Complexes of Viral-Based Therapeutic Agents and Modified Poly(Beta-Amino Ester)s

Abstract

Disclosed are complexes of virus-based therapeutic agents with polymers that are poly(beta-amino ester)s (PBAEs) modified with at least one oligopeptide. Also disclosed are methods of treatment using these complexes and methods of encapsulating said complexes to form nanoparticles.

Claims

1. A complex of a virus-based therapeutic agent with a polymer of formula I: ##STR00040## wherein each L.sub.1 and L.sub.2 is independently selected from the group consisting of: ##STR00041## O, S, NR.sub.x and a bond; wherein R.sub.x is independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, heteroalkyl, heterocycloalkyl, acyl, aryl or heteroaryl; L.sub.3 is independently selected from the group consisting of alkylene, alkenylene, heteroalkylene, heteroalkenylene, arylene or heteroarylene; or ##STR00042## at least one occurrence of L.sub.3 is wherein T.sub.1 is ##STR00043## and T.sub.2 is selected from H, alkyl or ##STR00044## wherein L.sub.T is independently selected from the group consisting of: ##STR00045## O, S, NR.sub.x and a bond, wherein R.sub.x is independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, heteroalkyl, heterocycloalkyl, acyl, aryl or heteroaryl, and the remaining L.sub.3 groups are independently selected at each occurrence from the group consisting of alkylene, alkenylene, heteroalkylene, heteroalkenylene, arylene or heteroarylene; L.sub.4 is selected from the group consisting of ##STR00046## L.sub.5 is independently selected from the group consisting of alkylene, alkenylene, heteroalkylene, heteroalkenylene, arylene or heteroarylene; R.sub.1 and R.sub.2 and R.sub.T (if present) are independently selected from an oligopeptide and R.sub.y; wherein at least one of R.sub.1 and R.sub.2 and R.sub.T (if present) is an oligopeptide; and wherein R.sub.y is selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, heteroalkyl, heterocycloalkyl, acyl, aryl or heteroaryl; each R.sub.3 is independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, heteroalkyl, heterocycloalkyl, acyl, aryl, heteroaryl and polyalkylene glycols, wherein said polyalkylene glycol is either bound directly to the nitrogen atom to which R.sub.3 is attached or bound to the nitrogen atom to which R.sub.3 is attached via a linker moiety, wherein said linker moiety is an alkylene, cycloalkylene, alkenylene, cycloalkenylene, heteroalkylene, heterocycloalkylene, arylene or heteroarylene group; and n is an integer from 5 to 1,000; or a pharmaceutically acceptable salt thereof.

2. The complex according to claim 1, wherein L.sub.3 is independently selected from the group consisting of alkylene, alkenylene, heteroalkylene, heteroalkenylene, arylene or heteroarylene.

3. The complex according to claim 1, wherein at least one occurrence of L.sub.3 is ##STR00047## wherein T.sub.1 is ##STR00048## and T.sub.2 is selected from H, alkyl or ##STR00049## wherein L.sub.T is independently selected from the group consisting of: ##STR00050## O, S, NR.sub.x and a bond; wherein R.sub.x is independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, heteroalkyl, heterocycloalkyl, acyl, aryl or heteroaryl, and the remaining L.sub.3 groups are independently selected at each occurrence from the group consisting of alkylene, alkenylene, heteroalkylene, heteroalkenylene, arylene or heteroarylene.

4. The complex according to any one of claims 1 to 3, wherein at least one R.sub.3 group is a polyalkylene glycol, preferably a polyethylene glycol.

5. The complex according to claim 4, wherein the at least one R.sub.3 group which is a polyalkylene glycol is bound to the nitrogen atom of an L.sub.4 group either directly or through a linker moiety.

6. The complex according to claim 4 or claim 5, wherein the at least one R.sub.3 group which is a polyalkylene glycol is bound to the nitrogen atom to which it is attached through a linker moiety which is an alkylene, alkenylene or heteroalkylene group.

7. The complex according to claim 4 or claim 5, wherein the at least one R.sub.3 group which is a polyalkylene glycol is bound directly to the nitrogen atom to which it is attached.

8. The complex of any one of the preceding claims, wherein the or each oligopeptide comprises from 3 to 20 amino acid residues.

9. The complex of any one of the preceding claims, wherein the or each oligopeptide has a net positive charge at pH 7.

10. The complex of claim 9, wherein the or each oligopeptide comprises amino acid residues selected from the group consisting of lysine, arginine and histidine.

11. The complex of any one of the preceding claims, wherein the or each oligopeptide is a compound of Formula VII: ##STR00051## wherein p is an integer from 2 to 19 and wherein R.sub.a is selected at each occurrence from the group consisting of H.sub.2NC(NH)NH(CH.sub.2).sub.3, H.sub.2N(CH.sub.2).sub.4 (1H-imidazol-4-yl)-CH.sub.2.

12. The complex of any one of the preceding claims, wherein R.sub.1 and R.sub.2 are both oligopeptides.

13. The complex of claim 12, wherein R.sub.1 and R.sub.2 are different oligopeptides.

14. The complex of any one of claims 1-11, wherein one of R.sub.1 and R.sub.2 is an oligopeptide and one of R.sub.1 and R.sub.2 is R.sub.y.

15. The complex of any one of the preceding claims, wherein n is from 10 to 700, or from 20 to 500.

16. The complex of any one of the preceding claims, wherein R.sub.y is selected from a group consisting of hydrogen, (CH.sub.2).sub.mNH.sub.2, (CH.sub.2).sub.mNHMe, (CH.sub.2).sub.mOH, (CH.sub.2).sub.mCH.sub.3, (CH.sub.2).sub.2(OCH.sub.2CH.sub.2).sub.mNH.sub.2, (CH.sub.2).sub.2(OCH.sub.2CH.sub.2).sub.mOH or (CH.sub.2).sub.2(OCH.sub.2CH.sub.2).sub.mCH.sub.3 wherein m is an integer from 1 to 20.

17. The complex of any one of the preceding claims, wherein each L.sub.3 is independently selected from the group consisting of C.sub.1-10 alkylene-(SS).sub.qC.sub.1-10 alkylene-, wherein q is 0 or 1.

18. The complex of any one of the preceding claims, wherein each R.sub.3 is independently selected from hydrogen, C.sub.1-6 alkyl, C.sub.1-6 alkenyl, C.sub.1-6 alkynyl, C.sub.1-6 hydroxyalkyl, hydroxyl, C.sub.1-6 alkoxy, halogen, aryl, heterocyclic, heteroaryl, cyano, O.sub.2CC.sub.1-6alkyl, carbamoyl, CO.sub.2H, CO.sub.2C.sub.1-6alkyl, C.sub.1-6 alkylthioether, thiol, ureido, and polyalkylene glycols, wherein said polyalkylene glycol is either bound directly to the nitrogen atom to which R.sub.3 is attached or bound to the nitrogen atom to which R.sub.3 is attached via a linker moiety, wherein said linker moiety is an alkylene, cycloalkylene, alkenylene, cycloalkenylene, heteroalkylene, heterocycloalkylene, arylene or heteroarylene group.

19. The complex of any one of the preceding claims, wherein L.sub.4 is selected from N(R.sub.3) and/or wherein L.sub.3 is selected from C1-6 alkylene groups.

20. The complex of any one of the preceding claims, wherein at least one R.sub.3 group is polyethylene glycol.

21. A composition comprising a virus-based therapeutic agent coated with polymeric material comprising or consisting of polymer(s) of Formula I as defined in any one of claims 1 to 20.

22. The composition of claim 21, wherein the composition comprises nanoparticles containing the virus-based therapeutic agent coated with polymeric material comprising or consisting of polymer(s) of Formula I as defined in any one of claims 1 to 20.

23. The complex of any one of claims 1 to 20 or the composition of claim 21 or 22 wherein the virus-based therapeutic agent and the polymer(s) are non-covalently linked.

24. The complex of any one of claim 1 to 20 or 23 or the composition of any of claims 21 to 23, wherein the surface of said virus-based therapeutic agent comprises binding sites suitable for binding to a polymer of Formula I wherein the or each oligopeptide has a net positive charge at pH 7.

25. The complex of any one of claims 1 to 20 or 23 to 24 or the composition of any of claims 21 to 24, wherein the virus-based therapeutic agent is selected from an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpex simplex viral vector, a vaccinia viral vector, a vesicular stomatitis viral vector, a reoviral vector, or a Semliki forest viral vector.

