Tumor Regression by Targeted Gene Delivery to the Neovasculature John D. Hood, Mark Bednarski, Ricardo Frausto, Samira Guccione, Ralph A. Reisfeld, Rong Xiang, and David A. Cheresh

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Scheme. Synthesis of the integrin antagonist that contains a linker to attach to a lipid for incorporation into polymerized vesicles for multivalent display. The amine of taurine 5 was protected as its benzyloxycarbonyl (CBZ) derivative to give 6 followed by formation of the sulfonyl chloride 7 and coupled to the methyl ester of tert-butoxycarbonyl diaminopropionic acid 8 to give compound 9. Saponification of 9 gave compound 10 and removal of the tert-butoxycarbonyl (BOC) group gave the key intermediate 11. Coupling of 11 to the benzoic acid derivative 12 gave compound 13 which was hydrogenated to deprotect the amine and simultaneously reduce the pyrimidine ring to give the integrin receptor antagonist-linker conjugate 14. Coupling of three equivalents of 14 to the tricarboxylic acid chelator lipid 15 using benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP) gave the key trivalent integrin antagonist lipid 16.The compound was designed to incorporate an ethylamine linker and retain the aminosulfonate that is necessary for binding to the integrins. Conditions: (a) 4N NaOH, 10%NaHCO₃; (b) PCl₅; (c) 8, NMM, THF; (d) LiOH, THF, H₂O; (e) TFA, CH₂Cl₂; (f) 12, EDC, HOBT, NMM; (g) H₂, 10%Pd/C, HOAc, HCl; (h) 15, BOP, Et₃N, DMF, CH₂Cl₂.

All solvents and reagents used were of reagent grade. Solvent evaporations were performed under reduced pressure provided from house vacuum or a Welch direct drive vacuum pump at 40 °C. NMR spectra were recorded on a JEOL FX90Q at 90MHz in CDCl3, CD3OD, D₂O or blends thereof as described for each case. (Note: although soluble in CDCl3, the addition of CD3OD to the lipids inhibits formation of inverted micelles and thus provided sharper spectra.) Spectra were referenced to residual CHCl3 (7.25 ppm) for 1H experiments. MALDI-TOF mass spectrometry was performed on PerSeptive DE instrument (Mass Spectrometry, The Scripps Research Institute, La Jolla, CA). TLC was performed on glass backed Merck 60 F254 (0.2 mm; EM Separations, Wakefield RI) and the developed plates routinely sprayed with ceric sulfate (1%) and ammonium molybdate (2.5%) in 10% aqueous sulfuric acid and heated to 150 °C. Other developers include iodine (general use), 0.5% ninhydrin in acetone (for amines), and ultraviolet light (for chromophores).

N-Benzyloxycarbonyl-taurine sodium salt (6).
Taurine, 5 (40 g, 320 mmoles) dissolved in 4N sodium hydroxide solution (80 mL) and water (200 mL). To this solution was added benzyloxycarbonyl chloride, (48 mL, 330 mmoles) drop wise, with vigorous stirring during a period of 4h. The pH was maintained alkaline by the addition of 10% sodium bicarbonate solution (300 mL) and 4N sodium hydroxide solution (45 mL). The reaction mixture was then washed with ether (1000 mL) and the aqueous layer was spin evaporated to dryness, dried under high vacuum over phosphorous pentoxide overnight to yield 12.70 g (14.1%) of 6. ¹H-NMR (D₂O): 7.50 (5H, s, Ar-H), 5.21 (2H, s, Ar-CH₂), 3.62 (2H, t, CH₂), 3.14 (2H, t, CH₂)

2-Benzyloxycarbonylaminoethanesulfonyl chloride (7).
N-CBZ-Taurine sodium 6 (12.7 g, 32 mmols) was suspended in dry diethyl ether (30 mL) under a positive pressure of argon and treated with phosphorous pentachloride (7 g, 33.6 mmoles) in 5 portions over 15 minutes. The reaction was stirred for 4h, at ambient temperature. The solvent was removed by spin evaporation. Ice water (10 g) was added and the residue was triturated after cooling the flask and the contents in an ice bath. More ice water (50 g) was added and the product solidified. The solids were collected by filtration washed with ice water (20 mL) and dried over phosphorous pentoxide overnight to yield 6.95 g (78.0%) of 7. ¹H-NMR (CDCl₃): 7.35 (5H, s, Ar-H), 5.12 (2H, s, Ar-CH₂), 3.89 (2H, t, CH₂) overlapping with 3.85 (2H, t, CH₂)

