WO2013165262A1 - Peptides, constructions et leurs utilisations - Google Patents

Peptides, constructions et leurs utilisations Download PDF

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Publication number
WO2013165262A1
WO2013165262A1 PCT/NZ2013/000076 NZ2013000076W WO2013165262A1 WO 2013165262 A1 WO2013165262 A1 WO 2013165262A1 NZ 2013000076 W NZ2013000076 W NZ 2013000076W WO 2013165262 A1 WO2013165262 A1 WO 2013165262A1
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Prior art keywords
peptide
cell
seq
nucleic acid
compound
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PCT/NZ2013/000076
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English (en)
Inventor
Geoffrey Wayne Krissansen
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Auckland Uniservices Limited
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Publication of WO2013165262A1 publication Critical patent/WO2013165262A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to novel peptides, constructs containing same and uses therefor.
  • the plasma membrane of eukaryotic cells has poor permeability to many chemical compounds, significantly reducing their efficacy, for example as therapeutics or experimental reagents.
  • Technologies have been developed to improve the cell permeability of chemical compounds, including the use of lipid-, polycationic-, nanoparticle- and peptide-based methods.
  • these technologies are not without problem.
  • cell permeable carrier peptides which are typically conjugated to a chemical compound for delivery to a target cell, may be large and expensive to manufacture, making them commercially non- viable. Large carrier peptides may also interfere with the conformation of the molecule which they carry, reducing efficacy of those compounds.
  • carrier peptides typically do not distinguish between adherent and non-adherent cells, or activated and unactivated cells.
  • difficulties can arise in ensuring the conjugated chemical compound is adequately delivered to target cells, particularly when used in vivo.
  • blood cells may mop up the conjugate before it is able to reach a target site.
  • it is often a requirement to deliver a chemical compound to the nucleus of a cell, and some carrier peptides do not localise to the nucleus.
  • the invention provides a peptide consisting of the amino acid sequence LCL (SEQ ID NO 1), LCLX (SEQ ID NO 2), XLCL (SEQ ID NO 3), XLCLX (SEQ ID NO 4), wherein X is any amino acid.
  • a peptide of the invention consists of the amino acid sequence LCLK (SEQ ID NO 5), LCLH (SEQ ID NO 6), LCLR (SEQ ID NO 7), LCLE (SEQ ID NO 8), LCLN (SEQ ID NO 9), LCLQ (SEQ ID NO 10), VLCLR (SEQ ID NO 11).
  • a peptide of the invention consists of the amino acid sequence LCLD (SEQ ID NO 12).
  • the invention provides a peptide consisting of the amino acid sequence MAARL (SEQ ID NO 15), MAARLX (SEQ ID NO 16), MAARLXX (SEQ ID NO 17), wherein the peptide is an L-isomer, and wherein X is any amino acid.
  • the invention provides a fusion peptide comprising a peptide of the first aspect of the invention.
  • the invention provides a nucleic acid encoding a peptide or a fusion peptide of the first, second or third aspects.
  • the invention provides a nucleic acid vector comprising a nucleic acid of the fourth aspect.
  • the invention provides the use of a peptide or fusion peptide of the first, second or third aspect as a cell membrane permeable carrier for a compound.
  • the invention provides a construct comprising:
  • a peptide of the first or second aspect and a compound desired to be delivered to a cell a fusion peptide of the third aspect and a compound desired to be delivered to a cell.
  • the construct may comprise more than one compound. In one embodiment, the construct may comprise more than one peptide or fusion peptide. In another embodiment, the construct may comprise a combination of one or more peptide and/or one or more fusion peptide of the invention. In one embodiment, a construct of the invention may comprise two or more copies of a particular peptide or fusion peptide of the invention.
  • the compound is chosen from a nucleic acid, a peptide nucleic acid, a polypeptide, a carbohydrate, a peptidomimetic, a small molecule inhibitor, proteoglycan, lipid, a lipoprotein, liposome, glycolipid, a natural product, dendrimer, imaging agent, micelle, nanoparticle, nanotube, polymeric particle, and a glycomimetic.
  • the invention provides a nucleic acid encoding a construct of the seventh aspect.
  • the invention provides a vector comprising a nucleic acid of the eighth aspect.
  • the invention provides a method for increasing the cell membrane permeability of a compound, the method comprising:
  • two or more compounds may be connected to a peptide or fusion peptide of the invention.
  • two or more peptides and/or fusion peptides of the invention may be connected to one or more compounds.
  • the invention provides a method of delivering a compound to a cell, the method comprising contacting one or a combination of a construct of the seventh aspect of the invention, a nucleic acid of the eighth aspect of the invention and a vector of the ninth aspect of the invention with the cell or a composition comprising the cell.
  • the invention provides a method of delivering a compound to a cell, the method comprising administering to a subject one or a combination of a construct of the seventh aspect of the invention, a nucleic acid of the eighth aspect of the invention and a vector of the ninth aspect of the invention.
  • the compound is delivered to the cytoplasm of a cell.
  • the compound is delivered to the nucleus of a cell.
  • the compound is delivered to the surface of a cell.
  • the invention provides a method for targeting delivery of a compound to adherent cells in a mixed population of adherent and non-adherent cells, the method comprising contacting one or a combination of a construct of the seventh aspect of the invention, a nucleic acid of the eighth aspect of the invention and a vector of the ninth aspect of the invention with a mixed population of cells or a composition comprising a mixed population of cells.