26. The complex or composition of claim 25, wherein the virus-based therapeutic agent is selected from an adenoviral vector, an adeno-associated viral vector, a retroviral vector, and a lentiviral vector, and preferably wherein the virus-based therapeutic agent is an adenoviral vector or adeno-associated viral vector.

27. A complex according to any one of claims 1 to 20 or 23 to 26 or a composition according to any one of claims 21 to 26 for use in medicine.

28. A complex according to any one of claims 1 to 20 or 23 to 26 or a composition according to any one of claims 21 to 26 for use in systemic viral gene therapy, particularly in the treatment of cancer, particularly liver cancer or pancreatic cancer.

29. A method of encapsulating a complex of a virus-based therapeutic agent and one or more polymers of Formula I according to any one of claims 1 to 20 to form nanoparticles, the method comprising the steps of: providing a virus-based therapeutic agent; providing the polymer(s) of Formula (I); and contacting the virus-based therapeutic agent and the polymer(s) under suitable conditions to form nanoparticles.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0222] FIG. 1 shows the results of coagulation assays.

[0223] FIG. 2 shows the results of assays to determine platelet activation. The polymer assays are shown as the left hand bar in each of the polymer and polymer+ADP results of FIG. 2. The DMSO assays are shown as the right hand bar in each of the polymer and polymer+ADP results of FIG. 2.

[0224] FIG. 3 depicts a schematic representation of the oncolytic adenovirus AdNuPARmE1A.

[0225] FIG. 4 depicts schematic representations of the adenoviruses AduPARmE1A and AdNuPARmE1A.

[0226] FIG. 5 shows a virus (1) without the polymer coating according to the invention, and the same virus attacked by a binding antibody (2)

[0227] FIG. 6 shows a coated virus (1) with a polymer coating (3) according to the invention. The antibody (4) is unable to bind to the virus due to the presence of the polymer coating.

[0228] FIG. 7 shows the results of assays to determine the masking capacity of the PBAE polymers against neutralizing antibodies.

[0229] FIG. 8 shows the results of assays to determine the activation of the adaptive immune response, specifically the determination of the ND50 discussed hereinbelow.

[0230] FIG. 9 shows the results of assays to determine the blood circulation kinetics.

[0231] FIG. 10 shows the results of assays to determine liver tropism where RLU refers to Relative Light Units.

[0232] FIG. 11 shows the results of assays to determine tumour tropism where RLU refers to Relative Light Units.

[0233] FIG. 12 shows the protocol of the anti-tumoral activity study set out in example 16.

[0234] FIG. 13 shows the results of the study performed in example 16.

[0235] FIG. 14 shows the results of the study performed in example 16.

[0236] FIG. 15 shows the results of the study performed in example 17.

[0237] FIG. 16 shows the results of the study performed in example 17.

[0238] FIG. 17 shows the results of the study performed in example 17.

[0239] FIG. 18 shows the results of the study performed in example 17.

[0240] FIG. 19 shows a transmission electron microscopy micrograph of SAG-101.

[0241] FIG. 20 shows the results of the study performed in example 19.

[0242] FIG. 21 shows a scanning electron microscopy micrograph of the nanoparticles in example 20.

[0243] The invention is further illustrated by the following examples. It will be appreciated that the examples are for illustrative purposes only and are not intended to limit the invention as described above. Modification of detail may be made without departing from the scope of the invention.

EXAMPLES

Example 1: Synthesis of PBAE Polymers

[0244] Poly(-aminoester)s were synthesized following a two-step procedure, described in the literature (e.g. in Montserrat, N. et al. J. Biol. Chem. 286, 12417-12428 (2011)). First, an acrylate-terminated polymer was synthesized by addition reaction of primary amines with diacrylates (at 1:1.2 molar ratio of amine:diacrylate). Finally, PBAEs were obtained by end-capping modification of the resulting acrylate-terminated polymer with different kinds of amine- and thiol-bearing moieties. Synthesized structures were confirmed by .sup.1H-NMR and FT-IR analysis. NMR spectra were recorded in a 400 MHz Varian (Varian NMR Instruments, Claredon Hills, Ill.) and methanol-d.sub.4 was used as solvent. IR spectra were obtained using a Nicolet Magna 560 (Thermo Fisher Scientific, Waltham, Mass.) with a KBr beamsplitter, using methanol as solvent in evaporated film. Molecular weight determination was conducted on a Hewlett-Packard 1050 Series HPLC system equipped with two GPC Ultrastyragel columns, 10.sup.3 and 10.sup.4 (5 m mixed, 300 mm19 mm, Waters Millipore Corporation, Milford, Mass., USA) and THF as mobile phase. The molecular weight was calculated by comparison with the retention times of polystyrene standards.

Example 2: Synthesis of Acrylate Terminated Intermediate (C32)

[0245] 1,4-butanediol diacrylate (8.96 g, 4.0710.sup.2 mol) and 5-amino-1-pentanol (3.5 g, 3.3910.sup.2 mol) were mixed in a vial. The mixture was stirred at 90 C. for 24 h, and then cooled to room temperature to form a slightly yellow viscous solid, the acrylate terminated intermediate (designated C32). Intermediate C32 was stored at 4 C. before being used in subsequent steps.

##STR00030##

Example 2A: Synthesis of Acrylate Terminated Intermediate (C6)

[0246] ##STR00031##

[0247] In a round-bottomed flask were mixed 5-amino-1-pentanol (3.9 g, 38 mmol), hexylamine (3.8 g, 38 mmol) and 1,4-butanediol diacrylate (18 g, 82 mmol) and the reaction was stirred at 90 C. under nitrogen for 18 h. After cooling down to room temperature, the product (designated C6) was collected as a yellow oil (25 g, n=8, Mw=2300). The product was analysed by NMR and GPC.

[0248] .sup.1H-NMR (CDCl.sub.3): 6.40 (dd, 2H, J 17.3, 1.5 Hz), 6.11 (dd, 2H, J 17.3, 10.4 Hz), 5.82 (dd, 2H, J 10.4, 1.5 Hz), 4.18 (m, 4H), 4.08 (m, 32H), 3.61 (m, 16H), 2.76 (m, 32H, J 7.2 Hz), 2.41 (m, 48H), 1.69 (m, 32H), 1.56 (m, 8H), 1.49-1.20 (m, 40H) and 0.87 (t, 12H, J 6.9 Hz) ppm.

Example 2B: Synthesis of Acrylate Terminated Intermediate Featuring Disulfide Bond

[0249] 4-amino-1-butanol or 5-amino-1-pentanol was polymerized with an equal molar mixture of hexane-1,6-diyl diacrylate and disulfanediylbis(ethane-2,1-diacrylate to form acrylate terminated intermediates featuring a disulfide bond.

Example 3: Synthesis of PBAEs End-Modified with Oligopeptides

[0250] In general, oligopeptide-modified PBAEs were obtained as follows: acrylate-terminated polymer C32 or C32SS and either amine- or thiol-terminated oligopeptide (for example, HS-Cys-Arg-Arg-Arg (CR3), H.sub.2N-Arg-Arg-Arg (R3) or HS-Cys-Glu-Glu-Glu (CE3)other oligopeptides are indicated by similar abbreviations using the standard one-letter code) were mixed at 1:2 molar ratio in DMSO.

[0251] The mixture was stirred overnight at room temperature and the resulting polymer was obtained by precipitation in diethyl ether:acetone (3:1).

[0252] (a) The following synthetic procedure to obtain tri-arginine end-modified PBAEs is shown as an example: Intermediate C32 was prepared as described in Example 2 above. A solution of intermediate C32 (0.15 g, 0.075 mmol) in DMSO (2 ml) was mixed with the corresponding solution of oligopeptide (Cys-Arg-Arg-Arg (CR3; 0.11 g, 0.15 mmol)) in DMSO (1 mL) in an appropriate molar ratio, 1:2 respectively. The mixture was stirred overnight at room temperature, then was precipitated in diethyl ether/acetone (3:1).