Methyl 3-butyloxycarbonylamino-2-(S)-benzyloxycarbonylaminoethylsulfonylaminopropionate (9).
A mixture of the sulfonyl chloride 7 (21.6 g, 78.0 mmoles) and Methyl-3-N-butoxycarbonylamine-2-aminopropionate (8, 9.96 g, 39.2 mmoles) in anhydrous tetrahydrofuran (150 mL) under a positive pressure of argon was cooled in an ice bath. To this solution was added N-methylmorpholine (16 mL, 145 mmoles) in anhydrous THF (275 mL) drop wise during a period of 30 min using a dropping funnel previously dried and under a positive pressure of argon. After 1h stirring in the ice bath, by TLC it was observed that all the sulfonyl chloride (R_f = 0.65) had disappeared (eluent: ethyl acetate/hexane 1:1). However there was unreacted diaminopropionic acid (R_f = 0.1, ninhydrin spray) still present. More sulfonyl chloride (5.0 g, 18 mmoles) was added during a period of 3h. The reaction was then filtered and spin evaporated to remove the solvent and dissolved in ethyl acetate (100 mL) and washed with cold dilute hydrochloric acid (20 mL), saturated sodium bicarbonate solution (20 mL) and saturated sodium chloride solution (20 mL) and dried over anhydrous sodium sulfate. The solvent removed by spin evaporation and dried under vacuum over night. The residue was recrystallized by first dissolving in ethyl acetate and then by adding equal volume of hexane to obtain 9 as a colorless solid 13.4 g (74.3%). ¹H-NMR (CDCl₃): 7.36 (5H, s, Ar-H), 5.83 (1H, d, NH), 5.55 (1H, t, NH), 5.12 (2H, s, Ar-CH₂), 5.06 (1H, t, NH), 4.26 (2H, m, CH), 3.79 (3H, s, CH₃), 3.70 (2H, dd, CH₂), 3.26 (2H, dd, CH₂), 1.43 (9H, s, (CH₃)₃)

3-butyloxycarbonylamino-2-(S)-benzyloxycarbonylaminoethylsulfonylaminopropionic acid (10).
A solution of the methyl ester 9 (13.3 g, 28.9 mmoles) in tetrahydrofuran (160 mL) was cooled in an ice bath and to this solution was added a solution of lithium hydroxide (5.42 g, 128 mmoles) in ice water (160 mL). The reaction mixture was slowly warmed to ambient temperature by removing the ice bath and the mixture was stirred at ambient temperature for 1h. The organic solvent was then removed by spin evaporation. The residual aqueous portion was washed with diethyl ether (20 mL) and then acidified to pH:4 using diluted hydrochloric acid. This solution was cooled in an ice bath and then mixed with ethyl acetate (100 mL) and then further acidified to pH 1 using ice-cold diluted hydrochloric acid and immediately extracted with ethyl acetate (2x200 mL). The ethyl acetate layer was washed with brine (50 mL) and dried over anhydrous sodium sulfate. The solvent was then removed by spin evaporation and dried under high vacuum over night to obtain 13.3 g of a foamy solid, which was recrystallized from hexane/ethyl acetate (1:1) to obtain 11.6 g (89.7%) of 10. ¹H-NMR (CDCl₃): 7.33 (5H, s, Ar-H), 6.12 (1H, d, NH), 5.68 (1H, t, NH), 5.26 (1H, t, NH), 5.1 (2H, s, Ar-CH₂), 4.24 (2H, m, CH₂), 3.67 (2H, t, CH₂), 3.27 (2H, t, CH₂), 1.45 (9H, s, C(CH₃)₃)

