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Acknowledgment: Authors would like to thank Endocyte Inc. (West Lafayette, IN) for funding

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this research work.

H-thymidine in cells using a scintillation counter (Packard, Packard Instrument Company). The


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References: 1. Azim, H. A. Jr., de Azambuja E., Colozza, M., Bines, J., Piccart, M. J. Long-term toxic effects of adjuvant chemotherapy in breast cancer. Ann Oncol. 22, 1939-1847 (2011). 2. Allen, T. M. Ligand targeted therapeutics in anticancer therapy. Nat Rev Cancer. 2, 750763 (2002). 3. Chillik, C., Acosta, A. The role of LHRH agonists and antagonists. Repro Biomed online. 2, 120-128 (2001). 4. Tolkach, Y., Joniau, S., Van Poppel, H. Luteinizing hormone-releasing hormone (LHRH) receptor agonists vs antagonists: a matter of the receptors? BJU Int. 111, 1021-1030 (2013). 5. Kwok, C. K., Treeck, O., Buchholz, S., Ortmann, O., Engel, J. B. Receptors for luteinizing hormone-releasing hormone (GnRH) as therapeutic targets in triple negative breast cancers (TNBC). Targ Oncol. 10, 365-373 (2015). 6. Seitz, S. et al. Triple Negative breast cancers express receptors for LHRH and are potential therapeutic targets for cytotoxic LHRH-analogs, AEZS 108 and AEZS 125. BMC Cancer. 14, 847-859 (2014). 7. Kakar, S. S., Jin, H., Hong, B., Eaton, J. W., Kang, K. A. LHRH receptor targeted therapy for breast cancer. Adv Exp Med Biol. 614, 285-296 (2008). 8. Limonta, P. et al. GnRH Receptors in Cancer: From Cell Biology to Novel Targeted Therapeutic Strategies. Endocr Rev. 33, 784–811 (2012). 9. Cook, T., Sheridan, W. P. Development of GnRH Antagonists for Prostate Cancer: New Approaches to Treatment. The Oncologist. 5, 162-168 (2000). 10. Shore, N. D., Abrahamsson, P. A., Anderson, J., Crawfors, E. D., Lange, P. New Considerations for ADT in Advanced Prostate Cancer and the Emerging Role of GnRH Antagonists. Prostate Cancer Prostatic Dis. 16, 7-15 (2013). 11. Liu, S. V. et al. Expression of Receptors for Luteinizing Hormone-Releasing Hormone (LH-RH) in Prostate Cancers following Therapy with LH-RH Agonists. Clin Cancer Res. 16, 4675-4680 (2010). 12. Nagy, A., Schally, A. V. Targeting of Cytotoxic Luteinizing Hormone-Releasing Hormone Analogs to Breast, Ovarian, Endometrial, and Prostate Cancers. Biol Reprod. 73, 851-859 (2005). 13. Reutter, M., Emons, G., Gründker, C. Starving tumors: inhibition of glycolysis reduces viability of human endometrial and ovarian cancer cells and enhances antitumor efficacy of GnRH receptor-targeted therapies. Int J Gynecol Cancer. 23, 34-40 (2013). 14. Engel, J. B. et al. Effective treatment of experimental human endometrial cancers with targeted cytotoxic luteinizing hormone-releasing hormone analogues AN-152 and AN207. Fertil Steril. 83, 1125-1133 (2005). 15. Cheung, L. W., Yung, S., Chan, T. M., Leung, P. C., Wong, A. S. Targeting Gonadotropin-Modulating Tumor-mesothelial Adhesion. Mol Ther. 21, 78-90 (2013). 16. Gründker, C., Nia, A H., Emons, G. Gonadotropin-releasing hormone receptor–targeted gene therapy of gynecologic cancers. Mol Cancer Ther. 4, 225-231 (2005).