  • the invention provides a method for targeting delivery of a compound to adherent cells in a subject, the method comprising administering to the subject one or a combination of a construct of the seventh aspect of the invention, a nucleic acid of the eighth aspect of the invention and/or a vector of the ninth aspect of the invention.
  • the adherent cells are epithelial cells. Accordingly, the invention provides a method for targeting delivery of a compound to epithelial cells, the method comprising contacting one or a combination of a construct of the seventh aspect of the invention, a nucleic acid of the eighth aspect of the invention and a vector of the ninth aspect of the invention with a mixed population of cells comprising epithelial cells or a composition comprising a mixed population of cells comprising epithelial cells.
  • the invention also provides a method for targeting delivery of a compound to epithelial cells in a subject, the method comprising administering to the subject one or a combination of a construct of the seventh aspect of the invention, a nucleic acid of the eighth aspect of the invention and a vector of the ninth aspect of the invention.
  • the adherent cells are activated immune cells.
  • the invention provides a method for targeting delivery of a compound to activated immune cells, the method comprising contacting one or a combination of a construct of the seventh aspect of the invention, a nucleic acid of the eighth aspect of the invention and a vector of the ninth aspect of the invention with a mixed population of cells comprising activated and unactivated immune cells or a composition comprising a mixed population of cells comprising activated and unactivated immune cells.
  • the invention also provides a method for targeting delivery of a compound to epithelial cells in a subject, the method comprising administering to the subject one or a combination of a construct of the seventh aspect of the invention, a nucleic acid of the eighth aspect of the invention and a vector of the ninth aspect of the invention.
  • the invention provides a host cell comprising a nucleic acid or vector of the invention.
  • the invention provides a composition comprising one or a
  • a peptide of the invention a fusion peptide of the invention, a nucleic acid or vector of the invention, and a construct of the invention, as herein before described, in combination with one or more carrier, excipient and/or diluent.
  • the invention provides the use of one or a combination of a peptide of the invention, a fusion peptide of the invention, a nucleic acid of the invention, a vector of the invention, and a construct of the invention in the manufacture of a medicament.
  • the invention provides the use of a nucleic acid encoding a peptide, fusion peptide or construct of the invention, or a nucleic acid vector comprising said nucleic acid, for any purpose mentioned herein before.
  • the invention provides methods as mentioned hereinbefore which utilise a nucleic acid encoding a peptide, fusion peptide or construct of the invention, or a nucleic acid vector comprising said nucleic acid.
  • the invention provides peptides comprising the sequence XCXR (SEQ ID NO. 34), wherein X is any hydrophobic amino acid.
  • the peptides comprise the sequence ICIR or VCVR.
  • the peptides consist the sequence XCXR (SEQ ID NO. 34), wherein X is any hydrophobic amino acid.
  • the peptides consist the sequence ICIR or VCVR.
  • the invention provides fusion peptides or proteins comprising the peptides of this aspect of the invention.
  • the invention also provides nucleic acids encoding these peptides and fusion peptides or proteins, vectors comprising said nucleic acids, constructs of the invention comprising said peptides or fusion peptides or proteins, and composition, uses and methods as herein before described which utilise the peptides and/or fusion peptides, constructs, nucleic acids, vectors of this aspect of the invention.
  • the invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
  • FIG. 1 A number of the figures illustrate cells or nuclei which have been labelled or stained in different colours. When reproduced in black and white, all the spots visible in these Figures represent cells or nuclei, in accordance with the label or stain used, unless otherwise stated. In some cases, colours became very dim or invisible when the figures were reproduced in black and white, hence contrast and brightness were increased, and in some figures the blue DAPI coloration was changed to gold. The changes were made to faithfully reproduce the colour photographs.
  • Figure 1 The core motif of the cell-permeable X-peptide LCLRPVG (SEQ ID 23) is the tripeptide LCL or the tetrapeptide LCLX.
  • SEQ ID 23 The FITC-labelled LCLRPVG peptide, and shortened and variant forms were incubated at 10 ⁇ with HepG2 (liver cancer), BT549 (breast cancer), WM-266-4 (melanoma), DU145 (prostate cancer), and H441 (lung cancer) cell lines, and their uptake by the cells recorded using a Nikon E600 fluorescence microscope. Cell nuclei were stained with DAPI.
  • A) FITC-labelled peptides MAARL, MAAR (SEQ ID 24), and RLCCQ (SEQ ID 20) derived from the N-terminal region of the X-protein were incubated at 10 ⁇ with HepG2, BT549, and WM-266-4 cell lines, and their uptake by the cells recorded using a Nikon E600 fluorescence microscope. Cell nuclei were stained with DAPI. Peptide uptake by cells was scored visually, as above.
  • FIG. 3 Properties of D-isomeric forms of the peptides LCLRPVG and LCLRP (SEQ ID 22).
  • a TAMRA-labelled D-isomer of LCLRPVG ie lclrpvg
  • the TAMRA-labelled D-isomeric peptide lclrpvg was incubated with HepG2 cells at the concentrations indicated and its uptake by the cells recorded by microscopy. Upper panels, DAPI stained nuclei; lower panels, TAMRA-labelled peptide.
  • FITC-labelled L- and D-isomeric forms of the peptide LCLRP were preincubated in serum for the times indicated and then incubated with HepG2 cells for 3 h. Peptide uptake by the cells was recorded by microscopy. Upper panels, DAPI stained nuclei; lower panels, FITC-labelled peptide.
