Optimizing Multi-step Delivery of PEGylated TRAIL-toxin Conjugates

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Optimizing Multi-step Delivery of PEGylated TRAILtoxin Conjugates for Improved Antitumor Activities Xiaoyue Wei, Xiaoyue Yang, Wenbin Zhao, Yingchun Xu, Liqiang Pan, and Shu Qing Chen Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00327 • Publication Date (Web): 11 Jul 2017 Downloaded from http://pubs.acs.org on July 12, 2017

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Bioconjugate Chemistry

Title page

Optimizing Multi-step Delivery of PEGylated TRAIL-toxin Conjugates for Improved Antitumor Activities

Xiaoyue Wei, Xiaoyue Yang, Wenbin Zhao, Yingchun Xu, Liqiang Pan* and Shuqing Chen* Institute of Drug metabolism and Drug analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China.

Corresponding author: Corresponding author: Liqiang Pan* and Shuqing Chen* Mailing address: Institute of Drug metabolism and Drug analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China. Tel.:+86-0571-88208411; Fax: +86-0571-88208410 E-mail: [email protected]; [email protected]

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ABSTRACT: Although TRAIL (tumor-necrosis-factor (TNF)-related apoptosis-inducing ligand) has been considered as a promising broad-spectrum anti-tumor agent, its further application was limited by poor drug delivery and TRAIL-resistant tumors. A three-step drug delivery strategy was applied to TRAIL for solving the above two obstacles in the form of PEG-TRAIL-MMAE (Monomethyl Auristatin E). PEGylation of TRAIL in first step was to improve its in vivo pharmacokinetics, while the interaction between TRAIL conjugates with death receptors in second step was to activate TRAIL extrinsic apoptosis pathway, and the further release of MMAE from lysosome was the third step for introducing another apoptosis pathway to overcome TRAIL resistance in some tumors. Herein, in order to reach a balance among the three steps, the PEG/MMAE ratio was optimized for PEG-TRAIL-MMAE conjugates. PEG-TRAIL-MMAE conjugates with various PEG/MMAE ratios were prepared and compared with each other on their pharmacokinetics (PK) and pharmacodynamics (PD). As a result, PEG-TRAIL-MMAE conjugates with a PEG/MMAE ratio of 1:2 showed prolonged half-life in rat (6.8 h), and the best antitumor activity in vitro(IC50 0.31 nM) and in vivo while no sign of toxicity in xenograft models, suggesting it’s a promising multi-step drug delivery and antitumor strategy after optimization.

KEYWORDS: TRAIL; multi-step anti-tumor drug delivery; site-specific hetero-modification; PEGylation; TRAIL resistance

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INTRODUCTION Homotrimeric tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis upon binding with two death receptors, death receptor 4 (DR4)1 and death receptor 5 (DR5),2 which are overexpressed on the cell membrane of various tumor tissues instead of most normal tissues.3-6 The ligand-receptor complex can be internalized by tumor cells rapidly (5~30 min),7,8 thus providing potent drug delivery strategy for death receptor-positive tumor cells via TRAIL. Although TRAIL showed promising anticancer activity in preclinical studies, the shortcomings, such as poor pharmacokinetics and drug resistance, impede further development of TRAIL in clinical trials. In some resistant cancers, TRAIL/TRAIL-Receptors can even promote metastasis via Rac1 and PI3K pathway, and TRAIL-induced cancer secretome was reported to promote a tumor-supportive immune microenvironment via CCR2 (C-C chemokine receptor type 2).9 Poor pharmacokinetics has been the major obstruction of clinical application of TRAIL, as half-life of TRAIL in rodents was 3 to 5 min and 23 to 31min in nonhuman primates.10 In our previous study, we prepared a PEGylated TRAIL and it exhibited prolong half-life (4.6h) and more therapeutic potentials in tumor xenograft model than TRAIL.11 To overcome the innate and acquired TRAIL resistance of tumor cells resulted from the defects of intracellular apoptotic pathway,12-14 the internalization process has been used to deliver the antimitotic agent (Monomethyl Auristatin E, MMAE) into cytoplasm in the form of TRAIL-MMAE conjugate.15 Extrinsic apoptosis pathway would be activated once the modified TRAIL binds with DR4 or DR5, followed by internalization into lysosome, where enzymes (e.g., cathepsin B) could release free MMAE from conjugates to inhibit tumor cell growth through blocking the polymerisation of tubulin.16 Because of the homotrimeric structure of TRAIL, PEG and MMAE could be conjugated with different TRAIL monomer via the same coupling strategy (e.g., mutated cysteine in N-terminus), to exert synergic effects of PEG and MMAE. As a proof of principle, a new PEG-TRAIL-MMAE conjugate was synthesized by simply conjugating PEG and MMAE in a sequential order. This site-specific hetero-modified TRAIL trimer conjugates (the molar ratio of PEG modified monomer: MMAE

modified

monomer:

unmodified

monomer

is

around

1:1:1,

abbreviated

as

PEG1-TRAIL-MMAE1) exhibited significantly improved half-life (11.54 h) and more potency on in vivo and in vitro antitumor activities.17 3



