Antibody-Assisted Delivery of a PeptideâDrug Conjugate for Targeted

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Antibody-assisted delivery of a peptidedrug conjugate for targeted cancer therapy Hyungjun Kim, Do Been Hwang, Minsuk Choi, Soyoung Lee, Sukmo Kang, Yonghyun Lee, Sunghyun Kim, Junho Chung, and Sangyong Jon Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b00924 • Publication Date (Web): 06 Dec 2018 Downloaded from http://pubs.acs.org on December 7, 2018

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Molecular Pharmaceutics

Antibody-assisted delivery of a peptide-drug conjugate for targeted cancer therapy

Hyungjun Kim†, Dobeen Hwang‡, Minsuk Choi†, Soyoung Lee†, Sukmo Kang†, Yonghyun Lee†, Sunghyun Kim§, Junho Chung‡,* and Sangyong Jon†,*

†KAIST

Institute for the BioCentury, Department of Biological Sciences, Korea Advanced

Institute of Science and Technology, 291 Daehak-ro, Daejeon 34141, Republic of Korea. ‡Department

of Biochemistry and Molecular Biology, Seoul National University College of

Medicine, 103 Daehak-ro, Seoul 03080, Republic of Korea. §Center

for Convergence Bioceramic Materials, Korea Institute of Ceramic Engineering and

Technology, 202 Osongsaengmyeong 1-ro, Cheongju 28160, Republic of Korea.

ACS Paragon Plus Environment

Molecular Pharmaceutics 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

ABSTACT A number of cancer-targeting peptide-drug conjugates (PDCs) have been explored as alternatives to antibody-drug conjugates (ADCs) for targeted cancer therapy. However, the much shorter circulation half-life of PDCs compared with ADCs in vivo has limited their therapeutic value and thus their translation into the clinic, highlighting the need to develop new approaches for extending the half-life of PDCs. Here, we report a new strategy for targeted cancer therapy of a PDC based on a molecular hybrid between an anti-hapten antibody and a hapten-labeled PDC. An anti-cotinine antibody (Abcot) was used as a model anti-hapten antibody. The anticancer drug SN38 was linked to a cotinine-labeled aptide specific to extra domain B of fibronectin (cot-APTEDB), yielding the model PDC, cot-APTEDB-SN38. The cotinine-labeled PDC showed specific binding to and cytotoxicity toward an EDBoverexpressing human glioblastoma cell line (U87MG) and also formed a hybrid complex (HC) with Abcot in situ, designated HC[cot-APTEDB–SN38/Abcot]. In glioblastoma-bearing mice, in situ HC[cot-APTEDB–SN38/Abcot] significantly extended the circulation half-life of cotAPTEDB-SN38 in blood, and enhanced accumulation and penetration within the tumor and, ultimately, inhibition of tumor growth. These findings suggest that the present platform holds promise as a new, targeted delivery strategy for PDCs in anticancer therapy. KEYWORDS: aptides, anti-cotinine antibody, cancer therapy, extra domain B of fibronectin, peptide-drug conjugates, SN38


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1. INTRODUCTION Antibody-drug conjugates (ADCs) have emerged as a platform for targeted cancer therapy, reflecting their extended circulation in blood and ability to deliver highly cytotoxic cargo specifically to target cancer cells, resulting in potent therapeutic efficacy.1-7 However, although a few ADCs have already been successfully used in the clinic,8-11 there are several drawbacks to using an antibody as an escort molecule for delivering drugs to tumors. These include limitations in production techniques, which generate a heterogeneous mixture of ADCs with variable numbers of drugs attached; the high cost of ADC manufacture and quality control; and poor penetration of ADCs deep into tumor tissue owing to the large size of the conjugated antibody, which limits their therapeutic efficacy, especially in solid tumors.12-14 As alternatives to ADCs, a number of cancer-targeting peptide-drug conjugates (PDCs) using somatostatin,1517

bombesin,18-20 cRGD,21-24 and aptides25,26 have been explored for targeted cancer therapy.27,28

