BR112016026581B1 - POLYPLEX OF A DOUBLE-STRANDED RNA (DSRNA) AND A POLYMERIC CONJUGATE, USE OF THE POLYPLEX AND PHARMACEUTICAL COMPOSITIONS COMPRISING IT - Google Patents

Abstract

POLYPLEX OF A DOUBLE-STRANDED RNA (DSRNA) AND A POLYMERIC CONJUGATE, PHARMACEUTICAL COMPOSITION COMPRISING THE SAME, AND USE THEREOF. A polyplex of a double-stranded RNA and a polymeric conjugate is provided, wherein the polymeric conjugate consists of a linear polyethyleneimine covalently linked to one or more polyethylene glycol (PEG) moieties, each PEG moiety being conjugated via a linker to a targeting moiety capable of binding to a cancer antigen.

Description

Field of invention

[0001] The present invention relates to polyplexes based on non-viral polyethyleneimines conjugated to a targeting moiety capable of binding to a cancer antigen.

Background of the invention

[0002] One of the obstacles facing molecular medicine is the targeted delivery of therapeutic agents, such as DNA or RNA molecules. One strategy under development is the construction of nonviral vectors, such as cationic polymers and cationic lipids, which bind and condense nucleic acids. These nonviral cationic vectors have many advantages over gene vectors, as they are nonimmunogenic, nononcogenic, and easy to synthesize [1-4]. Currently, several synthetic polycationic polymers are being developed for nucleic acid delivery. Among these, polyethyleneimines (PEIs) are considered promising agents for gene delivery [5].

[0003] PEIs are water-soluble organic macromolecules that are available as linear and branched structures [6]. PEIs change their degree of ionization over a wide pH range, since every third atom in their backbone is an amino nitrogen that can be protonated. Approximately 55% of the nitrogens in PEIs are protonated at physiological pH [7]. They possess high cationic charge density, and are therefore capable of forming non-covalent complexes with nucleic acids. Furthermore, their physicochemical and biological properties can be altered by various chemical modifications [8]. PEI-based complexes (also known as polyplexes) can be endocytosed by many cell types [9]. Following internalization of polyplexes, endosome release and high gene transfer efficiency are driven by the “proton sponge effect” [10]. The ability of PEI to condense DNA appears to be an important factor in the distribution of large DNA constructs in many cell types.

[0004] The main concern in the use of PEIs as delivery vehicles is toxicity, due to their high positive surface charge that can lead to nonspecific binding [11]. Recent attempts have been made to improve the selectivity and biocompatibility of nonviral vectors. This has led to the modification of PEI molecules with polyethylene glycol (PEG) in order to shield the PEI particle [12]. The conjugation of heterobifunctional PEG groups to PEI facilitates the coupling of PEI to a targeting ligand, which provides efficient gene delivery to cells containing the cognate receptor [12]. The generation of targeting vectors has been previously described, demonstrating the difference between branched PEI (brPEI-EGF) and linear PEI (LPEI) tethered to EGF as targeting vectors [13, WO 2004/045491, WO 2010/073247]. Current synthetic methods are unsatisfactory, as they result in insufficiently homogeneous products. There is therefore an urgent need for methods that can provide efficient conjugation of target moieties to LPEI-PEG in a reproducible manner to produce homogeneous batches of products that can be reliably used in cancer treatment methods.

Summary of the invention

[0005] In one aspect, the present invention relates to a polyplex of a double-stranded RNA (dsRNA) and a polymeric conjugate, wherein said polymeric conjugate consists of a linear polyethyleneimine (LPEI) covalently linked to one or more polyethylene glycol (PEG) moieties, each PEG moiety being conjugated via a linker to a targeting moiety capable of binding to a cancer antigen, provided that the targeting moiety is not mouse EGF (mEGF) or the peptide of the sequence YHWYGYTPQNVI (GE11) (SEQ ID NO: 1).

[0006] In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polyplex of the present invention as defined herein.

[0007] In yet another aspect, the present invention provides the polyplex of the present invention as defined herein, or pharmaceutical composition comprising the polyplex, for use in the treatment of a cancer selected from a cancer characterized by cells overexpressing EGFR, a cancer characterized by cells overexpressing HER2, and prostate cancer.

[0008] Yet another aspect, the present invention relates to a method for treating a cancer selected from the group consisting of a cancer characterized by cells overexpressing EGFR, a cancer characterized by cells overexpressing HER2, and prostate cancer, the method comprising administering to a subject in need thereof a polyplex of the present invention, as defined herein.

[0009] In another aspect, the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the polyplex of the present invention, for the treatment of a cancer selected from a cancer characterized by cells overexpressing EGFR, a cancer characterized by cells overexpressing HER2 and prostate cancer.

[0010] The polyplex of the present invention can be used in combination with immune cells.

Brief description of the drawings

[0011] Figure 1 shows that conjugation of LPEI (~22 kDa) with NHS-PEG-OPSS (~2 kDa) yielded primarily two copolymer networks that differed in the degree of PEGylation. The LPEI-(PEG2k-OPSS)3 copolymer (“1:3 di-conjugate”) consisted on average of 1 mole of LPEI and 3 moles of PEG, while the LPEI-PEG2k-OPSS copolymer (“1:1 di-conjugate”) consisted on average of a ratio of 1 mole of LPEI and 1 mole of PEG. (LPEI:PEG ratios were determined by 1H-NMR analysis.)

[0012] Figure 2 shows 1H-NMR analysis of the two diconjugates, LPEI-PEG2k-OPSS (1:1 diconjugate) and LPEI-(PEG2k-OPSS)3 (1:3 diconjugate). Coupling of the PEG groups to LPEI was indicated by the presence of chemical shifts that correlate to ethylene glycol hydrogens (a) at 3.7 ppm and ethyleneimine hydrogens at -3.0 ppm (b). The integral values of these peaks provide molar ratios of PEG to LPEI, from which the illustrated structures of the 1:1 diconjugate (A) and 1:3 diconjugate (B) were deduced.

[0013] Figure 3 shows a schematic for the conjugation of the copolymer networks (1:1 di-conjugate and 1:3 di-conjugate) to the Affibody (“Her-2”) via disulfide exchange, resulting in the generation of two differentially PEGylated tri-conjugates [20].

[0014] Figure 4 depicts an SDS/PAGE of purified Affibody, di-conjugate and tri-conjugate, in the absence and presence of DTT. In the presence of DTT, Affibody is released from the tri-conjugate, and migrates throughout the purified Affibody (slightly above 10 kDa).

[0015] Figure 5 shows particle sizing using DLS measurements of LPEI, the di-conjugates and tri-conjugates complexed with plasmid pGreenfire 1 in HBG buffer at pH 7.4.

[0016] Figure 6 depicts the Ç potential distributions of LPEI, di-conjugates and tri-conjugates complexed with the plasmid pGreenfire1. The zeta potentials were measured by DLS and calculated by the equation

Smoluchowski.

[0017] Figures 7A-B show atomic force microscope (AFM) images obtained from measurements performed in HGB buffer at pH 7.4 for both polyplexes. (A) 1:1 tri-conjugated polyplex (B) 1:3 tri-conjugated polyplex. Scale bar is 1 μm.

[0018] Figure 8 depicts an agar gel showing that differentially PEGylated polyplexes protect plasmid pGreenFire1 from DNase I degradation. 1 μg of plasmid (pGreenFirel) alone or in tri-conjugated polyplexes: 1:1 and 1:3 was treated with or without DNase I (2 IU). Supercoiled plasmid (s.c.), open circular plasmid (o.c.).

[0019] Figures 9A-C show Her-2-mediated gene transfer of pGFP-LUC using the 1:1 tri-conjugate polyplex and the 1:3 tri-conjugate polyplex containing LPEI:PEG ratios of 1:1 and 1:3, respectively. 10000 BT474 and MDA-MB-231 breast cancer cells/well were treated for 48 h with 1:1 and 1:3 tri-conjugates complexed with pGFP-LUC (1 μg/ml) to generate the two polyplexes. PEI/DNA phosphate nitrogen ratio of 6 (N/P=6) in HBS. (A) Luciferase activity measurements demonstrate significant decrease in pGreenFire1 delivery in MDA-MB-231 cells compared to BT474 and reduced gene delivery mediated by 1:3 tri-conjugated polyplexes compared to 1:1 tri-conjugated polyplexes (*p < 0.001). Luciferase activity was measured in triplicate after 48 h, shown as relative luciferase units (RLU) as mean + S.D. (B) Fluorescent images of polyplex-treated cells. Images are shown at X10 magnification and represent three experiments performed. (C) Methylene blue assay depicts percent cell survival compared to untreated cells (UT).

[0020] Figure 10 shows that PEI-PEG-Her2Affibody (PPHA) complexed with PolyIC inhibits BT474 breast cancer cells overexpressing HER2. The complexed vector inhibits cells overexpressing Her2, including Herceptin/trastusumab resistant cells.

[0021] Figure 11 shows the inhibition of Her2-overexpressing MCF7 cells injected into nude mice.

[0022] Figures 12A-B show the efficacy of (A) PEI-PEG-EGFRAffibody (PPEAffibody) compared to that of (B) PPE.

[0023] Figure 13 shows the activity of PolyIC/PPEaffibody in vivo as compared to that of untreated (UT), pIC/PPE, pI/PPE, pI/PPEA and lower pIC/PPEA (0.1 μg/μL pIC in the complex).

[0024] Figure 14 shows the survival of U87MG, U87MGwtEGFR cells after application of different concentrations of the PolyIC/LPEI-PEG-hEGF complex as compared with application of PolyIC/mPPE (mouse) as described in Schaffert D, Kiss M, Rodl W, Shir A, Levitzki A, Ogris M, Wagner E. (2011) Poly(I:C)-mediated tumor growth suppression in EGF- receptor overexpressing tumors using EGF-polyethylene glycol-linear polyethylenimine as carrier. Pharm Res. 28:731-41.

[0025] Figure 15 shows that PEI-PEG(PP)-DUPA(PPD)/PolyIC is highly efficient against LNCaP and VCaP cells. Viability was measured after 96 h of exposure.

[0026] Figures 16A-B show the production of cytokines (A) IP-10 and (B) RANTES by the PolyIC/PPD-transfected LNCaP cell.

[0027] Figure 17 shows that medium conditioned by LNCaP cells stimulates cytokine expression in PBMCs. Expression was measured after 24 h of incubation.

[0028] Figure 18 shows that co-incubation of PolyIC/PPD treated LNCaP cells with non-PSMA expressing PC3-Luciferase cells resulted in up to 70% killing of PC3-Luciferase cells via bystander effect. Addition of healthy human PBMCs strongly intensified the effect and led to 90% killing of PC3 cells.

[0029] Figure 19 shows the effect of PolyIC/PPD on subcutaneous LNCaP tumors in vivo. UT, untreated.