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However, the in vivo antitumor activities of PEG-TRAIL-MMAE conjugates requires three delivery steps: in vivo circulation of conjugates was the first step (PEGylation of TRAIL was to improve its in vivo pharmacokinetics); interaction between TRAIL conjugates with death receptors (TRAIL extrinsic apoptosis pathway) was the second step; subsequent release of MMAE from TRAIL conjugates after its internalization was the third step (another apoptosis pathway to overcome TRAIL resistance in some tumors). More PEGylation means better pharmacokinetics (PK) but poor pharmacodynamics (PD), on the other hand, more MMAE conjugation ensures powerful cell-killing activity but shorter half-life. A balance has to be reached among three steps to take full advantage of PEG-TRAIL-MMAE delivery strategy by optimizing PEG/MMAE ratio. The potential N-linked glycosylation site (Asn-109) within TRAIL95-281 was mutated to cysteine, resulting in a TRAIL95-281 mutant N109C. Thus, the trimeric TRAIL obtained three -SH at the same site of every monomer, which facilitates the conjugation of methoxy-polyethylene glycol-maleimide (mPEG-MAL, 5000 Da) and maleimidocaproyl-valine-citrulline MMAE (vcMMAE) to the Cys-109 in the same reaction system. Different sizes of PEG and MMAE (potential steric hindrance) and their nucleophiles allow us to manipulate PEG/MMAE molar ratio through the change of reaction feeding amount or order, generating TRAIL conjugates with different PEG/MMAE ratios, e.g., 0:3, 1:1, 1:2, 2:1 and 3:0 (Figure 1).

Figure 1. Strategy of the co-modification of N109C with mPEG-MAL5000 and vcMMAE. PEG refers to methoxy-polyethylene glycol-maleimide (mPEG-MAL, 5000 Da). MMAE is the abbreviation of Monomethyl Auristatin E, and linker (Val-Cit) represents maleimidocaproyl-valine-citrulline; spacer refers to p-aminobenzylcarbamate. N109C is a mutant of TRAIL95-281 (Asn-109 to Cys). 4


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TRAIL conjugates with different PEG/MMAE ratios would have different binding affinities, different endocytosis rates and drug release rates. Herein, we describe the preparation, analysis and evaluation of PEG-TRAIL-MMAE conjugates with serial PEG/MMAE ratios. We furthermore investigated the PK/PD characteristics of the TRAIL conjugates and evaluated their biological and antitumor activities in vitro and in vivo, for the purpose of optimizing three-step drug delivery strategy for TRAIL-based antitumor therapy.

RESULTS Preparation and characterization of PEG-TRAIL-MMAE conjugates with various PEG/MMAE ratios. In the present study, we used mPEG-MAL (5000 Da) and vcMMAE (1315.8 Da) for TRAIL conjugates preparation. TRAIL95-281 mutant N109C was reacted with mPEG-MAL first, followed by vcMMAE reaction. For the preparation of TRAIL-MMAE, N109C was conjugated with vcMMAE after reduction with TCEP (Tris(2-carboxyethyl)phosphine). TRAIL conjugates with different PEG/MMAE ratios were visualized by Coomassie blue staining in SDS-PAGE. As shown in Figure 2A, the modification yield of monomer was up to 89% for each conjugate. Pure PEGylated N109C (lane 2) was loaded as single positive control. The amount of conjugated TRAIL monomers were normalized by being divided with TRAIL monomer input, after which PEG/MMAE ratios were calculated based on normalized TRAIL monomer conjugate amount. As a result, serial TRAIL conjugates were successfully synthesized, with various PEG/MMAE ratios of 0, 1, 0.5, 2 according to lane 3- 6 in Figure 2A. As demonstrated by circular dichroism spectrum in Figure 2B, TRAIL mutant N109C maintained the majority of TRAIL’s secondary structure composition except for a small amount of changes in antiparallel (Table 1), which might be caused by the disulfide bond formation between cysteines at site 109). Comparing with N109C, α-helix increased in N109C conjugations while β-sheet content decreased equally. Indicating some structural changes had occurred as a result of PEG and MMAE co-modification.

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Figure 2. Characterization of PEG-TRAIL-MMAE conjugates. (A). Evaluation of the effect of different PEG/MMAE ratios on TRAIL conjugates synthesis by SDS-PAGE. Lane 2 and 3 represents the single modification of PEG and vcMMAE respectively. (B). Comparing the secondary structure of TRAIL95-281, N109C, TRAIL-MMAE and PEG-TRAIL-MMAEs via Circular dichroism (CD) spectroscopy.

Table 1. Comparison of secondary structure content in TRAIL and its derivatives Samples

Helix(%) Antiparallel(%) Parallel(%) Beta-Turn(%) Rndm. Coil(%)

TRAIL95-281 9.3

36.9

4.7

13.7

35.6

N109C

10.8

26.9

4.9

13.4

40.0

0：3

20.2

21.2

6.7

14.9

36.0

1：1

21.1

22.9

7.3

13.7

35.3

1：2

23.8

18.6

7.1

13.7

36.0

2：1

24.0

19.7

7.3

13.5

35.4

Mean±SD

18.2±6.5

24.37±6.8

6.33±1.2

13.82±0.5

36.38±1.8

Pharmacokinetics of TRAIL mutant and its conjugates. The pharmacokinetics of TRAIL and PEG-TRAIL-MMAE conjugates was studied on Sprague-Dawley (SD) rats. After conjugated with vcMMAE, the half-life of TRAIL-MMAE (PEG/MMAE=0:3) decreased to 4.7h (Table 2) in comparison with N109C (5.1h). The half-life of PEG/MMAE=1:1, 1:2 and PEG/MMAE=2:1 TRAIL conjugates were determined to be 7.4 h, 6.8 h and 8.9 h, respectively, and renal clearance were also calculated. It was observed that the modification with PEG conferred slower renal clearance and longer half-life to TRAIL, indicating the PK characteristics were improved along with increased PEGylation content (Figure 3 and Table 2). 6


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Figure 3. Pharmacokinetic profiles of N109C and its conjugates. Sprague-Dawley (SD) rats were administered an i.v. injection of N109C or its conjugates (1mg/rat), and their serum concentrations were determined by ELISA. Results are expressed as mean ± SD from 4 different rats (n=4).