Unlike ADCs, PDCs can be synthesized cost-effectively in large scale as a single chemical entity. Additionally, the much smaller size of PDCs should allow them to penetrate deeper into the tumor than do ADCs. However, a critical drawback of PDCs is their far shorter circulation half-life compared with ADCs owing to rapid renal clearance, limiting therapeutic efficacy and thus success in the clinic. Although high-molecular-weight polyethylene glycol (PEG; >~50,000 Da) can be attached to a PDC to improve its circulation half-life, the resulting PEGylated PDC no longer has an advantage over ADCs in terms of tumor penetration and costeffectiveness.29,30 Accordingly, there is a need to develop a new approach that can significantly extend the half-life of PDCs while retaining the expected tumor-penetrating ability associated with the small size of PDCs. It has recently been shown that the circulation half-life of an aptamer with the small molecular hapten cotinine can be significantly increased through non-covalent complexation with an anti-continine antibody (Abcot).31-33 Therefore, we reasoned that such interactions between a hapten tag and anti-hapten antibody could be employed as a possible strategy for overcoming the shortcomings of PDCs. Furthermore, we expected that the PDC could be slowly released from the antibody complex in the tumor and penetrate deep into the tumor more efficiently than the much larger-sized ADCs. In this study, we report a new strategy for targeted cancer therapy based on a molecular hybrid between an Abcot and a cotinine-labeled PDC. A model PDC containing a cotinine label was prepared using an aptide (a novel class of highaffinity peptides) specific to extra domain B of fibronectin (APTEDB) as a cancer-targeting (or escort) ligand25,26,34-38 and SN38 as an anticancer drug, a PDC referred to as cot-APTEDB-SN38. The extra domain B of fibronectin has been considered a promising cancer marker as it is



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specifically overexpressed around new blood vessels in tumors as well as in tumor-associated extracellular matrix and also it is undetectable in healthy adults.39 We hypothesized that i) the in situ hybrid complex (HC) between pre-injected Abcot and cot-APTEDB-SN38, designated as HC[cot-APTEDB–SN38/Abcot], could be formed during the blood circulation and ii) the resulting in situ complex would exhibit extended circulation in the bloodstream and consequently, accumulate at perivascular regions in the tumor via specific interaction between APTEDB and EDB-overexpressing tumor-associated endothelial cells/extracellular matrix, where it would begin to release either cot-APTEDB-SN38 or free SN38 that ultimately could penetrate deep into the tumor tissue to exert effective anticancer activity (Figure 1A). 2. EXPERIMENTAL SECTION 2.1. Materials SN38 and Irinotecan (CPT-11) were purchased from TCI Co., LTD (Tokyo, Japan). Succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]

ester

(NHS-OEG4-

maleimide) was purchased from Thermo Fisher Scientific (Rockford, IL, USA). Anti-EDB aptide-cys-cotinine (cot-APTEDB) was chemically synthesized and purchased from AnyGen Co. (Gwangju, South Korea). An anti-cotinine antibody (Abcot) was produced as described previously.31 All other solvents and reagents were from Sigma Chemical (St. Louis, MO, USA). EDB-positive murine Lewis lung carcinoma (LLC) and human glioblastoma (U87MG) cell lines were obtained from the Korean Cell Line Bank (Seoul, Korea). All animals were obtained from Orient Bio Inc. (Seoul, Korea) and maintained under pathogen-free conditions in the animal facility at Korea Advanced Institute of Science and Technology (KAIST). Animal experiments were approved by the KAIST Animal Care and Use Committee. 2.2. Preparation of Cot-APTEDB-SN38 conjugates A succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] derivative of SN38 (SN38OEG) was synthesized as described previously.26 For the synthesis of cot-APTEDB-SN38, SN38-OEG (50 mg, 1 mmol) and cotinine-modified aptide (15 mg, 1 mmol) were dissolved in anhydrous dimethylsulfoxide (DMSO; 0.5 mL, 100 mM). The mixture was stirred at room temperature overnight and then purified by high-performance liquid chromatography (HPLC). The product fraction was lyophilized to obtain the cot-APTEDB-SN38 conjugate as a yellow solid

(yield,

85–90%)

and

was

further

characterized

by

matrix-assisted

laser

desorption/ionization-time of flight (MALDI-TOF) mass spectrometry using a Bruker Autoflex system (Germany).