##STR00032##

[0253] IR (evaporated film): =721, 801, 834, 951, 1029, 1133 (CO), 1201, 1421, 1466, 1542, 1672 (CO, from peptide amide), 1731 (CO, from ester), 2858, 2941, 3182, 3343 (NH, OH) cm.sup.1

[0254] .sup.1H-NMR (400 MHz, CD.sub.3OD, TMS) (ppm): =4.41-4.33 (br, NH.sub.2O(O)CHNHC(O)CHNHC(O)CHNHC(O)CHCH.sub.2, 4.11 (t, CH.sub.2CH.sub.2O), 3.55 (t, CH.sub.2CH.sub.2OH), 3.22 (br, NH.sub.2C(NH)NHCH.sub.2, OH(CH.sub.2).sub.4CH.sub.2N), 3.04 (t, CH.sub.2CH.sub.2N), 2.82 (dd, CH.sub.2SCH.sub.2), 2.48 (br, NCH.sub.2CH.sub.2C(O)O), 1.90 (m, NH.sub.2C(NH)NH(CH.sub.2).sub.2CH.sub.2CH), 1.73 (br, OCH.sub.2CH.sub.2CH.sub.2CH.sub.2O), 1.69 (m, NH.sub.2C(NH)NHCH.sub.2CH.sub.2CH.sub.2), 1.56 (br, CH.sub.2CH.sub.2CH.sub.2CH.sub.2OH), 1.39 (br, N(CH.sub.2).sub.2CH.sub.2(CH.sub.2).sub.2OH).

[0255] (b) Tri-lysine modified oligopeptides (K3C-C32-CK3) were prepared according to the same protocol and characterized as follows:

[0256] IR (evaporated film): =721, 799, 834, 1040, 1132, 1179 (CO), 1201, 1397, 1459, 1541, 1675 (CO, from peptide amide), 1732 (CO, from ester), 2861, 2940, 3348 (NH, OH) cm.sup.1

[0257] .sup.1H-NMR (400 MHz, CD.sub.3OD, TMS) (ppm): =4.38-4.29 (br, NH.sub.2(CH.sub.2).sub.4CH), 4.13 (t, CH.sub.2CH.sub.2O), 3.73 (br, NH.sub.2CHCH.sub.2S), 3.55 (t, CH.sub.2CH.sub.2OH), 2.94 (br, CH.sub.2CH.sub.2N, NH.sub.2CH.sub.2(CH.sub.2).sub.3CH), 2.81 (dd, CH.sub.2SCH.sub.2), 2.57 (br, NCH.sub.2CH.sub.2C(O)O), 1.85 (m, NH.sub.2(CH.sub.2).sub.3CH.sub.2CH), 1.74 (br, OCH.sub.2CH.sub.2CH.sub.2CH.sub.2O), 1.68 (m, NH.sub.2CH.sub.2CH.sub.2(CH.sub.2).sub.2CH), 1.54 (br, CH.sub.2CH.sub.2CH.sub.2CH.sub.2OH), 1.37 (br, N(CH.sub.2).sub.2CH.sub.2(CH.sub.2).sub.2OH).

[0258] (c) Tri-histidine modified oligopeptides (H3C-C32-CH3) were prepared according to the same protocol and characterized as follows:

[0259] IR (evaporated film): =720, 799, 832, 1040, 1132, 1201, 1335, 1403, 1467, 1539, 1674 (CO, from peptide amide), 1731 (CO, from ester), 2865, 2941, 3336 (NH, OH) cm.sup.1

[0260] .sup.1H-NMR (400 MHz, CD.sub.3OD, TMS) (ppm): =8.0-7.0 (br N(CH)NHC(CH)) 4.61-4.36 (br, CH2-CH), 4.16 (t, CH.sub.2CH.sub.2O), 3.55 (t, CH.sub.2CH.sub.2OH), 3.18 (t, CH.sub.2CH.sub.2N, 3.06 (dd, CH.sub.2CH), 2.88 (br, OH(CH.sub.2).sub.4CH.sub.2N), 2.82 (dd, CH.sub.2SCH.sub.2), 2.72 (br, NCH.sub.2CH.sub.2C(O)O), 1.75 (br, OCH.sub.2CH.sub.2CH.sub.2CH.sub.2O), 1.65 (m, NH.sub.2CH.sub.2CH.sub.2(CH.sub.2).sub.2CH), 1.58 (br, CH.sub.2CH.sub.2CH.sub.2CH.sub.2OH), 1.40 (br, N(CH.sub.2).sub.2CH.sub.2(CH.sub.2).sub.2OH).

Example 3A: Synthesis of PBAEs End-Modified with Oligopeptides (R3C-C6-CR3)

[0261] ##STR00033##

[0262] To obtain the chlorhydrate of the peptide, 20 mL of 0.1 M HCl were added to peptide CRRR (200 mg) and the solution was freeze-dried.

[0263] In a round-bottomed flask were mixed a solution of the PBAE C6 (113 mg, 0.054 mmol) in dimethyl sulfoxide (1.1 mL) and a solution of peptide CRRR chlorhydrate (99 mg, 0.13 mmol) in dimethyl sulfoxide (1 mL). The reaction was stirred at room temperature under nitrogen for 24 h. The reaction mixture was added over diethyl ether-acetone (7:3) and a white precipitate was obtained. The suspension was centrifuged at 4000 rpm for 10 min and the solvent was take off. The solid was washed two times with diethyl ether-acetone (7:3) and dried under vacuum to obtain a white solid (233 mg). The product was analysed by NMR (MeOD) and the structure was in concordance.

Example 3B: Synthesis of Other PBAEs End-Modified with Oligopeptides

[0264] ##STR00034##

[0265] The same procedure described for the synthesis of PBAE R3C-C6-CR3 was used with PBAE C6 for the syntheses of: [0266] PBAE H3C-C6-CH.sub.3 with peptide CHHH. [0267] PBAE K3C-C6-CK3 with peptide CKKK. [0268] PBAE D3C-C6-CD3 with peptide CDDD. [0269] PBAE E3C-C6-CE3 with peptide CEEE.

Example 3C: Synthesis of Other PBARs End-Modified with Oligopeptides

[0270] The procedure described above for the synthesis of PBAE R3C-C6-CR3 can be used with PBAE C32 for the synthesis of: [0271] PBAE R3C-C32-CR3 with peptide CRRR. [0272] PBAE H3C-C32-CH3 with peptide CHHH. [0273] PBAE K3C-C32-CK3 with peptide CKKK. [0274] PBAE D3C-C32-CD3 with peptide CDDD. [0275] PBAE E3C-C32-CE3 with peptide CEEE.

Example 4: Synthesis of PBAEs with Asymmetric End Modifications

[0276] In general, asymmetric oligopeptide-modified PBAEs were obtained as follows: Acrylate-terminated polymer C32 (or C32SS) and either amine- or thiol-terminated oligopeptide (for example, CR3, R.sub.3 or CE3) were mixed at 1:1 molar ratio in DMSO. The mixture was stirred overnight at room temperature. Equimolar amount of a second amine- or thiol-terminated oligopeptide, or of a primary amine, was added and the mixture was stirred overnight at room temperature. The resulting asymmetric PBAE polymers were obtained by precipitation in diethyl ether/acetone (3:1).

[0277] The following synthetic procedure to obtain asymmetric end-modified B3-C32-CR3 PBAEs is shown as an example: a solution of intermediate C32 (0.15 g, 0.075 mmol) in DMSO (2 mL) was mixed with the corresponding solution of oligopeptide Cys-Arg-Arg-Arg (CR3; 0.055 g, 0.075 mmol) in DMSO (1 ml) and was stirred overnight at room temperature. Subsequently, 2-methyl-1,5-pentanediamine (0.017 g, 0.02 ml, 0.15 mmol) was added in the mixture for 4 h at room temperature in DMSO. A mixture of asymmetric end-modified polymer B3-C32-CR3 with B3-C32-B3 and R3C-C32-CR3 was obtained by precipitation overnight in diethyl ether/acetone (3:1). The asymmetric end-modified polymer B3-C32-CR3 may be separated from the mixture by standard methods.

##STR00035##

Example 5A: Synthesis of PEG Modified PBAE

[0278] Step 1: Synthesis of MeO-PEG-COOH To a solution of MeO-PEG (5 g, Mw=2000, 2.5 mmol) and succinic anhydride (0.275 g, 2.75 mmol) in dichloromethane (5 mL) was added triethylamine (0.174 mL, 1.25 mmol). The reaction mixture was stirred at room temperature for 4 h and washed with 1 M HCl (1 ml) twice. The organic phase was washed with brine twice and dried over MgSO.sub.4. The solid was filtered off and the solvent was evaporated under vacuum to obtain a white solid (4.47 g). The product was analysed by NMR (CDCl.sub.3) and the structure was in concordance.

