Prostate-Specific Membrane Antigen-Specific Antitumor Activity of a

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PSMA-specific antitumor activity of a self-immolative tubulysin conjugate Christopher P. Leamon, Joseph Reddy, Alicia Bloomfield, Ryan Dorton, Melissa Nelson, Marilynn Vetzel, Paul Kleindl, Spencer Hahn, Kevin Wang, and Iontcho R. Vlahov Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00335 • Publication Date (Web): 10 May 2019 Downloaded from http://pubs.acs.org on May 11, 2019

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Bioconjugate Chemistry

PSMA-specific antitumor activity of a selfimmolative tubulysin conjugate Christopher P. Leamon*, Joseph A. Reddy, Alicia Bloomfield, Ryan Dorton, Melissa Nelson, Marilynn Vetzel, Paul Kleindl, Spencer Hahn, Kevin Wang and Iontcho R. Vlahov Endocyte, Inc., 3000 Kent Ave., Suite A1-100, West Lafayette, IN 47906, USA

*To

whom correspondence should be addressed: Dr. Christopher P. Leamon 3000 Kent Ave. Suite A1-100 West Lafayette, IN 47906 Phone: (765)463-7175 FAX: (765)463-9271 Email: [email protected]

Running Title: Pre-clinical evaluation of PSMA-tubulysin Key Words: Prostate specific membrane antigen, targeted chemotherapy, cancer, tumor, tubulysin.

ACS Paragon Plus Environment

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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 Prostate-specific membrane antigen (PSMA) is a biomarker that is overexpressed on prostate cancer, and it’s 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. 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 PSMAnegative cells. Brief treatment of nude mice bearing PSMA-positive LNCaP human xenografts with EC1169 led to complete remissions and cures. Furthermore, this activity occurred in the absence of weight loss. 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 compound’s targeted specificity for PSMA-positive cells. Finally, treatment of LNCaP-bearing mice with docetaxel (the most active chemotherapeutic agent approved for late stage prostate cancer therapy), was found to produce only modest anti-tumor activity, and this outcome was also associated with severe weight loss. Taken together, these results strongly indicate that PSMA-positive tumors may be effectively treated using highly potent, PSMAtargeted small-molecule drug conjugates using regimens that do not cause undesirable side effects.


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INTRODUCTION Among males in the western society, prostate cancer (PCa) is the most prevalent, and deaths from this disease (>30,000/year in the US alone) is second only to lung cancer1. Patients are often treated surgically, through radical prostatectomy, external beam radiation, and then followed by adjuvant anti-androgen therapy. But most of these patients will eventually relapse and present with widespread systemic disease, with lesions in bones, rectum and bladder2-5. Chemotherapy (e.g. docetaxel) is often given following antiandrogen failure, but responses are limited and questionable given the level of associated toxicity, especially in elderly men. Better treatment options are clearly needed for end stage PCa patients. Prostate-specific membrane antigen (PSMA) is expressed at relatively high levels on PCa and the neovasculature within many human tumors; fortunately, its expression is limited to only a few normal tissues6. Although it is an active glutamate carboxypeptidase, PSMA is now viewed as a receptor since it binds small molecule antagonists (i.e. ligands) with high affinity and internalizes them into the cell via endocytosis7. Due to its restricted tissue expression and function, and available high affinity ligands like 2-[3-(1,3dicarboxypropyl)ureido]pentanedioic acid (DUPA) and L-Glu-urea-L-Lys (EuK), PSMA is viewed as an excellent target for the delivery of imaging and therapeutic agents (39) to PCa. The tubulysin family of secondary metabolites were originally isolated from the myxobacteria Archangium geophyra and Angiococcus disciformis. A variety of tubulysin analogs8-12 have been synthesized and tested for their activity towards different cancers1315. These compounds are potent microtubule destabilizing agents with IC values in the 50 picomolar range against many cancer cell lines16,17, including those with multidrug resistant properties18. Despite the powerful in vitro activity of tubulysins, they display limited in vivo therapeutic activity due to severe toxicity. In our laboratory, the natural tubulysin B drug proved to be inactive against a human cervical cancer tumor model when administered at doses near to or greater than the maximum tolerated dose (MTD)19. For this reason, we believe that tubulysins are ideal candidates to be incorporated into our small molecule drug conjugate (SMDC) delivery system. We have recently described the biological activity of EC030519, EC053120 and most recently EC145621, which are all folate conjugates of tubulysin B22,23. There is also an earlier report of a first-in-class DUPA-tubulysin SMDC from our collaborator at Purdue University24. Here we report on a detailed preclinical investigation of the PSMA-targeted tubulysin conjugate, EC1169, which is constructed with a modified EuK ligand and found to be highly active and specific against PSMA-positive tumors. RESULTS Design of EC1169, EC1453 and EC1555. Similar to folate-based SMDCs, EC1169 was constructed using a modular design25. The color-coded modularity of EC1169’s chemical structure is shown in Fig. 1. This SMDC contains the tumor-targeting ligand, L-Glu-urea-L-Lys (Module 1, shown in black). Module 2 in blue represents a hydrophilic peptide spacer consisting of an acyl-[4-(aminomethyl)phenyl]acetamide bridge connecting L-Asp-L-Asp-L-Cys. Module 3 in green is a bio-cleavable, self-


