NZ764423B2 - Improved polyethyleneimine polyethyleneglycol vectors - Google Patents
Improved polyethyleneimine polyethyleneglycol vectors Download PDFInfo
- Publication number
- NZ764423B2 NZ764423B2 NZ764423A NZ76442315A NZ764423B2 NZ 764423 B2 NZ764423 B2 NZ 764423B2 NZ 764423 A NZ764423 A NZ 764423A NZ 76442315 A NZ76442315 A NZ 76442315A NZ 764423 B2 NZ764423 B2 NZ 764423B2
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- New Zealand
- Prior art keywords
- polyplex
- peg
- lpei
- conjugate
- alkylene
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- 101700006494 nucA Proteins 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
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- 125000002971 oxazolyl group Chemical group 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
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- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 125000000843 phenylene group Chemical group C1(=C(C=CC=C1)*)* 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
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- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 230000000063 preceeding Effects 0.000 description 1
- OZAIFHULBGXAKX-UHFFFAOYSA-N precursor Substances N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
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- 239000003755 preservative agent Substances 0.000 description 1
- 239000003380 propellant Substances 0.000 description 1
- 125000006410 propenylene group Chemical group 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-M propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 108020001580 protein domains Proteins 0.000 description 1
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- 125000003373 pyrazinyl group Chemical group 0.000 description 1
- 125000003226 pyrazolyl group Chemical group 0.000 description 1
- 125000000714 pyrimidinyl group Chemical group 0.000 description 1
- 239000002510 pyrogen Substances 0.000 description 1
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- 238000011002 quantification Methods 0.000 description 1
- 125000002943 quinolinyl group Chemical group N1=C(C=CC2=CC=CC=C12)* 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 150000003335 secondary amines Chemical class 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
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Classifications
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/17—Immunomodulatory nucleic acids
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- C12N2310/00—Structure or type of the nucleic acid
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- C12N2310/53—Physical structure partially self-complementary or closed
Abstract
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. g to a cancer antigen.
Description
(12) Granted patent specificaon (19) NZ (11) 764423 (13) B2
(47) Publicaon date: 2021.12.24
(54) IMPROVED POLYETHYLENEIMINE POLYETHYLENEGLYCOL VECTORS
(51) Internaonal Patent Classificaon(s):
A61K 31/7115A61K 47/34 C12N 15/117 C07K 16/28 C07K 17/06
(22) Filing date: (73) Owner(s):
2015.05.14 TargImmune Therapeutics AG
(23) Complete specificaon filing date: (74) Contact:
2015.05.14 Griffith Hack
(62) Divided out of 727092 (72) Inventor(s):
LEVITZKI, Alex
(30) Internaonal Priority Data: JOUBRAN, Salim
US 61/993,110 2014.05.14 SHIR, Alexei
ZIGLER, Maya
TALHAMI, Alaa
LANGUT, Yael
(57) Abstract:
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) moiees, each PEG moiety being conjugated via a linker to a targeng moiety capable
of binding to a cancer angen.
NZ 764423 B2
IMPROVED POLYETHYLENEIMINE POLYETHYLENEGLYCOL VECTORS
FIELD OF INVENTION
The present invention relates to non-viral polyethylenimine-based polyplexes
conjugated to a targeting moiety capable of binding to a cancer antigen.
BACKGROUND OF THE INVENTION
One of the hurdles facing molecular medicine is the targeted delivery of therapeutic
agents such as DNA or RNA molecules. An emerging strategy is the construction of non-viral
vectors, such as cationic polymers and cationic lipids, which bind and condense nucleic acids.
These non-viral cationic vectors possess many advantages over viral gene vectors, as they are
non-immunogenic, non-oncogenic and easy to synthesize [1-4]. Currently, several synthetic
polycationic polymers are being developed for nucleic acid delivery. Among these,
polyethylenimines (PEIs) are considered promising agents for gene delivery [5].
PEIs are water-soluble, organic macromolecules that are available as both linear and
branched structures [6]. PEIs change their degree of ionization over a broad range of pH, since
every third atom in their backbone chain 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 the
polyplexes, endosome release and high efficiency gene transfer are driven by the "proton sponge
effect" [10]. The ability of PEI to condense DNA appears to be an important factor in delivering
large DNA constructs into many cell types.
The major concern in the utilization of PEIs as delivery carriers is toxicity, due to their
high positive surface charge, which may lead to non-specific binding [11]. Recent attempts have
been made to improve the selectivity and biocompatibility of non-viral 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 coupling of
the PEI to a targeting ligand, which provides efficient gene delivery into cells harboring the
17784661_1 (GHMatters) P42431NZ01
cognate receptor [12]. We have previously described the generation of targeting vectors,
demonstrating the difference between branched PEI (brPEI-EGF ) and linear PEI (LPEI)
tethered to EGF as targeting vectors [13, , ]. Current
methods of synthesis are unsatisfactory in that they result in insufficiently homogeneous
products. It would be desirable to provide methods that can provide efficient conjugation of
targeting moieties to the LPEI-PEG in a reproducible manner. These methods may be used to
produce homogenous batches of products that can be reliably used in methods for treating
cancer.
[0004a] It is to be understood that if any prior art publication is referred to herein, such reference
does not constitute an admission that the publication forms a part of the common general
knowledge in the art in Australia or any other country.
SUMMARY OF INVENTION
In one aspect, the present invention is related 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).
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.
In yet another aspect, the present invention provides the polyplex of the present
invention as defined herein, or the pharmaceutical composition comprising the polyplex, for use
in treatment of a cancer selected from a cancer characterized by EGFR-overexpressing cells, a
cancer characterized by HER2-overexpressing cells and prostate cancer.
In still another aspect, the present invention is related to a method for treating a cancer
selected from the group consisting of a cancer characterized by EGFR-overexpressing cells, a
cancer characterized by HER2-overexpressing cells and prostate cancer, the method comprising
administering to a subject in need a polyplex of the present invention as defined herein.
17784661_1 (GHMatters) P42431NZ01
In a further aspect, the present invention is related to a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and the polyplex of the present invention, for
treatment of a cancer selected from a cancer characterized by EGFR-overexpressing cells, a
cancer characterized by HER2-overexpressing cells and prostate cancer.
The polyplex of the present invention may be used in combination with immune cells.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows that conjugation of LPEI (~22 kDa) with NHS-PEG-OPSS (~2 kDa )
yielded mainly two co-polymeric networks that differ in the degree of PEGylation. The co-
polymer LPEI-(PEG -OPSS) (“di-conjugate 1:3”) consisted on average of 1 mole of LPEI
2k 3
and 3 moles of PEG, whereas co-polymer LPEI-PEG -OPSS (“di-conjugate 1:1”), consisted
on average of 1 mole ratio of LPEI and 1 mole of PEG. (Ratios of LPEI :PEG were determined
by H-NMR analysis.)
Figure 2 shows H-NMR analysis of the two di-conjugates, LPEI-PEG -OPSS (di-
conjugate 1:1) and LPEI-(PEG -OPSS) (di-conjugate 1:3). The coupling of PEG groups to
2k 3
LPEI was indicated by the presence of the 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 di-
conjugate 1:1 (A) and di-conjugate 1:3 (B) were deduced.
Figure 3 shows a scheme for conjugation of the co-polymeric networks (di-conjugate
1:1 and di-conjugate 1:3) to the affibody (“Her-2”) through disulfide exchange, resulting in the
generation of two differently PEGylated tri-conjugates [20].
Figure 4 depicts an SDS/PAGE of purified affibody, di-conjugate and tri-conjugate,
in the absence and in the presence of DTT. In the presence of DTT, the affibody is released
from the tri-conjugate, and migrates alongside purified affibody (slightly above 10 kDa).
Figure 5 shows particle sizing using DLS measurements of LPEI, the di-conjugates and
the tri-conjugates complexed with plasmid pGreenfire 1 in HBG buffer pH 7.4.
Figure 6 depicts ξ potential distributions of LPEI, di-conjugates and tri-conjugates
complexed with plasmid pGreenfire1. The zeta potentials were measured by DLS and calculated
by the Smoluchowski equation.
17784661_1 (GHMatters) P42431NZ01
Figs. 7A-B show atomic force microscopy (AFM) images obtained from measurements
performed in HBG buffer pH 7.4 for both polyplexes. (A) tri-conjugate 1:1 Polyplex (B) tri-
conjugate 1:3 Polyplex. Scale bar is 1µm.
Figure 8 depicts an agar gel showing that differentially PEGylated polyplexes protect
plasmid pGreenFire1 from DNase I degradation. 1 µg plasmid (pGreenFire1) alone or in tri-
conjugate polyplexes: 1:1 and 1:3 was treated with or without DNase I (2 IU). Supercoiled
plasmid (s.c.), open circular plasmid (o.c.).
Figs. 9A-C show Her-2 mediated gene transfer of pGFP-LUC using the tri-conjugate
1:1 polyplex and the tri-conjugate 1:3 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 tri-conjugates 1:1 and 1:3 complexed with pGFP-LUC (1µg/ml) to generate the two
polyplexes. PEI nitrogen/DNA phosphate ratio of 6 (N/P=6) in HBS. (A) Measurements of
luciferase activity demonstrate significant decreased pGreenFire1 delivery in MDA-MB-231
cells, compared to BT474 and reduced gene delivery mediated by tri-conjugate 1:3 polyplexes
as compared to tri-conjugate 1:1 (* p < 0.001). Luciferase activity was measured in triplicates
after 48 h shown as relative lucifarese units (RLU) as mean +S.D. (B) Fluorescent images of
cells treated with polyplexes. Images are shown at X10 magnification and are representative of
three experiments performed. (C) Methylene blue assay depicts percent of cell survival
compared to untreated (UT) cells.