409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450

17. Engel, J. B. et al. Targeted chemotherapy of endometrial, ovarian and breast cancers with cytotoxic analogs of luteinizing hormone-releasing hormone (LHRH). Arch Gynecol Obstet. 286, 437-442 (2012). 18. Gründker, E. J., Ernst, J., Reutter, M. D., Ghadimi, B. M., Emons, G. Effective targeted chemotherapy using AEZS-108 (AN-152) for LHRH receptor-positive pancreatic cancers. Oncol Rep. 26, 629-635 (2011). 19. Szepeshazi, K. et al. Powerful Inhibition of Experimental Human Pancreatic Cancers by Receptor Targeted Cytotoxic LH-RH analog AEZS-108. Oncotarget. 4, 751-760 (2013). 20. Treszl A, et al. Substantial expression of luteinizing hormone-releasing hormone (LHRH) receptor type I in human uveal melanoma. Oncotarget. 4, 1721-1728 (2013). 21. Jaszberenyi, M. et al. Inhibition of U-87 MG glioblastoma by AN-152 (AEZS-108), a targeted cytotoxic analog of luteinizing hormone-releasing hormone. Oncotarget. 4, 422432 (2013). 22. Curtis, K. K. et al. Novel LHRH‑receptor‑targeted cytolytic peptide, EP‑100: first‑in‑human phase I study in patients with advanced LHRH‑receptor‑expressing solid tumors. Cancer Chemother Pharmacol. 73, 931–941, (2014). 23. Wen, D. et al. LHRH-Conjugated Micelles for Targeted Delivery of Antiandrogen to Treat Advanced Prostate Cancer. Pharm Res. 31, 2784–2795 (2014). 24. Nukolova, N. V. et al. LHRH-targeted nanogels as a delivery system for cisplatin to ovarian cancer. Mol Pharm. 10, 3913-3921 (2013). 25. Aicher, B. et al. LHRH receptor targeting as mechanism of anti-tumor activity for cytotoxic conjugates of Disorazol Z with the LHRH receptor agonistic peptide D-Lys6LHRH. AACR annual meeting, Washington D.C. Abstract #5476, (2013). 26. Karampelas, T. et al. GnRH-Gemcitabine conjugates for the treatment of androgenindependent prostate cancer: pharmacokinetic enhancements combined with targeted drug delivery. Bioconjugate Chem. 16, 813-823 (2014). 27. Liu, H. et al. Ultrasound-Mediated Destruction of LHRHa-Targeted and PaclitaxelLoaded Lipid Microbubbles Induces Proliferation Inhibition and Apoptosis in Ovarian Cancer Cells. Mol Pharm. 11, 49–58 (2014). 28. Vegt, E. et al. Renal uptake of different radiolabelled peptides is mediated by megalin: SPECT and biodistribution studies in megalin-deficient mice. Eur J Nucl Med Mol Imaging. 38, 623–632 (2011). 29. Hosseinimehr, S. J., Tolmachev, V., Orlova, A. Liver uptake of radiolabeled targeting proteins and peptides: considerations for targeting peptide conjugate design. Drug Discov Today. 21/22, 1224-12232 (2012). 30. Tucci, F. C. et al. 3-[(2R)-Amino-2-phenylethyl]-1-(2,6-difluorobenzyl)-5-(2-fluoro-3methoxyphenyl)-6-methylpyrimidin-2,4-dione (NBI 42902) as a Potent and Orally Active Antagonist of the Human Gonadotropin-Releasing Hormone Receptor. Design, Synthesis, and in Vitro and in Vivo Characterization. J Med Chem. 48, 1169-1178 (2005). 31. Hao, D., Sun, L., Hu, X., & Hao, X. 99mTc-LHRH in tumor receptor imaging. Oncol. lett, 14, 569-578 (2017).


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32. Li, M., Tang, Z., Zhang, Y., Lv, S., Li, Q. and Chen, X. Targeted delivery of cisplatin by LHRH-peptide conjugated dextran nanoparticles suppresses breast cancer growth and metastasis. Acta biomaterialia, 18, 132-143 (2015). 33. Raffaele, C. et al. Total Synthesis and Biological Evaluation of Tubulysin Analogues. J Org Chem. 81, 10302-10320 (2016). 34. Srinivasarao, M., Galliford, C.V. and Low, P.S. Principles in the design of ligandtargeted cancer therapeutics and imaging agents. Nat Rev Drug Discov. 14, 203-219 (2015). 35. Srinivasarao, M. and Low, P.S. Ligand-Targeted Drug Delivery. Chem Rev. 117, 1213312164 (2017).

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306x111mm (149 x 149 DPI)

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A targeted tubulysin B hydrazide conjugate for the treatment of LHRH

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