Detailed description of the invention

[0030] Polycations, especially PEI, have been intensively investigated as agents for gene transfection. Optimal transfection efficiencies are achieved when the polymeric nanoparticle complexes possess a net positive charge, which allows them to bind to negatively charged heparin sulfate proteoglycans on the cell surface [28]. Previous studies have shown that linear PEI (LPEI) is more effective in gene transfection than branched PEI (brPEI) [29–31] [32, 33, WO 2010/073247], but that LPEI has a higher positive charge and is therefore more toxic. Several shielding entities, such as PEG [12], poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) [18, 34], and poly(ethylene oxide) [35], have been conjugated to cationic polymers in an attempt to decrease the positive charge and consequent toxicity. Indeed, shielding of PEIs with PEG groups of varying lengths significantly reduced toxicity while maintaining transfection efficiency [12,13, 36].

[0031] It has been discovered, in accordance with the present invention, that conjugation of LPEI with PEG2k yields diconjugated copolymers comprising various ratios of LPEI to PEG2K. These diconjugates could be separated from each other using cation exchange chromatography, due to differences in charge, which reflect different numbers of conjugated PEG2k groups. 1H-NMR analysis confirmed that the diconjugates differ from each other in the average number of PEG2k units per LPEI unit, where the 1:1 diconjugate had a LPEI:PEG2k ratio of 1:1 and the 1:3 diconjugate had a LPEI:PEG2k ratio of 1:3. The conjugation of the Affibody target Her-2 to each of the purified diconjugates yielded a triconjugate product of appropriate molecular weight, i.e., from the 1:1 diconjugate we obtained “1:1 triconjugate” with LPEI:PEG2k:Her-2 equal to 1:1:1 and from the 1:3 diconjugate we obtained “1:3 triconjugate”, with a ratio of 1:3:3. This protocol allowed to obtain homogeneous products, with almost complete conjugation of the target Affibody to LPEI-PEG, in a reproducible manner.

[0032] PEGylation was observed to strongly affect the size of the polyplex particles obtained by complexing the di- or tri-conjugates with plasmid DNA. Both the 1:3 di-conjugate and 1:3 tri-conjugate polyplexes had larger average particle sizes than the 1:1 di-conjugate and 1:1 tri-conjugate polyplexes. It is believed that increasing the amount of PEGylation on a single cationic chain leads to steric hindrance, which prevents the polymer chain from condensing the plasmid to a smaller particle. This is consistent with the finding that the naked LPEI polyplex had the smallest particles. Furthermore, while both the 1:1 and 1:3 tri-conjugates protected the complexed plasmid from DNase I, the 1:3 tri-conjugate polyplex provided better protection, possibly due to increased steric hindrance.

[0033] Previous studies suggested that increasing the molecular weight of PEG units conjugated to cationic polymers led to a decrease in the surface charge of polyplexes obtained upon complexation with nucleic acids [13]. The data showed that increasing the amount of PEG groups of similar molecular weights led to a decrease in the surface charge, as defined by the Ç potential distribution. Indeed, the highest surface charge was shown by naked LPEI complexed with plasmid. These results support the idea that the more neutral entities are present in a chemical vector, the lower the surface charge will be. Surprisingly, tri-conjugated polyplexes conjugated to Affibodies had a lower surface charge than di-conjugated polyplexes, showing that the Her-2 Affibody (which itself has a slightly positive charge) also reduced the surface charge of the particles. It was suspected that the Her-2 Affibody alters the topography of the particle with more target moieties masking the surface charge, leading to a decrease in surface charge.

[0034] The shape of a polyplex has a significant effect on its performance as a drug delivery candidate[37, 38], although it is not yet known which polyplex shapes are desirable for effective drug delivery. To date, the effect of PEGylation on a polyplex shape has not been investigated. In AFM images, the 1:1 tri-conjugate polyplex - which was most effective for gene delivery - showed shape homogeneity, although the 1:3 tri-conjugate was more heterogeneous with many asymmetric ellipsoidal particles.

[0035] Selective gene transfer using cationic polymers remains a major challenge. Previous studies have shown that targeting LPEI and LPEI-PEG conjugates with EGF or transferrin increased their selectivity and decreased nonspecific interactions both in vitro and in vivo [39,40]. For example, to examine the selectivity of Her-2-targeting 1:1 and 1:3 tri-conjugate polyplexes, two breast cancer cells that differentially express Her-2 were used. Gene delivery, as shown by luciferase activity and GFP expression, was significantly greater in BT474 cells, which highly overexpress the Her-2 receptor, than in MDA-MB-231 cells, which express 100-fold less Her-2 receptor on the cell surface. Thus, the data demonstrate that both 1:1 and 1:3 tri-conjugates are highly selective for cells overexpressing Her-2 (Figure 10).

[0036] Previous studies have shown that high levels of PEGylation can result in reduced gene transfection [41]. These results are in agreement with the observation that the highly PEGylated 1:3 tri-conjugate polyplex showed a significant reduction in gene delivery as compared to the less PEGylated 1:1 tri-conjugate polyplex, as shown by luciferase activity and GFP expression. The increased gene delivery by the lower PEGylated 1:1 tri-conjugate polyplex was accompanied by slight cellular toxicity, most likely due to its higher surface charge.

[0037] The working hypothesis prior to engaging in this study was that increasing the amount of targeting moieties per unit of LPEI would lead to improved gene delivery and/or selectivity. It was speculated that the 1:3 tri-conjugate having 3 moles of conjugated Her-2 Affibody molecules per mole of LPEI would show increased receptor-mediated particle internalization. However, the 1:3 tri-conjugate polyplexes showed lower ΔC potential, larger and heterogeneous particle size, and non-spherical shape, all characteristics that may contribute to the decreased transfection efficiencies currently observed. The results show that the less PEGylated 1:1 tri-conjugate is superior to the more PEGylated 1:3 tri-conjugate in mediating selective and efficient gene delivery in HER-2 overexpressing cells.

[0038] It has been discovered in accordance with the present invention that PEGylation of LPEI-based polyplexes leads to decreased surface charge, increased polyplex size and increased shape heterogeneity, and that these properties can have profound effects on targeted gene delivery. The simplified synthesis allows purification of homogeneous products in a reproducible format, which can now be expanded to generate different tri-conjugates using a variety of targeting moieties.

[0039] In view of the above, the present invention, in one aspect, provides a polyplex of a double-stranded RNA (dsRNA) and a polymeric conjugate, wherein said polymeric conjugate consists of a linear polyethyleneimine (LPEI) covalently linked to one or more polyethylene glycol (PEG) moieties, each PEG moiety being conjugated via a linker to a targeting moiety capable of binding to a cancer antigen, provided that the targeting moiety is not mEGF or the peptide of the sequence YHWYGYTPQNVI (GE11) (SEQ ID NO: 1).

[0040] In certain embodiments, the cancer antigen can be, but is not limited to, an epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), prostate surface membrane antigen (PSMA), an insulin-like growth factor 1 receptor (IGF1R), a vascular endothelial growth factor receptor (VEGFR), a platelet-derived growth factor receptor (PDGFR), or a fibroblast growth factor receptor (FGFR). The targeting moiety can be a native, modified, or natural ligand or a paralog thereof, or a non-native ligand, such as an antibody, a single-chain variable fragment (scFv), or an antibody mimetic such as an Affibody, to any of the cancer antigens. Affibodies are based on the Z domain (the immunoglobulin G binding domain) of protein A and unique binding properties are acquired by randomization of 13 amino acids located in two alpha helices involved in the binding activity of the matrix protein domain.

[0041] In certain embodiments, the dsRNA is polyinosinic-polycytidylic acid (poly I:C) double-stranded RNA, the polymeric conjugate consists of LPEI covalently linked to one PEG moiety (LPEI-PEG 1:1) or to three PEG moieties (LPEI-PEG 1:3), and the cancer antigen is EGFR, HER2, or PSMA.

[0042] The molecular weight of PEG may be in the range of 2 to 8 kDa, in particular 2 kDa; the molecular weight of LPEI may be in the range of 10 to 30 kDa, in particular 22 kDa; and the polyIC of the polyplex of the invention may be composed of RNA strands each comprising at least 22, preferably at least 45 ribonucleotides. In certain embodiments, each strand has an amount of ribonucleotides within the range of 20 to 300.

[0043] In certain embodiments, one or more PEG moieties each independently form a -NH-CO- bond with the LPEI and a bond selected from -NH-CO-, -CO-NH-, -S-C-, -S-S-, -O-CO-, or -CO-O- with said linker. In particular, each of the one or more of the PEG moieties forms -NH-CO- bonds with the LPEI and the linker.

[0044] In certain embodiments, the linker forms a -SS-, NH-CO-, -CO-NH-, -SC-, O-CO-, -CO-O-, or urea (-NH-CO-NH) bond with the target moiety. The linker can be selected from -CO-R2-Rx-R3 or a peptide moiety consisting of 3 to 7 amino acid residues, wherein R2 is selected from (C1-C8)alkylene, (C2-C8)alkenylene, (C2-C8)alkynylene, (C6-C10)arylenediyl, or heteroarylenediyl; Rx is absent or -S-; R3 is absent or of the formula R4 is selected from (C1-C8)alkylene, (C2-C8)alkenylene, (C2-C8)alkynylene, (C1-C8)alkylene-(C3-C8)cycloalkylene, (C2-C8)alkenylene-(C3-C8)cycloalkylene, (C2-C8)alkynylene-(C3-C8)cycloalkylene, (C6-C10)aryle no-diyl, heteroarylenediyl, (C1-C8)alkylene-(C6-C10)arylene-diyl, or (C1-C8)alkylene-heteroarylenediyl; wherein each of said (C1-C8)alkylene, (C2-C8)alkenylene, or (C2-C8)alkynylene is optionally substituted by one or more groups each independently selected from halogen, -COR5, -COOR5, -OCOOR5, -OCON(R5)2, -CN, -NO2, -SR5, -OR5, -N(R5)2, -CON(R5)2, -SO2R5, -SO3H, -S(=O)R5, (C6-C10)aryl, (C1-C4)alkylene-(C6-C10)aryl, heteroaryl, or (C1-C4)alkylene-heteroaryl, and further optionally interrupted by one or more identical or different heteroatoms selected from S, O, or N, and/or at least one group each independently selected from -NH-CO-, -CO-NH-, -N(R5)-, -N(C6-C10aryl)-, (C6-C10)arylene-diyl, or heteroarylenediyl; and R5 is H or (C1-C8)alkyl.

[0045] In certain embodiments, R2 is selected from (C1-C8)alkylene, preferably (C1-C4)alkylene, optionally substituted by one or more groups each independently selected from halogen, -COH, -COOH, -OCOOH, -OCONH2, -CN, -NO2, -SH, -OH, -NH2, -CONH2, -SO2H, -SO3H, -S(=O)H, (C6-C10)aryl, (C1-C4)alkylene-(C6-C10)aryl, heteroaryl, or (C1-C4)alkylene-heteroaryl, and further optionally interrupted by one or more identical or different heteroatoms selected from S, O, or N, and/or at least one group each independently selected from -NH-CO-, -CO-NH-, -NH-, -N(C1-C8alkyl)-, -N(C6-C10aryl)-, (C6-C10)arylene-diyl, or heteroarylenediyl. In particular, R2 is selected from (C1-C8)alkylene, preferably (C1-C4)alkylene.

