Bioconjugate Chem. 2007, 18, 821−828
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Use of Antibody as Carrier of Oligomers of Peptidomimetic rvβ3 Antagonist to Target Tumor-Induced Neovasculature In Soo Shin,† Beom-Su Jang,† S. Narasimhan Danthi,‡ Jianwu Xie,‡ Sarah Yu,† Nhat Le,† Jin-Soo Maeng,† In Sook Hwang,† King C. P. Li,‡,§ Jorge A. Carrasquillo,†,| and Chang H. Paik*,† Department of Nuclear Medicine and Department of Radiology, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892. Received November 7, 2006; Revised Manuscript Received January 19, 2007
Sulfhydryl selective reactions were explored to conjugate oligomers of a peptidomimetic integrin Rvβ3 antagonist, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-(S)-aminoethylsulfonylamino-β-alanine (IA) to monoclonal antibody (MoAb) to increase integrin Rvβ3 receptor-binding avidity. To generate sulfhydryl groups, N-succinimidyl-S-acetylthioacetate (SATA) was conjugated to both MoAb and IA. Sulfhydryl groups were then generated upon the deacetylation of the protecting acetyl group from the S-acetylthioacetato (ATA) moiety of MoAb-(ATA)n or IA-ATA with 0.02 M hydroxylamine in the presence of 1 mM EDTA at pH 7.2. The major focus was on optimizing the reaction concentrations, molar ratios, and reaction pH to conjugate high levels of IA-(A-SH) to MoAb-(A-SH)n without causing the inter- and intramolecular cross-linking of MoAb. Stepwise reactions of MoAb-(A-SH)n (15 µM MoAb) with a homobifunctional cross-linker, 1,8-bis(maleimido)diethylene glycol (BM[PEO]2) at a >50× molar excess to the -SH, followed by the reaction of the purified product MoAb(A-S-succinimidomaleimido-[PEO]2)n with IA-(A-SH) at pH 7.2 afforded monomeric MoAb-(A-S-succinimido[PEO]2-succinimido-S-A-IA)n with 70% bindability to 0.4 µM Rvβ3. When injected iv to nude mice with the receptor-positive M21 tumor, MoAb-IA10 radiolabeled with both 111In and 125I accumulated rapidly and was retained in the tumor for a 44 h period while the radioactivity cleared rapidly from the blood, thereby resulting in increasing tumor-to-blood ratios over time. The tumor uptake was similar between the 125I label and the 111In label for a 44 h period. In contrast, the blood radioactivity was lower, but liver and other organ uptakes were much higher for the 111In label than for the 125I. The 111In label produced higher tumor-to-blood ratios but much lower tumor-to-organ ratios than the 125I. The rapid blood clearance, a short peak tumor uptake time, and a low peak tumor uptake value with prolonged tumor retention of this macromolecule appear to support a hypothesis that MoAb-IA10 primarily binds to Rvβ3 receptors on angiogenic vessels, but not on the tumor. This hypothesis was substantiated by the fluorescence microscopic analysis of FITC-MoAb-IA10, which showed that FITCMoAb-IA10 outlined neovasculatures but not tumor cells at 4 and 21 h ex vivo. Additional proof was observed when blood vessels outlined with rhodamine-lectin, which specifically binds to blood vessels, were superimposable on neovasculatures outlined with FITC-MoAb-IA10.
1. INTRODUCTION Angiogenesis is the physiological process in which new blood vessels are formed from pre-existing blood vessels. This process is essential for a tumor to receive nutrients and oxygen for survival (1). Since tumor angiogenesis precedes the anatomical change of tumor size and the newly formed vessels are readily accessible, the detection of tumor angiogenesis should be a sensitive means of assessing the status of tumors. Antiangiogenesis therapy using cytotoxic R- and β-emitters may be an effective approach, because damaged endothelial cells and adjacent tumor cells can block blood flow to tumors, thereby killing a wide variety of solid tumors. Integrins are a family of heterodimeric endothelial transmembrane glycoproteins composed of R and β subunits (2). * Corresponding author. Tel.: +1 301 496 1426. Fax: +1 301 402 4548. E-mail address:
[email protected]. † Department of Nuclear Medicine. ‡ Department of Radiology. | Current address: Memorial Sloan-Kettering Cancer Center, Nuclear Medicine Service Mail Box 77, 1275 York Avenue, New York, NY 10021. § Current address: The Methodist Hospital, Department of Radiology, 6565 Fannin Street, MB1-066, Houston, TX 77030.