Table 2. Comparison of pharmacokinetic parameters after administering N109C or PEG/MMAE modifications to rats at 1 mg/rat i.v. (5 mg/kg) (n = 4) Pharmacokinetic parameters

N109C

P/M=0:3

AUC(0-∞)a （mg/L*h）

1122.807±580.508

45.401±18.126 124.28±52.051 99.554±31.624 736.579±100.144

5.098±3.632

4.697±0.751

7.372±1.511

6.818±1.857

8.918±0.822

0.007±0.006

0.13±0.069

0.049±0.029

0.054±0.017

0.007±0.001

P/M=1:1

P/M=1:2

P/M=2:1

b

t1/2 （h） c

CL (L/h/kg) a

AUC(0-∞): Area under the curve from zero to infinity.bt1/2: Half-life. cCL: Clearance.

Evaluation of binding affinities of TRAIL conjugates to cancer cells. As various PEG or MMAE modification of N109C might have different influence on the binding affinities to DR4/5 on cells. Binding abilities of TRAIL and its conjugates were measured on NCI-H460 cell lines by flow cytometry. It was shown in Figure 4 that PEG-TRAIL-MMAE conjugates had lower affinities than TRAIL on NCI-H460 cell line except for the PEG/MMAE=1:2 conjugates, which showed the highest affinity, and PEG/MMAE=0:3 and 2:1 conjugates showed weaker binding affinities.

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Figure 4. Binding affinity of TRAIL and TRAIL conjugates with death receptor-positive tumor cell line. NCI-H460 cells were incubated with 1% BSA-PBS alone (control) or 20 µg/mL TRAIL95-281 and PEGTRAIL-MMAE conjugates in 200 µL 1% BSA-PBS for 30 min on ice. Rabbit anti-TRAIL polyclonal antibody and FITC-labeled goat anti-rabbit antibody were used as primary and secondary antibody respectively. The stained cells were analyzed by flow cytometry on a Cytomics FC 500 MCL flow cytometer (Beckman Coulter).

In vitro antitumor activities of TRAIL and TRAIL derivatives. We evaluated the antitumor activities of TRAIL and its conjugates on non-small lung carcinoma cell line NCI-H460. As shown in Figure 5, conjugates with PEG/MMAE=0:3 showed the best apoptosis-inducing activity on NCI-H460 cells, followed by conjugates with PEG/MMAE=1:1 and 1:2. TRAIL conjugates with PEG/MMAE=2:1 did not benefit from MMAE modification, as a result of weaker binding affinity with death receptor-positive cells and fewer MMAE payload. The IC50 of N109C and PEG/MMAE=0:3, 1:1, 1:2, 2:1 conjugates were calculated to be 0.63 nM, 0.14 nM, 0.23 nM, 0.31 nM and 0.80 nM, indicating that the in vitro antitumor activities of TRAIL conjugates increased along with MMAE payload.

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Figure 5. In vitro antitumor activities of TRAIL, N109C and PEG-TRAIL-MMAE conjugates. NCI-H460 cells were exposed to serial concentrations (from 0.001 to 50 nM) of TRAIL, N109C or TRAIL conjugates for 72 h, and the cell viability was measured by Cell Counting Kit-8 (CCK-8). Results are expressed as mean ± S.D. from three independent experiments (n = 3). Relative Cell viability (%) = ((OD450 of cells exposure to N109C or N109C conjugates - OD450 of blank media) /(OD450 of cells exposure to blank media (control) OD450 of blank media)) ×100%.

Apoptotic mechanism of TRAIL conjugates. In our previous study, we identified that TRAIL-MMAE conjugates internalized into lysosome through the interaction with receptors (e.g., DR4 and DR5)15. DR4/5 ectodomain (ECD)-Fc fusion proteins were used to compete with PEG-TRAIL-MMAE conjugates on cell surface DR4/5 binding in order to study TRAIL conjugates cell entry pathway. As shown in Figure 6, both DRs showed specific inhibition of cell apoptosis induced by N109C and PEG-TRAIL-MMAE conjugates, and this results appeared to be dose-dependent on TRAIL-sensitive cell line NCI-H460. Competition of DR5 appeared to be more efficient on inhibiting cytotoxicity of N109C and TRAIL conjugates than DR4, which was consistent with what Akazawa et al reported, that was TRAIL-induced lysosomal permeabilization occurred predominantly via DR5, rather than DR4.18

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Figure 6. Competitive inhibition of binding between cell surface DR4/5 and TRAIL (or its conjugates) by DR4/5-Fc fusion proteins. NCI-H460 cells were treated with 200 ng/mL N109C or PEG-TRAIL-MMAE conjugates after being pre-mixed with serial concentrations of DR4-Fc/DR5-Fc. The results were expressed as mean ± S.D. from triplicates of one experiment (n = 3).

In order to determine the contribution of the payload MMAE in killing tumor cells, TRAIL extrinsic apoptosis-inducing pathway was blocked by inactivating caspase with pan-caspase inhibitor Z-VAD-FMK. In Figure 7A and 7B, almost all TRAIL mutant N109C induced apoptosis was blocked in the presence of Z-VAD-FMK (the blocking percentage was 77.1%, from 87.5% to 20%), demonstrating the vital role of extrinsic pathway in TRAIL killing cells. However, the blocking percentage dropped to 44.4%, 53.2%, 45.6% and 39.5% for PEG: MMAE=0:3, 1:1, 1:2 and 2:1 respectively, which were significantly lower than N109C (Figure 7C). For the negative group (without inhibitors), proportion of apoptotic cells (including early and late apoptosis) in PEG:MMAE=0:3, 1:1, 1:2 and 2:1 groups were 90.6%, 83.7%, 86.7% and 82.1%, respectively. The above results demonstrated that tumor cell apoptosis was largely induced by MMAE payload in the case of TRAIL conjugates, indicating the important role of MMAE payload in providing another apoptosis pathway. TRAIL, even conjugated with PEG and MMAE, could induce apoptosis through caspase dependent pathway when binding to death receptors on tumor cell surface. 10


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Figure 7. Blockage of the extrinsic TRAIL pathway by the pan-caspase inhibitor Z-VAD-FMK. (A). NCI-H460 cells were treated with TRAIL mutant N109C (20 ng/mL) for 24 h or PEG-TRAIL-MMAE conjugates (200 ng/mL with or without 10 µM Z-VAD-FMN) for 72h. The apoptosis of cell samples were detected by flow cytometry ((FC500MCL, Beckman)) after staining with Annexin V-FITC and PI. (B). Comparison of the percentage of apoptotic cells (including early and late apoptosis) among TRAIL conjugates with different PEG/MAME ratios, in the presence or absence of pan-caspase inhibitor. (C). Blocking percentage of each experimental group after Z-VAD-FMK inhibition.