[0046] In certain embodiments, Rx is -S-. 0

[0047] In certain embodiments, R3 is absent or wherein R4 is (C1-C8)alkylene-(C3-C8)cycloalkylene, preferably (C1-C4)alkylene-(C5-C6)cycloalkylene.

[0048] In certain embodiments, in the polyplex as defined above, R2 is -o .—. CH2-CH2-; Rx is -S-; and R3 is absent or in which

[0049] In certain embodiments, the linker is a peptide moiety comprising at least one, in particular two or three aromatic amino acid residues, such as phenylalanine, tryptophan, tyrosine, or homophenylalanine. In certain embodiments, the peptide moiety is -(NH-(CH2)7-CO)-Phe-Gly-Trp-Trp-Gly-Cys- (SEQ ID NO:2) or -(NH-(CH2)7-CO)-Phe-Phe-(NH-CH2-CH(NH2)-CO)-Asp-Cys- (SEQ ID NO:3), linked via its mercapto group to the target moiety.

[0050] In certain embodiments, the polymer conjugate is a diconjugate of formula (i)-(viii), attached to the target moiety/moieties: (iii) -(NH-(CH2)7-CO)-Phe-Phe-(NH-CH2-CH(NH2)-CO)-Asp-Cys-PEG2k- LPEI; (iv) -(NH-(CH2)7-CO)-Phe-Gly-Trp-Trp-Gly-Cys-PEG2k-LPEI; (v)

[0051] In particular embodiments, the polyplex is selected from (a) the polyplex, wherein the targeting moiety is HER2 Affibody, and the polymeric conjugate is that of formula (i) above, and the HER2 Affibody is linked via a mercapto group thereof, also referred to herein as LPEI-PEG2k-HER2; (b) the polyplex, wherein the targeting moiety is HER2 Affibody, and the polymeric conjugate is that of formula (v) above, and the HER2 Affibody is linked via a mercapto group thereof, also referred to herein as LPEI-(PEG2k-HER2)3; (c) the polyplex, wherein the targeting moiety is EGFR Affibody, and the polymeric conjugate is that of formula (i) above, and the EGFR Affibody is linked via a mercapto group thereof, also referred to herein as LPEI-PEG2k-EGFR; (d) the polyplex, wherein the targeting moiety is EGFR Affibody, and the polymeric conjugate is that of formula (v) above, and the EGFR Affibody is linked via a mercapto group thereof, also referred to herein as LPEI-(PEG2k-EGFR)3; (e) the polyplex, wherein the targeting moiety is human EGF (hEGF), and the polymeric conjugate is that of formula (ii) above, wherein the hEGF is linked via an amino group thereof, also referred to herein as LPEI-PEG2k-hEGF; (f) the polyplex, wherein the targeting moiety is hEGF, and the polymeric conjugate is that of formula (vi) above, wherein hEGF is linked via an amino group thereof, also referred to herein as LPEI-(PEG2k-hEGF)3; (g) the polyplex, wherein the targeting moiety is HOOC(CH2)2-CH(COOH)-NH-CO- NH-CH(COOH)-(CH2)2-CO- (DUPA residue), and the polymeric conjugate is that of formula (iii) above; (h) the polyplex, wherein the targeting moiety is HOOC(CH2)2-CH(COOH)-NH-CO- NH-CH(COOH)-(CH2)2-CO- (DUPA residue), and the polymeric conjugate is that of formula (vii) above; (i) the polyplex, wherein the target moiety is HOOC(CH2)2-CH(COOH)-NH-CO- NH-CH(COOH)-(CH2)2-CO- (DUPA residue), and the polymer conjugate is that of formula (iv) above; or (j) the polyplex, wherein the target moiety is HOOC(CH2)2-CH(COOH)-NH-CO- NH-CH(COOH)-(CH2)2-CO- (DUPA residue), and the polymer conjugate is that of formula (viii) above;

[0052] The size of the nanoparticles formed by the polyplex of the present invention may be in the range of 120 to 150 nm, in particular 135-148 nm, or 142 nm.

[0053] Non-limiting examples of procedures for preparing the polymer conjugates used in the present invention are exemplified in the Examples below.

[0054] In certain particular embodiments, the EGFR Affibody is of the amino acid sequence as set forth in SEQ ID NO:4, the HER2 Affibody is of the amino acid sequence as set forth in SEQ ID NO:5, and the hEGF is of the amino acid sequence as set forth in SEQ ID NO:6.

[0055] In another aspect, the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the polyplex as defined above.

[0056] In yet another aspect, the present invention provides the polyplex of the present invention as defined herein, or pharmaceutical composition comprising the polyplex, for use in the treatment of a cancer selected from a cancer characterized by cells overexpressing EGFR, a cancer characterized by cells overexpressing HER2, and prostate cancer.

[0057] In certain embodiments, the cancer characterized by cells overexpressing EGFR is selected from non-small cell lung carcinoma, breast cancer, glioblastoma, head and neck squamous cell carcinoma, colorectal cancer, adenocarcinoma, ovarian cancer, bladder cancer, or prostate cancer, and metastases thereof. In certain embodiments, the polyplex used for the treatment of cancer characterized by cells overexpressing EGFR is selected from the polyplex of (c), (d), (e), or (f) defined above.

[0058] In certain embodiments, the cancer characterized by cells overexpressing HER2 is selected from breast cancer, ovarian cancer, stomach cancer, and aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma. In certain embodiments, the cells overexpressing HER2 are Herceptin/trastusumab-resistant cells. Thus, the polyplex of the present invention may be for use in the treatment of Herceptin/trastusumab-resistant cancer, i.e., cells comprising cancer that do not respond, or respond to a lesser degree of exposure to Herceptin/trastusumab.

[0059] In particular, the polyplex used for the treatment of cancer characterized by cells overexpressing HER2 is selected from the polyplex of (a), (b), (e) or (f) defined above.

[0060] In certain embodiments, the cancer is prostate cancer and the polyplex used for treating prostate cancer is selected from (g), (h), (i), or (j) defined above.

[0061] In yet another aspect, the present invention relates to a method for treating a cancer selected from the group consisting of a cancer characterized by cells overexpressing EGFR, a cancer characterized by cells overexpressing HER2, and prostate cancer, the method comprising administering to a subject in need thereof a polyplex of the present invention, as defined herein.

[0062] In another aspect, the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the polyplex of the present invention, for the treatment of a cancer selected from a cancer characterized by cells overexpressing EGFR, a cancer characterized by cells overexpressing HER2 and prostate cancer.

[0063] In yet another aspect, the present invention is directed to the polyplex, method or pharmaceutical composition of the present invention for use in combination with immune cells.

[0064] In yet another aspect, the present invention is directed to a polyplex as defined above, in which the dsRNA is replaced by a DNA molecule, such as a plasmid comprising protein-coding nucleic acid sequences operably linked to control elements, such as appropriate promoters and terminators.

[0065] In certain embodiments, the immune cells are tumor-infiltrating T cells (T-TILs), tumor-specific engineered T cells, or peripheral blood mononuclear cells (PBMCs).

[0066] The term “polyplex” as used herein refers to a complex between a nucleic acid and a polymer. The nucleic acid is linked to the polymer via non-covalent or covalent bonds, in particular electrostatic bonds. The term “polyplex” refers to a vector, i.e., a non-viral vector useful for transporting and delivering nucleic acids into cells.

[0067] The terms “patient,” “subject,” or “individual” are used interchangeably and refer to either a human being or a non-human animal.

[0068] The term “(C1-C8)alkyl”, as used herein, typically means a straight or branched hydrocarbon radical having 1-8 carbon atoms and includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2,2-dimethylpropyl, n-hexyl, n-heptyl, n-octyl, and the like.

[0069] The term “(C1-C8)alkylene” refers to a linear or branched divalent hydrocarbon radical having 1-8 carbon atoms and includes, for example, methylene, ethylene, propylene, butylene, pentanylene, hexanylene, heptanylene, octanylene, and the like. The terms “(C2-C2)alkenylene” and “(C2-C8)alkynylene” typically mean linear or branched divalent hydrocarbon radicals having 2-8 carbon atoms and one or more double or triple bonds, respectively. Non-limiting examples of such radicals include ethenylene, propenylene, 1- and 2-butenylene, 1- and 2-pentenylene, 1-, 2- and 3-hexenylene, 1-, 2- and 3-heptenylene, 1-, 2-, 3- and 4-octenylene, ethynylene, propynylene, 1- and 2-butynylene, 2-methylpropylene, 1- and 2-pentynylene, 2-methylbutylene, 1-, 2- and 3-hexynylene, 1-, 2- and 3-heptynylene, 1-, 2-, 3- and 4-octynylene and the like.

[0070] The term “(C6-C10)aryl” denotes an aromatic carbocyclic group having 6-10 carbon atoms consisting of a single ring or multiple condensed rings, such as, but not limited to, phenyl and naphthyl; the term “(C6-C10)arylenediyl” denotes a divalent aromatic carbocyclic group having 6-10 carbon atoms consisting of a single ring or multiple condensed rings, but not limited to, phenylene and naphthylene.

[0071] The term "heteroaryl" refers to a radical derived from a 5-10 membered mono- or polycyclic heteroaromatic ring containing one to three, preferably 1-2, heteroatoms selected from N, O, or S. Examples of monocyclic heteroaryls include, but are not limited to, pyrrolyl, furyl, thienyl, thiazinyl, pyrazolyl, pyrazinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, 1,2,3-triazinyl, 1,3,4-triazinyl, and 1,3,5-triazinyl. Polycyclic heteroaryl radicals are preferably composed of two rings, such as, but not limited to, benzofuryl, isobenzofuryl, benzothienyl, indolyl, quinolinyl, isoquinolinyl, imidazo[1,2-a]pyridyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, pyrido[1,2-a]pyrimidinyl, and 1,3-benzodioxinyl. The heteroaryl may optionally be substituted by one or more groups each independently selected from halogen, -OH, -COOH, -CN, -NO2, -SH, or -CONH2. It should be understood that when a polycyclic heteroaryl is substituted, the substitution may be on any of the carbocyclic and/or heterocyclic rings. The term “heteroarylenediyl” denotes a divalent radical derived from a “heteroaryl” as defined herein by removal of two hydrogen atoms from either ring atom.

[0072] The term “halogen” as used herein refers to fluorine, chlorine, bromine or iodine.