  • Figure 4 Cell permeability of the D-isomeric peptide lclrpvg, and shortened and variant forms.
  • Examples herein below, which provides experimental data supporting the invention, specific examples of various aspects of the invention, and means of performing the invention.
  • the inventors have previously identified peptide motifs derived from the X-protein of the hepatitis B Virus (HBV) which are cell permeable. These peptides comprise at least the amino acid motif LCLRP or MAARLCCQ and are described in WO 2011/155853.
  • the inventors have now surprisingly identified peptides comprising a shorter amino acid motif that are also cell permeable.
  • peptides show unexpected efficacy in entering cells and may be used as cell-permeable carriers to deliver chemical compounds to cells (including the cytoplasm and nucleus of cells).
  • the small size of the peptides provides the advantage that they will be relatively economical to manufacture and the risk that they will interfere with the conformation and efficacy of the compound which they are to deliver to a cell may be reduced compared to larger peptides.
  • the inventors contemplate the use of the peptides for delivery of therapeutic compounds to subjects, as well as delivery of compounds to cells for research purposes. Skilled persons will readily appreciate that reference to delivery of a compound to a cell includes delivery to the surface of a cell or delivery within a cell.
  • treatment is to be considered in its broadest context. The term does not necessarily imply that a subject is treated until total recovery. Accordingly, “treatment” broadly includes, for example, the prevention, amelioration or management of one or more symptoms of a disorder, the severity of one or more symptoms and preventing or otherwise reducing the risk of developing secondary complications.
  • the invention provides a peptide consisting of the amino acid sequence LCL (SEQ ID NO 1), LCLX (SEQ ID NO 2), XLCL (SEQ ID NO 3), XLCLX (SEQ ID NO 4), wherein X is any amino acid.
  • the invention provides a peptide consisting of the amino acid sequence MAARL (SEQ ID NO 15), MAARLX (SEQ ID NO 16), MAARLXX (SEQ ID NO 17), wherein the peptide is an L-isomer, and wherein X is any amino acid.
  • X can be any amino acid, including naturally and non-naturally occurring amino acids. In the sequence listing, X is noted to be any naturally occurring amino acid, but the invention should not be construed to be limited in this way.
  • X may be chosen from G, A, V, L, I, S, C, T, M, F, Y, W, P, H, K, R, D, E, N, Q, taurine, ornithine, 5-hydroxylysine, e-N-methyllysine, and 3-methylhistidine.
  • X may also comprise a modified amino acid, including selenocysteine, hydroxyproline, selenomethionine, hypusine, carboxylated glutamate.
  • the primary amine group and primary carboxyl group may be modified to include nucleophilic addition, amide bond formation and imine formation for the amine group, and esterification, amide bond formation and decarboxylation for the carboxylic acid group.
  • Certain amino acid residues may have added hydrophobic groups for membrane localization or endosome release, or have undergone myristoylation, palmitoylation, isoprenylation or prenylation,
  • the invention provides a peptide comprising the amino acid sequence XCXR (SEQ ID NO. 34), wherein X is any hydrophobic amino acid.
  • the peptides may comprise an additional 1, 2, 3, 4, 5, 6, 7, 8 or up to 15 amino acids at their N- and/or C- terminii.
  • the hydrophobic amino acid of this embodiment of the invention may be naturally or non-naturally occurring or may comprise a modified amino acid (for example modified as described in the immediately preceding paragraph).
  • the hydrophobic amino acid may be chosen from L, V, I, M, F and W.
  • the invention provides a
  • peptide comprising or consisting of the amino acid sequence ICIR or VCVR.
  • a peptide of the invention consists of the amino acid sequence LCLK (SEQ ID NO 5), LCLH (SEQ ID NO 6), LCLR (SEQ ID NO 7), LCLE (SEQ ID NO 8), LCLN (SEQ ID NO 9), LCLQ (SEQ ID NO 10), VLCLR (SEQ ID NO 11).
  • a peptide of the invention consists of the amino acid sequence LCLD (SEQ ID NO 12).
  • the invention provides a peptide consisting of the amino acid sequence MAARLC (SEQ ID NO 18) or MAARLCC (SEQ ID NO 19), wherein the peptide is an L-isomer.
  • the invention also provides fusion peptides or proteins, in which a peptide of the invention is fused to one or more heterologous amino acids at its N- and/or C-terminus; that is amino acid residues that are not naturally found adjacent to the peptide of the invention in the X- protein.
  • the fusion peptide is functionally equivalent to the peptides of the invention.
  • the phrase "functionally equivalent" as used herein should be taken broadly to encompass any fusion peptide which has the ability to move across a cell membrane, preferably carry a compound across a cell membrane, to enter a cell.
  • fusion peptide may have more or less activity than the peptide which it comprises.
  • the fusion peptide has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of the level of activity of the peptide.
  • a fusion peptide will at least have substantially the same level of activity than the peptide it comprises.
  • peptide or fusion peptide of the invention Skilled persons will readily appreciate the desired function and be able to assess function and determine the level of activity of a peptide or fusion peptide of the invention, based on the information contained herein, and using techniques known in the art.
  • the peptide or variant will have the ability to move across a cell membrane, preferably carry the compound across a cell membrane, to enter a cell and this function and the level of activity may be assessed based on uptake of the peptide into the cell, for example, using the techniques described in the "Examples" section herein after.
