WO2017134148A1 - Peptides à pénétration cellulaire sélective - Google Patents

Peptides à pénétration cellulaire sélective Download PDF

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Publication number
WO2017134148A1
WO2017134148A1 PCT/EP2017/052223 EP2017052223W WO2017134148A1 WO 2017134148 A1 WO2017134148 A1 WO 2017134148A1 EP 2017052223 W EP2017052223 W EP 2017052223W WO 2017134148 A1 WO2017134148 A1 WO 2017134148A1
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Prior art keywords
arg
cell
modified
cpp
tat
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PCT/EP2017/052223
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English (en)
Inventor
Saskia Annerie BODE
Harmen Dolstra
Dennis Wilhelmus Petrus Maria Löwik
Cornelis Maria VAN HEST
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Stichting Katholieke Universiteit
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Publication of WO2017134148A1 publication Critical patent/WO2017134148A1/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
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus human T-cell leukaemia-lymphoma virus
    • C07K14/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • C07K14/16—HIV-1 ; HIV-2
    • C07K14/163—Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to the field of medicine, preferably to the field of cancer treatment.
  • CPPs Cell-penetrating peptides
  • PTDs protein transduction domains
  • CPPs Cell-penetrating peptides
  • various types of cargoes may be attached to CPPs to transport them inside cells. Because of these characteristics, CPPs are frequently included in drug delivery methods. However, an important drawback of CPPs is that they are hardly cell type specific, causing them to be taken up by almost any type of cell. [2] Such unselective CPPs cannot be used in effectively combatting cancer, as healthy cells are also targeted.
  • the CPP is combined with a homing device, such as an antibody or a homing peptide, which allows for the accumulation of the drug complex to the tumour surface through interactions with receptors or antigens.
  • a homing device such as an antibody or a homing peptide
  • An example of such active targeting system employs folate homing device for the delivery CPP decorated liposomes.
  • Cell- penetrating homing peptides that were identified by mRNA phage display technology are known from Kondo et al. [ ] These peptides were able to bind to tumour cell lines, and internalize into these cells afterwards. The specificity for tumour cells is encoded in the sequence of the homing peptide.
  • CPPs selectivity in CPPs
  • external stimuli such as a change in pH, [8 ' 9] by an enzymatic reaction/ 10 ' 11] or by a UV-light trigger.
  • activatable CPPs or ACPPs
  • ACPPs ACPPs
  • a useful method to block their activity is by interaction with anions.
  • cargos are selectively released from a CPP in liver cells using a liver protease cleavable linker.
  • PEG poly(ethylene glycol) chains
  • MMPs matrix metalloproteases
  • Other blocking moieties, such as anionic peptides, have been used for similar strategies, which have been applied in tumour detection [14] and imaging.
  • Liu et al. made use of a legumain cleavable linker.
  • This enzyme is upregulated in various solid tumours.
  • the tripeptide linker (Ala-Ala-Asn) was attached to a lysine in the CPP Tat, to create a branched moiety, which led to a substantial decrease in cellular uptake.
  • the branched peptide was used for the delivery of doxorubicin- loaded liposomes to legumain- expressing cells.
  • the downside of ACPP systems is that their methodology depends on the micro-environment of tumour tissue, e.g. the (increased) presence of specific enzymes or a decrease in pH, to function.
  • large alterations or additions in the CPP structure need to be included to induce an activatable and specific system, such as the incorporation of PEG chains, or enzyme cleavable linkers and shielding groups.
  • ACPP based targeted drug delivery may also suffer reduced selectivity, causing undesirable side-effects.
  • US2011009336 discloses chemically modified TAT peptides for use in the treatment of cancer. These modifications mainly concern amino acid substitutions, which would lead to enhanced immunostimulating characteristics.
  • the present invention provides in this need, by modification of at least one positively charged amino acid of a CPP.
  • modified CPPs are known from Bode et al, [10] wherein modified Tat-peptides were used for enzymatic activation and uptake in HEK cells. The lysine side-chain of Tat were elongated by addition of amino acids, which blocked the uptake of this cell-penetrating peptide, preventing cellular delivery in healthy mammalian cells.
  • US 2009/0099066 describes a screening method for identifying tissue-selective peptides, wherein libraries of modified CPPs are tested.
  • the screening occurs in vivo, by administration to a mammalian subject.
  • US 8524659 discloses R A virus-derived peptides having modified arginine side-chains, which are used for inhibiting RHA viruses.
  • the use as drug delivery carrier for therapeutic and diagnostic agents is disclosed.
  • US 2009/062178 discloses cargo/carrier peptides constructs which are useful for the prevention and treatment of pain, wherein the carrier may be Tat or other CPPs.
  • the cargo is a PKC inhibitory peptide of minimally five amino acids that may be covalently attached to a side-chain of one of the amino acids of the carrier.