[0073] The term “(C3-C8)cycloalkylene” denotes a mono- or bicyclic saturated divalent cyclic hydrocarbon radical containing three to eight carbons. Non-limiting examples of such radicals include 1,2-cyclopropanediyl, 1,2-cyclobutanediyl, 1,3-cyclobutanediyl, 1,2-cyclopentanediyl, 1,3-cyclopentanediyl, 1,2-cyclohexanediyl, 1,3-cyclohexanediyl, 1,4-cyclohexanediyl, 1,2-cycloheptanediyl, 1,3-cycloheptanediyl, 1,4-cycloheptanediyl, 1,2-cyclooctanediyl, 1,3-cyclooctanediyl, 1,4-cyclooctanediyl, 1,5-cyclooctanediyl and the like.

[0074] The term “amino acid residue,” as used herein, refers to any natural or synthetic, i.e., unnatural, amino acid residue in both L- and D-stereoisomers. Although a natural amino acid is any of the twenty amino acid residues typically occurring in proteins, the term synthetic/unnatural amino acid refers to any amino acid, modified amino acid, and/or an analogue thereof, that is not one of the twenty natural amino acids. Non-limiting examples of natural amino acids include glycine, alanine, valine, leucine, isoleucine, lysine, valine, phenylalanine, glutamic acid, aspartic acid, asparagine, glutamine, arginine, histidine, proline, serine, tyrosine, methionine, threonine, and tryptophan. Examples of unnatural amino acids include, but are not limited to, ornithine, homolysine, 2,4-diaminobutyric acid (DABA), 2,3-diaminopropionic acid (DAP), 8-aminooctanoic acid (EAO), homophenylalanine, homovaline, homoleucine, and the like.

[0075] The pharmaceutical compositions according to the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers and/or excipients. The carrier(s) must be “acceptable” in the sense of being compatible with other ingredients of the composition and not deleterious to the recipient thereof.

[0076] The following exemplification of vehicles, modes of administration, dosage forms, etc. are listed as known possibilities from which vehicles, modes of administration, dosage forms, etc. may be selected for use with the present invention. Those skilled in the art will understand, however, that any formulation provided and mode of administration selected should first be tested to determine that it will achieve the desired results.

[0077] Methods of administration include, but are not limited to, parenteral routes, e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, mucosal (e.g., oral, intranasal, buccal, vaginal, rectal, intraocular), intrathecal, topical, and intradermal. Administration may be systemic or local. In certain embodiments, the pharmaceutical composition is adapted for intracerebral administration.

[0078] The term “vehicle” refers to a diluent, adjuvant, excipient, or vehicle with which the active agent is administered. The carriers in the pharmaceutical composition may comprise a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate; a disintegrating agent, such as alginic acid, cornstarch and the like; a lubricant or surfactant, such as magnesium stearate, or sodium lauryl sulfate; and a glidant, such as colloidal silicon dioxide.

[0079] The compositions may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection may be presented in single dosage form, for example, in ampoules or in multidose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents, such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0080] For administration by inhalation, e.g. for nasal administration, the compositions according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurised packs or a nebuliser, using a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to dispense a metered quantity. Capsules and refills of, e.g. gelatin, for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0081] In certain embodiments, the pharmaceutical composition is formulated for administration by any method known as described above. Particular methods of administration contemplated herein are intravenous and intracerebral administration.

[0082] The pharmaceutical composition according to any of the embodiments defined above may be formulated for intravenous, intracerebral, oral, intradermal, intramuscular, subcutaneous, transdermal, transmucosal, intranasal or intraocular administration.

[0083] The invention will now be illustrated by the following non-limiting Examples.

Examples Materials and Methods (M&M) Chemicals

[0084] NHS-PEG-OPSS (Ortho-Pyridyldisulfide-Polyethyleneglycol-N-Hydroxylsuccinimide ester), also named PDP-PEG-NHS (PDP: pyridyl dithiopropionate), with molecular weight of -2 kDa was purchased from Creative PEGworks (Winston, USA). Poly(2-ethyl-2-oxazoline), average molecular weight (Mn) -50 kDa, and anhydrous dimethylsulfoxide (DMSO) were purchased from Sigma Aldrich (Israel). Absolute ethanol was purchased from Romical (Israel). All solvents were used without further purification.

Synthesis of LPEI -22 kDa (base free form)

[0085] The cationic polymer Linear Polyethyleneimine (LPEI) was synthesized as previously described [16]. Briefly, 8.0 g (0.16 mmol) of poly(2-ethyl-2-oxazoline) was hydrolyzed with 100 ml of concentrated HCl (37%) and refluxed for 48 h, yielding a white precipitate. Excess HCl was removed under reduced pressure and the remaining solid was dissolved in 50 ml of water and freeze-dried (5 g, 78%, 1H-NMR, D2O-d6, 400 MHz: singlet 3.5 ppm). The resulting LPEI hydrochloride salt (4.5 g) was made alkaline by the addition of aqueous NaOH (3 M) and the resulting white precipitate was filtered and washed with water until neutral. The solid was then dissolved in water and further lyophilized to afford white solid (2 g, 81%).

Synthesis of LPEI-PEG2k-OPSS di-conjugates

[0086] 174 mg (8 μmols) of LPEI was dissolved in 2.7 mL of absolute EtOH and stirred at room temperature for 15 min. A 5-fold molar excess of OPPS-PEG2k-CONHS (79 mg, 39.5 μmols) was dissolved in 500 μL of anhydrous DMSO and introduced in small portions into the LPEI mixture. The reaction mixture was stirred at -800 rpm on a vortex mixer at room temperature for 3 h. Different PEG-substituted LPEIs were separated by cation exchange chromatography using a HR10/10 column packed with MacroPrep High S resin (BioRad). The purity of the eluted fractions of the diconjugates was assessed using reversed-phase HPLC equipped with a 5 μm Vydac C-8 analytical monomer column (300 Å, 4.6 × 150 mm) using a linear gradient of 5%-95% acetonitrile over 25 min at a flow rate of 1 ml/min. Fractions with 95% purity or greater were combined. The combined fractions were further dialyzed into 20 mM HEPES at pH 7.4. The ratio of PEG2k groups conjugated to LPEI in the diconjugates was determined by 1H-NMR. The integral values of the hydrogens of polyethylene -(CH2-CH2-O)- and LPEI -(CH2-CH2-NH)- were used to determine the ratio between the two conjugated copolymers. From the various products obtained from cation exchange, two products, LPEI-PEG2k-OPSS (1:1 diconjugate, with LPEI to PEG molar ratio of -1:1) and LPEI-(PEG2k)3-(OPSS)3 (1:3 diconjugate, with molar ratio of -1:3), were chosen for the generation of tri-conjugates. A copper assay was used to evaluate the copolymer concentration [17]. Briefly, the copolymers were incubated with CuSO4 (23 mg dissolved in 100 ml of acetate buffer) for 20 min and their absorbance was measured at 285 nm.

Protein electrophoresis in SDS-polyacrylamide gel (SDS-PAGE)

[0087] Samples (30 μL) were diluted in SDS protein sample buffer with or without 100 mM DTT and then applied to Tricine gel (13% polyacrylamide). Electrophoresis was performed using cathode buffer (0.1 M Tris, 0.1 M Tricine, and 0.1% SDS pH 8.25) and anode buffer (0.21 M Tris pH 8.9) and protein bands were visualized by InstantBlue™ staining.

Purification and expression of Affibody

[0088] A HER-2 Affibody gene was cloned into plasmid pET28a, generating a vector encoding Z:2891 Affibody fused to an N-terminal hexa-histidyl tag (His6) and a Cys residue at the C-terminus. The Affibody was expressed in E. coli BL21(DE3) as follows: Cells were grown at 37 °C to OD600 - 0.7. IPTG was added to a final concentration of 0.5 mM, followed by incubation at 30 °C for 4 h. The cell pellet was stored at -80 °C. To purify Affibody, the cell pellet was resuspended in buffer A (20 mM HEPES pH 7.4, 500 mM NaCI, 10% glycerol, 10 mM imidazole, and 2 mM β-mercaptoethanol) and disrupted using an M-110EHI Microfluidized Processor according to the manufacturer's instructions. The soluble fraction was recovered by centrifugation at 12,000 X g for 10 min at 4 °C. The resulting fraction was loaded onto a Ni affinity column (Clontech, Mountain View, CA). The column was washed with buffer A for 14 column volumes (cv). Thereafter, a gradient step elution was performed using increasing concentrations of buffer B (20 mM HEPES pH 7.4, 500 mM NaCI, 10% glycerol, 500 mM imidazole, and 2 mM β-mercaptoethanol); 6% buffer B for 5 hp, 10% buffer B for 15 hp, 30% buffer B for 2 hp. Bound protein was eluted with 100% buffer B for 5 hp (protein purification facility, Wolfson center, Israel). Eluted fractions were then concentrated with an Amicon filter (3 kDa cutoff) and loaded onto the Superdex 30 Gel Filtration Column Preparation Grid (120 ml) (GE healthcare). Purified proteins were further analyzed by SDS-PAGE and confirmed using Western blot analysis with anti-Affibody antibody (Abcam). Purity was further assessed by reversed-phase HPLC (Merck-Hitachi model L-7100) as previously described.

Synthesis of PEI-PEG ligand affibody (1:1 and 1:3 tri-conjugate)

[0089] 4.97 mg of each di-conjugate (1:1 and 1:3) were dissolved in 940 μL of 20 mM HEPES pH 7.4. Then, 3.4 mg of HER-2 Affibody in HBS was added dropwise to the reaction. 4 ml of 20 mM HEPES plus 700 μL of acetonitrile (HPLC grade) were introduced to the reaction mixture for increased solubility. The reaction was further centrifuged (800 rpm) at room temperature until A343 indicated complete turnover. The resulting tri-conjugates were purified by cation exchange chromatography on a HR10/10 column packed with MacroPrep High S resin (BioRad) (using three-step gradient elution from 20 mM HEPES pH 7.4 to 20 mM HEPES containing 3 M NaCl). The eluted fractions were introduced to analytical RP-HPLC to assess the purity of the tri-conjugates, fractions with 95% purity and higher were combined and kept at -80 °C. The concentration of the tri-conjugate was determined by copper assay (as above). The amount of the conjugated protein was determined by A280 using Nano-Drop 2000. Verification and purity of chemical vectors conjugated to the target protein.

[0090] The tri-conjugates were electrophoresed on SDS-PAGE, and stained with InstantBlueTM, to confirm conjugation of Affibody to LPEI-PEG2k. The purity of the tri-conjugates was confirmed by reversed-phase HPLC using a 5 μm Vydac C-8 analytical monomer column (300 Å, 4.6 x 150 mm) at 1 ml/min while monitoring at 220 nm. A gradient elution with acetonitrile, 5%-95% in 25 min with triple distilled water (TDW) containing 0.1% TFA as mobile phase, was used for HPLC analysis.