Integrins expressed on activated endothelial cells regulate cell migration, differentiation, and proliferation during angiogenesis (3). One of these integrins, integrin Rvβ3, is overexpressed in tumor-induced angiogenic vessels, various malignant human tumors, and diseases such as osteoporosis, rheumatoid arthritis, and macular degeneration, whereas it is not expressed in blood vessels of normal mature tissues (4, 5). Integrin Rvβ3 is a receptor for extracellular proteins including vitronectin, fibronectin, and fibrinogen, which contain an arginine-glycineaspartic acid (RGD) sequence (6). Recently, RGD peptides and peptidomimetic antagonists (IA) specific for the integrin receptors have been synthesized and labeled with various γ and positron emitters (7-13) for scintigraphic detection, and γ- and β-emitters for radiotherapy of tumors (10, 14-24). Steady progress has been reported in optimizing the radiolabeling methods of RGD peptides to improve the receptor-binding and tumor-targeting pharmacokinetics (18, 25, 26). Recently, RGD peptides were conjugated to antibodies to increase the receptorbinding affinity (27). It was also reported that RGD-conjugated Fc fragments (26) and RGD-conjugated anti-CD3 antibodies (28) inhibited angiogenesis in the tumor and redirected the cytolytic property of cytotoxic T lymphocytes to Rvβ3-expressing endothelial cells, respectively.
10.1021/bc0603485 Not subject to U.S. Copyright. Published 2007 by American Chemical Society Published on Web 03/22/2007
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To increase the affinity, Burnett et al. (29) modified the ethylamine terminus of IA to a series of the corresponding carbamate derivatives and reported that the hydrophobic carbamate-linked appendages improved the Rvβ3 receptorbinding affinity of the parent compound IA by 2 to 20 times. The affinity of IA was also improved by coupling to a cationic polymerized lipid-based nanoparticle that was successfully used to deliver a gene (30) or 90Y (20) to neovasculature for tumor regression. We have undertaken the conjugation of high levels of IA molecules to monoclonal antibody T101 to improve the affinity of IA and to test a possibility of targeting Rvβ3 receptors on neovasculature. T101 (MoAb) is a murine anti-CD5 monoclonal antibody of IgG2a isotype and is nonspecific to Rvβ3 receptor. We used whole IgG as a carrier because this IgG has a large number (>60) of lysine residues, each of which contains a reactive -amino group. Thus, intact MoAb can be conjugated at the lysine residues with a large number of antagonists to increase receptor-binding avidity, and sequentially conjugated with other molecules such as pendant groups to radiolabel with γ or positron emitters for scintigraphic imaging or β-emitters for cancer therapy and fluorescein for optical imaging. We also used intact MoAb instead of antibody fragments, because we were interested in assessing the status of tumor-induced angiogenesis and because IgG does not readily cross the endothelial tight junctions due to its large molecular size (150 kDa), thus more suitable for specific imaging of tumor-induced neovasculature than antibody fragments. Another advantage of using antibody as a carrier is that it would also provide an opportunity to diversify into a pretargeting approach using an antibody specific for radiometal chelates as a carrier described initially by Meares et al. (31-36) or a three-step pretargeting approach involving injection of three components: MoAb conjugated with both IA and biotin, avidin, and radiolabeled biotin (37-42). These pretargeting approaches could potentially amplify tumorto-nontumor background ratios. This paper describes the conjugation of IA molecules to MoAb; the labeling of IAconjugated MoAb with FITC, 125I, and 111In; in vitro binding study with Rvβ3 receptor; and biodistribution and histochemistry studies using nude mice with the receptor-positive M21 tumor to demonstrate the accumulation of the IA-conjugated MoAb on tumor-induced neovasculature.