  • WO 2008/043366 concerns compounds for transmembrane delivery of a biological agent, containing a cationic, a lipophilic and a modulator domain.
  • the cationic domain may be a CPP such as Tat.
  • the lipophilic domain may be a fatty acid conjugated to the cationic peptide at a lysine residue.
  • US 8501193 concerns HIV vaccines containing Tat protein derivatives or fragments thereof that may be functionalized with hydrophobic groups at polar or charged amino acids, including lysine.
  • the inventors have surprisingly found that the cellular uptake capacity of the CPPs can be modulated, by introducing specific chemical modifications, such that the modified CPPs are capable of targeting a specific cell of interest, such as a leukaemia cell. This finding has been put to practice in the various aspects of the present invention.
  • the present invention provides a method for selectively targeting a cell of interest, comprising:
  • the present invention provides a method for screening for selective uptake of modified CPPs by a cell of interest, comprising:
  • the present invention provides a modified CPP, wherein at least one positively charged amino acid residue is modified in that the positive charge is (a) absent, (b) located at least one atom more distant from the backbone of the CPP, (c) located at least one atom less distant from the backbone of the CPP, or (d) linked to the backbone of the CPP via a modified linking moiety.
  • the present invention provides a composition, preferably a pharmaceutical composition, comprising a cell penetrating peptide according to any one of claims 14 to 20 and a pharmaceutically acceptable carrier.
  • a cell penetrating peptide according to the invention for use as a medicament for use as a medicament
  • a composition comprising the cell penetrating peptide according to the invention for use as a medicament for use as a medicament for the treatment, prevention, delay, diagnosis, or detection of cancer
  • a composition comprising the cell penetrating peptide according to the invention for use as a medicament for the treatment, prevention, delay, diagnosis, or detection of cancer are provided.
  • CPPs cell penetrating peptides
  • selectivity is typically obtained by (a) linking an otherwise unselective CPP to a dedicated targeting moiety, such that selectivity is obtained by the dedicated targeting moiety, or by (b) inactivating an otherwise unselective CPP, which is subsequently activated only in proximity to a target cell.
  • the active CPP itself involved in both these methods is not selective and can therefore enter any cell, including untargeted cells.
  • CPPs according to the invention are 'always on' for the target cell, yet they are 'always off for other cells, which greatly reduces the risk of collateral cell penetration in non-targeted cells.
  • CPPs according to the invention are not activated in a method or during use.
  • the cellular uptake of the modified CPP is slightly reduced compared to known CPPs by modulating the distribution of positive charges around the CPP.
  • the modified CPPs according to the invention are only slightly modified compared to known CPPs, wherein the modification slightly changes the positive charge distribution of the CPP, as such slightly reducing the uptake capacity of the CPP. As such, the modified CPP is no longer readily taken up by any cell, but only by a specific cell of interest.
  • the present invention relates in a first aspect to a method for selective targeting a cell of interest.
  • the present invention relates to a method for screening modified cell penetrating peptides (CPP) for selective uptake by a cell of interest.
  • CPP modified cell penetrating peptides
  • the present invention relates a modified cell penetrating peptide.
  • the present invention relates to a composition comprising the modified cell penetrating peptide according to the invention.
  • the invention relates to the use of the modified cell penetrating peptide according to the invention or the composition according to the invention as medicament, for the treatment of cancer, and for the selective targeting of a cell of interest.
  • a cell of interest is a cell that is the intended target for the modified CPP.
  • the cell of interest takes up the modified CPP, while cells of a different kind do not or to a lesser extent take up the modified CPP.
  • the cell of interest can be a single cell or a multiplicity of such cells of the same kind.
  • a cell of interest can be identified as being of a different kind from other cells (or "additional cells") which may be present in the same sample. Cells that are of a different kind than the cell of interest can be of a different cell type. For example, an epithelial cell is different from a white blood cell. Thus, the cell of interest is typically of a different type as any other cell.
  • the difference between the cell of interest and the other cells may reside in the state of the cell.
  • the cell of interest may be in a diseased state, i.e. it suffers from a condition, while other cells, form the same type or not, do not suffer from that condition.
  • An example of such a condition is cancer, such as leukaemia.
  • the cell of interest is a leukemic cell, such as a leukemic lymphoblast, while other cells include non-leukemic cells, including healthy lymphoblasts, neuronal cells and bone cells.
  • the modified CPPs are taken up by leukemic cells, while they are not taken up by healthy cells.
  • Non-limiting examples of possible cells of interest are epithelial cells, kidney cells, nerve cells, blood cells, white blood cells, lymphocytes, bone marrow derived cells, cancer cells, human blood cells, human lymphocytes, human lymphocytes that suffer from a condition, Chinese hamster ovary (CHO) cells, the human cervical epithelioid carcinoma cell line HeLa, or human epithelial kidney (HEK) 293 cells.
  • the cell of interest may be from any organism that normally comprises such cells.