Polyplex Formation

[0091] Plasmid pGreenFire1, encoding Firefly Luciferase and GFP (System Biosciences, Inc), was amplified in E. coli and purified by Qiagen Plasmid Maxi Kits (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. The 1:1 tri-conjugate or 1:3 tri-conjugate was complexed with plasmid at a ratio of N/P=6 in HEPES buffer glucose (where N=nitrogen of LPEI and P=DNA phosphate) generating two Polyplexes. To allow complete formation of polyplex particles, samples were incubated for 30 min at room temperature. The final plasmid concentration in the polyplex samples was 100 μg/ml, while for DNase protection assay and luciferase assay, the final plasmid concentration was 10 μg/ml.

Sizing and ^-potential measurements

[0092] The sizes of the polyplex particles obtained after dispersion in HBG buffer were measured at 25 °C by dynamic light scattering using a Nano-ZS Zetasizer (Malvern, UK) using volume distribution calculation. The instrument is equipped with a 633 nm laser, and light scattering is detected at 173° by backscattering technology (NIBS, Non-Invasive Backscattering). Each sample was run in triplicate. Z potential measurements were also performed at 25 °C using a Nano-ZS Zetasizer (Malvern, UK). The Z potential was evaluated after incubation of the polyplexes in HBG buffer (pH 7.4). Light scattering from the moving particles was detected at 17°, and the Smoluchowski Model was used to determine the value of the Henry function.

Atomic force microscope

[0093] For AFM measurements, polyplexes were placed on freshly cleaved Mica disks (V1 12 mm, Ted Pella USA). Imaging was performed in HBG buffer at 25 °C using a commercial AFM, a NanoWizard® 3 (JPK instrument, Berlin, Germany) with QITM mode. Si3N4 cantilevers (serial MSNL-10, Bruker) with spring constant varying from 10 to 30 pN nm-1 were calibrated by the thermal fluctuation method (included in the AFM software) with an absolute inaccuracy of approximately 10%. The QI™ settings were as follows: Z-length: 0.1 μm; applied force: 0.5 nN; speed: 50 μm/s.

DNase protection assay

[0094] DNase I protection assays were performed as previously described [18]. In brief, 1 μg of pGreenFire1 DNA alone, with 1:1 polyplex, or with 1:3 polyplex was mixed in a final volume of 50 μL in HBS solution. After 30 min of incubation at room temperature, 2 μL of DNase I (2 units) or PBS were added to 10 μL of each sample and incubated for 15 min at 37 °C. DNase I activity was terminated by the addition of 5 μL of 100 mM EDTA for 10 min at room temperature. To dissociate the plasmid from the tri-conjugates, 10 μL of 5 mg/mL heparin (Sigma, St. Louis, MO) was added, and the tubes were incubated for 2 h at RT. Samples were electrophoresed on 0.8% agarose gel and stained with ethidium bromide. Images were captured using a Gel Doc EZ Imager (Bio Rad Laboratories, Inc).

Cell culture

[0095] BT474 cells overexpressing HER-2 were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), 104 U/L penicillin, and 10 mg/L streptomycin at 37 °C in 5% CO2. MDA-MB-231 human breast carcinoma cells were cultured in Leibovitz L-15 medium with 10% FBS, 104 U/L penicillin, and 10 mg/L streptomycin at 37 °C without CO2. Cell lines were from ATCC and cell culture reagents were from Biological Industries, Bet Ha'emek, Israel.

Luciferase assay and confocal microscope

[0096] 10000 BT474 and MDA-MB-231 cells were plated in triplicate in 96-well plates. Cells were treated with 1:1 tri-conjugate and 1:3 tri-conjugate complexed with plasmid. 48 h after treatment, cells were washed with PBS and lysed with 30 μl of cell lysis buffer (Promega, Mannheim, Germany) per well. Luciferase activity was measured in 25 μl samples of the lysates using the Luciferase Assay System (Promega) according to the manufacturer's recommendations. Measurements were performed using a Luminoskan™ Ascent Microplate Luminometer (Thermo Scientific). Values, in relative light units (RLU), are presented as mean and standard deviation of the luciferase activity of triplicate samples. Confocal microscope (FV-1200 Olympus) was used to visualize GFP, which was considered to reflect the internalization of pGreenFire1 plasmid. Images were captured at X10 magnification.

Quantification of cell viability

[0097] Cell viability was measured using a colorimetric assay using methylene blue as previously described [19]. In brief, 10000 BT474 and MDA-MB-231 cells were plated in triplicate in 96-well plates. Cells were treated with 1:1 and 1:3 polyplexes containing 1 μg/ml pGreenFire1. 48 h after treatment, cells were fixed with 1% formaldehyde in PBS (pH 7.4), washed with DDW, and then stained with 1% (wt/vol) methylene blue solution in borate buffer for 1 h. After that, the stain was extracted with 0.1 M HCl, and the optical density of the dye solution was read at 630 nm on a microplate reader (ELx800 BIO-TEX instruments Inc.).

Example 1 - Synthesis of reactive thiol copolymers.

[0098] 3.1. Previous studies have demonstrated PEGylation of LPEI and its conjugation to a target moiety of EGFR [13]. However, the amount of PEGylation on a single LPEI chain has not been fully characterized. To generate differentially PEGylated copolymers, secondary amines in LPEI were conjugated to the orthogonal terminal NHS ester protecting group in PEG. The N-hydroxysuccinimide (NHS) ester is spontaneously reactive with secondary backbone amines of LPEI, providing efficient PEGylation of LPEI. Furthermore, the reaction of NHS-PEG OPSS with PEI amines results in the formation of stable, irreversible amide bonds (Figure 1).

[0099] The PEGylation products were purified by cation exchange chromatography. Two peaks were eluted at high NaCl concentrations, one at 120 mS/cm and the other at 132 mS/cm (data not shown). The two products were suspected to differ in their LPEI:PEG ratios and, consequently, in their net positive charges. 1H-NMR spectra were analyzed using the relative integral values of the hydrogen atoms in PEG (-CH2-CH2-O-) (Figure 2) and the integral values of the hydrogen atoms in LPEI (-CH2-CH2-NH- ) (Figure 2). This analysis indicated that the material eluted in the first peak consisted of a copolymer in which each mole of LPEI was conjugated to approximately three moles of PEG. This product was named LPEI-(PEG2k)3-(OPSS)3 (“1:3 di-conjugate”). The second peak consisted of a copolymer in which equal moles of PEG were conjugated to LPEI, and was named LPEI-PEG2k-OPSS (“1:1 di-conjugate”) (Figure 2).

Example 2. Synthesis of the tri-conjugates, LPEI-PEG2k-HER2 Affibody (tri-conjugate 1:1) and LPEI-(PEG2k)-(HER2)3 Affibody (tri-conjugate 1:3)

[00100] The main objective of this study was to develop a cationic polymer that could target tumor cells overexpressing HER-2. Since HER-2 is an “orphan receptor,” Affibody molecules targeting the HER-2 receptor (rather than the ligand) were used to generate the HER-2 targeting tri-conjugates. HER-2 Affibody was expressed and purified with a Cys residue at the C-terminal end to allow further conjugation. The thiol-reactive copolymers, 1:1 and 1:3 di-conjugate, were conjugated to HER-2 Affibody via their terminal Cys residue, generating 1:1 and 1:3 tri-conjugates, respectively (Figure 3). In order to generate the triconjugates the reaction had to be carried out with low concentration of Affibody (to avoid aggregation) and in the presence of 10% acetonitrile (ACN) as a polar organic solvent to increase solubility. The reaction yields for both triconjugate reactions were approximately 33% as determined by copper assay. To confirm conjugation of Affibody to the diconjugate, the triconjugate products were reduced with DTT and separated on SDS-PAGE. Coomassie blue staining confirmed that the reduced triconjugate released Affibody (Figure 4). The amount of HER-2 Affibody present in the triconjugates was determined by measuring A280. Using a copper assay, LPEI was quantified. As described above, 1H-NMR analysis showed that the ratios of LPEI:PEG in the purified diconjugates were either 1:1 or 1:3. By comparing the molar ratios of HER-2 Affibody and LPEI, it was determined that the average ratio of HER-2 Affibody to LPEI in the 1:1 tri-conjugate was 1:1, and in the 1:3 tri-conjugate the average ratio was 3:1. Thus, it was concluded that approximate complete conjugation of Affibody to LPEI-PEG was obtained.

[00101] To generate the polyplexes, the pure di-conjugates and tri-conjugates (1:1 and 1:3) were complexed with plasmid DNA as described in the Materials and Methods (Figure 3).

Example 3. Potential and dimensioning of polyplexes

[00102] The polyplexes were next characterized with respect to size and surface charge using dynamic light scattering (DLS). The size of a polyplex has a significant impact on the distribution properties [21]. In order to investigate the effect of the targeting ligand on the size of a polyplex, it was decided to complex both 1:1 and 1:3 diconjugates with plasmid and measure their size. The 1:1 diconjugate had an average particle size of 115.2 ± 8.2 nm and the 1:3 diconjugate had an average particle size of 253.1 ± 9.5 nm. The polyplex generated from the 1:1 tri-conjugate with plasmid gave an average particle size of 141 ± 5.8 nm, whereas the 1:3 tri-conjugate complexed with plasmid had an average particle size of 256 ± 24.2 nm (Figure 5). The smallest particles (73.9 ± 3.0 nm) were obtained in polyplexes generated by complexing the plasmid with LPEI alone. Conjugation of Affibody to the di-conjugates had only a small influence on the particle size. The number of PEG groups, however, affected the particle size, suggesting that the PEG groups caused steric hindrance, interfering with plasmid condensation.

[00103] A positive surface charge facilitates binding of the polyplex to the negatively charged cell surface, but excess positive charge can lead to nonspecific binding and significant toxicity [12]. The Ç potentials of the various complexes, shown in Figure 6, are in agreement with previous studies, which showed a decrease in Ç potential with increasing number of PEG units [13]. To evaluate the effect of PEG groups on the surface charge of chemical vectors, the Ç potentials of polyplexes formed by complexation of plasmid DNA with the precursors, 1:1 and 1:3 diconjugates, and with 1:1 and 1:3 triconjugates were measured. The 1:1 diconjugate polyplex had an average Ç potential of 27.0 ± 0.1 mV and the 1:3 diconjugate polyplex had an average Ç potential of 20.0 ± 1.0 mV. The 1:1 tri-conjugated polyplex showed a mean Ç potential of 17.1 ± 0.7 mV, while the 1:3 tri-conjugated polyplex showed a mean of 10.2 ± 0.44 mV (Figure 6). Unlike the sizes, the Ç potentials of the polyplexes were affected by both the number of PEG groups and the HER-2 Affibody conjugation. Although smaller, more positively charged polyplexes were obtained with naked LPEI, these particles are extremely toxic [22]. It was expected that the addition of the PEG groups and a targeting moiety would decrease toxicity, but because the polyplexes were still relatively small in size, it was expected that their efficiency as nucleic acid delivery vectors would not be compromised.