Figure 10 shows that PEI-PEG-Her2Affibody (PPHA) complexed with PolyIC inhibits
Her2 overexpressing breast cancer cells BT474. The complexed vector inhibits Her2
overexpressing cells, including Herceptin/trastusumab resistant cells.
Figure 11 shows inhibition of MCF7 cells overexpressing Her2 injected in nude mice.
Figs. 12A-B shows the efficacy of (A) PEI-PEG-EGFRAffibody (PPEAffibody) in
comparison to that of (B) PPE.
Figure 13 shows the activity of PolyIC/PPEaffibody in vivo as compared with untreated
(UT), pIC/PPE, pI/PPE, pI/PPEA and pIC/PPEA low (0.1µg/µl pIC in the complex).
Figure 14 shows survival of U87MG, U87MGwtEGFR cells after application of
different concentrations of PolyIC/LPEI-PEG-hEGF complex as compared with application of
PolyIC/mPPE (mouse) as described in Schaffert D, Kiss M, Rödl W, Shir A, Levitzki A, Ogris
17784661_1 (GHMatters) P42431NZ01
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.
Figure 15 shows that PEI-PEG (PP)-DUPA (PPD)/PolyIC is highly effective against
LNCaP and VCaP cells . Viability was measured after 96 hr of exposure.
Figs. 16A-B shows production of the cytokines (A) IP-10, (B) RANTES by LNCaP cell
transfected with PolyIC/PPD.
Fig. 17 shows that medium conditioned by LNCaP cells stimulates expression of
cytokines in PBMCs. expression after 24 hrs incubation.
Figure 18 shows that co-incubation of PolyIC/PPD treated LNCaP cells with PC3-
Luciferase cells which do not express PSMA, resulted in up to 70% killing of the PC3-
Luciferase cells via bystander effect. Addition of healthy human PBMCs strongly enhanced the
effect and lead to the killing of 90% of the PC3 cells.
Figure 19 shows effect of PolyIC/PPD on subcutaneous LNCaP tumors in vivo. UT,
untreated.
DETAILED DESCRIPTION OF THE INVENTION
Polycations, especially PEI, have been intensively investigated as agents for gene
transfection. Optimal transfection efficacies are obtained when the polymeric nanoparticle
complexes possess an overall positive charge, which allows them to bind to the negatively
charged heparin sulfate proteoglycans on the cell surface [28]. Previous studies showed that
linear PEI (LPEI) is more effective in gene transfection than branched PEI (brPEI) [29-31] [32,
33, ], but that LPEI has higher positive charge and hence is more toxic.
Various 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 lower the positive charge and the consequent
toxicity. Indeed, shielding of PEIs with PEG groups of varying lengths significantly lowered
toxicity while maintaining transfection efficiency [12, 13, 36].
It has been found in accordance with the present invention that the conjugation of LPEI
with PEG yields di-conjugate co-polymers comprising various ratios of LPEI to PEG . These
2k 2K
di-conjugates could be separated from one another using cation exchange chromatography, due
17784661_1 (GHMatters) P42431NZ01
to differences in charge, which reflect the different numbers of PEG groups conjugated. H-
NMR analysis confirmed that the di-conjugates differed from one another in the average number
of PEG units per LPEI unit, where di-conjugate 1:1 had an LPEI:PEG ratio of 1:1 and di-
2k 2k
conjugate 1:3 had an LPEI:PEG ratio of 1:3. The conjugation of the Her-2 targeting affibody
to each of the purified di-conjugates yielded a tri-conjugate product of the appropriate molecular
weight, i.e. from di-conjugate 1:1 we obtained “tri-conjugate 1:1” with LPEI:PEG :Her-2 equal
to 1:1:1 and from di-conjugate 1:3 we obtained “tri-conjugate 1:3”, with ratio 1:3:3. This
protocol enabled us to obtain homogeneous products, with nearly complete conjugation of
targeting affibody to LPEI-PEG, in a reproducible manner.
We observed that PEGylation strongly affects the size of the polyplex particles obtained
upon complexation of the di-conjugates or tri-conjugates with plasmid DNA. Both di-conjugate
1:3 and tri-conjugate 1:3 polyplexes had average particle sizes larger than di-conjugate 1:1 and
tri-conjugate 1:1 polyplexes. We believe that increasing the amount of PEGylation on a single
cationic chain leads to steric hindrance, which prevents the polymeric chain from condensing
the plasmid to a smaller particle. This is consistent with the finding that the naked LPEI polyplex
had the smallest particles. Moreover, while both tri-conjugates 1:1 and 1:3 protected complexed
plasmid from DNase I, the tri-conjugated 1:3 polyplex provided better protection, possibly due
to the increased steric hindrance.
Previous studies suggested that increasing the molecular weight of the PEG units
conjugated to cationic polymers led to decreased surface charge of the polyplexes obtained upon
complexation with nucleic acids [13]. Our data show that increasing the number of PEG groups
of similar molecular weights leads to decreased surface charge, as defined by ξ 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. Surprisingly, the tri-conjugate polyplexes conjugated to
affibodies had lower surface charge than the di-conjugate polyplexes, showing that the Her-2
affibody (which itself has slight positive charge) also reduced the surface charge of the particles.
We suspect that Her-2 affibody changes the topography of the particle with more targeting
moieties masking the charge on the surface, leading to a decrease in surface charge.
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
17784661_1 (GHMatters) P42431NZ01
drug delivery. The effect of PEGylation on polyplex shape has not been investigated, to our
knowledge, until now. In AFM pictures, the tri-conjugate 1:1 polyplex – which was more
effective in gene delivery – presented shape homogeneity, while the tri-conjugate 1:3 was more
heterogenic, with many asymmetrical, ellipsoidal particles.
Selective gene transfer using cationic polymers remains a major challenge. Previous
studies have shown that targeting of LPEI and LPEI-PEG conjugates, with EGF or transferrin,
increased their selectivity and decreased non-specific interactions both in vitro and in vivo [39,
40]. For example, to examine the selectivity of our Her-2 targeting tri-conjugates 1:1 and 1:3
polyplexes, we utilized two breast cancer cells that differentially express Her-2. Gene delivery,
as shown by luciferase activity and GFP expression, was significantly higher in BT474 cells,
which highly overexpress the Her-2 receptor, than in MDA-MB-231 cells, which express 100-
fold less Her-2 receptors on the cell surface. Thus, the data demonstrate that both tri-conjugates
1:1 and 1:3 are highly selective for Her-2 overexpressing cells (Figure 10).
Previous studies have shown that high levels of PEGylation can result in reduced gene
transfection [41]. These results are in accordance with our observation that highly PEGylated
tri-conjugate 1:3 polyplex showed a significant reduction in gene delivery, as compared to the
less PEGylated tri-conjugate 1:1 polyplex, as shown by luciferase activity and GFP expression.
The increased gene delivery by the lower PEGylated tri-conjugate 1:1 polyplex was
accompanied by slight cellular toxicity, most likely due to its higher surface charge.
Our working hypothesis before engaging in this study was that increasing the number
of targeting moieties per LPEI unit would lead to improved gene delivery and/or selectivity. We
speculated that tri-conjugate 1:3, which has 3 moles of Her-2 affibody molecules conjugated
per mole of LPEI, would show increased receptor-mediated particle internalization. However,
the tri-conjugate 1:3 polyplexes showed lower ξ potential, larger particle size and
heterogeneous, non-spherical shape, all of which characteristics might contribute to the
decreased transfection efficiencies actually observed. Our results show that the less PEGylated
tri-conjugate 1:1 is superior to the more PEGylated tri-conjugate 1:3 in mediating selective and
efficient gene delivery into Her-2 overexpressing cells.
It has been found 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.
17784661_1 (GHMatters) P42431NZ01
Our simplified synthesis allows purification of homogeneous products in a reproducible
fashion, which can now be expanded to generate different tri-conjugates, using a variety of
targeting moieties.
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).
In certain embodiments, the cancer antigen may 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 may be
a native, natural or modified 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 one 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 parent
protein domain.
In certain embodiments, the dsRNA is polyinosinic-polycytidylic acid double stranded
RNA (poly I:C), 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.
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 a
number of ribonucleotides within the range of 20 to 300.
In certain embodiments, the one or more PEG moieties each independently forms -NH-
CO- bond with the LPEI and a bond selected from -NH-CO-, -CO-NH-, -S-C-, -S-S-, -O-CO-
17784661_1 (GHMatters) P42431NZ01
or -CO-O- with the linker. In particular, each one of the one or more PEG moieties forms -NH-
CO- bonds with the LPEI and the linker.