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2.3. Evaluation of cell binding by confocal imaging U87MG human glioblastoma cells were seeded at 5 × 103 cells/well in 12-well plates and incubated for 2 d. The cells were then washed once with phosphate-buffered saline (PBS) and incubated with solutions of APTEDB and cot-APTEDB in serum-free RPMI-1640 medium at 37 °C for 1 h. Thereafter, cells were washed three times with PBS to remove drug and incubated with Abcot for 1 h at 37 °C. The cells were then washed three times with PBS, followed by incubation for 1 h with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG secondary antibody. The cells were mounted with mounting medium containing 4’,6-diamidino-2phenylindoe (DAPI; Vector Laboratories) and examined under an Olympus FluoView FV1000 fluorescence microscope (Olympus Imaging Co., Tokyo, Japan). 2.4. In vitro cytotoxicity assay U87MG human glioblastoma cell lines were used as EDB-positive cells. Cells (5 × 103/well) were seeded in 96-well plates and incubated overnight. CPT-11, cot-APTEDB-SN38 and HC[cot-APTEDB–SN38/Abcot] were dissolved in distilled water, and SN38 was dissolved in DMSO; each was diluted 100-fold from its starting concentration with growth medium, and then added to wells of a 96-well plate. After incubating cells for 72 h, 20 μL of MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazoniumbromide] solution (5 mg/mL) was added to each well of the plate and the absorbance of the solution was measured at 570 nm using a microplate reader (FL600; Bio-Tek Inc., Winooski, VT, USA). 2.5. Pharmacokinetics and tumor distribution ICR mice received intravenous injection (i.v.) of Abcot (5 mg/kg) via the tail vein and after 30 min, CPT-11 (1 mg/kg) or cot-APTEDB-SN38 (equivalent to 1 mg SN38/kg) was intravenously injected to the same mice. Those mice were sacrificed at 0.08, 0.5, 1, 3, 6, 12, 24, and 48 h post injection. Blood (450 μL) was then collected and mixed with 50 μL of sodium citrate (3.8% solution), and samples were immediately centrifuged at 13,000 rpm for 10 min. The active metabolite of CPT-11, SN38, was extracted from 100-μL aliquots of plasma using 200 μL of acetonitrile and its concentration in each sample was determined by HPLC. For the tumor distribution study, BALB/c nude mice bearing U87MG human glioma with tumor size of ~350 mm3 received i.v. injection of Abcot (10 mg/kg) and after 30 min, followed by another i.v. injection of CPT-11 (2 mg/kg) or cot-APTEDB-SN38 (equivalent to 2 mg SN38/kg). The mice were sacrificed at different time points (1, 6, and 12 h) post injection,