Example 4. Evaluation of polyplex shape using Atomic Force Microscopy.

[00104] The importance of particle shape and its influence on distribution properties is gaining recognition [23]. The morphology of the polyplexes obtained with the triconjugates in solution was analyzed using Atomic Force Microscopy (AFM). The diameters of the 1:1 and 1:3 triconjugate polyplexes were both in the nanosize range (Figures 7A–B), in agreement with the results obtained by DLS. The 1:1 triconjugate polyplex exhibited mainly elliptical particles. Most of the particles ranged in diameter from 101 nm to 178 nm, with an average particle diameter of 142 nm. A few particles were exceptionally large, with some even reaching >250 nm (Figure 7A). The 1:3 triconjugate polyplex was more heterogeneous in shape, and, in addition, yielded large aggregates with undefined particle shape (Figure 7B). These ranged in length from 150 nm to 650 nm, with an average particle length of 312 nm, and their width ranged from 85 nm to 400 nm, with an average width of 175 nm.

Example 5. DNAse protection assay.

[00105] Successful gene delivery in vivo depends on efficient protection from nucleases. To determine the ability of the tri-conjugates to protect plasmids from degradation and allow efficient gene delivery, polyplexes were treated with DNase I and analyzed using gel electrophoresis. As shown in Figure 8, naked pGreenFire1 plasmid DNA was completely degraded after 10 minutes of incubation with 2 units of DNase I. In contrast, when polyplexes were generated by mixing the plasmid with the tri-conjugates, the plasmid was protected from degradation by DNase I. Complete plasmid protection was observed for the 1:3 tri-conjugate polyplex, while some crossover occurred for the 1:1 tri-conjugate polyplex, as shown by the variations from supercoiled (s.c.) to open circular (o.c.) form of the plasmid. The stronger DNase I protection conferred by the 1:3 triconjugated polyplex may be attributed to the increased steric hindrance provided by the additional PEG protein units in these complexes. Indeed, previous studies have shown that PEGylation of PEI can stabilize polyplexes and enhance their circulation in the blood by preventing their interactions with enzymes and serum factors [24, 25].

Example 6. Biological activity of target triconjugate polyplexes

[00106] The size and potential Ç of the polyplex influence the efficiency of targeted DNA delivery and gene expression, but the effect of size appears to be dependent on the specific conjugate [2][21]. To evaluate the specificity and transfection efficiency of the 1:1 and 1:3 tri-conjugate polyplexes, two breast cancer cell lines that differentially express HER-2 were used. The 1:1 and 1:3 tri-conjugate polyplexes were formed with pGreenFire1 and transfected into MDA-MB-231 cells (expressing approximately 9x103 HER-2 receptors/cell [26]) and BT474 cells (expressing approximately 1x106 HER-2 receptors/cell [27]). Differential luciferase activity was observed 48 h after transfection. Both the 1:1 and 1:3 triconjugate polyplexes led to 300-fold higher luciferase activity in BT474 than in MDA-MB-231 cells (*p < 0.001) (Figure 9A). The more efficient gene delivery for BT474 was confirmed by GFP expression as seen by confocal microscopy (Figure 9B). These results show that the selectivity of the polyplex depends on HER-2 expression.

[00107] Targeted delivery to BT474 cells by the 1:1 triconjugate polyplex was 10-fold more efficient than delivery by the 1:3 triconjugate polyplex (Figure 9A, B), even though the 1:3 triconjugate had more targeted moieties. This may reflect the higher potential and smaller size of the 1:1 triconjugate polyplex.

[00108] Positively charged LPEI-based chemical vectors are associated with significant toxicity. Therefore, we next tested the survival of MDA-MB-231 and BT474 cells after treatment with the 1:1 and 1:3 triconjugate polyplexes. Neither polyplex showed cytotoxic effects on MDA-MB-231 cells in a methylene blue assay. Similar results were observed in BT474 cells treated with the 1:3 triconjugate polyplex. However, a small increase in cell cytotoxicity was observed in BT474 treated with the 1:1 triconjugate polyplex (Figure 9C). Overall, these results indicate that the small size and higher Ç potential of the 1:1 triconjugate polyplex confer efficient targeted delivery properties with only a small increase in toxicity. Thus, the 1:1 triconjugate polyplex is superior in gene distribution than the more shielded 1:3 triconjugate polyplex.

Example 7. Antitumor activity of PEI-PEG-HER2Affibody

[00109] PEI-PEG-HER2Affibody (PPHA) complexed with PolyInosine/PolyCytosine (PolyIC) has strong antitumor activity. Breast cancer cell lines overexpressing HER-2 were found to be strongly inhibited by a complex of PEI-PEG-HER2Affibody with PolyIC. Strong inhibition was also observed in breast cancer cell lines overexpressing trastuzumab-resistant HER2.

[00110] Figure 10 shows that the vector/polyplex inhibits HER2 overexpressing cells, including Herceptin/trastusumab resistant cells.

[00111] 0.5x106 MCF-7 HER-2 cells were injected s.c. into nude mice. After tumors reached and measured 100 mm3, treatment was initiated. 1 mg/kg pIC/PPHA was injected i.v. every 24 h. Trastuzumab was administered i.v. once a week (indicated by arrows) to two groups of mice. Tumor growth was measured twice a week. The PolyIC/PPHA complex was found to have strong antitumor activity in mouse models in which these cell lines were implanted into nude mice, as exemplified in Figure 11.

Example 8. Synthesis of LPEI-PEG-EGFR Affibody

[00112] 5 mg (2 x 10-4 mmols) of LPEI-PEG2k-OPSS (1:1 di-conjugate) was dissolved in 1 ml of 20 mM HEPES pH 7.4. Then, 3.4 mg (3.8 x 10-4 mmols, ~2 eq) of EGFR Affibody in HBS was added dropwise to the reaction. 4 ml of 20 mM HEPES plus 700 μL of acetonitrile (HPLC grade) was introduced to the reaction mixture for increased solubility. The reaction was best centrifuged (800 rpm) at room temperature and in low light conditions until A343 indicates complete turnover. The resulting tri-conjugate was purified by cation exchange chromatography on a HR10/10 column packed with MacroPrep High S resin (BioRad) (using three-step gradient elution from 20 mM HEPES pH 7.4 to 20 mM HEPES containing 3 M NaCl). The eluted fractions were introduced into analytical RP-HPLC to assess the purity of the LPEI-PEG-EGFR tri-conjugate, fractions with 95% purity and higher were combined and kept at -80 °C. The concentration of the tri-conjugate was determined by copper assay. The amount of the conjugated protein was determined by A280 using Nano-Drop 2000.

Example 9. Antitumor activity of PEI-PEG-EGFR Affibody

[00113] PEI-PEG-EGFRAffibody (PPEA) complexed with PolyIC has strong antitumor activity. A variety of cell lines overexpressing EGFR were found to be strongly inhibited by a complex of PEI-PEG-EGFRAffibody (PPEA) in complex with PolyInosine/PolyCytosine (PolyIC). It can be seen that the efficacy of PPEA is greater than that of PPE (Figure 12).

[00114] The PolyIC/PPEA complex was found to have strong antitumor activity in mouse models, in which these cell lines were implanted into hairless mice.

[00115] Sixty-five 5-week-old female hairless mice were injected s.c. with 2 million A431 cells. Seven days later, tumors of average volume 136 mm3 had grown, and the mice were divided into 6 groups (7-8 mice/group) as follows: UT; pIC/PPEA, 0.75 mg/kg=250 μL pIC per 25 g mouse, IC, 6/wk; pI/PPEA, 0.75 mg/kg IV, 6/wk; pIC/PPEA-low, 0.1 mg/kg=250 μL of 0.01 μg/μL pf pIC per 25 g mouse IC, 6/wk. pIC, pI and PPE and PPEA were diluted before mixing to obtain a lower than usual pIC concentration (0.1 μg/μL) in the complex. Figure 13 shows the activity of PolyIC/PPEAffibody in vivo. Again, the efficacy of PPEA/PolyIC is greater than that of PolyIC/PPE.

Example 10. Synthesis of LPEI-PEG-h/mEGF 10.1. Synthesis of the intermediate LPEI-PEG-SH

[00116] To 5 mg of LPEI-PEG-OPSS (0.2 μmol, according to 24000 g/mol) in 5 ml of 20 mM HEPES buffer (pH 7.4) was added 50-fold molar excess of dithiothreitol (DTT; 0.1 mmol, 1.5 mg) and mixed by centrifugation for 20 min at room temperature in a 15 ml plastic centrifuge tube. The reduced di-conjugate was separated on a Sephadex G-25 column (20 ml, 4x5 ml) using 5 ml sample repeat and elution performed with 20 mM HEPES, pH 7.4 at a flow rate of 1.0 ml/min and analyzed by HPLC using the same conditions and method as described above.

[00117] The Ellman assay was used to evaluate the concentration of the sulfhydryl group in the LPEI-PEG-SH intermediate. The concentration of the SH groups is directly proportional to the concentration of the chromophore 6-nitro-3-thioxocyclohexa-1,4-diene-1-carboxylic acid, released by free thiols that react quantitatively with Ellman's reagent. The chromophore was measured via absorbance at 412 nm, without any interference from the sample or Ellman's reagent.

[00118] Summary of the synthesis. h/mEGF (human/mouse epidermal growth factor) was modified into h/mEGF-MCC (EGF-4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid; MCC) and purified to be used later for conjugation with LPEI-PEG-SH. Activation of h/mEGF by attachment to the MCC group avoids protein reduction by DTT and allows avoiding exposure of h/mEGF to harsh conditions. In this way the efficiency of the conjugation reaction is improved, in addition -EGF-MCC is a stable material that can be stored at -80 °C for weeks.

[00119] Synthesis of hEGF-MCC: 1 mg of hEGF (160 nmols) was reconstituted in 0.5 ml of water, then degassed with argon. The amount of hEGF was determined at 280 nm using Nano-drop 2000. The solution was added to 0.5 ml of 200 mM sodium acetate buffer, pH 6.0, and 60% ethanol while mixing vigorously. The solution was mixed with 10 equivalents of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC) in 0.5 ml of 100% ethanol under argon. The slightly acidic pH of the reaction mixture (pH 6) was necessary to selectively modify the N-terminal amino group of hEGF. After 4 h at room temperature, the functionalized peptide was purified by dialysis bags (cutoff 3.5k) against HBS (100 mM) three times in 1 L for 1 h then 1 L overnight. hEGF-MCC was analyzed using HPLC-mass spectra (MS) and had a molecular weight of 6435.7 g/mol, which indicates that there is a conjugation of sulfo-SMCC to hEGF. HPLC-MS analysis was performed using Thermo SCIENTIFIC/LCQ FLEET, equipped with C-18 reversed-phase column (phenomenex, Aeris, 3.6 μm, 2.1 mm x 50 mm, 100 A0).