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immolative disulfide-based linker system, while Module 4 in red depicts the active agent, tubulysin B hydrazide (TubBH). CO2H

HO2C

AcO

HO

N H

N H

N H

O

O

CO2H

O

N H

O N H

EC1169

CO2H O H N

CO2H S

N H CO2H

O

H N

O

S

O

NH N H

O

N

N S

H N

N O

O O

O

CO2H O HO2C

N H

CO2H N H

O N H

HO N H

CO2H H N

O N H

O

N H CO2H

O

EC1453

AcO O

CO2H S

O N

O

O

O

HO

H N

EC1555 O

O

CO2H O H N

N H O CO2H

CO2H S

N H CO2H

O S

O

O

OAc O

H N HN

NH O

N O

O

HN

O

H N

N S

NH

NH

O

N

N

N S

H N

N O

O O

Figure 1. Chemical structures of EC1169, EC1453 and EC1555. The structures are color coded as follows: The PSMA targeting ligand, L-Glu-urea-L-Lys (Module 1, shown in black). Module 2 in blue represents a hydrophilic peptide-based spacer. Module 3 in green is a bio-cleavable, self-immolative disulfide-based linker system (EC1169) or a more stable thioether linker system (EC1453). Module 4 in red depicts the active agent, tubulysin B hydrazide.

EC1453 was synthesized as an EC1169 analog, but instead of a bioreleasable disulfide linker system, EC1453 was constructed with the more stable thioether linker system. As an untargeted control, EC1555 was synthesized to represent an EC1169 analog lacking the PSMA ligand. Relative PSMA binding affinity of EC1169 and associated structural analogs. Additional EC1169 analogs were prepared to determine the structural and stereochemical requirements for PSMA binding, as shown in Table 1. Using PSMA-positive LNCaP cells, the affinities of these compounds for PSMA were determined relative to the wellknown high affinity ligand, 2-phosphonomethyl pentanedioic acid (PMPA). While PMPA’s affinity for PSMA was set to unity, our results show that EC1169 binds ~ 6.5fold more tightly to this membrane “receptor”. Interestingly, the D-Glu analog of EC1169 (EC1192) as well as the D-Glu/D-Lys (EC1268) and L-Glu/D-Lys (EC1269) analogs were found to be non-binders. Substitution of EC1169’s L-Glu with L-Asp (EC1197) or L-Aad (EC1241) was found to reduce PSMA binding affinity by 17- and 6-fold from EC1169, respectively, indicating that the 2-methylene side chain of EC1169’s L-Glu residue is likely optimal.


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Table 1: Relative affinity of EC1169 analogs. Aad, 2-aminoadipic acid. Name/ EC# Ligand Changes PMPA (reference)