Anti-tumor activities on xenograft nude mice of TRAIL and TRAIL derivatives. In vivo antitumor activities of TRAIL, N109C and PEG-TRAIL-MMAE conjugates were studied on NCI-H460 lung cancer mouse xenograft model, PBS was used as negative control. TRAIL-sensitive tumor cells, such as NCI-H460, could acquire resistance to TRAIL-induced apoptosis after repeated administration of high-dose TRAIL.19,20 Therefore, NCI-H460 cells were chosen to facilitate the comparison of in vivo antitumor activities of TRAIL and PEG-TRAIL-MMAE conjugates with 11



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different PEG/MMAE ratios. When tumor volume reached ~190 mm3, mice were divided randomly into different groups and given the same dose of TRAIL or its conjugates (20 mg/kg). As depicted in Figure 8A, PEG/MMAE=1:2 conjugates showed the best in vivo antitumor activity, followed by PEG/MMAE=1:1 conjugates. Surprisingly, tumor disappeared on three mice from the PEG/MMAE=1:2 group, and no recurrence was observed throughout the entire experiment. TRAIL and TRAIL mutant N109C were incapable to inhibit tumor growth efficiently even after frequent administrations (once every three days for four times). Tumor growth was efficiently inhibited after two injections of PEG/MMAE=2:1 conjugates, but become uncontrolled after the third injection, implying that the recovered tumor growth could be attributed from induced TRAIL resistance and MMAE payload of PEG/MMAE=2:1 was not enough to exert another apoptosis pathway. Although TRAIL conjugated with PEG/MMAE=0:3 exhibited dramatic tumor growth inhibition activity, its equivalent MMAE concentration was 1.17mg/kg in vivo exceeded the MTD (maximum-tolerated dose) of free MMAE (between 0.5 and 1.0 mg/kg),16 resulting that some mice could not tolerate its toxicity. To further confirm the in vivo antitumor activities of TRAIL and its derivatives, the dissected tumor from xenograft mice were weighed, and the tumor inhibition rate (R=((W experimental)/

W

control)×100%,

control-W

R and W refer to tumor inhibition rate and dissected tumor weight

respectively) was calculated (Figure 8B).

Figure 8. In vivo antitumor activities of TRAIL, N109C and PEG-TRAIL-MMAE conjugates. (A). In vivo antitumor activities of TRAIL and its derivatives. Tumor volume was calculated by the formula: V = (L× W2)/2, L and W refer to longitudinal and transverse tumor diameters respectively. Results are expressed as mean ± SEM (n=5). (B), Tumor growth inhibition rate was calculated according to the tumor weight compared with control group. Results are expressed as mean ± SD (n=5).

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Toxicity studies of TRAIL and TRAIL derivatives. The toxicities of TRAIL and its derivatives were evaluated. Mice body weight was monitored once every three days, and the body weight of all groups (except for PEG/MMAE=0:3) was close to the control group (Figure 9A). Lesions of major organs and serum biochemical indexes were examined after mice were sacrificed. The biochemical indexes in serum of all groups appeared to be similar (Figure 9B). No obvious toxicities were found in H&E (Haematoxylin and eosin) staining histological sections of the major organs (Figure 9C), indicating the introduction of PEG into TRAIL-MMAE conjugates could significantly reduce or even eliminate the toxicity resulted from MMAE.

Figure 9. Toxicity studies of TRAIL and TRAIL derivatives. (A). Body weight monitoring throughout the in vivo antitumor experiment. Body weight was measured every three days. Results are expressed as mean ± SEM (n=5). (B). Serological detection of serum markers. The serum levels of total bilirubin (TBIL), albumin (ALB), alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN) and creatinine (CREA)) were analyzed in the end of in vivo antitumor study to determine potential irreversible liver and kidney damage. Results are expressed as mean±SD.(n=5). (C). Histological studies. Livers, lungs and kidneys were investigated histologically after H&E staining. P/M refers to PEG/MMAE. Magnification equals to 400×. 13