at which point tumors were weighed and homogenized with 200 μL acetonitrile and centrifuged at 20,000 × g for 10 min. Supernatants were analyzed by HPLC using the Agilent 1100 modules (Wilmington, DE, USA). The mobile phase consisted of a mixture of 25 mM NaH2PO4 (pH = 3.1) buffer and acetonitrile at a ratio of 50:50 v/v and the flow rate was set as 1 mL/min. UV detector at a wavelength of 265 nm and an Agilent Zorbax SB-C18 column (250 × 4.6 mm, 5 µm) were used. 2.6. Tumor penetration test BALB/c nude mice bearing U87MG tumors (~450 mm3) were injected intravenously via the tail vein with Abcot (10 mg/kg) or cot-APTEDB-SN38 (equivalent to 2 mg SN38/kg). Tumors were removed 12 h post-injection, sectioned, fixed with 4% paraformaldehyde for 20 min, and washed three times with PBS (3 min each). Blood vessels were stained by incubating sections for 1 h in a mixture of anti-CD31 antibody (Santa Cruz Biotechnology) and 20% fetal bovine serum (FBS). The distribution of cot-APTEDB-SN38 in tumor tissues was evaluated by incubating samples in a solution of anti-cotinine antibody (Acris) for 1 h, followed by incubation for 1 h with FITC-conjugated anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology). The slides were mounted with mounting solution containing Hoechst 33342 (Thermo Fisher Scientific) and examined under an Olympus FluoView FV1000 fluorescence microscope (Olympus Imaging Co., Tokyo, Japan). 2.7. In vivo antitumor activity The antitumor effects of HC[cot-APTEDB–SN38/Abcot] against xenograft tumors were assessed in female BALB/c nude mice subcutaneously injected into the back with 5 × 106 EDB-positive U87MG cells. When tumors had reached a size of at least 180 mm3, mice were divided into four groups (n = 5 mice/group): (1) PBS (control), (2) CPT-11 (2 mg/kg/d), (3) cot-APTEDBSN38 (2 mg/kg/d), and (4) HC[cot-APTEDB–SN38/Abcot] (2 mg/kg/d). Agents were administrated via the tail vein every day for a total of six injections, and Abcot was injected every 3 d for a total of two injections. Tumor dimensions were measured using calipers, and tumor volumes were calculated using the equation (1/2) (L×W2). 2.8. TUNEL assay After tumors were excised, they were sectioned and stained for apoptotic cells by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay using the DeadEnd Fluorometric TUNEL System (Promega) according to the manufacturer’s instructions.


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2.9. Statistical analysis The significance of differences among groups was calculated by one-way analysis of variance (ANOVA). A p-value < 0.001 was considered statistically significant.

3. RESULTS & DISCUSSION 3.1. Synthesis and characterization of cot-APTEDB-SN38 The overall synthetic scheme for cot-APTEDB-SN38 is shown in Figure 1B. First, a cotinine tag was introduced at the lysine residue of the β-hairpin loop in APTEDB, yielding cot-APTEDB. Second, alanine was linked via a cleavable ester bond to the tertiary alcohol of SN38, yielding ala-SN38. It has been shown that the ester bond between SN38 and the amino acid linker becomes cleaved under physiological conditions at pH 7.4 and 37 °C to release the intact SN38 with a half-life of ~14 h.40 Third, cot-APTEDB and ala-SN38 were connected to each other by reacting with a hetero-bifunctional linker containing amine-reactive NHS on one end and sulfhydryl-specific maleimide on the other end, yielding cot-APTEDB-SN38, the model PDC. All the resulting organic compounds obtained in each step were characterized by 1H-NMR (Figures S1-4). The MALDI-TOF mass spectrometry confirmed successful synthesis of the PDC, cot-APTEDB-SN38 (Figure S5 and S6). HPLC elution profiles of cot-APTEDB, SN38, and cot-APTEDB-SN38 are shown in Figure S7. The EDB binding affinity of in situ HC[cotAPTEDB–SN38/Abcot] measured by the surface plasmon resonance (SPR)-based assay was Kd of ~686 nM (Figure S8), which is approximately one-tenth of the unmodified APTEDB. The loss in the binding affinity might be due to the chemical modification around the original APTEDB.