Structure HO

HO2C

P

n/a

Fold Change

OH

1X

O CO2H

CO 2H

EC1169

HO 2C

AcO

HO

N H

O

O

CO 2H

O

L-Glu L-Lys

N H

N H

N H

CO 2H O H N

O N H

CO 2H S

N H CO 2H

O

H N

O

S

O

N H

N S

EC1192

HN

D-Glu L-Lys

O

CO2H

N H

N H

N H

CO2H S

N H CO2H

O

O HO2C

O

H N

AcO

O

S

N H

O

HN

N H

O

N H

N H

O

CO2H S

N H CO2H

O

O

HO2C

H N

O

S

O

HO2C

N H

Non Binder

O

AcO

NH N H

O

N

N S

H N

N O

O

0.37X

O

O

AcO

HO O

L-Aad L-Lys

N O

CO2H

HO2C

EC1241

H N

O

O

O CO2H H N

N H

HO2C

N S

HO

L-Asp L-Lys

6.5X

CO2H

O

EC1197

O

N

NH

H N

N O

O

O N H

H N

O

O

HO CO2H

O

N

NH

O

O

O

CO2H N H

N H

N H

O N H

CO2H O H N

CO2H S

N H CO2H

O

H N

O

S

N H

O

O

N

NH

N S

H N

N O

O

1.1X

O

O

CO2H

EC1268

O

D-Glu D-Lys

HO2C

N H

N H

N H

CO2H

O

H N O

O O

CO2H

O

S

N H CO2H

S

HO 2C

N S

N H

N H

OAc O

N H

CO2H

O N H

H N O

O

CO2H N H

O

S S

O

NH

NH HN

Non Binder

O

N

N S

H N

N O

O

Non Binder

O

O

CO2H

N O

O

O

O

CO2H N H

H N

O

HN

HO O

L-Glu D-Lys

O

N

NH

NH

O

CO2H

EC1269

OAc

HO

O

CO2H N H

N H

EC1169’s in vitro activity is dose-dependent and specific for the PSMA expression. As shown in Fig. 2, PSMA-positive LNCaP cells were found to be highly sensitive to EC1169 with membrane blebbing, loss of membrane integrity and cell death within 24 h yielding an in vitro IC50 of ~ 13 nM. 120 100

^ % Viability

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


^

80 60

***

40 20

****

****

-7

-6

0 -10

-9

-8

Concentration (Log M)

Figure 2. PSMA binding and a cleavable bond is required for EC1169 activity. PSMA-positive LNCaP cells (●,) or PSMA-negative A549 () and KB () cells were pulsed for 2 h with increasing concentrations of cleavable EC1169 (●,, ) or non-


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cleavable EC1453 (). Percent viability was assessed 72 h later using a 3H-thymidine incorporation assay. N = 3 +/- 1 S.D. (^P < 0.05, ***P < 0.001, ****P < 0.0001).

This activity was viewed as PSMA-specific since EC1169 was found to be benign against the PSMA-negative human lung adenocarcinoma cell line A549 as well as against PSMA-negative KB cells (Fig. 2) despite the fact both PSMA-negative cell lines are intrinsically sensitive to the base drug (IC50 for tubulysin B is 0.6 and 0.9 nM against KB and A549 cell lines, respectively; data not shown). Anti-tumor activity and specificity of EC1169 against PSMA-positive and PSMAnegative tumor xenografts. The activity of EC1169 against the PSMA-positive LNCaP model was assessed by treating tumor-bearing mice with a 2 µmol/kg dose following a three times per week (TIW), 2-week schedule. As shown in Fig. 3 Panel A, untreated control mice reached a tumor size of 1500 mm3 by approximately post tumor implant (PTI) day 34, whereas treatment with EC1169 lead to 100% complete response (CRs) by PTI day 31, an effect that persisted until Day 44 (see discussion). Thereafter, 29% of the mice were found to be tumor-free at the end of study on day 90. 20

B

A

1400

% Weight Change

Tumor Volume (mm3)

1600

1200 1000 800 600 400 200

10

0 32

47

62

77

92

PTI (Days)

-10

** ****

0 17

32

47

62

77

92

-20

PTI (days)

1600

C

1400

20

1200

% Weight Change

Tumor volume (mm3)

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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1000 800 600

*

400

* ^ ^ *******

200 0 5

15

25

35

45

55

65

75

85

95

D

10

0 15 -10

25

35

45

55

65

75

85

95

PTI (days)

-20

PTI (days)

Figure 3. EC1169 is highly active and specific for PSMA-positive tumors. Panel A, LNCaP tumor cells (3 x 106/animal) were inoculated subcutaneously into nu/nu mice and randomized. Mice were treated with EC1169, 2 µmol/kg, TIW x 2 weeks (●) or left untreated as a control () (**P < 0.01, PTI > 22 days; ****P < 0.0001, PTI > 27 days). Panel B, changes in test animal weights from the LNCaP study. Panel C, KB tumor cells (1 x 106/animal) were inoculated subcutaneously into nu/nu mice and treated with either EC1169 [●, (^P < 0.05, PTI < 16 days; *P < 0.05, PTI > 18 days)] or EC145 [, (***P < 0.001, PTI > 10 days; ****P < 0.0001, PTI > 17


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days)] at 2 µmol/kg, TIW for 5 doses. Panel D, changes in test animal weights from the KB study. Each curve shows the average tumor volume from individual mice (n ranges from 3 to 9).