3-amino-2-(S)-benzyloxycarbonylaminoethylsulfonylaminopropionic acid (11). N-BOC--amino acid 10 (11.5 g, 25.8 mmoles) was treated with trifluoroacetic acid (68 mL) in methylenechloride (350 mL) for 1.5h and then spin evaporated to dryness. The residue was dissolved in water (200 mL) and lyophilized to obtain 11 as a solid of 10.9 g (98.8%). ¹H-NMR (CDCl₃): 7.30 (5H, s, Ar-H), 6.07 (1H, d, NH), 5.61 (1H, t, NH), 5.20 (1H, t, NH), 5.17 (2H, s, Ar-CH₂), 4.11 (2H, m, CH₂), 3.53 (2H, t, CH₂), 3.32 (2H, t, CH₂). DCI-MS for C₁₃H₁₉N₃O₆S: m/z (ion) 346 (M+H) (calcd for C₁₃H₁₉N₃O₆S + H, m/z 346)

4-[2-(pyrimidin-2-ylamino)ethyloxy]benzoyl-2-(S)-benzyloxycarbonylaminoethylsulfonylamino--alanine (13).
The benzoic acid derivative 12 (6.4 g, 24.7 mmoles) and N-hydroxysuccinimide (3.6 g, 31 mmoles) were dissolved in anhydrous dimethylsulfoxide (110 mL), under a positive pressure of argon and cooled in an ice bath. To this solution was added 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (4.9 g, 25.6 mmoles). The solution was stirred at ice-cold temperature for 1h and then allowed to warm to ambient temperature and continued to stir at room temperature for another 24h. To this mixture was added a solution of the -amino acid 11 (12.2 g, 25.8 mmoles) followed by N-methylmorpholine and stirred under argon for 3 days. The mixture was then poured into water (1 L) and acidified with diluted hydrochloric acid to pH 1.5 and extracted with ethyl acetate (5x500 mL). The organic phase was washed with saturated sodium chloride solution (50 mL) and then dried over anhydrous sodium sulfate. The solvent was removed by spin evaporation and the residue was triturated in ethyl acetate, filtered and dried under high vacuum to obtain 10.5 g (72.5%) of 13. ¹H-NMR (DMSO-d₆): 8.30 (2H, d, Ar-H), 7.99 (2H, d, Ar-H), 7.34 (5H, s, Ar-H), 7.00 (2H, d, Ar-H), 6.60 (1H, dd, Ar-H), 5.01 (2H, s, CH₂), 4.15 (1H, t, CH), 3.67 (2H, t, CH₂), 3.56 (2H, t, CH₂), 3.17 (2H, t, CH₂)

4-[2-(3,4,5,6-Tetrahydropyrimidin-2-ylamino)ethyloxy]benzoyl-2-(S)-aminoethylsulfonylamino--alanine (14 = R’NH₂).
A solution of the pyrimidine derivative 14 (3.7 g, 6.4 mmoles) was dissolved in acetic acid (190 mL) and concentrated hydrochloric acid (17 mL). This solution was treated with 10% palladium over carbon (1.62 g) and hydrogenated at 45 psi of hydrogen gas for 5h. The mixture was then filtered through celite and washed with water. The solvent was removed by spin evaporation and dried under high vacuum. The residue was dissolved in water (100 mL) and pH adjusted to 7.0 with 1N sodium hydroxide solution and then spin evaporated to dryness. The residue was dissolved in methanol (20 mL) and filtered. The filtrate was spin evaporated and dissolved in water (275 mL) and lyophilized. The lyophilized material was then recrystallized from water to obtain 2.96 g (78.9%) of the product. ¹H-NMR (D₂O): 7.80 (2H, d, Ar-H), 7.14 (2H, d, Ar-H), 4.49 (1H, s, CH_aH_b), 4.28 (2H, t, CH₂), 3.94 (1H, dd, CH_aH_b), 3.61 (6H, m, CH₂), 3.32 (4H, t, CH₂), 1.90 (2H, t, CH₂). ES-MS for C₁₈H₂₈N₆O₆S: m/z (ion) 457 (M+H) (calcd for C₁₈H₂₈N₆O₆S + H, m/z 457)