2. EXPERIMENTAL PROCEDURES 2.1. Conjugation of SATA to MoAb. A heterofunctional cross-linker, N-succinimidyl-S-acetylthioacetate (SATA; Pierce, Rockford, IL) at a concentration ranging from 0.135 to 13 mM was reacted with MoAb (27 µM) in 0.1 M phosphate buffer, pH 7.2, at room temperature for 1 h. MoAb conjugated with several different levels of S-acetylthioacetyl moieties (ATA) was purified with size exclusion Zeba Desalt Spin Columns (Pierce, Rockford, IL). To determine the level of the S-acetylthioacetato moiety per MoAb, the S-acetyl protecting group was deacetylated with hydroxylamine (0.02 M) at room temperature in the presence of 1 mM EDTA to generate the mercaptoacetyl (ASH) group. The concentration of the sulfhydryl group was then titrated with Ellman’s reagent (Pierce, Rockford, IL) following manufacturer’s instructions. The MoAb concentration was determined by measuring the absorbance at 280 nm ( ) 1.4 mL/mg) or by Bradford assay with Coomassie plus protein assay reagent (Pierce, Rockford, IL) according to manufacturer’s instructions (43). 2.2. Preparation of S-Acetylthioaceto-IA. The synthesis of IA was previously reported (29). The amino terminus of IA (0.02 M) was reacted with SATA (0.2 M) in 0.1 M sodium phosphate buffer, pH 7.2, at room temperature for 1 h to modify IA with a moiety containing an ATA. The product mixture was
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loaded on a Sep-Pak C18 cartridge (Pierce, Rockford, IL) and eluted with a stepwise gradient of acetonitrile solution from 0% to 50% acetonitrile in water. The product IA-ATA was collected with 50% acetonitrile. The chemical purity of IAATA was confirmed by HPLC (Gilson, Middleton, WI) equipped with a reverse-phase ACE 5C-18 column (4.6 × 100 mm, 5 µm, MAC-MOD Analytical Inc., Chadds Ford, PA) and a UV monitor. The HPLC system was operated under the following gradient elution conditions: eluent A, 50 mM triethylammonium phosphate in water; eluent B, 100% acetonitrile; gradient, 0-2 min with 100% eluent A, 2-5 min with 100%-80% eluent A, 5-12 min with 80%-65% eluent A, and 12-20 min with 65%-0% eluent A; flow rate, 1.0 mL/min. The concentration of IA-SATA was determined by the Ellman’s reagent as described above. 2.3. Conjugation of IA-ATA to MoAb-(ATA)n. IA-ATA was conjugated to MoAb-(ATA)n using a homobifunctional cross-linker, 1,8-bis(maleimido)diethylene glycol (BM[PEO]2). MoAb-(ATA)n (20 µM) was reacted with BM[PEO]2 at a molar excess in the presence of hydroxylamine (0.02 M) and 1 mM EDTA in 0.1 M sodium phosphate, pH 7.2, at room temperature for 30 min. To ensure MoAb-(ATA)n (n ) 5 and 10) conjugation to only one maleimido group of the homobifunctional BM[PEO]2 and prevent the inter- and intramolecular cross-linking of MoAb caused by BM[PEO]2, MoAb-(A-SH)n (n ) 5 and 10) was reacted with BM[PEO]2 at g50× molar excess to the concentration of A-SH. The resulting product, MoAb-(acetyl-S-succinimidomaleimido[PEO]2)n, was purified using a size exclusion Zeba Desalt Spin Columns (Pierce, Rockford, IL). The purified product was immediately reacted with IA-A-SH at a 4× molar excess to the concentration of the maleimido[PEO]2. The level of IA-(A-SH) conjugated per MoAb was determined by titrating the concentration of IA-(ASH) before and after the conjugation reaction with the Ellman’s reagent as describe above. The product MoAb-(acetyl-Ssuccinimido-[PEO]2-succinimido-S-acetyl-IA)n (MoAb-(A-SS[PEO]2S-S-A-IA)n) was purified with a size exclusion Zeba Desalt. The protein concentration was determined as described above. 2.4. Radiolabeling. The final products MoAb-(A-S-S[PEO]2SS-A-IA)n with n ) 5 and 10 were conjugated with 2-(pisothiocyanatobenzyl)cyclohexyl-DTPA (CHX-A′′; Macrocyclic, Dallas, TX) and labeled with 111InCl3 (Perkin-Elmer, Boston, MA) as described previously (44, 45). The product was purified with a size exclusion Zeba Desalt Spin column (Pierce, Rockford, IL) or a microcon filter with a 30 kDa cutoff (Millipore, Bedford, MA). The purified product was labeled with 111In chloride in 0.2 M sodium acetate, pH 4.5, at room temperature for 1 h. DTPA (0.2 mM) was then added to the reaction solution to bind possible free 111In ion. The labeled product was purified with a size exclusion PD-10 column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The radiochemical purity was assessed by HPLC (Gilson, Middleton, WI) equipped with a size exclusion TSK gel G3000SWXL column (7.8 × 300 mm, 5 µm, TOSOH Bioscience, Japan; 0.067 M sodium phosphate/0.15 M sodium chloride, pH 6.8; 1.0 mL/ min), a UV monitor, and an on-line flow radioactivity detector (Bioscan Inc., Washington, DD). 2.5. Fluorescein Labeling. MoAb-(A-S-S[PEO]2S-S-AIA)10 (12 µM) was reacted with FITC (0.18-1.6 mM) in 0.1 M sodium bicarbonate buffer, pH 8.4, for 18 h at room temperature. The labeled product with four fluorescein molecules per MoAb (10 µM) was purified with a size exclusion Zeba Desalt Spin column (Pierce, Rockford, IL) or by HPLC equipped with a size exclusion TSK G3000SWXL column, and the fluorescein-labeled product with a molecular weight similar to monomeric MoAb was collected.