Importantly, EC1169-treated animals did not lose any significant weight throughout the dosing period and beyond (Panel B), which is similar to that seen with our previously reported microtubule inhibitor-based SMDCs.21,24,26-29 When tested against the PSMAnegative but folate receptor-positive KB model, EC1169 was found to be inactive under similar conditions where a folate-based SMDC (EC145; vintafolide)30 displayed strong anti-tumor effect (Panel C). We have also reported earlier that both tubulysin B (38) and its hydrazide counterpart (22) had no anti-tumor activity when tested at and above their maximum tolerated doses. Superiority of EC1169 over its structural analogs and the standard of care agent, docetaxel. To better define EC1169’s in vivo mechanism of action, structural analogs were prepared whereby the PSMA targeted ligand, EuK, was removed (EC1555) or the intramolecular disulfide bind was replaced with a thioether (EC1453; see Fig. 1). Although targeted to PSMA, EC1453 was confirmed prior to animal studies to be inactive against LNCaP cells (Fig 2). When tested against the LNCaP tumor model using our standard 2 µmol/kg, TIW x 2 week dosing regimen, both EC1555 and EC1453 were also found to be inactive (Fig. 4 Panels A and B). These results indicate that only a PSMA-targeted tubulysin SMDC containing a self-immolative, bioreleasable linker system can effectively kill a PSMA-expressing tumor. Recognizing that docetaxel is a standard of care agent routinely used to treat end stage metastatic castrate-resistant prostate cancer31, we also evaluated this agent’s activity and toxicity against our LNCaP model. As shown in Fig. 4 Panel C, tumors in mice treated with the maximum tolerated dosing regimen (10 mg/kg docetaxel, BIW for 2 consecutive weeks) disappeared in 1 of 4 mice, and 2 of the remaining 3 mice had partial responses (PRs). However, this activity did occur at the expensive of significant weight loss (Fig. 4 Panel D). Compared to EC1169’s strong anti-tumor activity without weight loss (see Fig. 3), docetaxel was determined to be the inferior agent. 1600 1400

1600

A Tumor Volume (mm3)

Tumor Volume (mm3)

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


1200 1000 800

^

600

Lacks PSMA ligand

400 200 0 15

* 25

^

B 1200

800

^

Lacks a releasable linker

^ 400

^

^

0

35

45

55

65

75

85

20 25 30 35 40 45 50 55 60 65 70 75

PTI (Days)

PTI (Days)


7


C

1400

20

D

1200 1000 800

Docetaxel at MTD

^^

600 400

^

200

^

% Weight Change

Tumor Volume (mm3)

1600

10

0 30

40

50

60

70

80

90

100 110 120

PTI (Days)

-10

** ^

0 30

40

50

60

70

80

90

-20

100 110 120

PTI (Days)

Figure 4. EC1169 is superior to analogs lacking either the PSMA targeting ligand, a bioreleasable linker, or even the standard of care agent, docetaxel. LNCaP tumor cells (3 x 106/animal) were inoculated subcutaneously into nu/nu mice and randomized. Mice were treated with 2 µmol/kg of EC1555 (; Panel A, (^P < 0.05, on all days except *P < 0.05 on PTI day24)] or EC1453 [●; Panel B (^P < 0.05, on all PTI days)] following a TIW x 2 week schedule. A separate cohort of LNCaP tumor-bearing mice were treated with 10 mg/kg docetaxel BIW for 2 consecutive weeks (▼; Panels C and D, (^P < 0.05, on all days except **P < 0.01 between days 40-50)]). Changes in test animal weights from the docetaxel study are shown in Panel D. Untreated animals served as a control (). Each curve shows the average tumor volume from individual mice (n = 5).