In certain embodiments, the linker forms an -S-S-, NH-CO-, -CO-NH-, -S-C-, O-CO-, -
CO-O- or urea (-NH-CO-NH) bond with the targeting moiety. The linker may be selected from
-CO-R -R -R or a peptide moiety consisting of 3 to 7 amino acid residues, wherein
2 x 3
R is selected from (C -C )alkylene, (C -C )alkenylene, (C -C ) alkynylene, (C -
2 1 8 2 8 2 8 6
C )arylene-diyl, or heteroarylenediyl;
R is absent or -S-;
R is absent or of the formula ;
R4 is selected from (C1-C8)alkylene, (C2-C8)alkenylene, (C2-C8) alkynylene, (C1-
C )alkylene-(C -C )cycloalkylene, (C -C )alkenylene-(C -C )cycloalkylene, (C -C )
8 3 8 2 8 3 8 2 8
alkynylene-(C -C )cycloalkylene, (C -C )arylene-diyl, heteroarylenediyl, (C -C )alkylene-
3 8 6 10 1 8
(C -C )arylene-diyl, or (C -C )alkylene-heteroarylenediyl;
6 10 1 8
wherein each one of said (C -C )alkylene, (C -C )alkenylene, or (C -C ) alkynylene is
1 8 2 8 2 8
optionally substituted by one or more groups each independently selected from halogen, -COR ,
-COOR , -OCOOR , -OCON(R ) , -CN, -NO , -SR , -OR , -N(R ) , -CON(R ) , -SO R , -
5 5 2 2 5 5 5 2 5 2 2 5
SO H, -S(=O)R , (C -C )aryl, (C -C )alkylene-(C -C )aryl, heteroaryl, or (C -C )alkylene-
3 5 6 10 1 4 6 10 1 4
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(R )-, -N(C -C aryl)-, (C -C )arylene-diyl, or heteroarylenediyl; and
6 10 6 10
R is H or (C -C )alkyl.
1 8
In certain embodiments, R is selected from (C -C )alkylene, preferably (C -
2 1 8 1
C )alkylene, optionally substituted by one or more groups each independently selected from
halogen, -COH, -COOH, -OCOOH, -OCONH2, -CN, -NO2, -SH, -OH, -NH2, -CONH2, -SO2H,
-SO H, -S(=O)H, (C -C )aryl, (C -C )alkylene-(C -C )aryl, heteroaryl, or (C -C )alkylene-
3 6 10 1 4 6 10 1 4
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(C -C alkyl)-, -N(C -C aryl)-, (C -C )arylene-diyl, or heteroarylenediyl.
1 8 6 10 6 10
In particular, R is selected from (C -C )alkylene, preferably (C -C )alkylene.
2 1 8 1 4
In certain embodiments, R is -S-.
17784661_1 (GHMatters) P42431NZ01
In certain embodiments, R is absent or , wherein
R is (C -C )alkylene-(C -C )cycloalkylene, preferably (C -C )alkylene-(C -
4 1 8 3 8 1 4 5
C )cycloalkylene.
In certain embodiments, in the polyplex as defined above, R is -CH -CH -; R is -S-;
2 2 2 x
and R is absent or , wherein R is .
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-(CH ) -CO)-Phe-Phe-(NH-CH -
2 7 2
CH(NH )-CO)-Asp-Cys- (SEQ ID NO: 3), linked via its mercapto group to the targeting moiety.
In certain embodiments, the polymeric conjugate is a diconjugate of the formula (i)-
(viii), linked to the targeting moiety/moieties:
N N OH
H N N N
(ii)
(iii) -(NH-(CH ) -CO)-Phe-Phe-(NH-CH -CH(NH )-CO)-Asp-Cys-PEG -LPEI;
2 7 2 2 2k
(iv) -(NH-(CH ) -CO)-Phe-Gly-Trp-Trp-Gly-Cys-PEG -LPEI;
2 7 2k
17784661_1 (GHMatters) P42431NZ01
(vi)
HN N N
O O O
O O O
HN HN HN
O O O
S S S
O O O
O O O
N N N
CO- CO- CO-
(vii)
17784661_1 (GHMatters) P42431NZ01
wherein R is
; or
(viii)
N N OH
HN N N
O O O
O O O
HN HN HN
O O O
S S S
R R R
7 7 7
wherein R is
HOOC
In particular embodiments , the polyplex is selected from
(a) the polyplex, wherein the targeting moiety is HER2 affibody, and the polymeric
conjugate is of the formula (i) above, and the HER2 affibody is linked via a mercapto group
thereof, herein also referred to as LPEI-PEG -HER2;
17784661_1 (GHMatters) P42431NZ01
(b) the polyplex, wherein the targeting moiety is HER2 affibody, and the polymeric
conjugate is of the formula (v) above, and the HER2 affibody is linked via a mercapto group
thereof, herein also referred to as LPEI-(PEG -HER2) ;
2k 3
(c) the polyplex, wherein the targeting moiety is EGFR affibody, and the polymeric
conjugate is of the formula (i) above, and the EGFR affibody is linked via a mercapto group
thereof, herein also referred to as LPEI-PEG -EGFR;
(d) the polyplex, wherein the targeting moiety is EGFR affibody, and the polymeric
conjugate is of the formula (v) above, and the EGFR affibody is linked via a mercapto group
thereof, herein also referred to as LPEI-(PEG -EGFR) ;
2k 3
(e) the polyplex, wherein the targeting moiety is human EGF (hEGF), and the
polymeric conjugate is of the formula (ii) above, wherein the hEGF is linked via an amino
group thereof, herein also referred to as LPEI-PEG -hEGF;
(f) the polyplex, wherein the targeting moiety is hEGF, and the polymeric conjugate
is of the formula (vi) above, wherein the hEGF is linked via an amino group thereof, herein also
referred to as LPEI-(PEG -hEGF) ;
2k 3
(g) the polyplex, wherein the targeting moiety is HOOC(CH ) -CH(COOH)-NH-
CO-NH-CH(COOH)-(CH ) -CO- (DUPA residue), and the polymeric conjugate is of the
formula (iii) above;
(h) the polyplex, wherein the targeting moiety is HOOC(CH ) -CH(COOH)-NH-
CO-NH-CH(COOH)-(CH ) -CO- (DUPA residue), and the polymeric conjugate is of the
formula (vii) above;
(i) the polyplex, wherein the targeting moiety is HOOC(CH ) -CH(COOH)-NH-
CO-NH-CH(COOH)-(CH ) -CO- (DUPA residue), and the polymeric conjugate is of the
formula (iv) above; or
(j) the polyplex, wherein the targeting moiety is HOOC(CH2)2-CH(COOH)-NH-
CO-NH-CH(COOH)-(CH ) -CO- (DUPA residue), and the polymeric conjugate is of the
formula (viii) above.
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 142nm.
Non-limiting examples of procedures for the preparation of polymeric conjugates used
in the present invention are exemplified in Examples hereinafter.
17784661_1 (GHMatters) P42431NZ01
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.
In another aspect, the present invention relates to a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and the polyplex as defined above.
In yet another aspect, the present invention provides the polyplex of the present
invention as defined herein, or the pharmaceutical composition comprising the polyplex, for use
in treatment of a cancer selected from a cancer characterized by EGFR-overexpressing cells, a
cancer characterized by HER2-overexpressing cells and prostate cancer.
In certain embodiments, the cancer characterized by EGFR-overexpressing cells is
selected from non-small-cell-lung-carcinoma, breast cancer, glioblastoma, head and neck
squamous cell carcinoma, colorectal cancer, adenocarcinoma, ovary cancer, bladder cancer or
prostate cancer, and metastases thereof. In certain embodiments, the polyplex used for treatment
of cancer characterized by EGFR-overexpressing cells is selected from the polyplex of (c), (d),
(e) or (f) defined above.
In certain embodiments, the cancer characterized by HER2-overexpressing cells 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 Her2
overexpressing cells 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. cancer
comprising cells that do not respond, or respond to a lesser extent to exposure to
Herceptin/trastusumab.
In particular, the polyplex used for treatment of cancer characterized by HER2-
overexpressing cells is selected from the polyplex (a), (b), (e) or (f) defined above.
In certain embodiments, the cancer is prostate cancer and the polyplex used for treatment
of the prostate cancer is selected from (g), (h), (i) or (j) defined above.
In still another aspect the present invention is related to a method for treating a cancer
selected from the group consisting of a cancer characterized by EGFR-overexpressing cells, a
cancer characterized by HER2-overexpressing cells and prostate cancer, the method comprising
administering to a subject in need a polyplex of the present invention as defined herein.
17784661_1 (GHMatters) P42431NZ01
In a further aspect the present invention is related to a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and the polyplex of the present invention, for
treatment of a cancer selected from a cancer characterized by EGFR-overexpressing cells, a
cancer characterized by HER2-overexpressing cells and prostate cancer.
In yet a further aspect, the present invention is directed to the polyplex, the method, or
the pharmaceutical composition of the present invention, for use in combination with immune
cells.
In still a further aspect, the present invention is directed to a polyplex as defined herein
above, in which the dsRNA is replaced with a DNA molecule, such as a plasmid comprising
protein-encoding nucleic acid sequences operably linked to control elements such as appropriate
promoters and terminators.
In certain embodiments, the immune cells are tumor-infiltrating T-cells (T-TILs), tumor
specific engineered T-cells, or peripheral blood mononuclear cells (PBMCs).
The term "polyplex" as used herein refers to a complex between a nucleic acid and a
polymer. The nucleic acid is bound 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 carrying and delivering nucleic acids into cells.
The term “patient”, “subject”, or “individual” are used interchangeably and refer to
either a human or a non-human animal.
The term “(C -C )alkyl”, as used herein, typically means a straight or branched
hydrocarbon radical having 1-8 carbon atoms and includes, e.g., 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.
The term “(C -C )alkylene” refers to a straight or branched divalent hydrocarbon radical
having 1-8 carbon atoms and includes, e.g., methylene, ethylene, propylene, butylene,
pentanylene, hexanylene, heptanylene, octanylene, and the like. The term “(C -C )alkenylene”
and “(C -C )alkynylene” typically mean straight 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-
17784661_1 (GHMatters) P42431NZ01
methylbutylene, 1-, 2- and 3-hexynylene, 1-, 2- and 3-heptynylene, 1-, 2-, 3- and 4-octynylene
and the like.