A

B

Figure 1. Schematic illustration for the preparation of in situ HC[cot-APTEDB–SN38/Abcot] and cot-APTEDB–SN38. (A) Illustration of how in situ HC[cot-APTEDB–SN38/Abcot] is formed and works for targeted cancer therapy. (B) A synthetic scheme for the synthesis of cot-APTEDBSN38. The drug SN38 is linked to the cotinine-labeled aptide via a cleavable bond of an ester. 3.2. Binding specificity and anticancer efficacy of cot-APTEDB-SN38 We first examined whether cot-APTEDB-SN38 retains its binding specificity toward EDBoverexpressing cancer cells.25 The human glioblastoma cell line, U87MG, used as an EDBpositive cancer cell model, was treated with cot-APTEDB-SN38 for 1 h. After washing, the presence of the PDC on cells was detected by sequential incubation with Abcot and a FITClabeled secondary antibody. As shown in Figure 2A, an intense fluorescence signal was seen around the cell membrane of EDB-positive glioblastoma cells in the cot-APTEDB-SN38–treated group, suggesting that a) cot-APTEDB-SN38 retained its specificity for EDB-positive cancer


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cells, and b) the cotinine tag on cot-APTEDB-SN38 is well recognized by Abcot. By comparison, little fluorescence signal was observed in the APTEDB–treated group (lacking the cotinine tag). The in vitro cytotoxicity of HC[cot-APTEDB–SN38/Abcot] against two EDB-positive cancer cell lines, human U87MG and murine LLC (Figure S9), was assessed and compared with that of CPT-11 (Irinotecan) and SN38 using MTT assays. Treatment with HC[cot-APTEDB– SN38/Abcot] for 72 h exerted cytotoxic effects similar to those of cot-APTEDB-SN38 (IC50 value for both in the 300-nM range) and much greater than that of free CPT-11 that showed little cytotoxicity at the range of concentrations from ~nM to ~µM (Figure 2B). Unlike SN-38, an active metabolite of CPT-11 in vivo, it is natural for us to observe such little cytotoxicity for CPT-11 because it is known to have IC50 values of ~tens of µM, ~two or three orders of magnitude higher than SN-38.41 However, HC[cot-APTEDB–SN38/Abcot] was less potent compared to free SN38 (IC50 value ≈ 38.7 nM). This observation suggests that the active drug, SN38, becomes released from cot-APTEDB-SN38 under physiological conditions.

Figure 2. (A) Laser-scanning confocal microscopic images of U87MG (EDB-positive) cells after incubation with APTEDB or cot-APTEDB-SN38. U87MG cells were treated with APTEDB or cot-APTEDB-SN38 for 1 h, washed, and incubated with Abcot. (B) In vitro cytotoxicity of CPT-11, SN38 and cot-APTEDB-SN38 against EDB-positive U87MG cell lines after treatment for 72 h at 37°C. Data are presented as means ± S.E. (n = 6). 3.3. Pharmacokinetics and tumor distribution of HC[cot-APTEDB–SN38/Abcot] To examine whether the pharmacokinetics of cot-APTEDB-SN38 are improved by complexation with Abcot, we sequentially administered Abcot and cot-APTEDB-SN38 through the tail vein at a ACS Paragon Plus Environment