[0279] Step 2: Synthesis of N-Boc 5-Amino-1-Pentanol

[0280] To a solution of 5-amino-1-pentanol (0.525 g, 5.1 mmol) and triethylamine (0.779 mL, 5.6 mmol) in dichloromethane (16 mL) was added a solution of di-tert-butyl dicarbonate (1.1 g, 5.1 mmol) in dichloromethane (5 mL). The mixture was stirred at room temperature for 1 h and then washed with 0.5 M HCl (1 ml) three times. The organic phase was dried over MgSO.sub.4. The solid was filtered off and the solvent was evaporated under vacuum to obtain a white solid (1.3 g). The product was analysed by NMR (CDCl.sub.3) and the structure was in concordance.

[0281] Step 3: Synthesis of MeO-PEG-NHBoc

[0282] To a solution of MeO-PEG-COOH (1 g, 0.49 mmol) in dichloromethane (14 mL) were added dicyclohexylcarbodiimide (151 mg, 0.74 mmol) and N,N-dimethylaminopyridine (9 mg, 0.074 mmol). After 5 min, a solution of N-boc 5-amino-1-pentanol (100 mg, 0.49 mmol) in dichloromethane (1 mL) was added to the mixture. The reaction mixture was stirred at room temperature for 6 h and then the solid was filtered off. The solvent was evaporated under vacuum and the residue was washed with diethyl ether (5 mL) three times. The product was dried under vacuum to obtain a white solid (0.940 g). The compound was analysed by NMR (CDCl.sub.3) and the structure was in concordance with the anticipated structure.

[0283] Step 4: Synthesis of MeO-PEG-NH.sub.2

[0284] To a solution of MeO-PEG-NHBoc (464 mg, 0.21 mmol) in dichloromethane (3 mL) was added trifluoroacetic acid (1.2 mL) at 0 C. The reaction mixture was stirred at 0 C. for 10 min and then it was stirred at room temperature for 2 h. The solvent was reduced under vacuum and the residue was washed with diethyl ether (5 mL) twice. The product was dissolved in dichloromethane (8 mL) and washed with 0.5 M NaOH (1 ml) twice. The organic phase was washed with brine and dried over MgSO.sub.4. The solid was filtered off and the solvent was evaporated under vacuum to obtain a white solid (319 mg). The product was analysed by NMR (CDCl.sub.3) and the structure was in concordance with the anticipated structure.

[0285] Step 5: Synthesis of PBAE C6-PEG

##STR00036##

[0286] 5-Amino-1-pentanol (42 mg, 0.41 mmol), hexylamine (41 mg, 0.41 mmol) and MeO-PEG-NH.sub.2 (314 mg, 0.14 mmol) were mixed in dichloromethane (2 mL) and the solvent was reduced under vacuum. To the residue was added 1,4-butanediol diacrylate (198 mg, 1 mmol) and the reaction mixture was stirred at 90 C. under nitrogen for 18 h. After cooling down to room temperature, the product was collected as a yellow solid (527 mg, n=7). The product was analysed by NMR (CDCl.sub.3) and the structure was in concordance.

[0287] .sup.1H-NMR (CDCl.sub.3): 6.40 (dd, 2H, J 17.3, 1.5 Hz), 6.11 (dd, 2H, J 17.3, 10.4 Hz), 5.82 (dd, 2H, J 10.4, 1.5 Hz), 4.18 (m, 4H), 4.08 (m, 28H), 3.63 (m, OCH.sub.2CH.sub.2O, PEG), 3.37 (s, CH.sub.3O, PEG), 2.76 (m, 28H), 2.43 (m, 42H), 1.80-1.20 (m) and 0.87 (t, 12H, J 6.9 Hz) ppm.

[0288] Step 6: Synthesis of PBAE R3C-C6-CR3-PEG

##STR00037##

[0289] To obtain the chlorhydrate of the peptide, 15 mL of 0.1 M HCl were added to peptide CRRR (150 mg) and the solution was freeze-dried.

[0290] In a round-bottomed flask were mixed a solution of PBAE C6-PEG (92 mg, 0.022 mmol) in dimethyl sulfoxide (1.2 mL) and a solution of peptide CRRR chlorhydrate (40 mg, 0.054 mmol) in dimethyl sulfoxide (1.1 mL). The reaction was stirred at room temperature under nitrogen for 20 h. The reaction mixture was added over diethyl ether-acetone (7:3) and a white precipitate was obtained. The suspension was centrifuged at 4000 rpm for 10 min and the solvent was taken off. The solid was washed two times with diethyl ether-acetone (7:3) and dried under vacuum to obtain a white solid (133 mg). The product was analyzed by NMR (MeOD) and the structure was in concordance.

Example 5B: Synthesis of PEG Modified PBAEs where the PEG is Bound to the PBAE Through a Linker Moiety

Step 1: Synthesis of Methoxy-PEG Acid

[0291] 1. Add methoxy-PEG (5 g, 2.5 mmol) into a round-bottom flask.

[0292] 2. Add dichloromethane (5 mL) to the flask.

[0293] 3. Add succinic anhydride (0.275 g, 2.75 mmol) to the solution.

[0294] 4. Add trietylamine (0.174 mL, 1.25 mmol) to the mixture.

[0295] 5. Then, stir the mixture at room temperature for 4 h.

[0296] 6. Wash the mixture reaction with 1M HCl (1 ml) twice.

[0297] 7. Wash the solution with brine twice.

[0298] 8. Dry the organic phase over MgSO.sub.4.

[0299] 9. Filter off the solid and evaporate the solvent under vacuum.

Step 2: Reaction of Esterification

[0300] ##STR00038##

[0301] 1. Add methoxy-PEG acid (230 mg, 0.11 mmol) in a screw tap tube.

[0302] 2. Add dichloromethane (1.5 mL) to the tube.

[0303] 3. Add dicyclohexylcarbodiimide (34 mg, 0.17 mmol) to the solution.

[0304] 4. Stir the solution for 20 min at room temperature.

[0305] 5. Add a solution of C6 PBAE (200 mg, 0.099 mmol) in dichloromethane (1 mL).

[0306] 6. Then, stir the mixture at room temperature for 20 h.

[0307] 7. Filter off the solid and evaporate the solvent under vacuum.

Step 3: Reaction with Peptides

##STR00039## [0308] 1. Add 0.1 M HCl (20 mL) to peptide Cys-Arg-Arg-Arg (200 mg). [0309] 2. Freeze the solution at 80 C. and freeze-dried the peptide. [0310] 3. Make a solution of C6-linkPEG PBAE (114 mg, 0.027 mmol) in dimethylsulfoxide (0.8 mL). [0311] 4. Make a solution of Cys-Arg-Arg-Arg (50 mg, 0.068 mmol) in dimethylsulfoxide (0.8 mL). [0312] 5. Mix the two solutions in a screw tap tube. [0313] 6. Stir the mixture solution at room temperature for 20 h. [0314] 7. Add dropwise the mixture to 7:3 diethyl ether/acetone (8 mL). [0315] 8. Centrifuge the suspension at 4000 rpm for 10 min and remove the solvent. [0316] 9. Wash the solid with 7:3 diethyl ether/acetone (4 mL) twice. [0317] 10. Dry the product under vacuum. [0318] 11. Make a solution of 100 mg/mL of the product in dimethylsulfoxide.

Example 6: Library of Compounds

[0319] A library of different oligopeptide end-modified PBAEs was synthesized by adding primary amines to diacrylates followed by end-modification. According to Formula I, the oligopeptide end-modified PBAEs shown in Table 1 were synthesized.