The term “(C -C )aryl” denotes an aromatic carbocyclic group having 6-10 carbon
6 10
atoms consisting of a single ring or condensed multiple rings such as, but not limited to, phenyl
and naphthyl; the term “(C -C )arylene-diyl” denotes a divalent aromatic carbocyclic group
6 10
having 6-10 carbon atoms consisting of either a single ring or condensed multiple rings such as,
but not limited to, phenylene and naphthylene.
The term “heteroaryl” refers to a radical derived from a 5membered mono- or poly-
cyclic heteroaromatic ring containing one to three, preferably 1-2, heteroatoms selected from
N, O, or S. Examples of mono-cyclic heteroaryls include, without being 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, benzthiazolyl, 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, -NO , -SH, or -CONH . It is to be understood that when a
polycyclic heteroaryl is substituted, the substitution may be in 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 any of the ring atoms.
The term "halogen" as used herein refers to fluoro, chloro, bromo or iodo.
The term "(C -C )cycloalkylene" denotes a mono- or bi-cyclic saturated divalent cyclic
hydrocarbon radical containing three to eight carbons. Non-limiting examples of such radicals
include 1,2-cyclopropane-diyl, 1,2-cyclobutane-diyl, 1,3-cyclobutane-diyl, 1,2-cyclopentane-
diyl, 1,3-cyclopentane-diyl, 1,2-cyclohexane-diyl, 1,3-cyclohexane-diyl, 1,4-cyclohexane-diyl,
1,2-cycloheptane-diyl, 1,3-cycloheptane-diyl, 1,4-cycloheptane-diyl, 1,2-cyclooctane-diyl, 1,3-
cyclooctane-diyl, 1,4-cyclooctane-diyl, 1,5-cyclooctane-diyl, and the like.
The term “amino acid residue”, as used herein, refers to any natural or synthetic, i.e.,
non-natural, amino acid residue in its both L- and D-stereoisomers. While a natural amino acid
is any one of the twenty amino acid residues typically occurring in proteins, the term
synthetic/non-natural amino acid refers to any amino acid, modified amino acid and/or an
17784661_1 (GHMatters) P42431NZ01
analog 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 non-natural amino acids,
without being limited to, include ornithine, homolysine, 2,4-diaminobutyric acid (DABA), 2,3-
diaminopropionic acid (DAP), 8-aminooctanoic acid (EAO), homophenylalanine, homovaline,
homoleucine, and the like.
Pharmaceutical compositions in accordance with 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 the other
ingredients of the composition and not deleterious to the recipient thereof.
The following exemplification of carriers, modes of administration, dosage forms, etc.,
are listed as known possibilities from which the carriers, modes of administration, dosage forms,
etc., may be selected for use with the present invention. Those of ordinary skill in the art will
understand, however, that any given formulation and mode of administration selected should
first be tested to determine that it achieves the desired results.
Methods of administration include, but are not limited to, parenteral, e.g., intravenous,
intraperitoneal, intramuscular, subcutaneous, mucosal (e.g., oral, intranasal, buccal, vaginal,
rectal, intraocular), intrathecal, topical and intradermal routes. Administration can be systemic
or local. In certain embodiments, the pharmaceutical composition is adapted for intra-brain
administration.
The term “carrier” 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, maize starch and the like; a lubricant or surfactant, such as magnesium stearate, or
sodium lauryl sulphate; and a glidant, such as colloidal silicon dioxide.
The compositions may be formulated for parenteral administration by injection, e.g., by
bolus injection or continuous infusion. Formulations for injection may be presented in unit
dosage form, e.g., 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
17784661_1 (GHMatters) P42431NZ01
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, e.g., sterile pyrogen free water, before use.
For administration by inhalation, for example for nasal administration, the compositions
according to the present invention are conveniently delivered in the form of an aerosol spray
presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or
other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by
providing a valve to deliver a metered amount. Capsules and cartridges 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.
In certain embodiments the pharmaceutical composition is formulated for administration
by any known method as described above. Particular methods of administration contemplated
here are intravenous and intra-brain (intracerebral) administration.
The pharmaceutical composition according to any one of the embodiments defined
above may be formulated for intravenous, intra-brain (intracerebral), oral, intradermal,
intramuscular, subcutaneous, transdermal, transmucosal, intranasal or intraocular
administration.
The invention will now be illustrated by the following non-limiting Examples.
[0083a] In the claims which follow and in the preceeding description of the invention, except
where the context requires otherwise due to express language or necessary implication, the word
“comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e.
to specify the presence of the stated features, integers, steps or components but not to preclude
the presence or addition of further features integers, steps, components or groups thereof in
various embodiments of the invention.
EXAMPLES
Material and Methods (M&M)
Chemicals
17784661_1 (GHMatters) P42431NZ01
NHS-PEG-OPSS (Ortho-Pyridyldisulfide-Polyethyene glycol-N-Hydroxylsuccinimide
ester), also named PDP-PEG-NHS (PDP: pyridyl dithio propionate), with molecular weight of
~2 kDa was purchased from Creative PEGworks (Winston, USA). Poly (2-ethyloxazoline),
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 ~22 kDa LPEI (free base form)
The cationic polymer Linear Polyethyleneimine (LPEI) was synthesized as described
previously [16]. Briefly, 8.0 g (0.16 mmols) of poly(2-ethyloxazoline) were 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%, H-NMR, D O-d6, 400 MHz: singlet 3.5 ppm). The resulting
LPEI hydrochloride salt (4.5 g) was made alkaline by adding aqueous NaOH (3 M) and the
resulting white precipitate was filtrated and washed with water until neutral. The solid was then
dissolved in water and further lyophilized to give white solid (2 g, 81%).
Synthesis of LPEI-PEG -OPSS di-conjugates
174 mg (8 µmol) of LPEI were dissolved in 2.7 ml of absolute EtOH and agitated at
room temperature for 15 minutes. A 5-fold molar excess of OPPS-PEG -CONHS (79 mg, 39.5
µmol) was dissolved in 500 µL of anhydrous DMSO and introduced in small portions into the
LPEI mixture. The reaction mix was agitated at ~800 rpm on a vortex stirrer at ambient
temperature for 3 h. Different PEG-substituted LPEIs were separated by cation-exchange
chromatography, using an HR10/10 column filled with MacroPrep High S resin (BioRad). The
purity of the eluted fractions of the di-conjugates was assessed using reverse phase HPLC
equipped with analytical Vydac C-8 monomeric 5 μm column (300 Å, 4.6 x 150 mm), using a
linear gradient of 5%-95% acetonitrile over 25 min at 1 ml/min flow. The fractions with 95%
purity or higher were combined. The combined fractions were further dialyzed against 20 mM
HEPES pH 7.4. The ratio of PEG groups conjugated to LPEI in the di-conjugates was
determined by H-NMR. The integral values of the hydrogens from the polyethylene -(CH -
CH -O)- and from the LPEI -(CH -CH -NH)- were used to determine the ratio between the two
2 2 2
17784661_1 (GHMatters) P42431NZ01
conjugated co-polymers. Of the various products obtained from the cation-exchange, two
products, LPEI-PEG -OPSS (di-conjugate 1:1, with molar ration of LPEI to PEG ~1:1) and
LPEI-(PEG ) -(OPSS) (di-conjugate 1:3, with molar ratio of ~1:3), were chosen for the
2k 3 3
generation of tri-conjugates. A copper assay was used to evaluate the co-polymer concentration
. Briefly, co-polymers were incubated with CuSO (23 mg dissolved in 100 ml of acetate
buffer) for 20 minutes and their absorbance at 285 nm was measured.
SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) of proteins
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 staining with InstantBlue™.
Affibody expression and purification
A Her-2 affibody gene was cloned into plasmid pET28a, generating a vector encoding
Z:2891 affibody fused to an N-terminal hexahistidyl (His6) tag and a Cys residue at the C
terminus. The affibody was expressed in E. coli BL21(DE3) as follows: The cells were grown
at 37°C to OD ~ 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 the affibody, the cell
pellet was resuspended in buffer A (20 mM HEPES pH 7.4, 500 mM NaCl, 10% glycerol, 10
mM imidazole and 2 mM β-mercaptoethanol) and disrupted using a Microfluidizer Processor
M-110EHI according to the manufacturer's instructions. The soluble fraction was recovered by
centrifugation at 12,000Xg for 10 min at 4°C. The resulting fraction was loaded onto a Ni
affinity column (Clontech, Mountain View, CA). Column was washed with buffer A for 14
column volumes (cv). Thereafter, a step gradient elution was performed using increasing
concentrations of buffer B (20 mM HEPES pH 7.4, 500 mM NaCl, 10% glycerol, 500 mM
Imidazole and 2 mM β-Mercaptoethanol); 6% buffer B for 5cv, 10% buffer B for 1.5cv, 30%
buffer B for 2cv. The bound protein was eluted with 100% buffer B for 5cv (protein purification
facility, Wolfson centre, Israel). The eluted fractions were then concentrated with an Amicon
filter (3 kDa cutoff) and loaded onto Gel filtration column Superdex 30 prep grade (120 ml)
(GE healthcare). The purified proteins were further analyzed by SDS–PAGE and confirmed
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using Western blot analysis with anti-affibody antibody (Abcam). The purity was further
assessed by reverse phase HPLC (Merck-Hitachi model L-7100) as described previously.