  • the cell of interest is a human cell.
  • the cell of interest suffers from a condition and differentiates from other cells in that the latter do not suffer from said condition.
  • cancer is a preferred condition, most preferably the condition is leukaemia.
  • leukaemia in the context of the present invention are acute myeloid leukaemia (AML) and B-cell lymphocytic leukaemia (B- ALL). These syndromes are of particular interest because AML is the type of leukaemia most prevalent in adults and B-ALL is a frequently occurring childhood cancer.
  • AML acute myeloid leukaemia
  • B- ALL B-cell lymphocytic leukaemia
  • the cell of interest can be present in a subject or in a sample.
  • the cell of interest can be present in a sample obtained from a subject, which may have been previously obtained from a subject, preferably from a human subject, although samples from a non- human subject are also encompassed in the present invention.
  • obtaining the sample is not part of the method according to the invention.
  • selectively targeting of a cell of interest, preferably a cancer cell, most preferably a leukaemia cell, within the (human) body is encompassed by the present invention.
  • the modified CPP according to the invention is administered to a (human) subject and within the (human) body the modified CPP is selectively taken up by the cell of interest, preferably a diseased cell, and not or to a lesser extent by other cells, typically healthy cells.
  • Selective targeting refers to the capacity of a substance, in particular the modified CPPs according to the invention, to selectively be taken up by the cell of interest.
  • the modified CPPs according to the invention are exclusively or particularly capable to penetrate the cell of interest, while other cells are not or to a lesser extent susceptible to penetration by the modified CPP.
  • a modified CPP according to the invention that is administered to said mammal will largely to exclusively be taken up by cells of interest comprised in the mammal, and not by other cells.
  • Selectivity for a cell of interest is to be understood as that uptake by a cell of interest is more prevalent than by other cells.
  • the inventors have found that when the cell of interest is a HeLa cell and the other cells are HEK cells, the uptake capacity for a particular modified CPP according to the invention (Tat-GP(l 1) as defined further below) increases from 20 % in HEK to 110 % in HeLa (measured against uptake of unmodified Tat). It is preferred that the uptake of a modified CPP according to the invention for the cell of interest is at least twice as high (e.g. 2 - 100 times as high) as for the other cell(s), more preferably at least 3 times as high (e.g. 3 - 50 times as high), most preferably at least 4 times as high (e.g. 4 - 10 times as high).
  • Uptake is a term that is known in the art. As defined herein, uptake is the uptake of a substance by a cell, in particular of a (modified) CPP by a cell. Uptake may also be referred to as "cellular uptake”. Uptake involves the active or passive transportation of a substance from outside of a cell to inside of a cell. In this context, inside a cell can also mean inside a compartment or membrane of a cell. Uptake can take place via myriad mechanisms.
  • Such mechanisms are sometimes referred to as pathways, and include micropinocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis, clathrin-caveolin-independent endocytosis, transitory pore formation, uptake via (hyper)nucleation zones, direct translocation and cytosolic delivery through nucleation zones.
  • entering a cell through endocytosis or via direct penetration is also considered uptake.
  • uptake is explicitly differentiated from association, which can involve binding of a substance to the outside of a cell.
  • (modified) CPPs are taken up by a cell without modification of the (chemical) structure of the (modified) CPP.
  • a peptide is known in the art and comprises at least two amino acid residues.
  • the terms 'residue' and 'amino acid' are often used interchangeably to indicate an amino acid residue within a peptide.
  • the main chain of a peptide or "backbone” is the chain of amino acid residues that are interconnected by amide bonds.
  • the backbone contains the interconnecting amide bonds and the a-carbon atom of each residue.
  • the side chains of the amino acid residues are not part of the backbone.
  • Peptides have a certain amino acid sequence.
  • peptides may contain naturally occurring amino acids as well as non- natural amino acids, peptidomimetics, unconventional linkages, and all known variations, including alkylated bonds, inverted bonds, or other types of bonds, such as esters, triazoles, carbamates, ureas, thioureas, imides, imines, halogenated bonds, alpha- halogenated bonds, ketones, or peptides comprising beta-amino acids, other extended amino acids, or peptoids where side chains of residues are attached to the backbone amide bonds instead of to the corresponding alpha carbon atoms, or bonds that involve side chains instead of backbone functional groups.
  • Peptides can comprise amino acids of any chirality, such as L-amino acids or D-amino acids, or mixtures thereof. Accordingly, the term 'amino acid' as used in this invention should be interpreted as any moiety that can constitute a residue in a peptide.
  • an amino acid contains a carboxylic acid moiety and an amine moiety at the alpha-carbon next to the carboxylic acid, with either a D or an L chiral configuration, preferably L.
  • the amine can also be more distant from the carboxylic acid.
  • amino acids are often characterized by the nature of their side chains.
  • Amino acids that are considered to be basic or positively charged amino acids include lysine, arginine, and histidine.