  • a peptide(s) of the invention and a fusion peptide containing a peptide of the invention may be referred to herein collectively as "peptide(s)". Accordingly, where not specifically mentioned, references to a "peptide” or “peptides” of the invention herein should be taken to include reference to fusion peptides comprising such peptide(s).
  • the phrases “move across a cell membrane”, “carry a compound across a cell membrane”, “cell membrane translocation” and like phrases should be taken broadly to encompass transport of the peptide, a compound to be delivered to a cell, and/or a conjugate comprising such peptide and compound from the outside of a cell to the inside of the cell. It should not be taken to imply a particular mode or mechanism of transport across or through the cell membrane. Similarly the phrase “increasing the cell membrane permeability of a compound” should be taken broadly to mean that there has been at least some increase or improvement in the ability of the compound to move across a cell membrane.
  • peptides of the invention may be composed of L-amino acids, D-amino acids or a mixture thereof and may include non-naturally occurring amino acids.
  • the peptide is an L isomer of LCLK (SEQ ID NO 5), LCLH (SEQ ID NO 6), LCLR (SEQ ID NO 7), LCLE (SEQ ID NO 8), LCLN (SEQ ID NO 9), LCLQ (SEQ ID NO 10) or LCLD (SEQ ID NO 10).
  • the peptide is a D-isomer of VLCLR (SEQ ID NO 11).
  • peptides of the invention are “isolated” or “purified” peptides.
  • An “isolated” or “purified” peptide is one which has been identified and separated from the environment in which it naturally resides, or artificially synthesized. It should be appreciated that these terms do not reflect the extent to which the peptide has been purified or separated from an environment in which it naturally resides.
  • a peptide of the invention may be isolated from natural sources, or preferably derived by chemical synthesis (for example, fmoc solid phase peptide synthesis as described in Fields GB, Lauer-Fields JL, Liu Q and Barany G (2002) Principles and Practice of Solid-Phase peptide Synthesis; Grant G (2002) Evaluation of the Synthetic Product. Synthetic Peptides, A User's Guide, Grant GA, Second Edition, 93-219; 220-291, Oxford University Press, New York) or genetic expression techniques, methods for which are readily known in the art to which the invention relates.
  • Fusion peptides may be produced using any number of techniques known in the art. For example, they may be derived by chemical synthesis or produced recombinantly (as herein after described). To the extent that a peptide or fusion peptide of the present invention may be produced by recombinant techniques the invention provides nucleic acids encoding peptides of the invention and vectors comprising nucleic acids encoding peptides of the invention, which may aid in cloning and expression of peptides. Certain nucleic acids and vectors may also be of use to a therapeutic end as herein after detailed.
  • nucleic acid in accordance with the invention is an “isolated” or “purified” nucleic acid.
  • An “isolated” or “purified” nucleic is one which has been identified and separated from the environment in which it naturally resides, or artificially synthesized. It should be appreciated that these terms do not reflect the extent to which the nucleic has been purified or separated from the environment in which it naturally resides.
  • Nucleic acids of use in accordance with the invention may be isolated from natural sources, or preferably derived by chemical synthesis or recombinant techniques which will be readily known to persons skilled in the art.
  • nucleic acids which encode the peptides of the invention on the basis of the amino acid sequences provided herein, the genetic code and the understood degeneracy therein and published X-protein nucleic acid sequences (for example, see Guo, Y. and Hou, J. Establishment of the consensus sequence of hepatitis B virus prevailing in the mainland of China. Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi 19: 189-2000, 1999).
  • nucleic acids are suitable: gtc ctt tgt eta cgt (peptide comprising VLCLR) (SEQ ID NO 13)
  • Nucleic acid vectors will generally contain heterologous nucleic acid sequences; that is nucleic acid sequences that are not naturally found adjacent to the nucleic acid sequences of the invention.
  • the constructs or vectors may be either RNA or DNA, either prokaryotic or eukaryotic, and typically are viruses or a plasmid. Suitable constructs are preferably adapted to deliver a nucleic acid of the invention into a host cell and are either capable or not capable of replicating in such cell.
  • Recombinant constructs comprising nucleic acids of the invention may be used, for example, in the cloning, sequencing, and expression of nucleic acid sequences of the invention. Additionally, recombinant constructs or vectors of the invention may be used to a therapeutic end.
  • cloning vectors such as pUC and pBluescript and expression vectors such as pCDM8, adeno-associated virus (AAV) or lentiviruses to be particularly useful.
  • AAV adeno-associated virus
  • the constructs may contain regulatory sequences such as promoters, operators, repressors, enhancers, termination sequences, origins of replication, and other appropriate regulatory sequences as are known in the art. Further, they may contain secretory sequences to enable an expressed protein to be secreted from its host cell. In addition, expression constructs may contain fusion sequences (such as those that encode a heterologous amino acid sequence) which lead to the expression of inserted nucleic acid sequences of the invention as fusion proteins or peptides.
  • Heterologous amino acid sequences of use may include, for example, those which can aid in subsequent isolation and purification of the peptide (for example, ubiquitin, his-tag, a c-myc tag, a GST tag, or biotin), or those which assist the activity of the peptide (for example, an additional sequence which aids in transport across a cell membrane, such as a poly arginine sequence, tat, or penetratin).