Synthesis of PEI-PEG-ligand affibody (tri-conjugate 1:1 and 1:3)
4.97 mg of each di-conjugate (1:1 and 1:3) were dissolved in 940 μl 20 mM HEPES pH
7.4. Then, 3.4 mg of Her-2 affibody in HBS were added dropwise to the reaction. 4 ml of 20
mM HEPES plus 700 μL of acetonitrile (HPLC Grade) were introduced to the reaction mix for
increased solubility. The reaction was further vortexed (800 rpm) at room temperature until A
indicated complete turnover. The resulting tri-conjugates were purified by cation exchange
chromatography on a HR10/10 column filled with MacroPrep High S resin (BioRad) (using
three step gradient elution of 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 tri-
conjugates, fractions with 95% purity and higher were combined and were kept at -80 C. The
concentration of the tri-conjugate was determined by copper assay (as above). The amount of
conjugated protein was determined by A using Nano-Drop 2000.
Verification and purity of chemical vectors conjugated to targeting protein.
The tri-conjugates were electrophoresed on SDS-PAGE, and stained with
InstantBlue , to confirm the conjugation of the affibody to LPEI-PEG . The purity of the tri-
conjugates was confirmed by reverse phase HPLC, using an analytical Vydac C-8 monomeric
μm 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 were used for the HPLC analysis.
Polyplex formation
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 tri-conjugate 1:1 or tri-conjugate 1:3 were
complexed with plasmid at a ratio of N/P=6 in HEPES buffer glucose (where N=nitrogen from
LPEI and P=phosphate from DNA) generating two Polyplexes. To allow complete formation
of the polyplexes particles, the samples were incubated for 30 min at room temperature. The
final plasmid concentration in polyplexes samples was 100 µg/ml whereas for DNase protection
assay and luciferase assay, the final concentration of the plasmid was 10 µg/ml.
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ξ-potential and sizing measurements
The sizes of the polyplex particles obtained after dispersal 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 173º by back scattering technology (NIBS, Non-Invasive Back-Scatter). Each
sample was run in triplicate. ζ potential measurements were also performed at 25°C using a
Nano-ZS Zetasizer (Malvern, UK). The ζ potential was evaluated after incubation of polyplexes
in HBG buffer (pH 7.4). Light scattering from the moving particles was detected at 17º, and the
Smoluchowski Model was used to determine the value of the Henry's function.
Atomic force microscopy
For AFM measurements, polyplexes were placed on freshly cleaved Mica disks (V1
12mm, Ted Pella USA). Imaging was carried out in HBG buffer at 25 C, using commercial
AFM, a NanoWizard® 3 (JPK instrument, Berlin, Germany) with QI mode. Si3N4 (MSNL-
series, Bruker) cantilevers with spring constants ranging from 10 to 30 pN nm-1 were
calibrated by the thermal fluctuation method (included in the AFM software) with an absolute
uncertainty of approximately 10%. QI™ settings were as follows: Z-length: 0.1 μm; applied
force: 0.5 nN; speed: 50 μm/s.
DNase protection assay
DNase I protection assays were conducted as described previously [18]. Briefly, 1 µg
of pGreenFire1 DNA alone, with polyplex 1:1 or with polyplex 1:3 was mixed in a final volume
of 50 µl in HBS solution. Following 30 minutes incubation at room temperature, 2 µL of DNase
I (2 unit) 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) were added, and the tubes were incubated for 2 h at RT. Samples were
electrophoresed on an 0.8% agarose gel and stained with ethidium bromide. Images were
acquired using a Gel Doc EZ Imager (Bio Rad Laboratories, Inc).
Cell culture
Her-2 overexpressing BT474 cells were cultured in RPMI medium supplemented with
% fetal bovine serum (FBS), 10 U/L penicillin, and 10 mg/L streptomycin at 37°C in 5%
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CO . MDA-MB-231 human breast carcinoma cells, were cultured in Leibovitz L-15 medium
with 10% FBS, 10 U/L penicillin, and 10 mg/L streptomycin at 37°C without CO . Cell lines
were from the ATCC and cell culture reagents were from Biological Industries, Bet Ha'emek,
Israel.
Luciferase assay and confocal microscopy
10000 BT474 and MDA-MB-231 cells were plated in triplicate in 96-well plates. Cells
were treated with tri-conjugate 1:1 and tri-conjugate 1:3 complexed with plasmid 48 h following
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 manufacturer’s
recommendations. Measurements were performed using a
Luminoskan™ Ascent Microplate Luminometer (Thermo Scientific). Values, in relative light
units (RLU), are presented as the mean and standard deviation of luciferase activity from the
triplicate samples. Confocal microscopy (FV-1200 Olympus) was used to visualize the GFP,
which was taken to reflect the internalization of plasmid pGreenFire1. Pictures were taken at
×10 magnification.
Quantification of cell viability
Cell viability was measured by means of a colorimetric assay using methylene blue, as
described previously [19]. Briefly, 10000 BT474 and MDA-MB-231 cells were plated in
triplicate in 96-well plates. The cells were treated with polyplexes 1:1 and 1:3 containing 1
µg/ml pGreenFire1. 48 h following treatment, the cells were fixed with 1% formaldehyde in
PBS (pH 7.4), washed with DDW and then stained with a 1% (wt/vol) solution of methylene
blue in borate buffer for 1 h. Thereafter, the stain was extracted with 0.1 M HCl and the optical
density of the stain solution was read at 630 nm in a microplate reader (ELx800 BIO-TEX
instruments Inc.).
Example 1 – Synthesis of thiol reactive co-polymers.
3.1. Previous studies have demonstrated the PEGylation of LPEI and its conjugation to
an EGFR targeting moiety [13]. However, the amount of PEGylation on a single LPEI chain
has not been fully characterized. To generate differentially PEGylated co-polymers, the
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secondary amines on LPEI were conjugated to the terminal NHS ester orthogonal protecting
group on PEG. The N-hydroxysuccinimide (NHS) ester is spontaneously reactive with the
secondary backbone amines of LPEI, providing efficient PEGylation of LPEI. Furthermore, the
reaction of the NHS-PEG OPSS with the amines of PEI results in formation of stable,
irreversible amide bonds (Figure 1).
The PEGylation products were purified by cation exchange chromatography. Two peaks
were eluted at high concentrations of NaCl, one at 120 mS/cm and the other at 132 mS/cm (data
not shown). The two products were presumed to differ in their ratios of LPEI:PEG and
consequently in their net positive charges. H-NMR spectra were analyzed using the relative
integral values of the hydrogen atoms on PEG (-CH -CH -O-) (Figure 2) and the integral values
of the hydrogen atoms on LPEI (-CH2-CH2-NH-) (Figure 2). This analysis indicated that the
material eluted in the first peak consisted of a co-polymer in which each mole of LPEI was
conjugated to approximately three moles of PEG. This product was named LPEI-(PEG ) -
2k 3
(OPSS) (“di-conjugate 1:3”). The second peak consisted of a co-polymer in which equal moles
of PEG were conjugated to LPEI, and was named LPEI-PEG -OPSS (“di-conjugate 1:1”)
(Figure 2).
Example 2. Synthesis of the tri-conjugates, LPEI-PEG -Her2 affibody (tri-conjugate 1:1)
& LPEI-(PEG )-(Her2) affibody (tri-conjugate 1:3)
2k 3
The primary aim of this study was to develop a cationic polymer that would
target Her-2 overexpressing tumor cells. Since Her-2 is an “orphan receptor”, affibody
molecules targeting the Her-2 receptor (rather than ligand) were used to generate Her-2
targeting tri-conjugates. We expressed and purified Her-2 affibody with a Cys residue at the C-
terminal end to allow further conjugation. The thiol reactive co-polymers, di-conjugate 1:1 and
1:3, were conjugated to Her-2 affibody through its terminal Cys residue, generating tri-
conjugates 1:1 and 1:3 respectively (Figure 3). In order to generate the tri-conjugates the
reaction had to be performed with low concentration of affibody (to prevent aggregation) and
in the presence of 10% acetonitrile (ACN) as an organic polar solvent for increased solubility.
The reaction yields for both tri-conjugate reactions were approximately 33% as determined by
copper assay. To confirm the conjugation of the affibody to the di-conjugate, the tri-conjugate
products were reduced with DTT and separated on SDS-PAGE. Coomassie blue staining
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confirmed that the reduced tri-conjugate released the affibody (Figure 4). The amount of Her-
2 affibody present in the tri-conjugates was determined by measuring A . Using copper assay,
we quantified the LPEI. As described above, H-NMR analysis showed that the ratios of
LPEI:PEG in the purified di-conjugates were 1:1 or 1:3. Comparing the molar ratios of Her-2
affibody and LPEI, we determined that the average ratio of Her-2 affibody to LPEI in tri-
conjugate 1:1 was 1:1, and in tri-conjugate 1:3 the average ratio was 3:1. Thus we conclude that
nearly complete conjugation of affibody to LPEI-PEG was achieved.
To generate 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. ξ potential and sizing of polyplexes
We next characterized the polyplexes, with respect to size and surface charge,
using dynamic light scattering (DLS). The size of a polyplex has a significant impact on its
delivery properties [21]. In order to investigate the effect of targeting ligand on the size of a
polyplex we decided to complex both di-conjugates 1:1 and 1:3 with plasmid and measure their
size. Di-conjugate 1:1 had an average particle size of 115.2 ± 8.2 nm and di-conjugate 1:3 had
an average particle size of 253.1 ± 9.5 nm. The polyplex generated from tri-conjugate 1:1 with
plasmid gave an average particle size of 141 ± 5.8 nm, whereas tri-conjugate 1:3 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. The conjugation of the affibody to the di-conjugates had only a minor affect on the
particle size. The number of PEG groups, however, did affect the particle size, suggesting that
the PEG groups cause steric hindrance, interfering with plasmid condensation.