TABLE-US-00001 TABLE 1 Library of oligopeptide end-modified PBAEs wherein at least one of R.sub.1 and R.sub.2 is an oligopeptide Polymer L.sub.3 L.sub.4 HL.sub.1-R.sub.1 R3-C32-R3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH H.sub.2N-Arg-Arg-Arg K3-C32-K3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH NH.sub.2-Lys-Lys-Lys H3-C32-H3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH NH.sub.2-His-His-His R3C-C32-CR3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Arg-Arg-Arg K3C-C32-CK3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Lys-Lys-Lys H3C-C32-CH3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-His-His-His B3-C32-R3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH H.sub.2NCH.sub.2(CH.sub.2).sub.2CH(CH.sub.3)CH.sub.2NH.sub.2 B3-C32-CR3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH H.sub.2NCH.sub.2(CH.sub.2).sub.2CH(CH.sub.3)CH.sub.2NH.sub.2 B3-C32-CK3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH H.sub.2NCH.sub.2(CH.sub.2).sub.2CH(CH.sub.3)CH.sub.2NH.sub.2 B3-C32-CH3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH H.sub.2NCH.sub.2(CH.sub.2).sub.2CH(CH.sub.3)CH.sub.2NH.sub.2 R3C-C32-CK3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Arg-Arg-Arg R3C-C32-CH3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Arg-Arg-Arg K3C-C32-CH3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Lys-Lys-Lys R3C-C32SS-CR3 CH.sub.2CH.sub.2SSCH.sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Arg-Arg-Arg K3C-C32SS-CK3 CH.sub.2CH.sub.2SSCH.sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Lys-Lys-Lys H3C-C32SS-CH3 CH.sub.2CH.sub.2SSCH.sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-His-His-His B3-C32SS-CR3 CH.sub.2CH.sub.2SSCH.sub.2CH.sub.2 >N(CH.sub.2).sub.5OH H.sub.2NCH.sub.2(CH.sub.2).sub.2CH(CH.sub.3)CH.sub.2NH.sub.2 B3-C32SS-CK3 CH.sub.2CH.sub.2SSCH.sub.2CH.sub.2 >N(CH.sub.2).sub.5OH H.sub.2NCH.sub.2(CH.sub.2).sub.2CH(CH.sub.3)CH.sub.2NH.sub.2 B3-C32SS-CH3 CH.sub.2CH.sub.2SSCH.sub.2CH.sub.2 >N(CH.sub.2).sub.5OH H.sub.2NCH.sub.2(CH.sub.2).sub.2CH(CH.sub.3)CH.sub.2NH.sub.2 R3C-C32SS-CK3 CH.sub.2CH.sub.2SSCH.sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Arg-Arg-Arg R3C-C32SS-CH3 CH.sub.2CH.sub.2SSCH.sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Arg-Arg-Arg K3C-C32SS-CH3 CH.sub.2CH.sub.2SSCH.sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Lys-Lys-Lys D3C-C32-CD3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Asp-Asp-Asp E3C-C32-CE3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Glu-Glu-Glu D3C-C32-CE3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Asp-Asp-Asp E3C-C32SS-CD3 CH.sub.2CH.sub.2SSCH.sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Glu-Glu-Glu E3C-C32SS-CE3 CH.sub.2CH.sub.2SSCH.sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Glu-Glu-Glu D3C-C32SS-CE3 CH.sub.2CH.sub.2SSCH.sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Asp-Asp-Asp R3C-C6-CR3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Arg-Arg-Arg >N(CH.sub.2).sub.5CH.sub.3 H3RC-C6-CRH3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Arg-His-His-His >N(CH.sub.2).sub.5CH.sub.3 R3C-C6-CR3- CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Arg-His-His-His linkPEG >N(CH.sub.2).sub.5CH.sub.3 >N(CH.sub.2).sub.5-PEG-OMe R3C-C6-CR3-PEG CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH HS-Cys-Arg-Arg-Arg >N(CH.sub.2).sub.5CH.sub.3 >N-PEG-OMe Polymer HL.sub.2-R.sub.2 R3-C32-R3 H.sub.2N-Arg-Arg-Arg K3-C32-K3 H.sub.2N-Lys-Lys-Lys H3-C32-H3 NH.sub.2-His-His-His R3C-C32-CR3 HS-Cys-Arg-Arg-Arg K3C-C32-CK3 HS-Cys-Lys-Lys-Lys H3C-C32-CH3 HS-Cys-His-His-His B3-C32-R3 H.sub.2N-Arg-Arg-Arg B3-C32-CR3 HS-Cys-Arg-Arg-Arg B3-C32-CK3 HS-Cys-Lys-Lys-Lys B3-C32-CH3 HS-Cys-His-His-His R3C-C32-CK3 HS-Cys-Lys-Lys-Lys R3C-C32-CH3 HS-Cys-His-His-His K3C-C32-CH3 HS-Cys-His-His-His R3C-C32SS-CR3 HS-Cys-Arg-Arg-Arg K3C-C32SS-CK3 HS-Cys-Lys-Lys-Lys H3C-C32SS-CH3 HS-Cys-His-His-His B3-C32SS-CR3 HS-Cys-Arg-Arg-Arg B3-C32SS-CK3 HS-Cys-Lys-Lys-Lys B3-C32SS-CH3 HS-Cys-His-His-His R3C-C32SS-CK3 HS-Cys-Lys-Lys-Lys R3C-C32SS-CH3 HS-Cys-His-His-His K3C-C32SS-CH3 HS-Cys-His-His-His D3C-C32-CD3 HS-Cys-Asp-Asp-Asp E3C-C32-CE3 HS-Cys-Glu-Glu-Glu D3C-C32-CE3 HS-Cys-Glu-Glu-Glu E3C-C32SS-CD3 HS-Cys-Asp-Asp-Asp E3C-C32SS-CE3 HS-Cys-Glu-Glu-Glu D3C-C32SS-CE3 HS-Cys-Glu-Glu-Glu R3C-C6-CR3 HS-Cys-Arg-Arg-Arg H3RC-C6-CRH3 HS-Cys-Arg-His-His-His R3C-C6-CR3- HS-Cys-Arg-His-His-His linkPEG R3C-C6-CR3-PEG HS-Cys-Arg-Arg-Arg

TABLE-US-00002 TABLE 2 Library of end-modified PBAEs (reference compounds) Polymer L.sub.3 L.sub.4 HL.sub.1-R.sub.1 B3 CH.sub.2(CH.sub.2).sub.2CH.sub.2 >N(CH.sub.2).sub.5OH H.sub.2NCH.sub.2(CH.sub.2).sub.2CH(CH.sub.3)CH.sub.2NH.sub.2 B3-C32SS-B3 CH.sub.2CH.sub.2SSCH.sub.2CH.sub.2 >N(CH.sub.2).sub.5OH H.sub.2NCH.sub.2(CH.sub.2).sub.2CH(CH.sub.3)CH.sub.2NH.sub.2 Polymer HL.sub.2-R.sub.2 B3 H.sub.2NCH.sub.2(CH.sub.2).sub.2CH(CH.sub.3) CH.sub.2NH.sub.2 B3-C32SS-B3 H.sub.2NCH.sub.2(CH.sub.2).sub.2CH(CH.sub.3) CH.sub.2NH.sub.2

Example 7: Coagulation Assay

[0320] The coagulation cascade is generally divided into three pathways. The effect of the polymers described in the present application on the three coagulation pathways was evaluated by measuring three representative parameters. Specifically, the intrinsic pathway was measured by the activated partial thromboplastin time, the extrinsic pathway was measured by the prothrombin time and the final common pathway was evaluated by measuring the thrombin time. The possible changes induced in the coagulation cascade due to binding or depletion of the coagulation factors with the polymers described in the present invention were evaluated measuring the time necessary to form a clot formation.

[0321] The polymer used contains 65% of R3C-C6-CR3 and 35% of R3C-C6-CR3-PEG. Three different concentrations were studied: 355 g/ml, 213 g/ml and 106.5 g/ml. Briefly, the polymers were incubated with human pool plasma from at least three donors. Clot formation was detected by a viscosity-based detection system, using a hemostasis analyzer, which measures in seconds. This system avoids interference due to physicochemical attributes of the sample. The reference values for each pathway are as follows: partial thromboplastin time (APTT) 34.1 sec, prothrombin time (PT) 13.4 sec and thrombin time (TT) 21 sec. There is no guidance on the degree of prolongation, but generally prolongation >2-fold versus normal control is considered physiologically significant.

[0322] As shown in FIG. 1, there were no alterations in the coagulation times of the pooled human plasma (negative control) with respect to the normal control. Normal and pathological (abnormal) controls were used as internal controls of the technique. Since the polymer stocks were dissolved in DMSO, a control of the dissolution was also included in the assays. DMSO alone did not show changes in coagulation times.

[0323] As shown in FIG. 1, the polymers did not induce significant alterations on coagulation times at any of the concentrations tested. The coagulation times were within the normal limits after exposure of the polymer at 355 g/mL, 213 g/mL and 106.5 g/mL. The polymer decreases the prothrombin time (PT) at the highest concentrations tested and it was statistically significant with respect to the negative control.