EC1169 is effective against a recurrent PSMA-positive tumor. To demonstrate that EC1169 is active against a second PSMA-positive model, mice bearing MDA-PCa 2b tumors were treated at 2 µmol/kg TIW x 2 weeks. As shown in Fig. 5, tumors quickly regressed over the first 2 week cycle of treatment, with a 100% PR rate. The MDA-PCa 2b tumors eventually recurred over the following three weeks without treatment. But, a second 2-week cycle of EC1169 (starting on Day 56; average tumor volume = 253 mm3) provided further activity to yield a 50% PR and 50% CR rate by Day 68. These results indicated that EC1169 can safely and effectively be administered over multiple cycles for a greater overall response. 1600 20

A 1200

B

Cycle 1

% Weight Change

Tumor Volume (mm3)

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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Cycle 2

800

400

0 23

**

**

33

43

**** 53

**** 63

73

83

10

0 30 -10

40

50

60

70

80

90

PTI (Days)

-20

PTI (days)

Figure 5. EC1169 is active against PSMA-positive MDA-PCa 2b tumors across multiple cycles. Panel A, MDA-PCa 2b tumor cells (3 x 106/animal) were inoculated subcutaneously into nu/nu mice and randomized. Mice were treated with EC1169, 2 µmol/kg, TIW x 2 weeks for 2 cycles separated by a three week drug holiday (●,(**P < 0.01, PTI of 30-44 days; ****P < 0.0001, PTI of 48-58 days) ), or left untreated as a control ().


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DISCUSSION Prior to selecting EC1169 as our lead PSMA-tubulysin SMDC, we synthesized and tested numerous structural analogs. Keeping the TubBH, disulfide-based linker system and the hydrophilic spacer regions constant (up to and including the lysyl urea moiety), components of the EuK ligand were varied and new test articles were evaluated for PSMA binding using a cell-based relative affinity assay. From this analysis we quickly determined that PSMA binding was strongly dependent on the ligand’s stereochemistry. Specifically, both the Glu and Lys residues in the EuK ligand must be in the natural L configuration. Hence, substitution of either or both residues with the D enantiomer simply eliminated PSMA binding potential (Table 1). Furthermore, increasing the L-Glu side chain to three methylenes (L-Aad) or shortening it to 1 (L-Asp) was found to decrease PSMA binding by 5.9- and 17.6-fold, respectively. L-Glu is clearly the preferred residue in the EuK ligand format. EC1169’s activity was confirmed to be PSMA specific, as supported by multiple lines of evidence. First, EC1169 is cytotoxic against PSMA-positive but not PSMAnegative cells, up to 1 µM (Fig. 2). A similar pattern was observed in vivo where EC1169 significantly decreased, or eliminated, PSMA-positive LNCaP tumors but had no effect against PSMA-negative KB tumors (Fig. 3). No anti-tumor activity was also observed if the EuK ligand in EC1169 was removed, as in EC1555, or its bioreleasable disulfide linker was replaced with a more stable thioether linkage, as in EC1453 (Fig. 4). Taken together, our data strongly indicate that EC1169 is highly active against and specific for PMSA-expressing tumors. Following a single 2-week course of intravenous EC1169 therapy, subcutaneous LNCaP tumor volume decreased to an unpalpable level in all test animals (Fig. 3). The tumor-free interval lasted for 2 additional weeks, after which the LNCaP tumors started to recur in 5 of the 7 treated animals. When tested against the MDA-PCa 2b model, once again a significant anti-tumor response was observed against this PSMA-positive model; but, recurrence happened in all but 1 test animal after the first cycle of EC1169 therapy. Following a three week drug holiday, we found that the MDA-PCa 2b tumors responded well to a second 2-week cycle of EC1169 therapy (Fig. 5). These results suggested i) resistance against EC1169, or more likely the tubulysin drug payload, did not occur after the first cycle of therapy, and ii) multiple cycles are required for prolonged anti-tumor responses. Knowing that new anti-cancer investigational agents typically enter clinical trials enrolling advanced, end-staged patients, and that the microtubule stabilizing agent, docetaxel, is commonly used to treat men with end-staged prostate cancer31, we assessed docetaxel’s activity against our subcutaneous LNCaP tumor model. We tested docetaxel at its MTD and found that only partial responses were possible (compare to EC1169’s ability to produce CRs and cures at sub-MTD doses against this model; Fig. 3). Plus, docetaxel therapy caused significant weight loss, whereas EC1169 did not. Admittedly, EC1169 is constructed with the more powerful, microtubule destabilizing payload, TubBH. But the data shown in Figs. 3 through 5 clearly indicate that EC1169 has a wider, more effective therapeutic range than the standard of care agent, docetaxel.