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Scheme 1. Synthesis of IA-Conjugated MoAb
2.6. SDS-PAGE Analysis. To estimate the extent of the interand intramolecular cross-linking caused by the conjugation reaction with BM(PEO)2, electrophoresis was performed with 14% Tris-glycine gel (Invitrogen, Carlsbad, CA) in a running buffer containing 25 mM Tris, 192 mM glycine, pH 8.3, and 0.1% (w/v) SDS (Bio-Rad, Hercules, CA). The MoAb conjugates from the reactions of MoAb-(acetylthiol)10 with BM(PEO)2 at a molar ratio of BM(PEO)2 to thiol ranging from 50 to 200 were treated with 5% β-mercaptoethanol (ICN Biomedicals Inc., Aurora, OH) at 90 °C for 5 min to reduce the interchain disulfide bonds. The samples were loaded on the gel together with a mixture of molecular weight standards (SeeBluePlus2, Invitrogen, Carlsbad, CA; the standards include myosin, phosphorylase B, BSA, glutamic dehydrogenase, alcohol dehydrogenase, carbonic anhydrase, myoglobin lysozyme, aprotinin, and insulin B chain). The runned gel was stained for proteins with colloidal Coomassie blue (Invitrogen, Carlsbad, CA). 2.7. Receptor-Binding Assay. To assess the receptor-binding ability of the 111In labeled MoAb-(A-S-S[PEO]2S-S-A-IA)n, 111In-labeled MoAb with n ) 10 (2.2 × 106 cpm, 0.04 µM)
was incubated with human integrin Rvβ3 (Mw 237 000; Chemicon, Temecula, CA) at a concentration ranging from 0.1 to 0.4 µM in 32 µL PBS at 37 °C for 3 h. To assess the percentage of nonspecific binding, the binding assay was performed in the presence of a 20× molar excess of cold MoAb-(A-S-S[PEO]2SS-A-IA)10 to integrin Rvβ3. In a separate experiment, 111Inlabeled MoAb-(A-S-S[PEO]2S-S-A-IA)n with n ) 5 and 10 (2.2 × 106 cpm, 0.04 µM MoAb) were incubated with 0.4 µM Rvβ3 to determine the effect of the level of IA conjugation on the receptor binding. The percent binding to the integrin Rvβ3 was analyzed by size exclusion HPLC. The binding of the 111In-labeled MoAb to R β shifted the HPLC peak (9.5 min) v 3 for the 111In-labeled MoAb to a higher molecular weight peak (6.8 min). The addition of the molar excess of MoAb-(A-SS[PEO]2S-S-A-IA)10 completely blocked the formation of the higher molecular weight complex. 2.8. Tumor Model. A human M21 melanoma cell line that expresses Rvβ3 was used to make an in vivo tumor model. Human M21 melanoma cells initially received from Dr. David Cheresh (Scripps Research Institutes, La Jolla, CA) were
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Figure 1. Relationship between level of SATA conjugation to MoAb and SATA to MoAb reaction molar ratios. [MoAb] ) 27 µM. The number of SATA conjugated per MoAb increased up to 40 as the reaction concentration of SATA was increased. The values represent means and standard deviations. N ) 5.
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Figure 3. Relationship between formation of MoAb dimer plus polymer and BM(PEO)2 to MoAb-(A-SH)10 reaction molar ratios. MoAb-(A-SH)10 at 15 µM MoAb was reacted with BM(PEO)2 in sodium phosphate buffer, pH 7.2, at room temperature for 0.5 h. The products were analyzed by size exclusion HPLC. The formation of higher molecular weight MoAb due to the intermolecular cross-linking decreased as the concentration of the homobifunctional cross-linker BM(PEO)2 was increased. The formation of the higher molecular weight MoAb became 70% binding ability to Rvβ3 at 0.4 µM were used for the biodistribution studies. The animals were euthanized at 4, 21, and 44 h by CO2 inhalation and exsanguinated by cardiac puncture before dissection. Blood and various organs were removed and weighed, and their decaycorrected radioactivity counts were measured with a γ-counter (Wallac, Inc., Perkin-Elmer, Inc., Boston, MA). The percentage of injected dose per gram (% ID/g) of the blood or each organ was calculated and was normalized to a 20 g mouse. The wholebody (WB) radioactivity count was obtained by adding the
Figure 4. SDS PAGE analysis of MoAb-(COCH2-S-SM[PEO]2)10. MoAb-(COCH2SH)10 (15 µM MoAb) was reacted with BM(PEO)2 in sodium phosphate buffer, pH 7.2, at room temperature for 30 min. The products were treated with 2-mercaptoethanol before the SDS PAGE analysis to reduce the interchain disulfide bridges. Lanes: (A) [BM(PEO)2]/[-SH] ) 50; (B) [BM(PEO)2]/[-SH] ) 100; (C) [BM(PEO)2]/[SH] ) 200; (D) MoAb; (M) molecular weight markers. All three samples showed bands corresponding to 25 and 50 kDa fragments indicating that the homobifunctional cross-linker BM(PEO)2 reacted at a [BM(PEO)2]/[-SH] ratio higher than 50 did not cause the interand intramolecular cross-linking of MoAb.