A positive surface charge facilitates polyplex binding to the negatively charged
cell surface, but excessive positive charge can lead to non-specific binding and significant
toxicity [12]. The ξ potentials of the various complexes, presented in Figure 6, are in agreement
with previous studies, which showed a decrease in ξ potential with increased number of PEG
units [13]. To assess the effect of PEG groups on the surface charge of our chemical vectors we
measured the ξ potentials of polyplexes formed by complexation of plasmid DNA with the
precursors, di-conjugates 1:1 and 1:3, and with the tri-conjugates 1:1 and 1:3. Di-conjugate
polyplex 1:1 had an average ξ potential of 27.0 ± 0.1 mV and di-conjugate polyplex 1:3 had an
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average ξ potential of 20.0 ± 1.0 mV. Tri-conjugate polyplex 1:1 showed ξ potential with an
average of 17.1 ± 0.7 mV, whereas tri-conjugate polyplex 1:3 showed an average of 10.2 ± 0.44
mV (Figure 6). Unlike the sizes, the ξ potentials of the polyplexes were affected by both the
number of PEG groups and the conjugation of the Her-2 affibody. Although the smallest, most
positively charged polyplexes were obtained with naked LPEI, these particles are extremely
toxic [22]. We expected that the addition of PEG groups and a targeting moiety would diminish
toxicity, but because the polyplexes were still relatively small in size, we hoped that their
efficiency as nucleic acid delivery vectors would not be compromised.
Example 4. Assessment of polyplex shape using Atomic Force Microscopy.
The importance of particle shape and its influence on delivery properties is
gaining recognition [23]. We analyzed the morphology of the polyplexes obtained with the tri-
conjugates in solution using Atomic Force Microscopy (AFM). The diameters of tri-conjugates
1:1 and 1:3 polyplexes were both in the nano-size range (Figs. 7A-B), in agreement with the
results obtained by DLS. The tri-conjugate 1:1 polyplex displayed mainly elliptical particles.
Most 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 (Fig. 7A).
Tri-conjugate 1:3 polyplex was more heterogenic in shape, and moreover, yielded large
aggregates with undefined particle shape (Fig. 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.
Successful in vivo gene delivery depends on efficient protection from nucleases.
To determine the ability of the tri-conjugates to protect plasmids from degradation and enable
efficient gene delivery, the polyplexes were treated with DNase I and analyzed using gel
electrophoresis. As shown in Figure 8, naked plasmid pGreenFire1 DNA was fully degraded
following 10 minutes of incubation with 2 units of DNase I. In contrast, when polyplexes were
generated by mixing plasmid with the tri-conjugates, the plasmid was protected from
degradation by DNase I. Complete protection of the plasmid was observed for tri-conjugate
polyplex 1:3, while some nicking did occur for tri-conjugate polyplex 1:1, as shown by the shift
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from the supercoiled (s.c.) to the open circular (o.c.) form of the plasmid. The stronger
protection from DNase I conferred by tri-conjugate polyplex 1:3 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
increase their circulation in the blood, by impeding their interactions with enzymes and serum
factors [24, 25].
Example 6. Biological activity of targeting tri-conjugate polyplexes
Polyplex size and ξ potential influence the efficiency of targeted DNA delivery
and gene expression, but the effect of size appears to be dependent on the particular conjugate
[21]. To evaluate the specificity and the efficiency of transfection of the tri-conjugate
polyplexes 1:1 and 1:3, two breast cancer cell lines that differentially express Her-2 were
utilized. Polyplexes of the tri-conjugates 1:1 and 1:3 were formed with pGreenFire1 and
transfected into MDA-MB-231 cells (expressing approximately 9x10 Her-2 receptors/cell [26])
and BT474 cells (expressing approximately 1x10 Her-2 receptors/cell [27]). Differential
luciferase activity was observed 48 h after transfection. Both tri-conjugate polyplexes 1:1 and
1:3 led to more than 300-fold higher luciferase activity in BT474 cells than in MDA-MB-231
(* p < 0.001) (Figure. 9A). More efficient gene delivery to BT474 was confirmed by GFP
expression, as seen by confocal microscopy (Figure 9B). These results show that polyplex
selectivity is dependent on Her-2 expression.
Targeted delivery to BT474 cells by tri-conjugate polyplex 1:1 was 10-fold more
efficient than delivery by tri-conjugate polyplex 1:3 (Figure 9A, B), even though tri-conjugate
1:3 has more targeting moieties. This may reflect the higher ξ potential and lower size of tri-
conjugate polyplex 1:1.
Positively charged LPEI-based chemical vectors are associated with significant
toxicity. Therefore, we next tested the survival of MDA-MB-231 and BT474 cells following
treatment with tri-conjugate polyplexes 1:1 and 1:3. Neither polyplex showed cytotoxic effects
in MDA-MB-231 cells, in a methylene blue assay. Similar results were observed in BT474 cells
treated with tri-conjugate polyplex 1:3. However, a slight increase in cell cytoxicity was
observed in BT474 treated with tri-conjugate polyplex 1:1 (Figure 9C). Altogether, these
results indicate that the small size and higher ξ potential of tri-conjugate polyplex 1:1 confer
17784661_1 (GHMatters) P42431NZ01
efficient targeted delivery properties, with only a slight increase in toxicity. Thus, polyplex of
the tri-conjugate 1:1 is superior in gene delivery to the more shielded tri-conjugate polyplex 1:3.
Example 7. Anti-tumor activity of PEI-PEG-Her2Affibody
The PEI-PEG-Her2Affibody (PPHA) complexed with
PolyInosine/PolyCytosine (PolyIC) has strong anti-tumor 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 observed also of trastuzumab resistant Her2
overexpressing breast cancer cell lines.
Figure 10 shows that the vector/polyplex inhibits Her2 overexpressing cells,
including Herceptin/trastusumab resistant cells.
0.5x10 MCF-7 HER-2 cells were injected s.c. into mude mice. After tumors
reached and average of 100mm , treatment began. 1mg/kg pIC/PPHA was injected i.v. at every
24 hrs. Trastuzumab was administered i.v. once a week (indicated by arrows), to two groups of
mice. Tumor growth was measured twice a week. The complex PolyIC/PPHA was found to
possess strong anti-tumor activity in mouse models in which these cell lines were implanted in
nude mice as exemplified in Figure 11.
Example 8. Synthesis of LPEI-PEG-EGFR affibody
5 mg (2 × 10 mmol) of LPEI-PEG -OPSS (di-conjugate 1:1) were dissolved
in 1 ml 20 mM HEPES pH 7.4. Then, 3.4 mg (3.8 × 10 mmol, ~ 2 eq) of EGFR affibody in
HBS were added dropwise to the reaction. 4 ml of 20 mM HEPES plus 700 μL of acetonitrile
(HPLC Grade) were introduced to the reaction mix for increased solubility. The reaction was
further vortexed (800 rpm) at room temperature and dark conditions until A indicated
complete turnover. The resulting tri-conjugate was purified by cation exchange chromatography
on a HR10/10 column filled with MacroPrep High S resin (BioRad) (using three step gradient
elution of 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 LPEI-PEG-EGFR tri-conjugate,
fractions with 95% purity and higher were combined and were kept at -80 C. The concentration
of the tri-conjugate was determined by copper assay. The amount of conjugated protein was
determined by A using Nano-Drop 2000.
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Example 9. Anti-tumor activity of PEI-PEG-EGFR affibody
The PEI-PEG-EGFRAffibody (PPEA) complexed with PolyIC has strong anti-
tumor activity. A variety 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 higher than that of PPE (Figure 12).
The complex PolyIC/PPEA was found to possess strong anti-tumor activity in
mouse models in which these cell lines were implanted in nude mice.
Sixty five female nude mice 5 weeks old were injected s.c. with 2 million A431
cells. Seven days later tumors of average volume of 136mm had grown and mice were divided
into 6 groups (7-8mice/group) as follows: UT; pIC/PPEA, 0.75 mg/kg=250µl of pIC for 25 gr
mouse., IC, 6/week; pI/PPEA, 0.75 mg/kg IV, 6/week; pIC/PPEA low, 0.1 mg/kg=250µl of
0.01 µg/µl pf pIC for 25 gr mouse IC, 6/week. pIC, pI and PPE and PPEA were diluted before
mixing to obtain lower than ususal (0.1µg/µl) concentration of pIC in the complex. Figure 13
shows the activity of PolyIC/PPEffibody in vivo. Again, the efficacy of PPEA/PolyIC is higher
than that of PolyIC/PPE.
Example 10. Synthesis of LPEI-PEG-h/mEGF
.1. Synthesis of LPEI-PEG-SH intermediate
To 5 mg of LPEI-PEG-OPSS (0.2 µmol, according to 24000 g/mol) in 5 ml of
mM HEPES (pH 7.4) buffer was added 50-fold molar excess of dithiothreitol (DTT; 0.1
mmol, 1.5 mg) and mixed by vortex for 20 min at room temperature in 15 ml plastic
centrifugation tube. The reduced diconjugate was separated on Sephadex G-25 column (20 ml,
4x5 ml) using 5 ml sample loop and the elution performed with 20 mM HEPES, pH 7.4 at 1.0
ml/min flow rate and were analyzed by HPLC using the same conditions and method as
described above.
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Ellman’s assay was used to evaluate the sulfhydryl group concentration in the
LPEI-PEG-SH intermediate. The concentration of SH groups is directly proportional to the
concentration of chromophor 6-nitrothioxocyclohexa-1,4-dienecarboxylic acid, released
by free thiols that react quantitatively with Ellman’s reagent. The chromophor was measured
via absorbance at 412 nm, without any interference from the sample or the Ellman’s reagent.