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Collectively, these exciting preclinical qualities have provided justification for a Phase 1 clinical trial of EC1169 in advanced prostate cancer patients. EXPERIMENTAL PROCEDURES Materials. Clinical vial solutions of vintafolide (formerly EC145) and EC1169 were used in all experiments. Docetaxel was obtained from the Purdue University Pharmacy, West Lafayette, IN. All other common reagents were purchased from Sigma or other major suppliers. All cell lines were obtained by ATCC. General Synthetic Methods and Instrumentation. All chemical reactions requiring dry conditions were performed using commercially available anhydrous solvents under an argon atmosphere. Ammonium bicarbonate buffers used for analytical and preparative reverse phase chromatography were prepared and adjusted to pH 7.0 upon addition of the appropriate amount of acetic acid. All compounds purified by reversed phase chromatography were lyophilized to remove buffer and ACN. Compounds were lyophilized for no fewer than two days. Reactions were monitored with a Waters Alliance 2695 separations module equipped with a 2996 PDA detector and Waters ZQ mass spectrometer using a Waters XBridge RP18 3.5 µm, 3.0 x 50 mm column. Preparative HPLC was performed on a Waters 2545 Binary Gradient Module with PDA detector and Waters 2767 Sample Manager using a Waters XBridge Prep C18 5 µm, 19 x 250 mm column. General synthetic procedure for EC1169, EC1192, EC1197, EC1241, EC1268, EC1269, and EC1555. The syntheses of EC1169, EC1192, EC1197, EC1241, EC1268, EC1269, and EC1555, have been previously described,32,33 and all follow the same synthetic route: 1) synthesis of a modified PSMA-targeting ligand, 2) attachment of the targeting ligand to the spacer, and 3) synthesis of the PSMA-targeted tubulysin B hydrazide conjugate via a disulfide-exchange reaction. The synthesis of EC1169, which serves as an illustrative example, is shown in Fig. 6. Protected PSMA-ligand- 4-aminomethylphenyl acetic acid adduct 1 is synthesized in five steps from di-tert-butyl protected glutamic acid (Fig. 6, I). PSMA spacer 2 was synthesized using standard solid phase peptide synthesis techniques (Fig 6, II).


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I. H-Glu(Ot-Bu)-Ot-Bu•HCl salt

CO2t-Bu

1. 4-nitrophenyl chloroformate, DIPEA, DCM, 0 °C to rt 2. H-Lys(Z)-Ot-Bu•HCl, DIPEA, DCM, rt 3. H2, Pd/C, MeOH 4. 4-nitrophenyl chloroformate, DIPEA, DCM, rt 5. 4-aminomethylphenyl acetic acid, DIPEA, DMF, rt

O N H

t-BuO2C

CO2t-Bu

O

N H

N H

N H

O OH

1

II. O

O

H2N

STrt

H-Cys(Trt)2-Cl-Trt-resin

CO2H

1. Fmoc-Asp(Ot-Bu)-OH, PyBOP, DIPEA, DMF 2. 20% piperidine in DMF 3. Fmoc-Asp(Ot-Bu)-OH, PyBOP, DIPEA, DMF 4. 20% piperidine in DMF 5.1, PyBOP, DIPEA, DMF 6. TFA/H2O/TIPS (95:2.5:2.5), DTT

