Polyethyleneimine polyethyleneglycol vectors
Inventors
Levitzki, Alex • Joubran, Salim • Shir, Alexei • Zigler, Maya • Talhami, Alaa • LANGUT, Yael
Assignees
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Publication Number
US-10278991-B2
Publication Date
2019-05-07
Expiration Date
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) moieties, each PEG moiety being conjugated via a linker to a targeting moiety capable of binding to a cancer antigen.
Core Innovation
The invention relates to non-viral targeted polyplexes comprising double stranded RNA (dsRNA), including poly I:C, complexed with a linear polyethyleneimine (LPEI) covalently PEGylated polymer conjugate. The polymer conjugate includes polyethylene glycol (PEG) moieties that are linked via linkers to targeting moieties capable of binding to a cancer antigen.
A central aspect is that the PEG moieties are covalently attached to LPEI through defined linker chemistries and that the resulting PEGylated conjugate architecture is provided as LPEI-PEG diconjugates and triconjugates, including defined LPEI:PEG diconjugate ratios. PEGylation is described as affecting polyplex physicochemical properties, including particle size, ζ-potential, nuclease protection, particle shape heterogeneity, and targeted transfection and toxicity.
The disclosure further specifies representative conjugate formula architectures (i–viii) with targeting-moiety conjugation, including targeting moieties such as EGFR/HER2/PSMA using affibodies or hEGF and DUPA. Linker embodiments include specific bond types and peptide linker sequences (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3), and the examples include targeted selectivity (Her-2 or EGFR) and anti-tumor activity in vivo using PolyIC (dsRNA analog), including bystander killing with PBMCs.
Claims Coverage
The document provides three independent claim themes that cover targeted polyplexes of dsRNA with a linear polyethyleneimine (LPEI) covalently linked to PEG moieties, where each PEG is conjugated via a linker to a targeting moiety binding a cancer antigen. The independent claims further constrain the conjugate architecture via formula (i)–(viii), specific linker bond types, specified peptide linker sequences, and specific named targeting moieties and dsRNA identity.
LPEI-PEG targeting polyplex with PEG-to-cancer-antigen linker
A polyplex of dsRNA and a polymeric conjugate where linear polyethyleneimine (LPEI) is covalently linked to one or more PEG moieties, each PEG moiety conjugated via a linker to a targeting moiety capable of binding to a cancer antigen, with the polymeric conjugate as a diconjugate of formula (i)–(viii) linked to the targeting moiety, where m and n are ≥1.
Peptide-linked PEG-to-targeting moiety within LPEI-PEG polyplex
A polyplex of dsRNA and a polymeric conjugate where linear polyethyleneimine (LPEI) is covalently linked to one or more PEG moieties, each PEG moiety conjugated via a linker to a targeting moiety capable of binding to a cancer antigen, wherein 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, and wherein the linker is a peptide moiety selected from —(NH—(CH2)7—CO)-Phe-Gly-Trp-Trp-Gly-Cys (SEQ ID NO: 2) or —(NH—(CH2)7—CO)-Phe-Phe-(NH—CH2—CH(NH2)—CO)-Asp-Cys (SEQ ID NO: 3).
hEGF-targeted polyplex with poly I:C and formula (ii) architecture
A polyplex of dsRNA and a polymeric conjugate where linear polyethyleneimine (LPEI) is covalently linked to one PEG moiety, with the PEG moiety conjugated via a linker to a targeting moiety capable of binding to a cancer antigen; wherein the polymeric conjugate is a diconjugate of formula (ii) linked to the targeting moiety; and wherein the targeting moiety is hEGF linked via an amino group to the linker, and wherein the dsRNA is polyinosinic-polycytidylic acid double stranded RNA (poly I:C).
DUPA-targeted polyplex with poly I:C and formula (iv) architecture
A polyplex of dsRNA and a polymeric conjugate where linear polyethyleneimine (LPEI) is covalently linked to one PEG moiety, with the PEG moiety conjugated via a linker to a targeting moiety capable of binding to a cancer antigen; wherein the polymeric conjugate is a diconjugate of formula (iv) linked to the targeting moiety and includes a specified PEG2k–LPEI architecture as recited in the claim; and wherein the targeting moiety is HOOC(CH2)2—CH(COOH)—NH—CO—NH—CH(COOH)—(CH2)2—CO (DUPA residue), and wherein the dsRNA is polyinosinic-polycytidylic acid double stranded RNA (poly I:C).
Overall, the independent claims cover dsRNA-containing polyplexes formed with an LPEI covalently PEGylated polymer conjugate where PEG is linked to cancer-antigen targeting moieties. The claims define (i–viii) diconjugate formula architectures, specify linker bond types and peptide linker sequences (SEQ ID NO: 2 and SEQ ID NO: 3), and in specific claim embodiments fix the targeting moiety to hEGF or the DUPA residue while specifying dsRNA as poly I:C.
Stated Advantages
A PEGylation-dependent effect on polyplex physicochemical properties, including particle size, ζ-potential, nuclease protection, and particle shape heterogeneity.
Changes in targeted transfection and toxicity.
Cell-line selectivity (Her-2 or EGFR) and anti-tumor activity in vivo using PolyIC (dsRNA analog).
Bystander killing with PBMCs.
Documented Applications
Targeted transfection/cancer-related activity using dsRNA (including PolyIC as a dsRNA analog) with HER2-, EGFR-, and PSMA-targeted constructs, including in vivo anti-tumor activity.
Immune cell-related activity combining with PBMCs for bystander killing.
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