  • Amino acids that are considered to be acidic or negatively charged amino acids include aspartic acid, glutamic acid, and tyrosine.
  • Amino acids that are considered to be polar uncharged amino acids include serine, threonine, cysteine, asparagine, and glutamine.
  • Amino acids that are considered to be hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, proline, and tryptophan.
  • Proline is considered to be a conformationally restrained amino acid.
  • Glycine is achiral and can thus be part of both D-peptides and L-peptides.
  • Further possible amino acids include ornithine (Orn), which is analogous to lysine with one fewer methylene moiety in its side chain, 2,4-diaminobutyric acid (Dab), which is analogous to lysine with two fewer methylene moieties in its side chain, 2,3-diaminopropionic acid (Dap), which is analogous to lysine with three fewer methylene moieties in its side chain, and 2,7- diaminoheptanoic acid (Dah), which is analogous to Lysine with one more methylene moiety in its side chain.
  • Orn ornithine
  • Dab 2,4-diaminobutyric acid
  • Dap 2,3-diaminopropionic acid
  • Dah 2,7- diaminoheptanoic acid
  • the peptides in particular the (modified) CPPs, in the context of the present invention, may optionally be comprised in larger peptides, or in larger molecules.
  • Peptides may comprise capping groups such as terminal amides, acetamides, methyl esters, other terminating esters, or other terminal moieties that are known to a person skilled in the art.
  • Peptides may possibly comprise one or more protecting groups as known in the art, such as t-butyl carbamate (tBoc), 9-fluorenylmethyl carbamate (Fmoc), benzyl carbamate (Z), benzyl ester, t-butyl ester, methyl ester, azides, 4- methyltrityl (Mt).
  • a terminal amino acid is the last or the first amino acid of a sequence of amino acids or of a peptide.
  • the terminal amino acid is linked to its sloe neighbouring residue via its carboxylic acid moiety, then that terminus is an N- terminus.
  • the terminal amino acid is linked to its sloe neighbouring residue via its amine moiety, then that terminus is a C-terminus
  • Peptides in the context of the present invention may be conjugated to a further moiety, such as a cargo, optionally via a linker moiety.
  • a linker moiety as known in the art may be used, such as 6-aminohexanoic acid (ahx), beta-alanine (also known as beta-aminopropionic acid (bAla)), 4-aminobutyric acid (also known as piperidinic acid (4Abu)), 3-aminoisobutyric acid (bAib).
  • ahx 6-aminohexanoic acid
  • acp 6-aminocaproic acid
  • Ahx or acp is a linker moiety which is capable to connect two moieties together.
  • Peptide sequences as defined herein are to be interpreted as defining (part of) the chain of amino acids only, and such peptides may possibly feature various end group modifications, unless such moieties are expressly represented.
  • the peptide in question may contain the amino acid sequence as part of a larger sequence.
  • Amino acid sequences are represented herein with the N-terminus on the left and with the C-terminus on the right.
  • Side chain modifications may be designated in between parentheses behind the three letter code of the residue. For example, Lys(Boc) represents a lysine residue wherein a Boc group is attached to its side chain amine.
  • At least one of the amino acid residues of the peptide is a natural amino acid, preferably an L-amino acid.
  • at least two of said residues are natural amino acids.
  • each of the residues, except for the modified residues as further defined below are natural amino acids.
  • the amino acid residues may be either L-amino acids and/or D-amino acids.
  • the amino acid residues are all L-amino acids.
  • the amino acid residues are all D-amino acids. D-amino acids may be preferred for their reduced sensitivity towards proteolytic enzymes, while they typically are equally capable of entering cells.
  • Salt of peptides preferably a pharmaceutically acceptable salts, are also considered peptides in the context of the present invention.
  • the peptide may be bought, synthesized or obtained by isolating it from a natural product.
  • Peptides can be obtained through isolation from a digest of a larger protein.
  • peptides according to the invention are of synthetic origin.
  • a preferred method for peptide synthesis is solid phase peptide synthesis (SPPS), which is well-known to a person skilled in the art. Advantages of obtaining short peptides through SPPS are the ease of synthesis, the low component cost, the speed of synthesis, and the possibility for automation using synthesis robots, synthesizers, semiautomatic synthesizers, or automatic synthesizers.
  • SPPS strategies known in the art allow both N-terminal and C-terminal modification, such as alkylation, amidation, or labelling.
  • CPPs Cell penetrating peptides
  • Modified CPPs in the context of the present invention are modified with respect to known CPPs, such as those defined here.
  • CPPs are (highly) cationic (often referred to as "oligocationic” or "poly cationic”).
  • the CPP is typically an oligocationic or a polycationic CPP, more preferably a polycationic CPP.
  • (oligo- or poly)cationic CPP typically contain at least four cationic amino acid residues, preferably at least five, at least six or even at least seven cationic amino acid residues.