O N H

HO2C

CO2H

O

N H

N H

N H

O N H

CO2H O H N

CO2H SH

N H CO2H

O

2

III. AcO

HO

N

S

H N

O

S

O

NO2

O

N S

N H

N H O

O

N

H N

N O

O O

3 2, sodium phosphate buffer (20 mM, pH = 7) DMSO

CO2H O HO2C

N H

CO2H N H

N H

AcO

HO

O N H

O N H

CO2H O H N O

CO2H S

N H CO2H

S

H N

O O

O

N H O

N H

O

N

N S

H N

N O

O O

EC1169

Figure 6. Synthesis of EC1169. H-Cys(Trt)-2-Cl-Trt-resin was treated with Fmoc-Asp(Ot-Bu)-OH in the presence of PyBOP and DIPEA, followed by Fmoc removal with 20% piperidine in DMF (coupling and deprotection repeated two times). Adduct 1 was then coupled to the N-terminus of the resin bound peptide chain with PyBOP and DIPEA. After cleavage from the resin/global deprotection and reverse phase chromatographic purification, pure 2 was recovered. Nitro-pyridyldisulfanyl activated tubulysin B hydrazide 312,32-34 is then treated with 2 in a solution of phosphate buffer and DMSO under inert atmosphere to give EC1169 (Fig. 6, III). Synthesis of EC1453. The synthetic scheme for EC1453 is shown in Fig. 7. A suspension of tubulysin B12,34-36 (44.2 mg, 53.5 µmol), pentafluorophenol (PFP, 19.6 mg, 2 eq.), and dicyclohexylcarbodiimide (DCC) on resin (14 mg, 1.9 mmol/g, 5 eq.), in anhydrous dichloromethane (DCM, 5 mL) was stirred at room temperature under argon for 17 h.


11


AcO

HO

O

N

N S

N H

HO

O

H N

N

1. PFP, DCC-resin, DCM 2. NH2NH2 3. NSM, DIPEA

HN

O N

O

O

AcO

HO

O

O

Page 12 of 18

tubulysin B

O

O

H N

N S

N H

H N

O

N

N O

O O

O

4

O

2, sodium phosphate buffer (20 mM, pH = 7) MeOH

O HO2C

N H

AcO

HO

CO2H CO2H N H

O

O N H

N H

O N H

CO2H O H N O

CO2H S

N H CO2H

HN

O N

N H

H N O

O

O

N

N S

H N

N O

O O

O

EC1453

Figure 7. Synthesis of EC1453. The resin was removed by filtration and washed with DCM. To the combined filtrate was added anhydrous hydrazine (2.4 µL, 1.4 eq.) and the reaction was stirred at room temperature for 15 min. N-succinimidyl 3-maleimidopropionate (NSM, 28 mg, 2 eq.) was then added, followed by DIPEA (28 µL, 3 eq.). The reaction was stirred for 2.5 h at room temperature and then concentrated. The residue was purified by preparative HPLC (A: 50 mM ammonium bicarbonate buffer pH = 7, B: acetonitrile; 10% B to 100% B in 20 min) to give the maleimidopropionate tubulysin B hydrazide adduct 4 (21 mg, 39% yield) as a white solid after lyophilization. Spacer 2 (6.8 mg, 8.0 µmol) was dissolved in 20 mM phosphate buffer (2 mL, pH = 7, argon purged) and added dropwise to a stirring solution of tubulysin B hydrazide adduct 4 (8.0 mg, 1 eq.) in anhydrous methanol (2 mL). The solution was left to stir for 30 min. The reaction was diluted with water and purified by preparative HPLC (A: 50 mM ammonium bicarbonate buffer pH = 7, B: acetonitrile; 10% B to 100% B in 20 min) to give EC1453 (8.4 mg, 57% yield) as a white solid after lyophilization. Relative PSMA binding affinity assay. The relative affinity of EC1169 and associated analogs was determined according to a previously published procedure adapted for PSMA37. Briefly, PSMA-positive LNCaP cells in 24-well Falcon plates (> 75% confluent) were incubated with RPMI medium supplemented with 10% fetal bovine serum containing 1% glucose, 1% sodium pyruvate (medium) and 100 nM of 3H-PMPA (Moravek) in the absence and presence of increasing concentrations of unlabeled test articles. Cells were incubated for 1 h in an incubator at 37 C and then rinsed 3 times with 0.5 mL of PBS. Five hundred microliters of 1% sodium dodecylsulfate in PBS were added to each well; after 5 min, cell lysates were collected, transferred to individual vials containing 5 mL of scintillation cocktail, and then counted for radioactivity. Cells exposed to only the 3H-PMPA in RPMI (no competitor) were designated as Negative Controls, whereas cells exposed to the 3H-PMPA plus 1 mM unlabeled PMPA served as Positive Controls; DPMs measured in the latter samples (representing non-specific binding of label)