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DISCUSSION The in vivo antitumor activities of PEG-TRAIL-MMAE conjugates require three delivery steps, in vivo circulation of conjugates, interaction between TRAIL conjugates with death receptors (TRAIL extrinsic apoptosis pathway), and subsequent release of MMAE from TRAIL conjugates after its internalization. But all these processes would be affected PEG/MMAE ratio as a consequent result from altered affinity and overall shape. The PEG/MMAE ratio therefore was tuned carefully and its effect on antitumor activities of conjugates was also studied. We first prepared multiple TRAIL conjugates with serial PEG/MMAE ratios (0:3, 1:1, 1:2 and 2:1), whose modification yield was all around 89% via a two-step reaction within 3h. PEG is well-known for its solubility, thus it stabilized N109C after treatment with reducing agent, even though the reduced mutant was easy to precipitate during reaction with vcMMAE. It was demonstrated that TRAIL and its conjugates had similar secondary structure composition, indicating successful hetero-modification of TRAIL mutant N109C without disturbing trimeric structure (Figure 2B and Table 1). TRAIL conjugates with PEG/MMAE=0:3 or 2:1 showed slightly lower affinity comparing to other TRAIL conjugates, suggesting either too much PEG or MMAE modification would affect the affinity. This could be resulted from strong hydrophobicity of MMAE that could change the physical and chemical properties of TRAIL, or steric hindrance effect of PEG which could affect the binding process. The in vivo circulation behavior of TRAIL conjugates could be expressed as their PK characteristics. In the first delivery step, PEG played the most important role in improving the PK of TRAIL conjugates as expected, the more PEG was coupled with TRAIL trimer, the longer half-life the conjugates would have (Figure 3). During cell entry level (second delivery step), PEG-TRAIL-MMAE (PEG/MMAE=1:2) showed the best binding affinity with death receptor positive NCI-H460 cells, leading to its superior in vitro tumor cell growth inhibition (Figure 4 and Figure 5). With the most MMAE payload, PEG/MMAE=3 conjugates appeared to have the best in vitro antitumor activities, implying additional benefit of attaching more MMAE in vitro. Since the binding of TRAIL mutant and its conjugates could be specifically blocked by DR4/DR5-Fc fusion proteins in a dose-dependent manner, the subsequent cell entry (internalization) could be mediated via death receptors (Figure 6). The intracellular activities of TRAIL conjugates, triggered by the release of MMAE as the third delivery step, were confirmed 14


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through the inhibition of TRAIL extrinsic apoptosis pathway by pan-caspase inhibitors (Figure 7). Because after the inhibition of caspase activities, MMAE was the only part of PEG-TRAIL-MMAE that could exert apoptosis-inducing activity. The combined effects of different PEG/MMAE co-modification during three delivery steps were reflected as in vivo antitumor activities. Intriguingly, TRAIL conjugates with PEG/MMAE ratio of 0:3 and 1:2 showed similar in vivo tumor growth inhibition (Figure 8), suggesting that improved PK and cell binding affinity compensated the slightly weaker in vitro antitumor activity of PEG/MMAE=1:2 TRAIL conjugates. Severe in vivo toxicity was observed in PEG/MMAE=0:3 group, which even led to the death of several mice, while no toxicity in PEG/MMAE=1:2 group. It was amazing that the in vivo toxicity of TRAIL-MMAE conjugates could even be eliminated by reducing MMAE loading (e.g., replacement of one MMAE by PEG) while maintain the same in vivo antitumor activities. The in vivo antitumor activities of other PEG-TRAIL-MMAE conjugates (e.g., PEG/MMAE=1:1 or 2:1) were relatively limited by attenuated cell binding affinity and in vitro antitumor activities, indicating the importance of second delivery step (interaction with tumor cells). In summary, we sought to optimize PEG/MMAE ratio of TRAIL conjugates to obtain better in vivo antitumor activities, which required the balance-reach among three delivery steps in vivo. Too much PEGylation would affect TRAIL binding with death receptors, and excess MMAE modification could also introduce additional in vivo toxicity. After optimization, when TRAIL trimer was conjugated with one PEG and two MMAE molecules (PEG/MMAE=1:2) on different monomers, improved PK, cell binding affinity, additional apoptosis pathway (MMAE) and in vivo antitumor activities were obtained simultaneously without introducing MMAE-related toxicity. According to the above results, PEG-TRAIL-MMAE conjugates could serve as efficient multi-step drug delivery system, and a promising broad-spectrum anti-tumor drug.

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EXPERIMENTAL PROCEDURES Material. All reagents were purchased from Sigma Aldrich unless otherwise noted. TRAIL95-281 and TRAIL95-281 mutant N109C were expressed and purified as previously reported.15 Mouse anti-MMAE monoclonal antibody was prepared through hybridoma technique.21 Maleimidocaproyl-valine-citrulline-monomethyl auristatin E (Mal-Val-Cit-MMAE) was prepared by Dr. David Miao as previously described.22-24 Methoxy-PEG-maleimide (mPEG-MAL, 5000Da) was purchased from SINOPEG (Xiamen, China). Amicon Ultra-4 mL 10K (Millipore) was used to desalt and concentrate samples.

Cell lines and animals. NCI-H460 were obtained from the Cell bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai). Cell lines were cultured in a humid atmosphere at 37 °C and 5% CO2, and were grown in RPMI-1640 medium (GIBCO, life technologies), supplemented with 10% FBS (GIBCO, life technologies), 100U/mL penicillin and 100 µg/mL streptomycin. Balb/c athymic nude mice (female, 4-6 weeks) were purchased from Slaccas (Shanghai, China), and Sprague-Dawley (SD) rats (male and female, 170-190g) were from Zhejiang Academy of Medical Sciences (China). All animal experiments were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. The protocols were approved by the Committee on the Ethics of Animal Experiments of the Zhejiang University, China (SCXK 2007–0029).

Preparation and characterization of PEG-TRAIL-MMAE conjugates with serial PEG/MMAE modification ratios. The PEG-TRAIL-MMAE conjugates were prepared and characterized according to the method previously reported.17 To synthesize PEG-TRAIL-MMAE conjugates, certain molar equivalents (see Table 3 ) of methoxyl-PEG-maleimide (mPEG-MAL, 5000 Da) or vcMMAE was added to the reactions at room temperature, in which TRAIL mutant N109C was reduced by TCEP-HCl. N109C reacted with mPEG-MAL first for 0.5 h, prior to the following addition of vcMMAE. After 1 h reaction between PEG-TRAIL and vcMMAE, 20-fold molar-excess N-Acetyl-L-cysteine was added to quench the reaction. All PEG-TRAIL-MMAE conjugates were purified by centrifugal ultrafiltration and analyzed by 12% SDS-PAGE under 16