[00120] Synthesis of mEGF-MCC: 0.5 mg (total amount of 0.088 μmol) of Murine EGF (PeproTech) was dissolved in 2100 μL of 20 mM HEPES buffer forming a clear viscous solution. One mg of sulfo-SMCC (30 eq., Ornat, IL) was dissolved in 0.9 mL of absolute EtOH and slowly mixed with EGF solution to reach the final concentration of 30% EtOH in the total volume of 3.0 mL. Brief mixing resulted in a clear solution. The reaction vessel was shaken at room temperature for 4 h. After this period of time, the solution remained clear. mEGF-MCC was first separated on Sephadex G-25 column (4x5 ml) and elution performed with 20 mM HEPES, pH 7.4 at flow rate of 1.0 ml/min and further purified and analyzed by HPLC using the same conditions and standard method as described above for LPEI-PEG-OPSS. 10.3 Synthesis of LPEI-PEG-h/mEGF

[00121] One and a half equivalents of hEGF-MCC or mEGF-MCC was mixed with 1.0 equivalent of LPEI-PEG-SH. The reaction mixture was initially stirred at room temperature for 2 hours and then incubated for 4 days at +4 °C with slow shaking. The reaction product (LPEI-PEG-hEGF or LPEI-PEG-mEGF) was separated by cation exchange chromatography (7.8 cm MacroPrep High S resin (BioRad) on a 10/1 cm Tricorn column from GE Healthcare) using the gradient pump via the buffer A18 inlet, at a flow rate of 0.5 ml/min and using solvent A: 20 mM HEPES pH 7.4 and solvent B: 20 mM HEPES pH 7.4 3.0 M NaCl. The purified LPEI-PEG-hEGF or LPEI-PEG-mEGF tri-conjugate was analyzed by HPLC and quantified by a “copper assay” and a photometric EGF assay to measure the corresponding [LPEI] and [EGF] concentrations. 10.4 Biological activity of LPEI-PEG-hEGF.

[00122] Using the cellular assay, the activity of the PolyIC/LPEI-PEG-hEGF (PPEm) complex was compared with PolyIC/mPPE (mouse) described in Schaffert D, Kiss M, Rodl W, Shir A, Levitzki A, Ogris M, Wagner E. (2011) Poly(I:C)-mediated tumor growth suppression in EGF-receptor overexpressing tumors using EGF-polyethylene glycol-linear polyethylenimine as carrier. Pharm Res. 28:731-41). It can be seen that the new HEGF conjugate is more effective in killing cells overexpressing EGFR (Figure 14). Cells expressing moderate amounts of EGFR molecules on their surface (U87MG cells express 80,000 EGFRs/cell) are less sensitive to treatment than cells overexpressing massive amounts (U87MGwtEGFR cells express 1,000,000 EGFRs/cell).

Example 11. Synthesis of DyLight 680 analogous to DUPA

[00123] The peptide was synthesized using standard Fmoc solid-phase peptide synthesis (SPPS) procedures on Wang Fmoc-Cys(trt) resin as the solid support. Swelling: the resin was swelled for at least 2 h in dichloromethane. Fmoc removal: the resin was first treated with a 20% piperidine solution in dimethylformamide (DMF) (2 × 20 min), then washed with DMF (5 × 2 min). Fmoc-Asp(OtBu)-OH coupling: 3 eq. of Fmoc-Asp(OtBu)-OH, 3 eq. of (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) (HATU) were dissolved in 15 ml of DMF, and 8 eq. of N,N-diisopropylethylamine (DIEA or DIPEA) were added to the mixture. The solution was mixed (pre-activated) for 10 min at room temperature before being added to the resin for 1 h. Coupling was repeated twice with the new mixture in order to ensure complete coupling of the aspartic acid. A Keiser test was performed to ensure complete coupling. The resin was washed with DMF (3 × 2 min) and dichloromethane (DCM) (2 × 2 min). Leveling: The resin was treated with a solution of anhydrous acetic acid (10 eq.) and DIPEA (8 eq.) in DMF for 20 min and washed with DMF (3 × 2 min). Fmoc removal: The resin was first treated with a 20% piperidine solution in DMF (2 × 20 min), then washed with DMF (5 × 2 min). Fmoc-diaminopropionic acid (DAP) coupling: 3 eq. of Fmoc-diaminopropionic acid (DAP), 3 eq. of HATU, and 8 eq. of DIEA were dissolved in 15 mL of DMF. The solution was mixed (preactivated) for 10 min at room temperature before being added to the resin for 1 h. A Keiser test was performed to ensure complete coupling. The resin was washed with DMF (3 × 2 min) and DCM (2 × 2 min). Peptide elongation: The following compounds were coupled to the resin in the following order (1) Fmoc-Phe-OH, (2) Fmoc-Phe-OH, (3) Fmoc-8-aminooctanoic acid (EAO), and (4) OtBu-Glu(Fmoc)-OH. Fmoc removal: The resin was treated with a 20% piperidine solution in DMF (2 × 20 min) and then washed with DMF (5 × 2 min) and DCM (3 × 2 min). DUPA ligand coupling: 0.9 mL of triethylamine (6.6 mmol) was combined with 0.9 g (3 mmol) of L-glutamic acid di-tert-butyl ester hydrochloride in 15 mL of DCM. This solution was added dropwise over 45 min to a solution of 5 mL of DCM and triphosgene (0.35 g, 1.1 mmol) at 0 °C. After stirring for an additional 50 min, the mixture was added to the resin with an additional 0.9 mL of triethylamine. The reaction mixture with the resin was stirred for 3 h and washed with DMF (3 × 2 min). Complete cleavage: The resin was washed with DCM (3 × 2 min) and dried under vacuum. A solution of 2.5% TDW and 2.5% triisopropylsilane in trifluoroacetic acid (TFA) at 0 °C was added. The reaction was allowed to proceed for 4 h at room temperature, filtered and treated with a cold 1:1 ether/hexane solution, and the peptides were precipitated by centrifugation. The crude peptides were dissolved in a 1:1 acetonitrile/TDW solution and lyophilized. The crude was purified by preparative reversed-phase (RP) HPLC. DyLight 680 coupling: Under an argon atmosphere, 1 mg of DyLightTM 680 (Life Technologies, Cat. No. 46418) was dissolved in anhydrous dimethyl sulfoxide (DMSO; 100 μL) containing 50 equivalents of anhydrous diisopropylethylamino. A two-fold molar excess of the peptide ligand DUPA dissolved in anhydrous DMSO (100 μL) was added to the above mixture and stirred at room temperature. Product formations were confirmed by liquid chromatography-mass spectrometry (LC-MS). Crude DUPA near-infrared (NIR) probes were then purified by preparative RP-HPLC.

Example 12. Synthesis of Precursor Drug Analogous to Dupa

[00124] The peptide was synthesized using standard Fmoc SPPS procedures on Wang Fmoc-Cys(trt) resin as the solid support. Swelling: The resin was swelled for at least 2 h in dichloromethane. Fmoc removal: The resin was treated with a 20% piperidine solution in DMF (2 × 20 min), then washed with DMF (5 × 2 min). Fmoc-Gly-OH coupling: 3 eq. of Fmoc-Gly-OH, and 3 eq. of HATU were dissolved in 15 mL of DMF, followed by the addition of 8 eq. of DIEA. The solution was mixed (preactivated) for 10 min at room temperature before being added to the resin for 1 h. Coupling was repeated twice with the fresh mixture. A Keiser test was performed to ensure complete coupling. The resin was washed with DMF (3 × 2 min) and DCM (2 × 2 min). Leveling: The resin was treated with a solution of anhydrous acetic acid (10 eq.) and DIPEA (8 eq.) in DMF for 20 min and washed with DMF (3 × 2 min). Fmoc removal: The resin was treated with a solution of 20% piperidine in DMF (2 × 20 min) then washed with DMF (5 × 2 min). Fmoc-Trp(Boc)-OH coupling: 3 eq. of Fmoc-Trp(Boc)-OH, 3 eq. of HATU, and 8 eq. of DIEA were dissolved in 15 mL of DMF. The solution was mixed (preactivated) for 10 min at room temperature before being added to the resin for 1 h. A Keiser test was performed to ensure complete coupling. The resin was washed with DMF (3 × 2 min) and DCM (2 × 2 min). Peptide elongation: The following compounds were coupled to the resin in the following order (1) Fmoc-Trp(Boc)-OH, (2) Fmoc-Gly-OH, (3) Fmoc-Phe-OH, (4) Fmoc-8-aminooctanoic acid (EAO), and (5) OtBu-Glu(Fmoc)-OH. Fmoc removal: The resin was treated with a 20% piperidine solution in DMF (2 × 20 min), then washed with DMF (5 × 2 min) and DCM (3 × 2 min). DUPA ligand coupling: 0.9 mL of triethylamine (6.6 mmol) was combined with 0.9 g (3 mmol) of L-glutamic acid di-tert-butyl ester hydrochloride in 15 mL of DCM. This solution was added dropwise over 45 min to a solution of 5 mL of DCM and triphosgene (0.35 g, 1.1 mmol) at 0 °C. After stirring for an additional 50 min the mixture was added to the resin with an additional 0.9 mL of triethylamine. The reaction mixture with the resin was stirred for 3 h and washed with DMF (3 × 2 min). Cleavage complete: the resin was washed with DCM (3 × 2 min) and dried under vacuum. A solution of 2.5% TDW and 2.5% triisopropylsilane in trifluoroacetic acid (TFA) at 0 °C was added. The reaction was allowed to proceed for 4 h at room temperature, filtered and treated with a cold 1:1 ether/hexane solution, and the peptides containing the DUPA analogue were precipitated by centrifugation. The crude peptides were dissolved in a 1:1 acetonitrile/TDW solution and lyophilized. The crude was purified by preparative RP-HPLC. Synthesis of PEI-PEG-DUPA analogue: 4.37 mg (1.2 x 10-4 mmol) of 1:1 or 1:3 diconjugate (prepared as explained in Example 1 above) was dissolved in 940 μl of 20 mM HEPES pH 7.4. Then, 1 mg (9.1 x 10-4 mmol, -5 eq) of DUPA analogue, dissolved in 2 mL of acetonitrile was added dropwise to the reaction (ACN; HPLC grade)/(20 mM HEPES pH 7.4) at a ratio of 1:1. Then, to reach an approximate total concentration of -10% ACN in the reaction, an addition of 4 mL of 20 mM was introduced into the reaction mixture. The reaction was further centrifuged (800 rpm) in the dark at room temperature until the absorbance at wavelength 343 (A343) indicated a complete turnover. The resulting tri-conjugate was purified by cation exchange chromatography on a HR10/10 column packed with MacroPrep High S resin (BioRad) (using a three-step gradient elution from 20 mM HEPES pH 7.4 to 20 mM HEPES containing 3 M NaCl). The eluted fractions were introduced into an analytical RP-HPLC to assess the purity of the tri-conjugate. Fractions with 95% purity and higher were combined and kept at -80 °C. The concentration of the tri-conjugate was determined by copper assay. The amount of DUPA analog conjugate was determined by chromophore absorption (Trp amino acid).