  • CPPs can be naturally occurring, they can be derived from naturally occurring polypeptides or they are synthetic. Such derived CPPs are typically derived from polypeptides that are capable of independently entering cells. Cell penetrating subsequence of said polypeptide were identified by screening. Other CPPs are synthetic in origin, i.e. they were designed de novo.
  • Preferred CPPs to be used according to the present invention have at least one lysine residue, preferably at least two lysine residues, more preferably two lysine residues.
  • Non-limiting examples of CPPs in the context of the present invention are oligoarginines R H , wherein n is an integer in the range 4 - 17, preferably in the range 7 - 10 (e.g. R 9 (SEQ ID NO: 1, nona-arginine), R 8 (SEQ ID NO: 2, octa-arginine), R 7 (SEQ ID NO: 3, hepta-arginine)); oligolysines Km, wherein m is an integer in the range 4 - 17, preferably in the range 7 - 9 (e.g. K9 (SEQ ID NO: 4, nona- lysine)); a Tat (trans- activating transcriptional activator) variant (e.g.
  • GRKKRRQRRRPQ (SEQ ID NO: 5), RKKRRQRRR (SEQ ID NO: 6), YGRKKRRQRRRPQ (SEQ ID NO: 7, also referred to as Tyr-Tat), GRRRRRRRPPQ (SEQ ID NO: 8, also known as R 9 -Tat)); RGGRLSYSRRRFSTSTGR (SEQ ID NO: 9, which is also referred to as SynBl); RRLSYSRRRF (SEQ ID NO: 10 which is also referred to as SynB3); PIRRRKKLRRLK (SEQ ID NO: 11, also known as PTD-4); RRQRRTSKLMKR (SEQ ID NO: 12, also known as PTD-5); RRRRNRTRRNRRRVR (SEQ ID NO: 13, also known as FHV Coat (35-49)); KMTRAQRRAAARRNRWTAR (SEQ ID NO: 14, also known as BMV Gag (7-25)); TRRQRTRRARRNR (SEQ ID NO: 15, also known as HTLV
  • the CPP of SEQ ID NO: 6 is referred to as "Tat”, while the CPP of SEQ ID NO: 5 is referred to as "extended Tat”.
  • Preferred CPPs in the context of the present invention are any of the Tat variants (e.g. SEQ ID NO: 5, 6, 7 or 8) and oligo-arginine (e.g. SEQ ID NO: 1 , 2 or 3), most preferably a Tat variant.
  • X 1 absent.
  • Most preferably X 6 absent.
  • X 7 absent.
  • sequence of SEQ ID NO: 25 can be incorporated in a larger peptide, which is preferably represented by X tractX 1 X 2 RX 3 X 4 RRX 5 R RX 6 X 7 X m , wherein each of X 1 , X 2 , X 3 , X 4 , X 5 , X 6 and X 7 are as defined above for SEQ ID NO: 25, and each X is individually an amino acid residue and n and m are each individually integers in the range of 0 - 10.
  • Preferred variants of Tat are GRKKR QRRRPQ (SEQ ID NO: 5), RKKR QRR (SEQ ID NO: 6), YGRKKRPvQPvRRPQ (SEQ ID NO: 7) and GRRRRRRRRRPPQ (SEQ ID NO: 8).
  • Especially preferred CPPs in the context of the present invention are SEQ ID NO: 5 and SEQ ID NO: 6. Most preferred is SEQ ID NO: 6.
  • a CPP can be comprised in a larger molecule, e.g. can be part of a larger peptide or can be linked to other moieties.
  • CPPs may be loaded with a cargo, such as a detectable label or a pharmaceutically active substance.
  • Cargo is a term that is known in the art to refer to substances linked to a CPP, with the intent to effectuate uptake of the cargo by a cell. Cargo may also be referred to as payload.
  • the term cargo may refer to each part of the cargo-CPP complex that is not the CPP itself, which thus includes linking moieties.
  • the term "loaded with” may be replaced with "linked to" or "conjugated to”.
  • the cargo may be associated with the CPP through covalent chemical linkage or through non-covalent interactions.
  • Covalent linkage can be reversible, such as linkage through dithiol-bridge formation. Without limitation, examples of covalent linkage are through amide bond formation, through C-N bond formation, through C-S bond formation, or through C-0 bond formation.
  • Non-limiting examples of non-covalent linkage include linkage through H-bonding, through charge-charge interactions, through molecular recognition such as coiled-coil interaction, leucine zipper interaction, or through joint bilayer or micelle formation and related entrapment.
  • a CPP linked to a liposome or comparable vesicle is considered to also be linked to substances encapsulated in said vesicle, or in membranes or hydrophobic compartments thereof.
  • peptides in general and CPPs in general specifically applies to the modified CPPs according to the invention. As they typically contain one or more non- natural amino acids, they are still classified as peptides. Also in conjugated form, i.e. when conjugated to a cargo, the modified CPP according to the invention classifies as a peptide as defined herein.