12

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were subtracted from the DPM values from all samples. Relative affinities were defined as the inverse molar ratio of compound required to displace 50% of 3H-PMPA bound to PSMA on LNCaP cells, and the relative affinity of PMPA for PSMA was set to 1. Final data were tabulated as a fold change from the PMPA value. Dose-Dependent PSMA Specific Activity of EC1169. PSMA-positive LNCaP (~ 4 x 106 binding sites per cell) and PSMA-negative A549 cells were seeded in individual 12well Falcon plates and allowed to form nearly confluent monolayers overnight in RPMI/5%FCS. Each well then received 1 mL of medium containing increasing concentrations of EC1169 (3 wells per sample). Cells were pulsed (40) for 2 h at 37 C, rinsed 4 times with 0.5 mL of medium and then chased in 1 mL of fresh medium up to 72 h. Spent medium was aspirated from all wells and replaced with fresh medium containing 5 µCi/mL of 3H-thymidine. Following a 2 h incubation at 37 C, cells were washed 3 times with 0.5 mL of PBS and then treated with 0.5 mL of ice-cold 5% trichloroacetic acid per well. After 15 min, the trichloroacetic acid was aspirated and the cells solubilized by the addition of 0.5 mL of 0.25 N sodium hydroxide for 15 min at room temperature. Each solubilized sample was transferred to a scintillation vial containing 3 mL of Ecolume scintillation cocktail and counted in a liquid scintillation counter. Final results were expressed as the percentage of 3H-thymidine incorporation relative to untreated controls. In vivo antitumor experiments. All animal housing, care, and procedures were followed according to Purdue University Animal Care and Use Committee-approved animal care and use protocols (West Lafayette, IN campus). Four- to eight-week-old male and female nu/nu mice (Harlan Sprague-Dawley, Inc.) were maintained on a standard 12-h light-dark cycle and fed ad libitum with Teklad Global 18% Rodent diet (Harlan Teklad diet #2018S) for the duration of the experiment. PSMA-expressing human prostate LNCaP or MDA-PCa 2b tumor cells (3 x 106 per nu/nu mouse) in 100 µL were mixed with 4.25 mg/ml (1:1) Matrigel and inoculated in the subcutis dorsal medial area of male nude mice. PSMA-negative KB cells (1 x 106 per nu/nu mouse) in 100 µL were injected into the subcutis of female nude mice. Mice were divided into groups of 5, and freshly prepared test articles were injected through the lateral tail vein under sterile conditions in a volume of 200 µL PBS. Intravenous treatments were typically initiated when LNCaP tumors were visible over the Matrigel plug or when KB tumors were approximately 100-150 mm3 in volume. The mice in the control groups received no treatment. Growth of each subcutaneous tumor was followed by measuring the tumor 3 times per week during treatment and twice per week thereafter, until a volume of 1500 mm3 was reached. Tumors were measured in 2 perpendicular directions using Vernier calipers, and their volumes were calculated as V = 0.5 x L x W2, where L = measurement of longest axis in mm and W = measurement of axis perpendicular to L in mm. As a general measure of gross toxicity, changes in body weights were determined on the same schedule as tumor volume measurements. Survival of animals was monitored daily. Animals that were moribund (or unable to reach food or water) were euthanized by CO2 asphyxiation. Cures were defined as CRs without tumor regrowth within the 90-day study time frame. PR was defined as tumor shrinkage below 50% at any point during the study.


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Statistical analysis. Statistical analysis for cell viability and antitumor activity (compared with untreated controls) was carried out as per the standard procedure using student’s ‘t’ test. P values were calculated to determine significant differences. All graphs and statistics were performed using GraphPad Prism. ACKNOWLEDGMENTS We wish to thank Dr. Philip S. Low for thoughtful discussions related to this work. ABBREVIATIONS PCa, prostate cancer; PSMA, prostate-specific membrane antigen; MTD, maximum tolerated dose; DUPA, 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid; EuK, L-Gluurea-L-Lys; SMDC, small molecule drug conjugate; TubH, tubulysin B hydrazide; TIW, three times per week; BIW, twice a week; CR, complete response; PR, partial response.


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Prostate-Specific Membrane Antigen-Specific Antitumor Activity of a

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