Determination of chiral purity of 4-[2-(3,4,5,6-Tetrahydropyrimidin-2-ylamino)ethyloxy]benzoyl-2-(S)-aminoethylsulfonylamino--alanine (14).
To 1mL of a solution of 14 (1.4 mg in 636 L of water and 636 L of acetone) was added 1 mL of a solution of Marfey’s reagent (1.4 mg/mL). To the turbid solution was added 500 L of acetone, 1.5 mL of water, and 400 L of 1M NaHCO₃ solution and incubated at 40¹C for 24h. The solution was then neutralized with 200 L of 2M hydrochloric acid solution and injected into HPLC. A control solution made without 14 was also treated similarly and injected into HPLC. A sample of 14 was epimerized by heating it to melt and then cooled to room temperature. The epimerized compound was treated similar to that of 14. Compound 14 showed only one peak corresponding to the SS diastereoisomer and the SR diastereoisomer was completely absent indicating the %ee was >99.9% (t_R = 12.2 min for SS diastereoisomer and 10.8 min for SR diastereoisomer)

[(PDA-PEG3)₂-DTPA-(CONHPM)₃] (16)
Compound 15 (69 mg, 50 mole) was dissolved in anhydrous CH₃CN (5 mL), anhydrous CH₂Cl₂ (2 mL) and Et₃N (1 mL) in a 3-neck RB flask, previously flame dried and filled with argon. To this solution was added the BOP reagent (134 mg 150 mole) and stirred well for 5 minutes. A solution of 14 (69 mg, 150 moles) was prepared in a dry vial filled with argon, in a mixture of anhydrous CH₃CN (5 mL) and anhydrous DMF (2 mL). The cloudy solution of 14 was added to the lipid solution using a dry syringe with continuous stirring. The reaction was allowed to stir for 10h in dark. TLC (solvent: CHCl₃, CH₃OH, H₂O, and CH₃COOH (73:27:4:1) showed complete disappearance of the starting material (R_f = 0.53). There was one major product (R_f = 0.2) and 5 minor products (R_f < 0.16). The solvent was removed by spin evaporation and dried under high vacuum for 24 hours. The crude product was purified by normal phase HPLC using a semi preparative silica column, flow rate 5 mL/min. Gradient starting with 100% CHCl₃ for 5 min, then 75% CHCl₃/25% CH₃OH for 10 min, then 50% CHCl₃/50% CH₃OH for 10 minutes, then 25% CHCl₃/75% CH₃OH for 10 minutes, and finally for 20 minutes with 100% CH₃OH. The fractions (t_R = 35 to 37 minutes) that contained the major product were combined and spin evaporated to remove the solvent, dried under high vacuum for 24h to obtain 35.5 mg (26.5%) of the desire product. ¹H-NMR (CDCl₃/CD₃OD(1/1)): 7.74 (6H, bm, Ar-H), 6.96 (6H, bm, Ar-H), 4.59, 4.20, 3.80, 3.61, 3.32, 2.68, 2.20, 2.05, 1.94, 1.42 (overlapping peaks, 167H, bm, all CH and CH₂), 0.84 (6H, t, CH₂). High resolution MALDI-FTMS: m/z 2681.4711 (calcd for C₁₃₀H₂₀₉N₂₅O₂₉S₃ + H, m/z 2681.4882)

Preparation of Polymerized Nanoparticles (NP1 through NP3).

Appropriate amounts of purified lipid components (1–4) dissolved in organic solvents (CHCl3 and CH3OH in a ratio 1:1) were combined. The solvents were evaporated and the residue dried in vacuo for 24h while shielded from light. Distilled and deionized water was added to yield a heterogeneous solution 30 mM in lipid concentration. The lipid/water mixture was then sonicated with a probe-tip sonicator for at least one hour and the solution became clear. Throughout sonication, the pH of the solution was maintained between 7.0 and 7.5 with 0.1N NaOH solution, and the temperature was maintained above the gel-liquid crystal phase transition point (Tm). To polymerize the liposomes, the liposome solution was transferred to a petri dish resting on a bed of wet ice, cooled to 0 °C, and irradiated at 254 nm for at least one hour with a hand-held UV lamp placed ~1 cm above the petri dish, yielding NPs. The NPs were then filtered through a 0.2 m filter and collected.

Supplemental Table 1.