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reducing condition. PEG-TRAIL-MMAE conjugates were diluted in double-distilled H2O to a final concentration of 0.2 mg/mL for far-UV (190~260 nm) circular dichroism (CD) measurements. Table 3. Feeding amounts of mPEG-MAL and vcMMAE in various PEG/MMAE conjugate ratios PEG/MMAE

0:3

1:1

1:2

2:1

mPEG-MAL(eq)a 0

2

1

3

vcMMAE(eq)

2

2

1.5

conjugate ratios

4

PEG/MMAE conjugate ratios were measured by Quantity One 4.6.6 band analysis of SDS-PAGE bands using Gauss Model. aeq: molar equivalents of TRAIL monomer Pharmacokinetics of TRAIL mutant and PEG-TRAIL-MMAE conjugates. Twenty Sprague-Dawley (SD) rats were divided randomly into 5 groups (n=4), and each rat was administered intravenously (i.v.) with 1 mg N109C or PEG-TRAIL-MMAE conjugates (5 mg/kg). Blood samples were obtained at different time points after administration, and then were incubated at 37°C for 30 min. Serum was collected after centrifugation (3000 rpm, 30 min) and stored at -80°C. The N109C concentration in serum were determined by direct enzyme-linked immunosorbent assay (ELISA) according to a previous protocol.25 Double antibody sandwich ELISA was applied to measure the conjugates concentration in serum. Microtiter plates (96 wells, Corning) were coated with a mouse monoclonal anti-MMAE antibody at 1 µg/mL in coating buffer (0.05 M carbonate/bicarbonate buffer, pH 9.6) overnight at 4°C. Nonspecific binding sites were blocked by adding blocking buffer (5% defatted milk in PBST buffer (PBS, pH 7.4, containing0.05 % of Tween-20)) for 2 h at 37°C, after the plates were washed. Then all samples diluted in blocking buffer were incubated in the plates for 1 h at 37°C. After plate washing step, Rabbit anti-TRAIL polyclonal antibody diluted in blocking buffer was added into the plates and incubated for 1 h at 37°C. After plate washing, HPR (horseradish peroxidase) conjugated Goat anti-rabbit secondary antibody diluted in blocking buffer was added for detection. After 45 min incubation at 37°C, all the plates were visualized by tetramethyl benzidine peroxidase substrate (Sangon Biotech (Shanghai) Co., Ltd. China) for 20-30 min at 37°C, and then stopped by adding 2 N H2SO4. Absorbance was measured at 450 nm. To generate a standard curve, PEG-TRAIL-MMAE 17



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conjugates standard were diluted to serial concentrations in blocking buffer, and the linear range of the sigmoidal titration curve was used to calculate the PEG-TRAIL-MMAE conjugates concentration in rat blood. Serum samples were diluted to several final concentrations to locate their concentrations in the linear range. Pharmacokinetic parameters were obtained from serum conjugates concentration profiles using non-compartment model analysis in DAS 2.0 software (China).

Determination of the binding of TRAIL, N109C and PEG-TRAIL-MMAE conjugates to cancer cells. NCI-H460 cells in exponential phase were collected and divided into 1×106 cells/test. Cells were rinsed twice by ice-cold PBS (pH7.4), and then incubated with 1% BSA-PBS only (control group) or 20µg/mL TRAIL95-281 and PEG-TRAIL-MMAE conjugates in 200 µL 1% BSA-PBS for 30 min on ice. After cold PBS wash, cells in all group were incubated with 16.7µg/mL anti-TRAIL rabbit polyclonal antibody in 300µL 1% BSA-PBS for 30 min on ice. FITC-labeled goat anti-rabbit antibody (1:300, Beyotime, China) in 200 µL 1% BSA-PBS was used for immunostaining for 30 min on ice. After cold PBS wash, treated cells were resuspended in 500µL PBS and immediately analyzed by flow cytometry (FC500MCL, Beckman).

In vitro antitumor activities of TRAIL, N109C and PEG-TRAIL-MMAE conjugates. NCI-H460 were seeded at a density of 5×103cells/100µL/well in 96-well cell culture plates (Corning). After overnight culture, cells were treated with serial concentrations of TRAIL, N109C or

various

PEG-TRAIL-MMAE

conjugates

(PEG/MMAE=0:3,

PEG/MMAE=1:1,

PEG/MMAE=1:2, PEG/MMAE=2:1). After 72 h, cell viability was determined by a Cell Counting Kit-8 (Dojindo, Osaka, Japan). The absorbance at 450 nm was measured by BioRad Model 680 Microplate Reader. The results were expressed as mean ± standard deviation (S.D.) from three independent experiments (n = 3).

Study on the apoptotic mechanism of PEG-TRAIL-MMAE conjugates. To determinate the inhibition of DR-mediated internalization of conjugates, NCI-H460 cells were seeded at a density of 5×103cells/100µL/well in 96-well cell culture plates. Twenty four hours post culture, cells were treated with 200 ng/mL TRAIL mutant N109C and PEG-TRAIL-MMAE conjugates, serial 18


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concentrations of DR4-Fc/DR5-Fc were mixed with TRAIL derivatives. After 3-day culture, cell viability was determined as above. Experiments were performed in triplicates. For the Blockage of the extrinsic (TRAIL) pathway via pan-caspase inhibitor Z-VAD-FMK. NCI-H460 cells were seeded at a density of 1.5×106 cells/well (3 mL) in 6-well plates. After incubation for 24 h, cells were either treated with TRAIL mutant N109C (20 ng/mL) for 24 h or various PEG-TRAIL-MMAE conjugates (200 ng/mL) for 72 h. As in caspase inhibition group, cells were treated exactly as above with the addition of 10 µM Z-VAD-FMN (Selleck chemicals, United States). The apoptosis of cell samples was detected by flow cytometry (FC500MCL, Beckman) after staining with Annexin V-FITC Apoptosis Detection Kit (Beyotime, China).