Example 8: Platelet Activation Assay

[0324] Platelet activation comes with degranulation and activation of endothelial cells, leukocytes and other platelets, which ultimately cause formation of a thrombus. The platelets are small anucleate discoid cells involved in primary hemostasis. Their internal structure and membrane play a central role in platelet activation. One of the most reliable markers for platelet activation is CD62P. This is a platelet-specific selectin protein, which is expressed on the internal -granule membrane of resting platelets. This receptor mediates tethering and rolling of platelets on the surface of activated endothelial cells. Upon platelet activation and granule secretion, the -granule membrane fuses with the external plasma membrane and the CD62P antigen is expressed on the surface of the activated platelet.

[0325] The effect of the polymers described in the present application to induce or inhibit platelet activation was measured by the expression of CD62P on the surface of the activated platelet by flow cytometry. The polymer used contained 65% of R3C-C6-CR3 and 35% of R3C-C6-CR3-PEG. Three different concentrations were studied: 355 g/ml, 213 g/ml and 106.5 g/ml. The results were normalized with respect to basal level (negative control). A result was considered positive if the relative fluorescent intensity was >2.0 with respect to the negative control.

[0326] As shown in FIG. 2, the polymers did not induce platelet activation at any of the concentrations tested when it was incubated with a pool of platelet-rich plasma (PRP). Since the polymer stock was dissolved in DMSO, a control of the dissolution was also included in the assays. As shown in FIG. 2, DMSO did not induce platelet activation.

[0327] As a control of the potential inhibitory effect of the polymer on platelet activation, the assay was also performed with ADP (adenosine diphosphate). The polymer did not inhibit the platelet activation in presence of ADP. The results suggest that polymers described in the present application do not induce or inhibit platelet activation under the conditions tested.

Example 9: AdNuPARmE1A Virus

[0328] Structure

[0329] Notch-responsive genes are characterized by a DNA-binding domain, recognizing the CSL transcription factor, in the promoter region. The presence of dual sequence-paired CSL-binding sites (SPS) orientated head-to-head and separated by 16 nt promotes the dimerization of the Notch transcriptional complex, leading to transcriptional activation of Notch target genes, such as Hes1 (Nam Y, Sliz P, Pear W S, Aster J C, Blacklow S C. Cooperative assembly of higher-order Notch complexes functions as a switch to induce transcription. Proc Natl Acad Sci USA. 2007; 104:2103-2108).

[0330] The AdNuPARmE1A contains a synthetic promoter, engineered with three sequences that respond to Notch signalling activation (SPS) and a minimal uPAR promoter, controlling E1A expression. Moreover, the 214 bp short interspersed nuclear element B2 from the growth hormone boundary region (SINEB2) is inserted upstream the uPAR promoter to act as an insulator sequence to avoid any possible unspecific transcriptional activation of E1A by the ITR viral promoter, which would lead to a decrease in tumour selectivity. Expression of the E1A adenoviral gene is controlled by the 3SPSuPARm promoter. SINEB2 insulator sequence was cloned upstream the promoter sequence (see FIG. 3).

[0331] Production and Analysis

[0332] The oncolytic adenovirus AdNuPARmE1A is generated by first cloning the 3SPSuPARm promoter into a pShuttle vector and inserting the SINEB2 insulator upstream the promoter to generate pShSINE3SPSuPARmE1A. Homologous recombination of pShSINE3SPSuPARmE1A vector with the adenoviral genome, is performed following the standard protocol to generate pAdNuPARmE1A. Recombinant genomes are then transfected in HEK293 cells and amplified in A549 cells and purified by standard caesium chloride banding (Mato-Berciano A1, Raimondi G, Maliandi M V, Alemany R, Montoliu L, Fillat C. A NOTCH-sensitive uPAR-regulated oncolytic adenovirus effectively suppresses pancreatic tumor growth and triggers synergistic anticancer effects with gemcitabine and nab-paclitaxel. Oncotarget. 2017; 8(14) 22700-22715).

[0333] Adenoviral concentration is determined by two different methods: [0334] a) the physical particle concentration (vp/ml) is determined by optical density reading (OD260) [0335] b) the plaque forming units (pfu/ml) are determined on HEK293 cells by the anti-hexon staining-based method.

Example 10: AduPARmE1A and AdNuPARmE1A Viruses

[0336] An oncolytic AduPARmE1A virus in which the DA gene expression was regulated by the uPAR promoter was generated. A Kozak sequence was engineered upstream of the E1A gene to increase its replication potency (this sequence on an mRNA molecule is recognized by the ribosome as the translational start site, from which a protein is coded by that mRNA molecule). A DNA fragment from the myotonic dystrophy locus (DM-1), with enhancer-blocking insulator activity, was introduced upstream the uPAR promoter to isolate it from enhancer and transcriptional units from the adenovirus genome (see FIG. 4).

[0337] The AdNuPARmE1A has the same structure, but a shorter version of the uPAR promoter and three responsive elements capable of binding to the Notch intracellular domain (NICD) (see FIG. 2).

Example 11: Formation and Characterization of Polymer-Virus Complexes

[0338] Considerations Before Preparing the Complex

[0339] The virus stock must be titrated by vp/ml and by pfu/ml and the ratio between vp/pfu must be under 100. If this quality acceptance criteria is not reached, the virus production needs to be repeated. It is necessary to have the physical Vp/ml titer before proceeding with this procedure.

[0340] The main formulation used is R3C-C6-CR3/R3C-C6-CR3-PEG with a ratio of 65/35 but this protocol can be adapted to use other formulations. The only must is to maintain the ratio 4e6 molecules PBAE/vp.

[0341] Procedure for In Vitro Scale [0342] 1. Take a 2 l virus aliquot from the 80 C. freezer (The freeze-thawing process of adenoviral vectors is not recommended, since they lose infection efficiency. For this reason, when producing the virus, it is strongly recommended to aliquot it in small aliquots, in order to thaw only the fraction intended for use). The virus should be thawed very slowly on ice. [0343] 2. Thaw both polymers and prepare a mixture at 65/35 v/v. The polymer mixture can be stored at 20 C. [0344] 3. When preparing coated virus to be used in vitro, an entire 2 l aliquot is coated. Prepare a 1/50 dilution of virus stock in PBS (2 l of virus+98 l PBS). The resulting solution is labelled as VS. [0345] 4. Calculate the amount of polymer needed (l PBAE stock) to coat all viral particles in VS following equation 1.

[00001]$\begin{array}{cc}\mathrm{mPBAEs}=\left(\frac{\mathrm{VPt}}{1000}*V.Math..Math.s.Math..Math.\mathrm{tock}.Math..Math.\mathrm{ul}\right)*4.Math..Math.e.Math..Math.6.Math..Math.X.Math..Math.\mathrm{ul}.Math..Math.\mathrm{PBAE}.Math..Math.\mathrm{stock}=\frac{\mathrm{mPBAEs}*3287.Math.\text{,}.Math.52.Math.\frac{g}{\mathrm{mol}}*1.Math..Math.e.Math..Math.4}{6.Math.\text{,}.Math.023.Math..Math.e.Math..Math.23}.Math..Math.\mathrm{VPt}.Math..Math.\mathrm{refers}.Math..Math.\mathrm{to}.Math..Math.\mathrm{the}.Math..Math.\mathrm{physical}.Math..Math.\mathrm{titer}.Math..Math.\mathrm{in}.Math..Math.\mathrm{VP}.Math.\text{/}.Math.\mathrm{ml}.& \mathrm{Equation}.Math..Math.1\end{array}$ [0346] 5. Prepare polymer solution (PS) by mixing PBS with the calculated volume of PBAE stock to a final volume of 100 l. [0347] 6. Mix PS and VS by adding PS over VS and pipetting up & down slowly at least 10 times. [0348] 7. Incubate the sample for 30 minutes at room temperature to enable the electrostatic interaction between the negative surface charge of the virus and the positive charge of the polymer. [0349] 8. Now the sample is ready to be used taking into account that the resulting titer is 100 fold diluted. It can be diluted with complete medium to reach desired working concentration.