  • Heterologous amino acid sequences may also include peptide linkers which aid in linking the peptide to another compound to form a construct of the invention
  • transformation of a nucleic acid vector into a host cell can be accomplished by any method by which a nucleic acid sequence can be inserted into a cell.
  • transformation techniques include transfection, electroporation, microinjection, lipofection, adsorption, cell-penetrating carrier peptide delivery, and biolistic bombardment.
  • transformed nucleic acid sequences of the invention may remain extrachromosomal or can integrate into one or more sites within a chromosome of a host cell in such a manner that their ability to be expressed is retained.
  • host cells such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors; yeast transformed with recombinant yeast expression vectors; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); animal cell systems such as CHO (Chinese hamster ovary) cells using the pEE14 plasmid system; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid).
  • microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors; yeast transformed with recombinant yeast expression vectors; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); animal cell systems such as CHO
  • a recombinant peptide in accordance with the invention may be recovered from a transformed host cell, or culture media, following expression thereof using a variety of techniques standard in the art. For example, detergent extraction, sonication, lysis, osmotic shock treatment and inclusion body purification.
  • the peptides (including fusion peptides) of the invention may be used as carriers to transport compounds across a cell membrane into a cell.
  • the invention provides methods for delivering a compound to a cell as well as methods for increasing the cell membrane permeability of a compound to be delivered to a cell by connecting it to a peptide of the invention. It also provides constructs comprising a carrier peptide and at least one compound desired to be delivered to a cell.
  • the at least one compound may be any compound desired to be delivered to a cell.
  • Such compounds include those which may provide a therapeutic or diagnostic benefit, or compounds of use for research purposes.
  • the compounds are chosen from nucleic acids, peptide nucleic acids, peptides, polypeptides (including for example, fusion proteins), proteins, carbohydrates, peptidomimetics, small molecule inhibitors, chemical compounds, drugs, therapeutic compounds, chemotherapeutic drugs, anti-inflammatory drugs, antibodies, single chain Fv fragments (SCFV), lipids, proteoglycans, glycolipids, lipoprotein, liposomes, glycomimetics, natural products, radioisotopes, dendrimers, micelles, nanoparticles, liposomes, nanotubes, polymeric particles, imaging agents (for example, paramagnetic ions) and molecules, or fusion proteins.
  • SCFV single chain Fv fragments
  • the compound is a nucleic acid it may be DNA, RNA, cDNA, double- stranded, single-stranded, sense, antisense, or circular, including DNAzymes, iR A, siRNA, miRNA, piRNA, lcRNA, and ribozymes, phagemid, aptamer for example. Skilled persons may readily appreciate further examples of compounds in accordance with this embodiment of the invention.
  • the carrier peptide and at least one compound to be delivered to a cell may be "connected" to each other by any means which allows the peptide to carry the compound across a cell membrane into a cell while retaining at least a level of the function and structure of the compound.
  • the word "connected” or like terms should be taken broadly to encompass any form of attachment, bonding, fusion or association between the carrier peptide and the at least one compound (for example, but not limited to, covalent bonding, ionic bonding, hydrogen bonding, aromatic stacking interactions, amide bonds, disulfide bonding, chelation) and should not be taken to imply a particular strength of connection.
  • the carrier peptide and the at least one compound may be connected in an irreversible or a reversible manner, such that upon entry into a cell the compound is released from the carrier peptide.
  • the at least one compound may be connected to the carrier peptide at its N-terminus, its C- terminus or at any other location.
  • the compound is connected to the carrier peptide at its N-terminus.
  • the compound is connected to the carrier peptide at its C-terminus.
  • the constructs may be produced in the form of fusion proteins using known recombinant expression or chemical synthesis techniques (as herein before described).
  • the carrier peptide and the peptide (compound) to be delivered to a cell may also be manufactured separately and later connected to one another.
  • the carrier peptide and the nucleic acid may be made separately (using chemical synthesis or recombinant techniques, for example) and then connected via one of a number of known techniques.
  • the carrier peptide and the carbohydrate may be made separately and then connected via one of a number of known techniques.
  • the carrier peptide and the lipid may be made separately and then connected via one of a number of known techniques.
  • the methodology described in WO 91/09958, WO 03/064459, WO 00/29427, WO 01/13957 may be used to manufacture various constructs of the invention.
  • linker molecules which connect the at least one compound to the carrier peptide.
  • the linker molecule may be a peptide. Examples of appropriate linker molecules are also provided in WO 91/09958, WO 03/064459, WO 00/29427, and WO 01/13957.
  • the construct may further comprise at least one additional heterologous molecule.
  • the heterologous molecule may be a molecule which may assist the activity of the construct (for example, the activity of the carrier peptide or the compound to be delivered to a cell, or both), aid in release of the peptide from endosomes (for example, fusogenic lipids and membrane-disruptive peptides or polymers), enable targeting to a particular intracellular compartment or organelle, protect the construct from degradation or otherwise increase the half life of the construct, aid in isolation and purification of the construct during manufacture, or aid in the binding of a cargo.
  • the heterologous molecule is a molecule that may assist with cell membrane permeability (such as poly-arginine, Tat, or penetratin, for example).
  • the molecule may be a his-tag, a c-myc tag, a GST tag, or biotin, which may aid in isolation of a construct expressed recombinantly.
  • the heterologous molecule is a molecule that may assist in targeting the construct to a specific cell type.