Example 13. PolyIC/LPEI-PEG-DUPA targeting prostate cancer.

[00125] Prostate surface membrane antigen (PSMA) is overexpressed in metastatic prostate cancer. It is also found in the neovasculature of most solid tumors. Since PSMA is internalized upon ligand binding, it was chosen as a target, we designed and synthesized a PolyIC PSMA targeting vector. Indeed, DUPA-Daylight680 is internalized into PSMA-overexpressing cells (LNCaP) but not into MCF7 (HER2-overexpressing) (not shown).

[00126] Figure 15 shows that PEI-PEG-DUPA(PPD)/PolyIC is highly efficient against LNCaP and VCaP cells. Viability was measured after 96 h of exposure. PPD also induces cytokine production (Figure 16). In Figure 17, it is demonstrated that the medium conditioned by LNCaP cells stimulates the expression of the cytokines INF-γ, IL-2 and TNF-α in PBMCs growing in the conditioned medium. It was then shown that the coincubation of PolyIC/PPD treated LNCaP cells with PC3-Luciferase cells that do not express PSMA resulted in up to 70% killing of PC3-Luciferase cells via bystander effect. The addition of healthy human PBMCs strongly intensified the effect and led to the killing of 90% of PC3 cells (Figure 18).

Example 14. In vivo cancer treatment.

[00127] PolyIC/PPD has significant antitumor effects when tested in SCID mice (Figure 19). Tumor inhibition is not complete due to the lack of an immune system in the experimental mouse. REFERENCES 1. Boussif, O., et al., A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci U S A, 1995. 92(16): p. 7297-301. 2. Wagner, E., et al., Transferrin-polycation-DNA complexes: the effect of polycations on the structure of the complex and DNA delivery to cells. Proc Natl Acad Sci U S A, 1991. 88(10): p. 4255-9. 3. Little, S.R., et al., Poly-beta amino ester-containing microparticles enhance the activity of nonviral genetic vaccines. Proc Natl Acad Sci U S A, 2004. 101(26): p. 9534-9. 4. Davis, M.E., Z.G. Chen, and D.M. Shin, Nanoparticle therapeutics: an emerging treatment modality for cancer. Nat Rev Drug Discov, 2008. 7(9): p. 771-82. 5. 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Claims (31)

1. A polyplex of a double-stranded RNA (dsRNA) and a polymeric conjugate, characterized in that said polymeric conjugate consists of a linear polyethyleneimine (LPEI) covalently linked to one or more polyethylene glycol (PEG) moieties, each PEG moiety being conjugated via a linker to a targeting moiety capable of binding to a cancer antigen, provided that the targeting moiety is not mouse EGF or the peptide of the sequence SEQ ID NO: 1, wherein: the polymeric conjugate is a diconjugate of formula (i)-(viii), linked to said targeting moiety/moieties: (iii) -(NH-(CH2)7-CO)-Phe-Phe-(NH-CH2-CH(NH2)-CO)-Asp-Cys-PEG2k-LPEI; (iv) -(NH-(CH2)7-CO)-Phe-Gly-Trp-Trp-Gly-Cys-PEG2k-LPEI; (v) where m and n are > 1. 2. Polyplex according to claim 1, characterized by the fact that each strand of said dsRNA has 20 to 300 ribonucleotides. 3. The polyplex of claim 1 or 2, wherein the dsRNA is polyinosinic-polycytidylic acid (poly I:C) double-stranded RNA, and the cancer antigen is epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), or prostate surface membrane antigen (PSMA). 4. The polyplex of any one of claims 1 to 3, wherein: (a) said targeting moiety is HER2 affibody, and said polymeric conjugate is of formula (i), and the HER2 affibody is linked via a mercapto group thereof; (b) said targeting moiety is HER2 affibody, and said polymeric conjugate is of formula (v), and the HER2 affibody is linked via a mercapto group thereof; (c) said targeting moiety is EGFR affibody, and said polymeric conjugate is of formula (i), and the EGFR affibody is linked via a mercapto group thereof; (d) said targeting moiety is EGFR affibody, and said polymeric conjugate is of formula (v), and the EGFR affibody is linked via a mercapto group thereof; (e) said targeting moiety is hEGF, and said polymeric conjugate is of formula (ii), wherein hEGF is linked via an amino group thereof; (f) said targeting moiety is hEGF, and said polymeric conjugate is of formula (vi), wherein hEGF is linked via an amino group thereof; (g) said targeting moiety is HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-(DUPA residue), and said polymeric conjugate is of formula (iii); (h) said targeting moiety is HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-(DUPA residue), and said polymeric conjugate is of formula (vii); (i) said target moiety is HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-(DUPA residue), and said polymer conjugate is of formula (iv); or (j) said target moiety is HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-(DUPA residue), and said polymer conjugate is of formula (viii). 5. Polyplex according to claim 4, characterized by the fact that said EGFR affibody has the amino acid sequence as set forth in SEQ ID NO: 4 and said HER2 affibody has the amino acid sequence as set forth in SEQ ID NO: 5. 6. Polyplex according to claim 1, characterized in that said polymeric conjugate is a diconjugate of formula (ii) linked to said target moiety. 7. Polyplex according to claim 6, characterized by the fact that said targeting moiety is hEGF, and hEGF is linked via an amino group thereof. 8. The polyplex of claim 6 or 7, wherein said dsRNA is polyinosinic-polycytidylic acid (poly I:C) double-stranded RNA and the cancer antigen is epidermal growth factor receptor (EGFR). 9. Polyplex according to claim 1, characterized in that said polymeric conjugate is a diconjugate of formula (vi) linked to said target moieties. 10. The polyplex of claim 9, wherein said targeting moiety is hEGF, and hEGF is linked via an amino group thereof. 11. The polyplex of claim 9 or 10, wherein said dsRNA is polyinosinic-polycytidylic acid (poly I:C) double-stranded RNA and the cancer antigen is epidermal growth factor receptor (EGFR). 12. Polyplex according to claim 1, characterized in that said polymeric conjugate is a diconjugate of formula (iv) or (viii) linked to said target moiety. 13. Polyplex according to claim 12, characterized in that said target moiety is HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-(DUPA residue). 14. The polyplex of claim 12 or 13, wherein said dsRNA is polyinosinic-polycytidylic acid (poly I:C) double-stranded RNA and the cancer antigen is prostate surface membrane antigen (PSMA). 15. Polyplex according to any one of claims 2 to 14, characterized in that it is for use in the treatment of a cancer selected from a cancer characterized by cells overexpressing EGFR, a cancer characterized by cells overexpressing HER2 and prostate cancer. 16. Polyplex according to claim 15, characterized by the fact that said cancer having as a characteristic cells overexpressing EGFR is selected from non-small cell lung carcinoma, breast cancer, glioblastoma, head and neck squamous cell carcinoma, colorectal cancer, adenocarcinoma, ovarian cancer, bladder cancer or prostate cancer, and metastases thereof. 17. Polyplex according to claim 16, characterized in that: - said targeting moiety is EGFR affibody, and said polymeric conjugate is of formula (i), and the EGFR affibody is linked via a mercapto group thereof; - said targeting moiety is EGFR affibody, and said polymeric conjugate is of formula (v), and the EGFR affibody is linked via a mercapto group thereof; - said targeting moiety is hEGF, and said polymeric conjugate is of formula (ii), wherein the hEGF is linked via an amino group thereof; or - said targeting moiety is hEGF, and said polymeric conjugate is of formula (vi), wherein the hEGF is linked via an amino group thereof. 18. Polyplex according to claim 15, characterized by the fact that said cancer having as a characteristic cells overexpressing HER2 is selected from breast cancer, ovarian cancer, stomach cancer, and aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma. 19. Polyplex according to claim 18, characterized by the fact that said cancer that has as a characteristic cells overexpressing HER2 is cancer resistant to Herceptin/trastuzumab. 20. Polyplex according to claim 18 or 19, characterized in that: - said targeting moiety is HER2 affibody, and said polymeric conjugate is of formula (i), and the HER2 affibody is linked via a mercapto group thereof; - said targeting moiety is HER2 affibody, and said polymeric conjugate is of formula (v), and the HER2 affibody is linked via a mercapto group thereof; - said targeting moiety is hEGF, and said polymeric conjugate is of formula (ii), wherein the hEGF is linked via an amino group thereof; or - said targeting moiety is hEGF, and said polymeric conjugate is of formula (vi), wherein the hEGF is linked via an amino group thereof. 21. The polyplex of claim 15, wherein said cancer is prostate cancer and: - said targeting moiety is HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-(DUPA residue), and said polymeric conjugate is of formula (iii); - said targeting moiety is HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-(DUPA residue), and said polymeric conjugate is of formula (vii); - said targeting moiety is HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-(DUPA residue), and said polymeric conjugate is of formula (iv); or - said target moiety is HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-(DUPA residue), and said polymeric conjugate is of formula (viii). 22. Polyplex according to any one of claims 15 to 21, characterized in that it is for use in combination with immune cells. 23. The polyplex of claim 22, wherein said immune cells are tumor-infiltrating T cells (T-TILs), tumor-specific engineered T cells, or peripheral blood mononuclear cells (PBMCs). 24. Use of a polyplex defined in any one of claims 2 to 14, characterized in that it is for the manufacture of a medicament to treat a cancer selected from the group consisting of a cancer that has as a characteristic cells overexpressing EGFR, a cancer that has as a characteristic cells overexpressing HER2 and prostate cancer. 25. Use of a polyplex according to claim 24, characterized in that it is in combination with immune cells. 26. Use of a polyplex according to claim 25, characterized in that said immune cells are tumor-infiltrating T cells (T-TILs), tumor-specific engineered T cells, or peripheral blood mononuclear cells (PBMCs). 27. Pharmaceutical composition, characterized by the fact that it comprises a pharmaceutically acceptable vehicle and a polyplex defined in any one of claims 1 to 14. 28. Pharmaceutical composition according to claim 27, characterized in that it is for use in the treatment of a cancer selected from a cancer that has as a characteristic cells overexpressing EGFR, a cancer that has as a characteristic cells overexpressing HER2 and prostate cancer. 29. Pharmaceutical composition, characterized by the fact that it comprises a pharmaceutically acceptable vehicle and a polyplex defined in any one of claims 2 to 14, for the treatment of a cancer selected from a cancer that has as a characteristic cells overexpressing EGFR, a cancer that has as a characteristic cells overexpressing HER2 and prostate cancer. 30. Pharmaceutical composition according to claim 29, characterized in that it is for use in combination with immune cells. 31. Pharmaceutical composition according to claim 30, characterized in that said immune cells are tumor-infiltrating T cells (T-TILs), tumor-specific engineered T cells, or peripheral blood mononuclear cells (PBMCs).