[0350] Procedure for In Vivo Scale [0351] 1. Calculate the Total VPs needed taking into account n of animals (with an excess of 1 animal every 4 animals) and dose (typically 410.sup.10 vp/animal injected I.V. in 100 l bolus injection through the tail vein). [0352] 2. Calculate the volume of virus stock needed (Vstock) to prepare an injectable solution of 4e11 VPs/ml in final volume of 1 ml physiologic saline solution 0.9% sodium chloride. (The concentration of the injectable solution depends on the working dose, in this case 410.sup.10 VP/animal) [0353] 3. Calculate the total amount of PBAE stock solution needed to coat all viral particles.

[00002]$\mathrm{mPBAEs}=\left(4.Math..Math.e.Math..Math.11.Math..Math.\mathrm{total}.Math..Math.\mathrm{VPs}\right)*4.Math..Math.e.Math..Math.6$$X.Math..Math.\mathrm{ul}.Math..Math.\mathrm{PBAE}.Math..Math.\mathrm{stock}=\frac{\mathrm{mPBAEs}*3287.Math.\text{,}.Math.52.Math.\frac{g}{\mathrm{mol}}*1.Math..Math.e.Math..Math.4}{6.Math.\text{,}.Math.023.Math..Math.e.Math..Math.23}.Math.$ [0354] 4. Prepare polymer solution (PS) by mixing 0.9% saline with the calculated volume of PBAE stock to a final volume of 200 l [0355] 5. Prepare virus solution (VS) by mixing 0.9% saline with the calculated volume of virus stock to a final volume of 200 l. [0356] 6. Mix PS and VS by adding PS over VS and pipetting up & down slowly at least 10 times. [0357] 7. Incubate the sample for 30 minutes at room temperature to enable the electrostatic interaction between the negative surface charge of the virus and the positive charge of the polymer. [0358] 8. Add 600 l of 0.9% saline and mix pipetting slowly 5 times. [0359] 9. The sample is ready to be injected (e.g. the sample can treat 8 animals).

Examples 12-16

[0360] All non-clinical data, except for the anti-tumoral activity results (example 16), have been obtained with a recombinant serotype 5 Adenovirus, called AdTrackluc (AdTL), which expresses two reporter genes, GFP and luciferase. Furthermore, in the in vivo studies, this virus has been combined with two different polymeric coatings: C6Ad, which corresponds to a 100% R3C-C6-CR3 coating and CPEGAd, which stands for a combination of 65% R3C-C6-CR3 and 35% R3C-C6-CR3-PEG.

Example 12: Masking Capacity Against Neutralizing Antibodies

[0361] In order to determine which polymer combination was the best one to protect adenoviruses from anti-Ad5 neutralizing antibodies (Nabs), viral particles were coated with different combinations of R3C-C6-CR3 with H3C-C6-CH.sub.3 and R3C-C6-CR3-PEG polymers, and the naked and coated samples were then incubated with Nabs during 30 minutes. Naked adenoviruses were used as sample control. Then, viral preparations (MOI 50) were added to 96 well plates containing 1.510.sup.4 PANC-1 cells. After 2 h, the media was changed and cells were incubated 48 h. Finally, GPF positive cells were quantified by flow cytometry analysis.

[0362] The polymer combinations tested are as follows:

TABLE-US-00003 Designation R3C-C6-CR3 R3C-C6-CR3-PEG H3C-C6-CH3 C6CR3-25% 75% 25% 0% C6CR3-35% 65% 35% 0% C6CR3-45% 55% 45% 0% C6CRH3 60% 0% 40% C6CRH3-25% 35% 25% 40%

[0363] As FIG. 7 shows, when naked adenoviruses are incubated in the presence of neutralizing antibodies (+Naked), their transduction capacity is significantly reduced from 80% to 55%. Among the different coating combinations, it was observed that one coating containing 65% of R3C-C6-CR3 and 35% of R3C-C6-CR3-PEG (named C6CR3-35% in the graph) conferred an outstanding protection against the anti-Ad5 neutralizing antibodies (see the single result highlighted in the rectangle). Furthermore, the coating not only did not decrease the infectivity of the virus, but induced an increase in its potency (see the multiple results highlighted in the larger rectangle).

Example 13: Activation of the Adaptive Immune Response

[0364] The method by which naked and coated viral particles activated the adaptive immune response after two intravenous administrations was also studied. C57BL/6J mice (n=6) were divided into three groups (Naked Ad, R3C-C6-CR3 Ad, and R.sub.3C-C6-CR3-35% PEG-Ad). 110.sup.10 vp/animal were injected, at days 0 and 14, in the tail vein of C57BL/6J mice (n=5) and one week later, at day 21, animals were sacrificed and blood was collected by intracardiac puncture. Next, sera were extracted and heat inactivated and they were used to perform neutralization assays in the presence of naked Adenoviruses.

[0365] In order to compare the antibody concentration of each sample, the neutralizing dilution 50 (ND50) for each anti-serum was calculated. The ND50, defined as the dilution of the serum needed to neutralize half of the viral transduction, was determined as follows. Naked Ads (MOI 0.25) were incubated for 1 hour in 96-well plates with serial dilutions of sera from mice immunized with naked or coated Ads. Then, 110.sup.5 HEK293 cells were added to each well. After 24 h, luciferase activity was quantified and ND50 was calculated.

[0366] As shown in FIG. 8, animals injected with CPEGAd produced a three times less neutralizing sera than those injected with naked Ads, indicating that the CPEG-coating strategy (65% R3C-C6-CR3+35% R.sub.3C-C6-CR3-PEG) did reduce the activation of the adaptive immune response. In the case of C6Ad, this difference was less striking.

Example 14: Increase in Blood Circulation Time

[0367] In order to compare the blood circulation kinetics of naked and coated viral particles, 110.sup.10 vp/animal were injected in the tail vein of CC57BL/6J mice (n=5). Three groups were established, naked-Ad, C6Ad and CPEGAd, and blood samples were extracted from the saphenous vein at two time points post injection: 2 minutes and 10 minutes. Next, genomic DNA extraction was performed from each blood sample and quantified viral genomes/l using hexon specific primers by qPCR.

[0368] The blood circulation kinetics were determined as follows. Genomic DNA was extracted from each blood sample and viral genomes copies were quantified using hexon specific primers by qPCR. The 100% condition (equivalent to the injected dose) was analyzed by diluting the administered dose in 2 ml of whole mice blood before DNA extraction. The area under the curve was calculated from 2 minutes to 10 minutes and fold-change transformed.

[0369] As FIG. 9 shows, coated viral particles had an improved circulation time, especially those coated with the CPEG combination. In particular, their area under the curve, from 2 minutes to 10 minutes, was 3-fold bigger when compared to naked viral adenoviruses.

Example 15A: Liver Tropism

[0370] One of the main problems associated with adenoviruses is their high tropism towards the liver, which is responsible for their significant hepatotoxicity. In order to determine if the polymeric coating of the invention could decrease this natural behaviour, 110.sup.10 vp of naked and coated (C6Ad and CPEGAd) adenoviruses were administered in the tail vein of C57BL/6J mice (n=5) and 5 days later, whole body bioluminescent images were taken and luciferase activity was quantified from liver homogenates of mice treated with naked Ads or coated with two different polymers (C6Ad, CPEGAd).

[0371] As FIG. 10 shows, both types of coated viral particles significantly reduced hepatocytes transducing capacity, indicating that the polymeric coating could indeed modify the natural adenoviral tropism.

Example 15B: Tumour Tropism

[0372] To determine if the decreased liver tropism observed with coated Ads also takes place in tumour bearing mice the following study was performed. 110.sup.6 PANC-1 cells (derived from human pancreatic adenocarcinoma) were administered subcutaneously in immunodeficient Balb/C nu/nu mice and when tumours reached a volume around 150 mm.sup.3, 110.sup.10 vp of naked and coated (C6Ad and CPEGAd) adenoviruses were administered in the tail vein (n=6). Five days post injection, animals were sacrificed and luciferase activity (used as reporter gene) was quantified from tumours and liver homogenates of mice treated with naked Ads or Ads coated with one of two different polymers (C6Ad, CPEGAd).

[0373] As can be seen in FIG. 11, apart from the previously observed significant decreased transduction in the liver, when Ads were coated with the polymeric coating named CPEG a marked increased infection in the tumours was detected. This trend was not observed with the C6Ad, probably due to the lack of PEG in the formulation. Altogether, these results led to a significantly increased tumour-liver ratio when CPEGAd were administered in comparison to naked Ads.