  • Physiological conditions are known to a person skilled in the art, and typically comprise aqueous solvent systems, atmospheric pressure, pH-values between 5 and 8, a temperature ranging from room temperature to about 37 °C (from about 20 °C to about 40 °C), and a suitable concentration of buffer salts or other components. Conditions in a subject are always considered to be physiological conditions in the context of this invention.
  • a positively charged moiety such a positively charged amino acid side chain
  • this moiety is positively charged at physiological conditions. It is understood that charge is often associated with equilibrium.
  • a moiety that is said to carry or bear a positive charge is a moiety that will be found in a state where it bears or carries such a charge more often than that it does not bear or carry such a charge.
  • the primary amino moiety of the side chain of lysine and the guanidino moiety in arginine is positively charged at physiological conditions.
  • the side chain of histidine, having a ⁇ ⁇ of about 6, is considered positively charged in the context of the present invention.
  • the present invention provides a method for selectively targeting a cell of interest, comprising: (i) providing a modified CPP, wherein at least one positively charged amino acid residue is modified in that that positive charge is (a) absent, (b) located at least one atom more distant from the backbone of the CPP, (c) located at least one atom less distant from the backbone of the CPP, or (d) linked to the backbone of the CPP via a modified linking moiety, and
  • This method may also be referred to as a method for improving or enhancing the uptake selectivity of a CPP.
  • any mode of contacting a cell with a peptide as known in the art can be employed, such as adding the modified CPP according to the invention to a medium, buffer or solution in which the cell of interest is cultured, suspended or present.
  • Other preferred methods of contacting a cell comprise exposing a sample or subject, containing the cell of interest, to the peptide. Such exposing a subject may also be referred to as "administering a peptide to a subject”. The contacting may thus be performed by administering the modified CPP according to the invention to a subject containing the cell of interest.
  • the CPP that is contacted in step (ii) can also be a composition comprising the CPP.
  • contacting the CPP according to the invention with the cell of interest may entail contacting a composition comprising the CPP with the cell of interest.
  • Preferred compositions are defined herein below.
  • a cell of interest is contacted, this cell of interest can be comprised in a sample. As such, the entire sample is understood to be contacted. This can mean that other cells that are comprised in the sample which are not the cell of interest are considered to be contacted as well.
  • the contacting of step (ii) preferably results in cellular uptake of the modified CPP according to the invention by the cell of interest.
  • the modified CPP is selectively taken up by the cell of interest.
  • the modified CPP is taken up by the cell of interest exclusively when said sample is contacted with the modified CPP.
  • only the cell of interest is present in a sample. In such cases, all cells are penetrated by the modified CPP. This can find use in applications where the homogeneity of a sample is under investigation. It is also possible that the sample does not comprise the cell of interest, wherein no cells are penetrated by the modified CPP upon the contacting of step (ii). This can find use in applications where detection of the cell of interest is desirable.
  • a method as described above wherein the cell of interest is comprised in a sample that further comprises additional cells, wherein uptake of the modified CPP is selective for the cell of interest.
  • uptake of the modified CPP by the cell of interest occurs.
  • the additional cells are typically of a different type than the cell of interest.
  • uptake of the modified CPP by the cell of interest is the only detectable uptake, thus the ratio of uptake by the cell of interest over the average uptake by other cells in the sample is (close to) 1 and (close to) 100 % of the detected uptake is by the cell of interest.
  • At least 10% of the total detectable uptake is by the cell of interest.
  • the additional cells that are comprised in the sample and that are not or to a lesser extent penetrated by the CPP according to the invention are cells of a different kind as the cell of interest.
  • Selective uptake thus also includes preferential uptake of the modified CPP according to the invention by the cell of interest over the additional cells, wherein uptake of the modified CPP is 1% more prevalent than uptake by the additional cells, preferably 5% more prevalent, more preferably 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% or more percent more prevalent than uptake by the additional cells.
  • the uptake selectivity of the modified CPP according to the invention is measured in comparison to a template CPP which is the unmodified variant of the modified CPP.
  • uptake selectivity of the modified CPP by the cell of interest may be more prevalent, e.g. 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75% or 100% more prevalent, than uptake of the template CPP.
  • uptake selectivity for the modified CPP can also be provided when the uptake of the modified CPP for the cell of interest is less prevalent when compared to the uptake of the template CPP for the cell of interest, as the uptake of the modified CPP for additional cells is even further reduced compared to the uptake in additional cells by the template CPP.
  • each of the at least one positively charged amino acid residue that is modified is lysine or arginine.
  • the modified amino acid residues can be referred to as a modified lysine or a modified arginine.
  • the amino acid residue(s) in question in the template CPP are lysine and/or arginine.
  • at least one of the positively charged amino acid residues is lysine, most preferably all are lysine.