In vivo antitumor activities of TRAIL and TRAIL derivatives. Forty Balb/c athymic nude mice (female, 4-6 weeks) were used for in vivo antitumor activities of TRAIL and TRAIL derivatives. To build the tumor-bearing mice model, freshly harvested NCI-H460 cells (5×106cells per mouse) were inoculated subcutaneously in the right flank of three nude mice. When tumor volumes reached 1500 mm3, tumors were eviscerated and cut into small pieces of 1 mm3 and implanted into the right flank of the other thirty-seven nude mice. Tumor bearing nude mice were divided into 7 groups (PBS, TRAIL95-281, N109C, PEG/MMAE=0:3, PEG/MMAE=1:1, PEG/MMAE=1:2 and PEG/MMAE=2:1, 5 mice/group ). Thirteen days after inoculation, mice were treated with 20 mg/kg of the above samples (PBS 200 µl/mouse) once every three days for four times (q3d ×4) intravenously. Tumor volumes were continuously monitored until the end of the experiment and calculated by the formula V = (L×W2)/2, L and W refer to longitudinal and transverse tumor diameters respectively. The body weight of mice was also monitored.

Toxicity evaluation of TRAIL and TRAIL derivatives. For the investigation of chronic hepatic and renal injury, mice were kept from eating (while water unlimited) for 12 h before blood sampling and then were sacrificed. Serum prepared from blood samples was analyzed by Cobas C 311 analyzer (Roche) to determine the levels of total bilirubin (TBIL), albumin (ALB), alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN) and creatinine (CREA). Serological detection of these markers could indicate abnormal hepatic (TBIL, ALB, ALT and AST) and renal (BUN and CREA) function of mice after the treatment of TRAIL and TRAIL 19



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derivative. Liver, Lung and kidney tissues were also obtained from the same mice and immediately fixed by 10% neutral-buffered formalin for the following acute toxicity evaluation, which was studied through H&E (Hematoxylin and Eosin) staining for the morphological examination of tissue cells. In brief, the fixed tissues were paraffin-embedded and stained by H&E, the sections were examined by light microscopes at the magnification of 400 ×.

AUTHOR INFORMATION

Corresponding author: E-mail: Liqiang Pan*: [email protected]; Shuqing Chen*: [email protected] Tel.:+86-0571-88208411; Fax: +86-0571-88208410 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant No. 81502971), the State Key Program of National Natural Science of China (Grant No. 81430081), China Postdoctoral Science Foundation Funded Project (Project No. 2015M570519 and 2016T90549), and the Fundamental Research Funds for the Central Universities (Project No. 2016QNA7024).

ABBREVIATIONS TRAIL,

tumor-necrosis-factor

methoxy-polyethylene

(TNF)-related

glycol-maleimide;

apoptosis-inducing

vcMMAE,

ligand;

mPEG-MAL,

maleimidocaproyl-valine-citrulline

monomethyl auristatin E; DR, death receptor; CCR2, C-C chemokine receptor type 2; PK, pharmacokinetics; PD, pharmacodynamics; TCEP, Tris (2-carboxyethyl) phosphine; H&E, Haematoxylin and eosin; FBS, fetal bovine serum; ELISA, enzyme-linked immunosorbent assay; 20


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HPR, horseradish peroxidase; TBIL, total bilirubin; ALB, albumin; ALT, alanine transaminase; AST, aspartate transaminase; BUN, blood urea nitrogen; CREA, creatinine.

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Gynecol. Oncol. 101, 46-54. (7) Austin, C. D., Lawrence, D. A., Peden, A. A., Varfolomeev, E. E., Totpal, K., De Mazière, A. M., Klumperman, J., Arnott, D., Pham, V., Scheller, R. H. et al. (2006) Death-receptor activation halts clathrin-dependent endocytosis. Proc. Natl. Acad. Sci. U. S. A. 103, 10283-88. (8) Kohlhaas, S. L., Craxton, A., Sun, X. M., Pinkoski, M. J., and Cohen, G. M. (2007) Receptor-mediated endocytosis is not required for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. J. Biol. Chem.282, 12831-41. (9) Hartwig, T., Montinaro, A., von Karstedt, S., Sevko, A., Surinova, S., Chakravarthy, A., Taraborrelli, L., Draber, P., Lafont, E., Arce Vargas, F. et al. (2017) The TRAIL-Induced Cancer Secretome Promotes a Tumor-Supportive Immune Microenvironment via CCR2. Mol. cell 65, 730-42. (10) Kelley, S. K., Harris, L. A., Xie, D., DeForge, L., Totpal, K., Bussiere, J., and Fox, J. A. (2001) Preclinical studies to

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apoptosis-inducing ligand in humans: characterization of in vivo efficacy, pharmacokinetics, and safety. J. Pharmacol. Exp. Ther. 299, 31-38. (11) Pan, L. Q., Wang, H. B., Lai, J., Xu, Y. C., Zhang, C., and Chen, S. Q. (2013) Site-specific PEGylation of a mutated-cysteine residue and its effect on tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). Biomaterials 34, 9115-23. (12) Eggert, A., Grotzer, M. A., Zuzak, T. J., Wiewrodt, B. R., Ho, R., Ikegaki, N., and Brodeur, G. M. (2001) Resistance to Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis in Neuroblastoma Cells Correlates with a Loss of Caspase-8 Expression. Cancer Res. 61, 1314-19. (13) Sanlioglu, A. D., Dirice, E., Aydin, C., Erin, N., Koksoy, S., and Sanlioglu, S. (2005) Surface 22