.2 Synthesis of m/hEGF-MCC intermediate
Synthesis outline. The h/mEGF (human/mouse Epidermal Growth Factor) was
modified into h/mEGF-MCC (EGF(N-Maleimidomethyl)cyclohexanecarboxylic acid;
MCC) and purified to be later used for the conjugation with the LPEI-PEG-SH. Activating
h/mEGF by attaching the MCC group circumvents reduction of the protein by DTT and allows
to avoid exposure of h/mEGF to harsh conditions. This way the conjugation reaction efficiency
is improved, moreover - EGF-MCC is a stable material that can be stored at -80 C for weeks.
Synthesis of hEGF-MCC: 1 mg of hEGF (160 nmol) was reconstituted in 0.5
ml 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)cyclohexanecarboxylic 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
(3.5k cutoff) against HBS (100 mM), three times in 1 L for 1 hour, then 1 L for overnight. The
hEGF-MCC was analyzed using HPLC-mass spectra (MS) and had a molecular weight of
6435.7 g/mol, which indicates there is one conjugation of sulfo-SMCC to the hEGF. The HPLC-
MS analysis was performed using Thermo SCIENTIFIC/ LCQ FLEET, equipped with reverse
phase C-18 column (phenomenex, Aeris, 3.6 µm, 2.1 mm x 50 mm, 100 A ).
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Synthesis of mEGF-MCC: 0.5 mg (0.088 µmol total amount) 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 absolute EtOH
and slowly mixed with EGF solution to reach the final concentration of 30% EtOH in total 3.0
ml volume. Brief mixing resulted in a clear solution. The reaction vessel was shaken at ambient
temperature for 4 hours. After that period of time the solution remained clear. The mEGF-MCC
was first separated on Sephadex G-25 column (4x5 ml) and the elution performed with 20 mM
HEPES, pH 7.4 at 1.0 ml/min flow rate and further purified and analyzed by HPLC using the
same conditions and standard method as described above for LPEI-PEG-OPSS.
.3 Synthesis of LPEI-PEG-h/mEGF
One and a half of equivalents of hEGF-MCC or mEGF-MCC were mixed with
1.0 equivalent of LPEI-PEG-SH. The reaction mixture was initially agitated 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) in 10/1 cm Tricorn GE Healthcare
column) using the gradient pump via A18 buffer inlet, at 0.5 ml/min flow rate and using solvent
A: 20 mM HEPES pH 7.4 and solvent B: 20 mM HEPES pH 7.4 NaCl 3.0 M. The purified
LPEI-PEG-hEGF or LPEI-PEG-mEGF triconjugate was analyzed by HPLC and quantified by
a “copper assay” and an EGF photometric assay to measure [LPEI] and [EGF] concentrations,
correspondingly.
.4 Biological activity of LPEI-PEG-hEGF.
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Using the cellular assay we usually employ we compared the activity of the
PolyIC/LPEI-PEG-hEGF complex (PPEm) with the PolyIC/mPPE (mouse) described in
Schaffert D, Kiss M, Rödl 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 EGFR over-expressing cells (Figure
14). Cells that express moderate amounts of EGFR molecules on their surface (U87MG cells
express 80,000 EGFRs/cell) are less sensitive to the treatment than cells over-expressing
massive amounts (U87MGwtEGFR cells express 1,000,000 EGFRs/cell).
Example 11. Synthesis of DUPA analog-DyLight 680
The peptide was synthesized using standard Fmoc solid-phase peptide synthesis
(SPPS) procedures on Fmoc-Cys(trt) wang 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
solution of 20% piperidine in dimethylformamide (DMF) (2 x 20 min), then washed with DMF
(5 x 2 min). Coupling of Fmoc-Asp(OtBu)-OH: 3 eq. of Fmoc-Asp(OtBu)-OH, 3 eq. of (1-
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid
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 minutes at room temperature before it was added to the resin for 1 hour.
The coupling was repeated twice with the new mixture in order to ensure complete coupling of
aspartic acid. A Keiser test was performed to ensure a complete coupling. The resin was washed
with DMF (3 x 2 minutes) and dichloromethane (DCM) (2 x 2 min). Capping: The resin was
treated with a solution of acetic anhydride (10 eq.) and DIPEA (8 eq.) in DMF for 20 min and
washed with DMF (3 x 2 min). Fmoc removal.: The resin was first treated with a solution of
% piperidine in DMF (2 x 20 min), then washed with DMF (5 x 2 min). Coupling of Fmoc-
diaminopropionic(DAP) acid: 3 eq. of Fmoc-diaminopropionic (DAP) acid, 3 eq. of HATU and
8 eq. of DIEA were dissolved in 15 ml of DMF. The solution was mixed (pre-activated) for 10
minutes at room temperature before it was added to the resin for 1 hour. A Keiser test was
performed to ensure a complete coupling. The resin was washed with DMF (3 x 2 minutes) and
DCM (2 x 2 min). Peptide elongation: the following compounds were coupled to the resin in
17784661_1 (GHMatters) P42431NZ01
the following order (1) Fmoc-Phe-OH, (2) Fmoc-Phe-OH, (3) Fmocaminooctanoic (EAO)
acid, and (4) OtBu-Glu(Fmoc)-OH. Fmoc removal: the resin was treated with a solution of 20%
piperidine in DMF (2 x 20 min), then washed with DMF (5 x 2 min) and DCM (3 x 2 min).
Coupling of the DUPA ligand: 0.9 mL of triethylamine (6.6 mmol) was combined with 0.9 gr
(3 mmol) of L-glutamic acid di-tertbutyl ester hydrochloride in 15 mL DCM. This solution was
added dropwise over 45 minutes to a solution of 5 mL DCM and triphosgene (0.35 g, 1.1 mmol)
at 0° C. After stirring for additional 50 min the mixture was added to the resin with additional
0.9 mL of triethylamine. The reaction mixture with the resin was shaked for 3 hours and washed
with DMF (3 x 2 min). Full Cleavage: the resin was washed with DCM (3 x 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 proceeded for 4 h at room temperature, filtered and
treated with a cooled solution of ether/hexane 1:1, and the peptides were precipitated by
centrifugation. The crude peptides were dissolved in acetonitrile/TDW 1:1 solution and
lyophilized. The crude was purified by preparative reverse phase (RP) HPLC. DyLight 680
Coupling: under argon atmosphere, 1 mg of DyLight 680 (Life Technologies, Cat. No.
46418) was dissolved in anhydrous dimethyl sulfoxide (DMSO; 100 µL) containing 50
equivalents of anhydrous diisopropylethylamine. A two-fold molar excess of a DUPA peptide
linker dissolved in anhydrous DMSO (100 µL) was added to the above mixture and stirred at
room temperature. The formations of products were confirmed by liquid chromatography-mass
spectrometry (LC-MS). The crude DUPA near-infrared (NIR) probes were then purified by
preparative RP-HPLC.
Example 12. Synthesis of Dupa analog-Drug Lead
The peptide was synthesized using standard Fmoc SPPS procedures on Fmoc-
Cys(trt) wang 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 solution of 20% piperidine in
DMF (2 x 20 min), then washed with DMF (5 x 2 min). Coupling of Fmoc-Gly-OH: 3 eq. of
Fmoc-Gly-OH, and 3 eq. of HATU were dissolved in 15 ml of DMF, followed by addition of 8
eq. of DIEA. The solution was mixed (pre-activated) for 10 minutes at room temperature before
it was added to the resin for 1 hour. The coupling was repeated twice with the new mixture. A
Keiser test was performed to ensure a complete coupling. The resin was washed with DMF (3
17784661_1 (GHMatters) P42431NZ01
x 2 minutes) and DCM (2 x 2min). Capping: The resin was treated with a solution of acetic
anhydride (10 eq.) and DIPEA (8 eq.) in DMF for 20 min and washed with DMF (3 x 2 min).
Fmoc removal: the resin was treated with a solution of 20% piperidine in DMF (2 x 20 min),
then washed with DMF (5 x 2 min). Coupling of Fmoc-Trp(Boc)-OH: 3 eq. of the Fmoc-
Trp(Boc)-OH, 3 eq. of HATU and 8 eq. of DIEA were dissolved in 15 ml DMF. The solution
was mixed (pre-activated) for 10 minutes at room temperature before it was added to the resin
for 1 hour. A Keiser test was performed to ensure a complete coupling. The resin was washed
with DMF (3 x 2 min) and DCM (2 x 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) Fmocaminooctanoic(EAO) acid, and (5) OtBu-Glu(Fmoc)-OH. Fmoc
removal: the resin was treated with a solution of 20% piperidine in DMF (2 x 20 min), then
washed with DMF (5 x 2 min) and DCM (3 x 2 min). Coupling of the DUPA ligand: 0.9 mL of
triethylamine (6.6 mmol) was combined with 0.9 gr (3 mmol) of L-glutamic acid di-tertbutyl
ester hydrochloride in 15 mL DCM. This solution was added dropwise over 45 minutes to a
solution of 5 mL DCM and triphosgene (0.35 g, 1.1 mmol) at 0° C. After stirring for additional
50 min the mixture was added to the resin with additional 0.9 mL of triethylamine. The reaction
mixture with the resin was shaked for 3 hours and washed with DMF (3 x 2 min). Full Cleavage:
the resin was washed with DCM (3 x 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
proceeded for 4 h at room temperature, filtered and treated with a cooled solution of
ether/hexane 1:1, and the peptides containing the DUPA analog were precipitated by
centrifugation. The crude peptides were dissolved in acetonitrile/TDW 1:1 solution and
lyophilized. The crude was purified by preparative RP-HPLC. Synthesis of PEI-PEG-DUPA
analog: 4.37 mg (1.2 × 10 mmol) of di-conjugate 1:1 or 1:3 (prepared as explained in Example
1 herein above) were dissolved in 940 μl of 20 mM HEPES pH 7.4. Then, 1 mg (9.1 × 10
mmol, ~ 5 eq) of the DUPA analog, was dissolved in 2 ml of acetonitrile (ACN; HPLC
grade)/(20 mM HEPES pH 7.4) at a ratio of 1:1, were added dropwise to the reaction. Then, to
reach an approximate ~10% total concentration of ACN in the reaction, an addition of 4 mL of
mM were introduced into the reaction mixture. The reaction was further vortexed (800 rpm)
in the dark at room temperature until the absorption at wavelength 343 (A ) indicated a
complete turnover. The resulting tri-conjugate was purified by cation exchange chromatography
17784661_1 (GHMatters) P42431NZ01
on a HR10/10 column filled with MacroPrep High S resin (BioRad) (using a three-step gradient
elution of 20 mM HEPES pH 7.4 to 20 mM HEPES containing 3 M NaCl). The eluted fractions
were introduced to an analytical RP-HPLC to assess the purity of the tri-conjugate. Fractions
with 95% purity and higher were combined and were kept at -80 C. The concentration of the
tri-conjugate was determined by the copper assay. The amount of conjugated DUPA analog was
determined by the chromophores' (Trp amino acid) absorption.