Physical Characterstics of v3-NP

Material					Size (nm)	Zeta Potential (mv)	Cell Adhesion Assay IC50 (M of v3 ligand on NPs)	Effect of Multivalency IC50 (Free [v3 ligand]/[v3 ligand] on NPs)
v3-NP	41.7 ± 2.2	35	0.35	183
Cell adhesion inhibition study was done on plates coated with vitronectin using a human melonoma cell line M21. The multivalent particle complex v3-NP as well as the monomeric v3 ligand were separately incubated with M21 cells and applied onto the 48 well plates coated with vitronectin. After 1h incubation, the wells were washed and the cells that adhered were stained with a solution of crystal violet and the OD at 590 nm was measured. The OD measured was proportional to the number of cells bound to the vitronectin plate and was plotted against the concentration of 10 on the surface of the NPs in different formulations to calculate the IC50. The reported values are average of quadruplicate values and have a maximum standard error of ± 0.05. The multivalency effect was calculated by dividing the IC50 for free v3 ligand by the IC50 of the concentration of v3-NP.

Generation of v3-NP-DNA Particles
DNA and v3-NP were mixed in a ratio of 25 g of CsCl purified DNA: 450 nanomoles of v3-NP/200 l injection volume. Prior to mixing all solutions were warmed to 37°C. For each mouse injected, 15 l of 30 mM v3-NP and 25 g of DNA were diluted into seperate100 l volumes of 5% dextrose (pH 7.2). The DNA mixture was then rapidly pipetted into the v3-NP solution. Precipitation as observed visually or by light scattering was not detected in any solutions in which the DNA had been CsCl purified. DNA-v3-NP was generally injected into the treatment mice within 48 hours of preparation.

Immunostaining
At time of tumor resection, animals were first anesthetized followed by animal followed by opening the right atria and intracardial perfusion with 1 ml of heparinized saline. Tumors are then resected and fixed in Zn-formalin buffer followed by paraffin embedding and sectioning.

To further reduce autofluorescence, slides were incubated 3X for 10 minutes following paraffin removal in a fresh ice-cold 1 mg/ml solution of sodium borohydride solution in PBS on ice immediately before use. Slides were further pretreated using a microwave citrate protocol described at:
http://www.bdbiosciences.com/pharmingen/protocols/Microwave_Citrate_Pretreatment.shtml

Slides were then rinsed in PBS and treated for TUNEL staining according to manufacturer’s directions (Apoptag kit, Serologicals, Inc., GA) with the exception that primary antibodies directed against FLAG (Zymed, OR) and VE-Cadherin (Santa Cruz Biotechnologies, CA) were applied in conjunction with the anti-digoxigenin for TUNEL followed by blocking in normal goat serum, and exposure to secondary antibodies conjugated to Alexa fluors (Molecular Probes, OR). Slides were then mounted and visualized using confocal microscopy.

Supplemental Figure 1. ATP-Raf blocks FGF and VEGF-mediated Raf activation and angiogenesis (A) Bovine Aortic Endothelial Cells (BAECs) were transfected with pCI-ATP-Raf (pCI, Promega, Madison, WI) using lipofectamine under manufacture’s directions. After 24 hours cells were exposed to 50 ng/ml bFGF or VEGF for 5 minutes and Raf kinase was determined as described before (Hood et al., J. Biol.Chem. 273, 23504-8 (1998)). (B)Chick CAMs (10 days) were exposed to filter paper disks saturated with RCAS-ATP-Raf followed by stimulation with either bFGF or VEGF (2 mg/ml) for 72 hours. Blood vessels were enumerated by counting vessels branch points in a double blinded manner. Each bar represents the mean ± SEM of 24 replicates.

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Supplemental Figure 2. Representative treatment animals and organs. Animals were treated as described in the primary text. At the designated timepoints anmals were euthanized, organs resected, and photographed. Panels A & B are representative animals from the subcutaneous melanoma model (Fig. 4A). Panels C & D are representative lungs and livers from the established syngeneic pulmonary and hepatic metastases models (figures 4B, C & D).

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Supplemental Figure 3. Dose-dependent regression of pulmonary metastases in response to v3-NP-Raf(–). Pulmonary metastases were formed by tail vein injection of CT-26 colon carcinoma cells (5 x 10⁵) into syngeneic Balb/C mice (23). Mice (n = 5) were injected after establishment of tumors at days 10 and 17 with the indicated quantity of DNA conjugated to v3-NP. Lungs were harvested at day 24 and weighed.

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