  • the molecule is a nucleic-acid binding peptide which may assist in the delivery of RNA DNA/nucleic acids to a cell.
  • the heterologous molecule is a molecule that may assist in targeting the construct to a specific molecular target.
  • targeting the construct to a specific molecular target “specifically target a desired molecule” and like phrases should not be taken to require 100% specificity, although this may be preferred. It should be appreciated that a combination of two or more different heterologous molecules may be used in a construct of the invention.
  • heterologous molecules may be connected to the carrier peptide and/or compound to be delivered to a cell, or synthesised as a part of the construct, using any appropriate means (as described herein before in relation to manufacture of the constructs of the
  • heterologous molecules are peptide-based.
  • alternative molecules are provided, for example, in WO 91/09958, WO 03/064459, WO 00/29427, and WO
  • Constructs of the invention may be produced using recombinant cloning and expression techniques and accordingly the invention should be taken to include nucleic acids encoding the constructs and vectors comprising such nucleic acids.
  • nucleic acids encoding peptides/constructs of the invention could be used therapeutically or in vitro.
  • the nucleic acid/expression vector could be administered to a subject, with the peptide/construct subsequently being expressed and delivered to a relevant cell or tissue.
  • the invention includes nucleic acids and nucleic acid vectors suitable for these purposes.
  • nucleic acid vectors for example, pUC vectors, adeno-associated virus, lentivirus
  • techniques detailed elsewhere herein including those described in WO 91/09958, WO 03/064459, WO 00/29427, and WO 01/13957 for example
  • the invention also provides compositions comprising the peptides and/or constructs (or nucleic acids encoding the peptides and/or constructs) of the invention in association with one or more diluents, carriers and/or excipients and/or additional ingredients.
  • delivery or administration of a peptide, construct or nucleic acid of the invention is to include reference to delivery or administration of a composition comprising a peptide, construct and/or nucleic acid of the invention.
  • the one or more diluents, carriers and/or excipients are suitable for use in vitro. In another embodiment, the one or more diluents, carriers and/or excipients are suitable for use in vivo (in this instance they may be referred to as "pharmaceutically acceptable").
  • “Pharmaceutically acceptable diluents, carriers and/or excipients” is intended to include substances that are useful in preparing a pharmaceutical composition, may be coadministered with a peptide, construct, or nucleic acid encoding a peptide or construct of the invention while allowing it to perform its intended function, and are generally safe, non-toxic and neither biologically nor otherwise undesirable.
  • Pharmaceutically acceptable diluents, carriers and/or excipients include those suitable for veterinary use as well as human pharmaceutical use. Examples of pharmaceutically acceptable diluents, carriers and/or excipients include solutions, solvents, dispersion media, delay agents, emulsions and the like.
  • a composition in accordance with the invention may be formulated with one or more additional constituents, or in such a manner, so as to enhance the activity of a peptide, construct, nucleic acid encoding a peptide or construct, and/or compound to be delivered to a cell, help protect the integrity or increase the half life or shelf life of such agents, or provide other desirable benefits, for example.
  • the composition may further comprise constituents which provide protection against proteolytic degradation, enhance bioavailability, decrease antigenicity, or enable slow release upon administration to a subject.
  • slow release vehicles include macromers, poly(ethylene glycol), hyaluronic acid, poly(vinylpyrrolidone), or a hydrogel.
  • the compositions may also include preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifying agents, sweetening agents, colouring agents, flavouring agents, coating agents, buffers and the like. Those of skill in the art to which the invention relates will readily identify further additives which may be desirable for a particular purpose.
  • cell permeability of the peptides, constructs, nucleic acids encoding the peptides or constructs and/or compounds of the invention may be increased, or facilitated, through formulation of the composition.
  • the peptides, constructs, nucleic acids encoding the peptides or constructs and/or compounds of the invention may be formulated into liposomes. Further examples are provided in WO 91/09958, WO 03/064459, WO 00/29427, and WO 01/13957.
  • a pharmaceutical composition in accordance with the invention may be formulated with additional active ingredients which may be of benefit to a cell or a subject in particular instances.
  • additional active ingredients may be of benefit to a cell or a subject in particular instances.
  • Persons of ordinary skill in the art to which the invention relates will readily appreciate suitable additional active ingredients having regard to the description of the invention herein and the purposes for which the delivery of the peptide, compound and/or construct is required, including, for example, the nature and progression of any disease to be treated.
  • compositions of the invention may be formulated into any customary form such as solutions, orally administrable liquids, injectable liquids, tablets, coated tablets, capsules, pills, granules, suppositories, trans-dermal patches, suspensions, emulsions, sustained release formulations, gels, aerosols, and powders, for example. Additionally, sustained release formulations may be utilised.
  • the form chosen will reflect the purpose for which the composition is intended and the mode of delivery or administration to a sample or a subject.
  • the dosage forms exemplified in WO 91/09958, WO 03/064459, WO 00/29427, and WO 01/13957 may be used where the compositions are formulated for administration to a subject, for example for the treatment of a disease.). Skilled persons will readily recognise appropriate formulation methods. However, by way of example, certain methods of formulating compositions may be found in Gennaro AR: Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins, 2000.
  • the invention provides methods of delivering one or more compounds to a cell using a carrier peptide or construct of the invention.
  • the invention provides a method for targeting delivery of a compound to adherent cells in a mixed population of adherent and non-adherent cells.