radioactivity of all organs and that of the carcass measured by the γ-counter, and the whole-body radioactivity was expressed as the percentage of the injected dose (% ID). 2.10. Ex Vivo Fluorescence Microscopy. Mice with M21 tumor were injected iv with the fluorescein-labeled MoAbIA10 (200 µg) in 0.2 mL PBS, pH 7.2, containing 1% BSA. The animals were euthanized at 4 and 21 h by CO2 inhalation and exsanguinated by cardiac puncture before dissection. The animals were injected iv with rhodamine-lectin (1 mg) in 0.2
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Figure 5. Size exclusion HPLC profile for binding of 111In-labeled MoAb-IAn to integrin Rvβ3. (A) 25% of the total 111In-labeled MoAb-IA5 was bound to the receptor at 0.4 µM, whereas (B) 73% of the total 111In-labeled MoAb-IA10 was bound. (C) The receptor binding was completely blocked when 111In-labeled MoAb-IAn was incubated with 0.4 µM Rvβ3 in the presence of 8 µM cold MoAb-IA10.
mL PBS 5 min before the animals were euthanized to delineate vessels during observation with fluorescence microscopy (Olympus, Melville, NY). Tumors were dissected, mounted, and counterstained with 4,6-diamidino-2-phenylindole (DAPI). 2.11. Statistical Analysis. Statistical analysis was performed using ANOVA for comparing multiple groups, and the Student’s t test was performed for unpaired data between two groups. All tests were two-sided, and a probability value (P) of less than 0.05 was considered significant.
3. RESULTS AND DISCUSSION 3.1. Chemistry. This research was undertaken to explore the use of antibody as a carrier of peptidomimetic antagonists specific for integrin Rvβ3 receptor to improve its receptorbinding affinity and to target Rvβ3 receptor on neovasculature. To achieve this goal, T101, an irrelevant MoAb to Rvβ3 receptor, was used as a carrier of a peptidomimetic integrin Rvβ3 antagonist, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-(S)-aminoethylsulfonylamino-β-alanine (IA). Sulfhydryl selective reactions were used to conjugate IA to MoAb, because the sulfhydryl group could be readily quantified with Ellman’s reagent. High levels of IA molecules were conjugated to MoAb in three steps (Scheme 1). In the first step, a large number of S-acetylthioacetato (ATA) moieties were conjugated to MoAb upon the reaction with N-succinimidyl-S-acetylthioacetate (SATA) at a molar excess. The number of ATA molecules conjugated per MoAb increased proportionally to the concentration of SATA, and the conjugation was then leveled off at approximately 40 ATA molecules conjugated per MoAb (Figure 1). The products MoAb-(ATA)n with n ) 5 and 10 were used for the further reactions. In a separate reaction, the S-acetylthioaceto moiety was conjugated to IA. The concentration of the S-acetylthioaceto moiety was estimated by titrating the sulfhydryl group by the Ellman’s reagent following removal of the protecting acetyl group from the S-acetylthioaceto moiety. Hydroxylamine was used for this deacetylation reaction. The optimum concentration of hydroxylamine to complete the deacetylation was determined to be 0.02 M (Figure 2). Hydroxylamine at 0.02 M in phosphate buffer, pH 7.2, completed the deacetylation within 15 min at room temperature without interfering with the addition of the mercaptoacetyl (A-SH) of MoAb-(A-SH)n with n ) 5 or 10 to the maleimido group of BM[PEO]2. Then, the MoAb-(A-SH)n and IA-(A-SH) were
Figure 6. Percent binding of 111In-labeled MoAb-IA10 to Rvβ3. 111In-labeled MoAb-IA10 (