Example 13. PolyIC/LPEI-PEG-DUPA targets prostate cancer.
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 we have chosen it as a target, design and synthesize a PolyIC
PSMA targeting vector. Indeed, DUPA-Daylight680 is internalized to PSMA overexpressing
cells (LNCaP) but not to MCF7 (overexpressing Her2) (not shown).
Figure 15 shows that PEI-PEG-DUPA (PPD)/PolyIC is highly effective against
LNCaP and VCaP cells. Viability was measured after 96 hr of exposure. PPD also induces the
production of cytokines (Figure 16). In Figure 17, it is demonstrated that medium conditioned
by LNCaP cells stimulates expression of the cytokines INF-γ, IL-2 and TNF-α in PBMCs grown
in the conditioned medium. It was then shown that co-incubation of PolyIC/PPD treated LNCaP
cells with PC3-Luciferase cells which do not express PSMA, resulted in up to 70% killing of
the PC3-Luciferase cells via bystander effect. Addition of healthy human PBMCs strongly
enhanced the effect and lead to the killing of 90% of the PC3 cells (Figure 18).
Example 14. Cancer treatment in vivo.
PolyIC/PPD has significant anti-tumor effects when tested in SCID mice
(Figure 19). Tumor inhibition is not complete due to the lack of immune system in the
experimental mice.
17784661_1 (GHMatters) P42431NZ01
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17784661_1 (GHMatters) P42431NZ01
Claims (18)
1. 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, wherein said cancer antigen is prostate surface membrane antigen (PSMA), provided that the targeting moiety is not mouse EGF or the peptide of the sequence YHWYGYTPQNVI (GE11) (SEQ ID NO: 1).
2. The polyplex of claim 1, wherein said targeting moiety is HOOC(CH ) -CH(COOH)- NH-CO-NH-CH(COOH)-(CH ) -CO- (DUPA residue).
3. 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, wherein said targeting moiety is HOOC(CH ) -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ) -CO- (DUPA residue). 2 2 2 2
4. The polyplex of claim 3, wherein said cancer antigen is prostate surface membrane antigen (PSMA).
5. The polyplex of any one of the preceding claims, wherein the dsRNA is polyinosinic- polycytidylic acid double stranded RNA (poly I:C).
6. The polyplex of any one of the preceding claims, wherein 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).
7. The polyplex of any one of the preceding claims, wherein said one or more PEG moieties each independently forms a bond selected from -NH-CO-, -CO-NH-, -S-C-, -S-S-, -O-CO- or - 17784661_1 (GHMatters) P42431NZ01 CO-O- with said linker, and wherein said linker forms an -S-S-, NH-CO-, -CO-NH-, -S-C-, O- CO-, -CO-O- or urea (-NH-CO-NH) bond with said targeting moiety.
8. The polyplex of any one of the preceding claims, wherein said linker is a peptide moiety consisting of 3 to 7 amino acid residues.
9. The polyplex of claim 8, wherein said peptide moiety is -(NH-(CH ) -CO)-Phe-Gly- Trp-Trp-Gly-Cys- (SEQ ID NO: 2) or -(NH-(CH ) -CO)-Phe-Phe-(NH-CH -CH(NH )-CO)- 2 7 2 2 Asp-Cys- (SEQ ID NO: 3), linked via its mercapto group to said targeting moiety.
10. The polyplex of any one the claims 1 to 7, wherein said linker is selected from -CO-R2- R -R , wherein R is selected from (C -C )alkylene, (C -C )alkenylene, (C -C ) alkynylene, (C - 2 1 8 2 8 2 8 6 C )arylene-diyl, or heteroarylenediyl, wherein preferably R is selected from (C -C )alkylene, 10 2 1 8 further preferably (C -C )alkylene; R is absent or -S-, preferably R is -S-; R is absent or of the formula ; R is selected from (C -C )alkylene, (C -C )alkenylene, (C -C ) alkynylene, (C - 4 1 8 2 8 2 8 1 C )alkylene-(C -C )cycloalkylene, (C -C )alkenylene-(C -C )cycloalkylene, (C -C ) 8 3 8 2 8 3 8 2 8 alkynylene-(C -C )cycloalkylene, (C -C )arylene-diyl, heteroarylenediyl, (C -C )alkylene- 3 8 6 10 1 8 (C -C )arylene-diyl, or (C -C )alkylene-heteroarylenediyl, wherein preferably R is (C - 6 10 1 8 4 1 C )alkylene-(C -C )cycloalkylene, preferably (C -C )alkylene-(C -C )cycloalkylene; 8 3 8 1 4 5 6 wherein each one 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, -COR , -COOR , -OCOOR , -OCON(R ) , -CN, -NO , -SR , -OR , -N(R ) , -CON(R ) , -SO R , - 5 5 5 2 2 5 5 5 2 5 2 2 5 SO H, -S(=O)R , (C -C )aryl, (C -C )alkylene-(C -C )aryl, heteroaryl, or (C -C )alkylene- 3 5 6 10 1 4 6 10 1 4 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(R )-, -N(C -C aryl)-, (C -C )arylene-diyl, or heteroarylenediyl; and 5 6 10 6 10 17784661_1 (GHMatters) P42431NZ01 R is H or (C -C )alkyl. 5 1 8
11. The polyplex of claim 10, wherein R is -CH -CH -; 2 2 2 R is -S-; and R is absent or , wherein R is .
12. The polyplex of any one of claims 1 to 11, wherein the polymeric conjugate is a diconjugate of the formula (i)-(viii), linked to said targeting moiety/moieties: N N OH H N N N (ii) (iii) -(NH-(CH ) -CO)-Phe-Phe-(NH-CH -CH(NH )-CO)-Asp-Cys-PEG -LPEI; 2 7 2 2 2k (iv) -(NH-(CH ) -CO)-Phe-Gly-Trp-Trp-Gly-Cys-PEG -LPEI; 2 7 2k 17784661_1 (GHMatters) P42431NZ01 (vi) N N OH HN N N O O O O O O HN HN HN O O O S S S O O O O O O N N N CO- CO- CO- (vii) wherein R is ; or 17784661_1 (GHMatters) P42431NZ01 (viii) N N OH HN N N O O O O O O HN HN HN O O O S S S R R R 7 7 7 wherein R is HOOC
13. The polyplex of claim 12, wherein (a) said targeting moiety is HOOC(CH ) -CH(COOH)-NH-CO-NH-CH(COOH)- (CH2)2-CO- (DUPA residue), and said polymeric conjugate is of the formula (iii) of claim 12; (b) said targeting moiety is HOOC(CH ) -CH(COOH)-NH-CO-NH-CH(COOH)- (CH ) -CO- (DUPA residue), and said polymeric conjugate is of the formula (vii) of claim 12; (c) said targeting moiety is HOOC(CH ) -CH(COOH)-NH-CO-NH-CH(COOH)- (CH ) -CO- (DUPA residue), and said polymeric conjugate is of the formula (iv) of claim 12; 17784661_1 (GHMatters) P42431NZ01 (d) said targeting moiety is HOOC(CH ) -CH(COOH)-NH-CO-NH-CH(COOH)- (CH ) -CO- (DUPA residue), and said polymeric conjugate is of the formula (viii) of claim 12.
14. The polyplex of any one of the preceding claims, wherein each strand of said dsRNA has 20-300 ribonucleotides.
15. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polyplex of any one of claims 1 to 14.
16. Use of the polyplex of any one of claims 1 to 14, or the pharmaceutical composition of claim 15, for the manufacture of a medicament for the treatment of prostate cancer.
17. The use of claim 16, wherein the polyplex is to be administered or used in combination with immune cells.
18. The use of claim 17, wherein said immune cells are tumor-infiltrating T-cells (T-TILs), tumor specific engineered T-cells, or peripheral blood mononuclear cells (PBMCs). 17784661_1 (GHMatters) P42431NZ01
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US201461993110P | 2014-05-14 | 2014-05-14 | |
US61/993,110 | 2014-05-14 | ||
NZ727092A NZ727092A (en) | 2014-05-14 | 2015-05-14 | Improved polyethyleneimine polyethyleneglycol vectors |
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