Example 16: Increasing Antitumoral Activity

[0374] According to the data obtained with the two different coated recombinant Adenovirus (AdTL), C6Ad and CPEGAd, the latter coating consisting of 65% of R3C-C6-CR3+35% R3C-C6-CR3-PEG was chosen to be combined with the oncolytic adenovirus AdNuPARmE1A in order to form SAG-101.

[0375] In order to study the effect of the polymeric coating on the therapeutic effect of the virus, an efficacy study in tumour bearing mice was performed. In particular, the efficacy of the coated AdNuPARmE1A (SAG-101) after systemic administration was compared with that of naked AdNuPARmE1A in nave or pre-immune mice. To generate a pre-immune status in nude mice, the mice were passively immunized by an intraperitoneal injection of anti-Ad5 neutralizing serum from C57BL6 mice (nude mice bearing subcutaneous PANC-1 tumours were injected intraperitoneally with either PBS (nave groups) or anti-Ad5 neutralizing mice serum (pre-immune groups)). The next day, nave or passively immunized nude mice bearing PANC-1 tumours were injected intravenously with PBS, or 410.sup.10 vp of AdNuPARmE1A naked or coated (SAG-101) (n=8) and the tumour volume was monitored. FIG. 12 summarizes the protocol of this experiment performed to determine the effect of the polymeric coating on the therapeutic effect of the AdNuPARmE1A. Each group had n=8.

[0376] As shown in FIG. 13, a significant reduction in tumour growth was observed in three of the treated groups (naked and coated nave AdNuPARmE1A and pre-immunized coated AdNuPARmE1A) compared with the saline control and the pre-immunized naked virus groups (Results are expressed as mean+/SEM (*p<0.05; **p<0.01; ***p<0.001)). Importantly, the pre-immunized coated group showed a very similar effect when compared to the nave naked virus, indicating that the polymeric coating protected the virus from the pre-existing neutralizing antibodies. Moreover, the nave coated group was significantly more efficacious than the nave naked group. An overview of the median survival results and change in the fraction survival over time for each group tested is set out in FIG. 14.

[0377] The experimental data demonstrate that the coated viral particles unexpectedly exhibit the following properties:

[0378] 1. a reduced tendency to be neutralized by antibodies;

[0379] 2. a reduced de novo adaptive immune response generation capacity;

[0380] 3. improved bloodstream kinetics; and

[0381] 4. a decreased liver tropism, to the benefit of tumor transduction.

Example 17

[0382] In order to study the toxicity of the coated AdNuPARmE1A (SAG-101), a toxicity study in mice was performed. The coating consisted of CPEGAd, which stands for a combination of 65% R3C-C6-CR3 and 35% R3C-C6-CR3-PEG. Doses of SAG-101 after intravenous administration were compared with that of naked AdNuPARmE1A (Ad) in immunocompetent BALB/c mice. The immunocompetent mice were injected intravenously with PBS, or 410.sup.10 vp of AdNuPARmE1A naked or coated (i.e. low-dose), or 7.510.sup.10 vp of AdNuPARmE1A naked or coated (i.e. high-dose).

Example 17A: Body Weight

[0383] As shown in FIG. 15, no significant (p>0.05) changes in body weight were observed between groups, although mice receiving high-dose naked AdNuPARmE1 (Ad) did show an important body weight decrease at day five post-injection. The group which was administered high-dose SAG-101 showed a decrease in body weight, indicating that less toxicity was associated with the coated viral particles.

Example 17B: Transaminases Levels

[0384] Serum enzymatic transaminases activity was determined at day 7 post virus IV injection. Specifically, aspartate transaminase (AST) and alanine transaminase (ALT) levels were measured from blood taken from an intracardiac puncture.

[0385] Aminotransferases (AST, ALT) are commonly analyzed in serum to assess and monitor liver damage and possible viral infections of the liver. These enzymes are elevated in many forms of liver disease, presumably as a result of leakage from damaged cells. ALT is mainly found in the liver, but also in smaller amounts in the kidneys, heart, muscles, and pancreas. AST is present in the liver but also in considerable amounts in other tissues including the muscles.

[0386] As shown in FIG. 16, transaminases levels were significantly (*p<0.05) higher when a high-dose of naked virus (AdNu) was administered, indicating liver damage. This effect was not observed in mice that receive the same dose of SAG-101. Thus, the experimental data demonstrate that the coated viral particles provide a decrease in transaminase levels.

Example 17C: Hemogram and Platelet Count

[0387] A hemogram analysis and platelet count was conducted. The platelet count was based on blood extraction from the tail vein of the mice every other day from day 1 until day 7. As shown in FIG. 17B, no significant changes in the hemogram were detected at day 7 between mice receiving naked AdNuPARmE1A (Ad) or coated AdNuPARmE1A (CPEGAd).

[0388] Furthermore, no thrombocytopenia was observed, as shown in FIG. 17A. There were no significant differences between the number of platelets in mice receiving naked AdNuPARmE1A (Ad) or coated AdNuPARmE1A (SAG101).

Example 17D: Cytokine Quantification

[0389] Levels of cytokines were measured. At six hours and three days post-injection, blood aliquots were collected and cytokine concentration was evaluated using the Luminex xMAP technology platform.

[0390] As shown in FIG. 18, no significant differences in the levels of cytokines were observed after the administration of naked AdNuPARmE1A and SAG-101, indicating that the SAG-101 polymers did not increase the toxicity of the virus.

Example 18:TEM Micrograph

[0391] FIG. 19A provides a TEM micrograph showing SAG-101 at a low magnification (scale bar 20 m) and FIG. 19B provides a TEM micrograph showing SAG-101 at a high magnification (scale bar 200 nm). As observed in FIGS. 19A and 19B, no big aggregates were observed when analyzing the samples at low magnification.

Example 19: Surface Charge Change

[0392] Adeno-associated viral (AAV) particles were coated with 65% of R3C-C6-CR3+35% R3C-C6-CR3-PEG polymers. The polymeric coating formation was tracked by assessing the surface charge change in order to determine suitable coating concentrations i.e. suitable ratios of AAV to polymer.

[0393] The surface of AAVs are negatively charged (as can be seen in FIGS. 20A and 20B), whereas the polymer used offered a net positive charge. Thus, once the polymer effectively covered the virus, the surface charge of the complex was positive. This surface charge change was therefore assessed to track polymeric coating formation. Different ratios were tested in order to follow their surface charge change. FIG. 20B shows a plateau in the surface charge measurement. Thus, adding additional polymer was illogical. In FIG. 20A, closer spaced polymer/virus ratios were tested in order to see a progressive change of the surface charge.

[0394] As can be seen in FIG. 20, when ratios of 1e-9 mg PBAE/AAV viral particle (VP) or higher were used, positive surfaces were observed, as indicated by the positive Z potential value measurements, which indicates that the AAV particles were coated. As can be seen in FIG. 20B, the variability in Z potential value measurements for the free polymer is very high compared to the values when forming a coating of AAV.

Example 20: Scanning Electron Microscopy

[0395] Adeno-associated viral (AAV) particles (naked AAV), AAV particles coated C6Ad, which corresponds to a 100% R3C-C6-CR3 coating and AAB particles coated with CPEGAd, which stands for a combination of 65% R3C-C6-CR3 and 35% R3C-C6-CR3-PEG were characterized by scanning electron microscopy, as shown in FIG. 21. Briefly, 200 l of each sample (naked and coated virus) were prepared (as described in other examples) at a final concentration of 10.sup.11 vp/ml (which was the required concentration to obtain an optimal visualization). Samples were deposited on the grid and incubated there for one minute. Next, they were irradiated with gold for 40 seconds to create a thin layer over them. Finally, they were analysed with a Field Emission Scanning Electron Microscope.

[0396] Furthermore, scanning electron microscopy was used to determine the nanoparticle diameter (in nm):

TABLE-US-00004 Aggregates (all in nm) Naked AAV C6-AAV CPEG-AAV of C6-AAV Mean 24.81 50.32 68.12 150.40 Desvest 4.52 16.79 13.24 53.59 Max 39.05 119.65 118.46 372.55 Min 10.98 17.21 27.75 49.48 Counts 1000 1000 1000 1000

[0397] A broader distribution appears for the C6-AAV nanoparticles due to the presence of aggregates. As shown by the data above, all aggregates were smaller than 500 nm and thus this sample could also be used for intravenous administration.