  • adherent cells refers to those cells attached to a substrate, matrix, other cells or surface, including cells that normally adhere in such a fashion.
  • Adherent cells include epithelial cells and activated immune cells.
  • Non-adherent cells refers to cells that are not attached to a substrate, matrix, other cells, or surface (for example, resting circulating cells in the blood), including cells that do not normally attach in such a fashion unless they are activated.
  • Non-adherent cells include unactivated resting immune cells. The inventors believe that the peptides and constructs of the invention can differentially target activated proinflammatory immune cells leaving the bulk of the resting immune system unaffected, and thereby have application in treating inflammation.
  • the invention provides a method for targeting delivery of a compound to epithelial cells, epithelium, or epithelial cell surfaces.
  • the methods comprise contacting a peptide or construct of the invention with an epithelial cell or epithelium or administering a peptide or construct of the invention to a subject.
  • the methods may comprise administering to a subject a nucleic acid encoding relevant peptides or constructs of the invention and/or a vector comprising such nucleic acid. Delivery of the peptides and/or constructs (or nucleic acids encoding same) of the invention may occur in vivo or in vitro, depending on the purposes for which delivery is required. Such methods may be used for research purposes or in the treatment of disease.
  • an in vitro method may comprise bringing the construct and/or peptide (or nucleic acids or vectors encoding same) into contact with one or more cells or a composition comprising one or more cells or proteins of interest; for example, contacting the construct or peptide (or nucleic acids or vectors encoding same) with a sample, composition or media in which the one or more cells are contained (such as mixing a composition of the invention with a liquid sample containing one or more cells).
  • a method of the invention comprises administering a construct and/or peptide (or nucleic acids or vectors encoding same) to a subject.
  • a "subject” includes any animal of interest. However, in one particular embodiment the "subject” is a mammal, more particularly human.
  • the carrier peptides and related constructs of the invention comprising the carrier peptides are not taken into nonadherent cells (for example, resting blood cells).
  • administration to a subject may occur by any means capable of delivering the agents of the invention (peptides, compounds, constructs or nucleic acids or vectors encoding same) to target cells within the body of a subject.
  • agents of the invention may be administered by one of the following routes: oral, topical, systemic (eg.
  • transdermal, intranasal, or by suppository parenteral (eg. intramuscular, subcutaneous, or intravenous injection), by administration to the CNS (eg. by intraspinal or intracisternal injection), by administration to the liver (eg by intraportal injection), by implantation, and by infusion through such devices as osmotic pumps, transdermal patches, and the like.
  • parenteral eg. intramuscular, subcutaneous, or intravenous injection
  • administration to the CNS eg. by intraspinal or intracisternal injection
  • administration to the liver eg by intraportal injection
  • implantation e.g. by infusion through such devices as osmotic pumps, transdermal patches, and the like.
  • Skilled persons may identify other appropriate administration routes.
  • Exemplary administration routes are also outlined in WO 91/09958, WO 03/064459, WO 00/29427, and WO 01/13957 for example.
  • the dose of an agent administered, the period of administration, and the general administration regime may differ between subjects depending on such variables as the reason for delivery of the agent, the target cells to which the agent is to be delivered, and the severity of any symptoms of a subject to be treated, the type of disorder to be treated, the mode of administration chosen, and the age, sex and/or general health of a subject.
  • administration may include a single daily dose, administration of a number of discrete divided doses, or continuous administration as may be appropriate.
  • Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in cell cultures or animal models to achieve a cellular concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch.l, p.l).
  • a method of the invention may further comprise additional steps such as the delivery of additional agents or compositions to a sample, cell or subject.
  • Peptides were ordered from Peptide 2.0, 14100 SuUyfield Circle, Suite 200, Chantilly, VA 20151, USA.
  • the cell lines HepG2 liver cancer
  • BT549 breast cancer
  • WM-266-4 melanoma
  • DU145 prostate cancer
  • H441 lung cancer
  • the HepG2, WM-266-4 and DU145 cells lines were propagated in full MEM medium at 37°C and 5% C0 2 from cells that had been frozen in a freezing solution containing full MEM and 5% DMSO.
  • the BT549 and H441 cells lines were propagated in full RPMI medium at 37°C and 5% C0 2 from cells that had been frozen in a freezing solution containing full RPMI and 5% DMSO.
  • Cells were plated into 8-well chamber slides at 1 x 10 5 cells per well in MEM or RPMI with 10% FCS and PSG. The cells were then incubated overnight at 37° and 5% C0 2 , and washed thrice with media without FCS (L-isomers) or containing 10% FCS (D-isomers). The X-protein peptides and variants in 250 ⁇ of appropriate media without FCS (L- isomers) or containing 10% FCS (D-isomers) were added to the cells at indicated final concentrations (10-100 ⁇ ), and incubated for 3 h at 37°C and 5% C0 2 .
  • the cells were washed with PBS, fixed with 4% formaldehyde in PBS for 30 min, and washed thrice with PBS.
  • the plastic frame was removed from the slides, and a drop of Prolong Gold anti-fade reagent with DAPI (Invitrogen cat# P36931) was added to each sample.
  • the slides were dried overnight in the dark, and examined by microscopy by using a Nikon E600 fluorescent microscope, and the appropriate excitation and emission wavelengths for each fluorophore. Photos were taken using Nikon ACT-1 software.