30-min interval. As soon as cot-APTEDB-SN38 enters the bloodstream, it is expected to form a stable complex in situ with the pre-injected Abcot.32 CPT-11 was used as a surrogate for SN38 that cannot be used directly for cancer therapy because of its poor solubility. As SN38 is an active metabolite formed from CPT-11 and is also released from both cot-APTEDB-SN38 and in situ HC[cot-APTEDB–SN38/Abcot] regimen under the in vivo conditions, we measured concentrations of SN38 in plasma using HPLC for pharmacokinetic study. Figure 3A shows the mean concentrations of SN38 in plasma as a function of time; the major pharmacokinetic parameters are summarized in Table 1. With a one-compartment model, the clearance half-life (t1/2) of the in situ complex, HC[cot-APTEDB–SN38/Abcot], in blood was calculated to be 6.02 h, which was much higher than that of CPT-11 (0.48 h) or cot-APTEDB-SN38 alone (2.01 h). The area-under-the-curve (AUC0-∞) value of in situ HC[cot-APTEDB–SN38/Abcot] was also significantly higher than that of CPT-11 or cot-APTEDB-SN38 alone, indicating that the pharmacokinetic profile of cot-APTEDB-SN38 was considerably improved by forming an in situ complex with Abcot in blood. On the other hand, we also measured the concentrations of the intact form CPT-11 and cot-APTEDB-SN38 in plasma using HPLC after i.v. injection of each regimen (Figure S10). For in situ HC[cot-APTEDB-SN38/Abcot], the concentration of cotAPTEDB-SN38 that is released from the complex was measured as a surrogate. The main pharmacokinetic parameters are summarized in Table S1. This direct measurements of the intact unchanged drug, not SN38, showed a similar trend of the increase in the blood circulation time after in situ complexation as shown in Table 1. Next, we measured the amount of drug accumulated in tumor tissues of human glioblastoma (U87MG)-bearing mice after i.v. injection of CPT-11, cot-APTEDB-SN38, and in situ HC[cot-APTEDB–SN38/Abcot], respectively, as a function of time. As SN38 is an active metabolite formed from CPT-11 and is released from cot-APTEDB-SN38, or in situ HC[cotAPTEDB–SN38/Abcot] under the in vivo conditions, the amount of SN38 in each tumor was measured using HPLC. As seen in Figure 3B, at 1 h post-injection, tumors in all three groups showed similar levels of drug accumulation. However, at 6 h post-injection, no detectable SN38 remained in tumors of mice treated with CPT-11, whereas the amount of drug decreased gradually in tumors in the other two groups, with a larger amount of drug remaining in the in situ HC[cot-APTEDB–SN38/Abcot] group (171.2 ng) than in the cot-APTEDB-SN38 group (104.2 ng). Interestingly, 12 h after injection, the drug was no longer detectable in tumors treated with cot-APTEDB-SN38 alone, whereas an appreciable amount of SN38 remained in tumors of in situ HC[cot-APTEDB–SN38/Abcot]-treated mice. We speculate that the increased accumulation of


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drug in tumors of mice in the HC[cot-APTEDB–SN38/Abcot] group may be a result of Abcotmediated improvements in the pharmacokinetic profile.

Figure 3. In vivo performance of in situ HC[cot-APTEDB–SN38/Abcot] in blood circulation and tumor accumulation. (A) Concentrations of SN38 in plasma after i.v. injection of each drug group as a function of time. (B) Tumor distribution of CPT-11, cot-APTEDB-SN38, and in situ HC[cot-APTEDB–SN38/Abcot] after i.v. injection, which was measured by the amounts of SN38 formed or released from each drug regimen. Data are presented as means ± S.E. (n = 3). (C) Sections from U87MG xenograft tumors were dual-immunostained with an anti-CD31 Ab (to detect blood vessels), followed by a FITC-labeled anti-rabbit IgG 2nd Ab (upper and middle images). For the lower image, the tumor section was immunostained with an anti-CD31 Ab and Abcot, then followed by a FITC-labled 2nd Ab.



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Table 1. Pharmacokinetic parameters of SN38 in ICR mice after intravenous injection of CPT-11, cot-APTEDB-SN38, and HC[cot-APTEDB–SN38/Abcot] (n = 3/group) CPT-11 (1 mg/kg)

cot-APTEDB-SN38 (1 mg/kg)

HC[cot-APTEDB–SN38/Abcot] (1 mg/kg)

t1/2 (h)a

0.48

2.01

6.02

AUC0-∞ (μg·h/mL)b

93.37

745.69

2397.32

CL (mL/min)c

78.47

6.71

1.89

Cmax (ng/mL)d

267.66

494.09

692.72

at , 1/2

half-life; bAUC, area under the plasma concentration-time curve, cCL, clearance, dCmax, the peak plasma concentration of a drug after administration.