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TRAIL decoy receptor-4 expression is correlated with TRAIL resistance in MCF7 breast cancer cells. BMC cancer 5, 54. (14) Zhang, L., and Fang, B. (2005) Mechanisms of resistance to TRAIL-induced apoptosis in cancer. Cancer Gene Ther. 12, 228-37. (15) Pan, L. Q., Wang, H. B., Xie, Z. M., Li, Z. H., Tang, X. J., Xu, Y. C., Zhang, C., Naranmandura, H., and Chen, S. Q. (2013) Novel conjugation of tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) with monomethyl auristatin E for efficient antitumor drug delivery. Adv. Mater. 25, 4718-22. (16) Francisco, J. A., Cerveny, C. G., Meyer, D. L., Mixan, B. J., Klussman, K., Chace, D. F., Rejniak, S. X., Gordon, K. A., DeBlanc, R., Toki, B. E. et al. (2003) cAC10-vcMMAE, an anti-CD30-monomethyl auristatin E conjugate with potent and selective antitumor activity. Blood 102, 1458-65. (17) Pan, L. Q., Zhao, W. B., Lai, J., Ding, D., Wei, X. Y., Li, Y. Y., Liu, W. H., Yang, X. Y., Xu, Y. C., and Chen, S. Q. (2015) Hetero-modification of TRAIL trimer for improved drug delivery and in vivo antitumor activities. Sci. Rep. 5, 14872. (18) Akazawa, Y., Mott, J. L., Bronk, S. F., Werneburg, N. W., Kahraman, A., Guicciardi, M. E., Meng, X.-W., Kohno, S., Shah, V. H., Kaufmann, S. H. et al. (2009) Death receptor 5 internalization is required for lysosomal permeabilization by TRAIL in malignant liver cell lines. Gastroenterology 136, 2365-76. (19) Lee, T.-J., Lee, J. T., Park, J.-W., and Kwon, T. K. (2006) Acquired TRAIL resistance in human breast cancer cells are caused by the sustained cFLIP L and XIAP protein levels and ERK activation. Biochem. Biophys. Res. Commun. 351, 1024-30. (20) Yoshida, T., Zhang, Y., Rosado, L. A. R., and Zhang, B. (2009) Repeated treatment with 23



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subtoxic doses of TRAIL induces resistance to apoptosis through its death receptors in MDA-MB-231 breast cancer cells. Mol. Cancer Res. 7, 1835-44. (21) G. KÖHLER, C. M. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256(5517), 495-97. (22) Pettit, G. (1997) The dolastatins, in Fortschritte der Chemie organischer Naturstoffe Progress in the Chemistry of Organic Natural Products pp 1-79, Springer. (23) Doronina, S. O., Toki, B. E., Torgov, M. Y., Mendelsohn, B. A., Cerveny, C. G., Chace, D. F., DeBlanc, R. L., Gearing, R. P., Bovee, T. D., and Siegall, C. B. (2003) Development of potent monoclonal antibody auristatin conjugates for cancer therapy. Nat. Biotechnol. 21, 778-84. (24) Dubowchik, G. M., Firestone, R. A., Padilla, L., Willner, D., Hofstead, S. J., Mosure, K., Knipe, J. O., Lasch, S. J., and Trail, P. A. (2002) Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. Bioconjugate Chem. 13, 855-69. (25) Pan, L. Q., Xie, Z. M., Tang, X. J., Wu, M., Wang, F. R., Naranmandura, H., and Chen, S. Q. (2013) Engineering and refolding of a novel trimeric fusion protein TRAIL-collagen XVIII NC1. Appl. Microbiol. Biotechnol. 97, 7253-64.

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Table of Contents graphic Optimizing Multi-step Delivery of PEGylated TRAIL-toxin Conjugates for Improved Antitumor Activities

Xiaoyue Wei, Xiaoyue Yang, Wenbin Zhao, Yingchun Xu, Liqiang Pan* and Shuqing Chen* Institute of Drug metabolism and Drug analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China.

PEG-TRAIL-MMAE conjugates with various PEG/MMAE ratios were prepared and compared with each other on their pharmacokinetics (PK) and pharmacodynamics (PD). As a result, PEG/MMAE=1:2 showed prolonged half-life in rat (6.8 h), and the best antitumor activity in vitro (IC50 0.31 nM) and in vivo while no sign of toxicity in xenograft models, suggesting it’s a promising multi-step drug delivery and antitumor strategy after optimization.

25



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Figure 1:Strategy of the co-modification of N109C with mPEG-MAL5000 and vcMMAE. 190x107mm (300 x 300 DPI)


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Figure 2. Characterization of PEG-TRAIL-MMAE conjugates. 85x26mm (300 x 300 DPI)



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Figure 3. Pharmacokinetic profiles of N109C and its conjugates. 106x81mm (300 x 300 DPI)


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Figure 4. Binding affinity of TRAIL and TRAIL conjugates with death receptor-positive tumor cell line. 187x225mm (300 x 300 DPI)



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Figure 5. In vitro antitumor activities of TRAIL, N109C and PEG-TRAIL-MMAE conjugates. 77x57mm (300 x 300 DPI)


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Figure 6. Competitive inhibition of binding between cell surface DR4/5 and TRAIL (or its conjugates) by DR4/5-Fc fusion proteins. 226x257mm (300 x 300 DPI)



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Figure 7. Blockage of the extrinsic TRAIL pathway by the pan-caspase inhibitor Z-VAD-FMK. 284x203mm (300 x 300 DPI)


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Figure 8. In vivo antitumor activities of TRAIL, N109C and PEG-TRAIL-MMAE conjugates. 93x31mm (300 x 300 DPI)



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Figure 9. Toxicity studies of TRAIL and TRAIL derivatives. 190x130mm (300 x 300 DPI)


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Table of Contents graphic 170x50mm (300 x 300 DPI)


Optimizing Multi-step Delivery of PEGylated TRAIL-toxin Conjugates

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