  • the core motif of the cell-permeable X-peptide LCLRPVG is the tripeptide LCL or the tetrapeptide LCLX
  • peptides were tested for their ability to be taken up by various mammalian cell lines including the human BT549 breast cancer, WM-266-4 melanoma, DU145 prostate cancer, and H441 non-small cell lung cancer cell lines. Cells were incubated with FITC-labelled LCLRPVG and LCLRP for 3 h in medium not containing FCS.
  • FIG. 1A illustrates the uptake scoring system (-, little or no uptake; +, weak uptake; ++, moderate uptake; +++, strong uptake) in respect of peptide uptake by WM-266-4 melanoma cells. Removal of the N-terminal leucine residue rendered the X-peptide 17-20 (CLRP) completely inactive in that it was not cell-permeable to HepG2 cells.
  • LCLK, LCLH, and LCLE The role of the C-terminal basic arginine residue was tested by its substitution with conservative (lysine and histidine) and non-conservative (glutamic acid) residues (LCLK, LCLH, and LCLE, respectively).
  • the LCLK and LCLH peptide variants were cell- permeable, but surprisingly LCLE was also cell-permeable (Fig. 1A, B).
  • the variant peptides were cell permeable to different degrees in the cell types tested.
  • LCLH was readily taken up by HepG2, BT549, DU145 and H441 cells, but less well by WM-266-4 cells, whereas LCLE was readily cell-permeable to all cell lines tested except HepG2.
  • the N-terminal peptide MAARLCCQ (aa positions 1-8) from the X-protein of the hepatitis B virus is cell-permeable to HepG2 cells (Fig. 2A, B).
  • the peptide was divided to determine whether peptides MAARL and RLCCQ retained cell-permeability.
  • the MAARL peptide penetrated HepG2 and BT549 cells, but not WM-266-4 cells (Fig. 2A, B). Removal of the C-terminal leucine greatly reduced its cell-permeability (very high concentrations of peptide were required to achieve uptake by some cells, data not shown).
  • the peptide RLCCQ was not cell-permeable.
  • MAARL appears to be the core cell-permeable motif, but additional C-terminal residues are required for peptide uptake by some cells.
  • D-isomeric forms of the peptide LCLRPVG are cell permeable: the core motif is lcl or lclx
  • the D-isomer of LCLRPVG appeared to be taken up as readily by HepG2 cells as the L-isomer (Fig. 3A; Fig. 4A, B). Cell uptake was independent of the fluorophore label as both the TAMRA-labelled (Fig. 3 A) and FITC-labelled (Fig. 4B) D-isomer of LCLRPVG were readily cell-permeable. The D-isomer was readily taken up by HepG2 cells in a concentration-dependent fashion (Fig. 3A).
  • the L-isomer of the shortened peptide LCLRP was unstable in serum such that after one hour of pre-incubation in serum it was no longer able to permeate HepG2 cells (Fig. 3B).
  • the protease-resistant D-isomer (lclrp) remained cell-permeable even after 4 hours pre-incubation in serum.
  • D- isomeric variants of the LCLRP peptide and the LCLX core were tested for cell- permeability.
  • D-isomeric peptides lclrp, and lclr remained cell-permeable (Fig. 4A, B), irrespective of the presence of serum.
  • the earlier experiments had indicated that the cysteine in position 2 was essential for cell-permeability.
  • Variants of lclr were synthesized which retained the cysteine, but the leucines were conservatively substituted with other hydrophobic residues to give the peptides icir and vcvr. Substitution of the leucines reduced the cell-permeability of the lclr peptide (Fig. 4A, B). The peptide vcvr retained low level cell permeability, whereas the permeability of icir was very low and inconsistent. Addition of a valine residue to the N-terminus of the lclr peptide to give vlclr, according to the original X-protein sequence, had no major effect.
  • the tripeptide lcl appears to be the core motif, where the addition of a C-terminal residue (lclx) improves cell- permeability, and also appears to have an effect on cell-type-specificity.
  • L-isomers While in some cases, the results suggest that L-isomers have less or no activity compared to D-isomers, this does not mean that the L-isomers are of no use in the invention.
  • L- isomers are typically susceptible to degradation. However, they can be rendered less labile using standard techniques, for example by pegylation or incorporation of modified or unnatural amino acids.
  • the use of L-isomers versus D-isomers may depend on the nature of use or application. L-isomers may be preferable where rapid turnover of the carrier peptide is desirable, whereas D-isomers may be preferable in situations where prolonged exposure to the carrier peptide and/or its cargo is required to deliver maximum therapeutic benefit.
  • peptides of different embodiments of the invention will provide carrier peptides for delivery of compounds to any desired cell type.

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Abstract

L'invention concerne de nouveaux peptides et de nouvelles constructions. De plus, l'invention concerne des procédés d'administration d'un ou plusieurs composés à une cellule. Les peptides et constructions peuvent être utilisés pour l'administration des composés à des cellules in vitro et in vivo.
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CN105664173B (zh) * 2016-01-29 2018-09-04 山东省医学科学院药物研究所 透膜短肽-苦参碱及其制备方法与应用
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CN105664173B (zh) * 2016-01-29 2018-09-04 山东省医学科学院药物研究所 透膜短肽-苦参碱及其制备方法与应用
US11466069B2 (en) 2017-04-28 2022-10-11 Auckland Uniservices Limited Methods of treatment and novel constructs

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