  • the modified CPP comprises one or two modified amino acid residues, which are each modified according to one of the modifications (a), (b), (c) and (d) as defined below.
  • the modified CPP differs from a known CPP in that one or two positively charged amino acid residues are modified.
  • the modified CPP comprises one amino acid residue is modified.
  • the modified CPP comprises two amino acid residues are modified.
  • the modified CPP is loaded with a cargo, preferably wherein the cargo is a detectable label or a pharmaceutically active substance.
  • the cargo is a detectable label.
  • Detectable labels such as a fluorophore, a chromophore, a radioactive tracer, a specific isotope (e.g. technetium, optionally introduced via a chelator such as DOTA), a diagnostic marker, or a hapten are known in the art.
  • Preferred fluorescent labels are selected from the group consisting of fluorescein and its derivatives such as fluorescein isothiocyanate (FITC) or Tokyo green, ASP (preferably 4-(4-(didecylamino)styryl)-N-methylpyridinium iodide), rhodamine, Cy3, Cy5, Atto dyes, Alexa dyes, calcein and IR dyes (e.g. CW8000).
  • fluorescein and its derivatives such as fluorescein isothiocyanate (FITC) or Tokyo green
  • ASP preferably 4-(4-(didecylamino)styryl)-N-methylpyridinium iodide
  • rhodamine preferably 4-(4-(didecylamino)styryl)-N-methylpyridinium iodide
  • Cy3 Cy5 preferably 4-(4-(didecylamino)styryl)-N-methylpyridin
  • the fluorescent labels are selected from the group consisting of Tokyo green, ASP (preferably 4-(4-(didecylamino)styryl)-N-methylpyridinium iodide), rhodamine, Cy3, Cy5, Atto dyes, Alexa dyes, calcein and IR dyes (e.g. CW8000).
  • the cargo does not comprise fluorescein diacetate 5-maleimide.
  • the cargo is a pharmaceutically active substance, such as a drug, prodrug or a drug candidate.
  • a pharmaceutically active substance can be a small molecule, a drug, a prodrug, a peptide, a depsipeptide, an acyldepsipeptide, an antibiotic, an antimicrobiotic, a polypeptide, a protein, a protein fragment, an oligooxopiperazine, a nucleic acid, a nucleic acid analogue, or parts thereof, a monosaccharide, oligosaccharide or polysaccharide, a chemotherapeutic, a nanoparticle, a polymeric drug carrier, a dendritic drug carrier, a liposome, a decoy molecule, or any other entity or combination thereof.
  • the cargo is a selected from the group of anti-cancer agents, cytotoxins, antiviral agents, antibacterials agents, peptides, oligonucleotides and nucleoic acids, in particular siRNA.
  • cytotoxins include colchicine, vinca alkaloids, anthracyclines, camptothecins, doxorubicin, daunorubicin, taxanes, calicheamycins, tubulysins, irinotecans, an inhibitory peptide, siRNA, amanitin, deBouganin, duocarmycins, maytansines, auristatins or pyrrolobenzodiazepines (PBDs).
  • PBDs pyrrolobenzodiazepines
  • anti-cancer agent are present as cargo, such as daunorubicin and doxorubicin.
  • the anti-cancer agent is preferably an anti- leukaemia agent, such as chlorambucil, cyclophosphamide, fludarabine, pentostatin, tretinoin, cladribine, prednisone, vincristine, anthracycline, imatinib, cyclophosphamide, doxorubicin, etoposide and bleomycin. All of these agents are used in the treatment of leukemia.
  • a method as described above wherein the modified CPP is a modified CPP according to the third aspect of the present invention.
  • a method as described above is provided, wherein the modified amino acid residue contains a side chain wherein the positive charge located at least one atom more distant from the backbone of the CPP.
  • Such a modification corresponds to a type (b) modification, which are further defined below.
  • a modification corresponds to a type (b) modification, which are further defined below.
  • SEQ ID NO: 27 refers to X 1 X 2 RX 3 X 4 RRX 5 RRRX 6 X 7 , wherein X 1 , X 2 , X 5 , X 6 and X 7 are as defined for Tat (see SEQ ID NO: 25) and at least one of X 3 and X 4 is not lysine (K), preferably not lysine (K) or arginine (R), most preferably at least one of X 3 and X 4 is a lysine residue that is modified according to any one of modifications (a), (b), (c) or (d) as defined below.
  • SEQ ID NO: 28 refers to RX 3 X 4 RRX 5 RRR, wherein X 5 is as defined for Tat (see SEQ ID NO: 25) and at least one of X 3 and X 4 is not K, preferably not K or R, most preferably at least one of X 3 and X 4 is a lysine residue that is modified according to any one of modifications (a), (b), (c) or (d) as defined below.
  • SEQ ID NO: 29 refers to RX 3 X 4 RRQRRR, wherein at least one of X 3 and X 4 is not K, preferably not K or R, most preferably at least one of X 3 and X 4 is a lysine residue that is modified according to any one of modifications (a), (b), (c) or (d) as defined below.