3.4. Tumor-penetration ability of in situ HC[cot-APTEDB–SN38/Abcot] We hypothesized that, given its enhanced circulation in blood, in situ HC[cot-APTEDB– SN38/Abcot] could accumulate at perivascular regions of the tumor, where cot-APTEDB-SN38 would become dissociated from the HC and subsequently penetrate deeper into the tumor tissue because of its much smaller size compared with conventional ADCs. ADCs could only target outside the surface of solid tumor because of large size of antibody. However, when HC[cotAPTEDB–SN38/Abcot] target solid tumor, cot-APTEDB-SN38 were dissociated from HC and then would penetrate solid tumor more deeply than ADCs itself. In situ HC[cot-APTEDB– SN38/Abcot] was formed via sequential i.v. injection of each component, as described above, into human glioblastoma (U87MG)-bearing mice; after 12 h, tumors were dissected for analysis. For comparison, Abcot alone was used as a control. The tendency of each formulation to penetrate tumors was evaluated by staining excised tumor tissues with a FITC-labeled secondary antibody capable of detecting Abcot (Figure 3C). As expected, little Abcot accumulated in EDB-positive tumor tissue when administered alone (Figure 3C, top), whereas in situ HC[cot-APTEDB–SN38/Abcot] showed appreciable fluorescence intensity in the vicinity of blood vessels (perivascular areas) within the tumor (Figure 3C, middle), suggesting APTEDB-mediated accumulation of the HC in the tumor. Next, to detect free cot-APTEDB-SN38 released from the HC, we stained excised tumor tissues, by treating at first with Abcot and then with a FITC-labeled secondary antibody. Interestingly, fluorescence signals associated with cot-APTEDB-SN38 were observed not only around near vessels, but also in the tumor areas away from the vessels (Figure 3C, bottom). This finding suggests that, once it arrives at the target


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tumor site, cot-APTEDB-SN38 was able to dissociate from in situ HC[cot-APTEDB–SN38/Abcot] and further diffuse into deeper tumor areas. 3.5. Antitumor efficacy of in situ HC[cot-APTEDB–SN38/Abcot] in vivo The antitumor efficacy of in situ HC[cot-APTEDB–SN38/Abcot] was assessed in EDB-positive human glioblastoma-bearing mice. When tumors reached ~180 mm3 in size, mice received each therapeutics intravenously and tumor growth and changes in body weight were monitored. For in situ generation of HC[cot-APTEDB–SN38/Abcot] in vivo, Abcot was i.v. injected to mice prior to administration of cot-APTEDB–SN38 in which the PDC would form a complex with the Ab during circulation. As shown in Figure 4A, in situ HC[cot-APTEDB–SN38/Abcot] at an SN38/kg dose-equivalent of 2 mg effectively suppressed tumor growth and showed much greater antitumor activity (49.8% inhibition) than both cot-APTEDB-SN38 alone (23.9% inhibition) and CPT-11 (10.6% inhibition). Moreover, mice treated with in situ HC[cot-APTEDB–SN38/Abcot] showed no significant loss in body weight as similar as the saline control group (Figure 4B), indicating low apparent toxicity. Drug-treated tumors were excised and stained for apoptosis using TUNEL assays. Tumors treated with in situ HC[cot-APTEDB–SN38/Abcot] showed considerably greater TUNEL-positive apoptotic areas than tumors treated with either cotAPTEDB-SN38 alone or CPT-11 (Figure 4C). Collectively, these results indicate that complexation with Abcot in situ significantly enhances the tumor targeting ability, tumor distribution and penetration, and antitumor efficacy of cot-APTEDB-SN38 when compared with the corresponding PDC alone.



Figure 4. Antitumor activity of in situ HC[cot-APTEDB–SN38/Abcot] in nude mice bearing U87MG human glioblastomas (A, B) tumors. (A) Drugs (2 mg/kg) were injected intravenously at the indicted times (arrows). Data are expressed as means ± S.E. (n = 5; *p

Antibody-Assisted Delivery of a PeptideâDrug Conjugate for Targeted

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