  • the modification over Tat thus resides in at least one of X 3 and X 4 not being lysine, preferably not being lysine or arginine.
  • the second one of X 3 and X 4 is lysine or arginine, preferably lysine.
  • Modified CPPs are further defined below, and preferred candidates are Tat-A(l 1), Tat-A(Ol), Tat-A(lO), Tat-GP(l 1), Tat-GP(Ol), Tat-GP(lO), Tat-G(l 1), Tat-G(lO), Tat-G(Ol), Tat-V(lO), Tat-L(lO), Tat-bA(l l), Tat-bA(lO), Tat-bA(Ol), Tat- AA(10), Tat-ObA(l l), Tat-ObA(lO), Tat-ObA(Ol), Tat-OA(l l), Tat-OA(l l), Tat-OA(IO), Tat- OA(01), Tat-F(l 1), Tat-F(lO), Tat-F(Ol), Tat-DpbA(l 1), Tat-DpbA(lO), Tat-DpbA(Ol), Tat-DbbA(l l), Tat-DbbA(lO), Tat-
  • Tat-A(Ol), Tat-A(lO), Tat-A(l l), Tat-GP(Ol), Tat-GP(lO), Tat-GP(l l), Tat-G(Ol), Tat- G(10) and Tat-G(l 1) are most preferred.
  • the modified CPP that is selective for a particular cell of interest is identified by the method according to the second aspect.
  • a method is provided as described above, wherein the cell of interest suffers from a condition.
  • cells that are of the same type as the cell of interest yet that do not suffer from the same condition are considered cells of a different kind and thus not cells of interest.
  • the cell of interest is a cancer cell, more preferably a leukaemia cell, most preferably an acute myeloid leukemia (AML) cell or a B-cell lymphocytic leukemia (B- ALL) cell.
  • the cell of interest is not a HEK cell or a pancreatic cancer cell (AsPC-1).
  • cancers include a cancer of epithelial origin or neuronal origin or a carcinoma or a solid tumour or a sarcoma or a liquid tumour such a leukaemia or a lymphoma.
  • Cancer cells may be from the bladder; brain; breast; colon; esophagus; gastrointestine; head; kidney; liver; lung; nasopharynx; neck; ovary; prostate; skin; stomach; testis; tongue; neuron or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm; malignant; carcinoma; carcinoma undifferentiated; giant and spindle cell carcinoma; small cell cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma; malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma; familial polyposis coli; solid carcinoma; carcinoid tumor; malignant; branchiolo-alveolar carcinoma; papillary carcinoma; squamous cell carcinoma; basal adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic a
  • a leukaemia includes any of: Acute lymphoblastic leukaemia (ALL) such as precursor B acute lymphoblastic leukaemia, precursor T acute lymphoblastic leukaemia, Burkitt's leukaemia, and acute biphenotypic leukaemia, Chronic lymphocytic leukaemia (CLL) such as B-cell prolymphocytic leukaemia, a more aggressive disease, Acute myelogenous leukaemia (AML) such as acute promyelocytic leukaemia, acute myeloblastic leukaemia, and acute megakaryoblastic leukaemia, Chronic myelogenous leukaemia (CML) such as chronic monocytic leukaemia, Hairy cell leukaemia (HCL), Tcell prolymphocytic leukaemia (T-PLL), Large granular lymphocytic leukaemia and Adult T-cell leukaemia.
  • ALL Acute lymphoblastic leukaemia
  • a lymphoma includes any of: Small lymphocytic lymphoma, Lymphoplasmacytic lymphoma (such as Waldenstrom macro globulinemia), Splenic marginal zone lymphoma, Plasma cell myeloma, Plasmacytoma, Extranodal marginal zone B cell lymphoma (MALT lymphoma), Nodal marginal zone B cell lymphoma (NMZL), Follicular lymphoma, Mantle cell lymphoma Diffuse large B cell lymphoma, Mediastinal (thymic) large B cell lymphoma, Intravascular large B cell lymphoma, Primary effusion lymphoma, Burkitt lymphoma/leukaemia, T cell prolymphocytic leukaemia, T cell large granular lymphocytic leukaemia, Aggressive NK cell leukaemia, Adult T cell leukemia/lymphoma, Extranodal NK/T cell lymphoma - nasal type,
  • a method as described above wherein the modified CPP has an increased uptake selectivity by the cell of interest as compared to the uptake selectivity of a template CPP.
  • said uptake selectivity is preferably expressed as a parameter selected from the group consisting of:
  • the ratio of uptake by the cell of interest relative to uptake by other cells than the cell of interest which is greater than 1, preferably greater than 10;
  • a method for screening is provided. This method is for screening modified cell penetrating peptides (CPPs) for selective uptake by a cell of interest, comprising:
  • the modified CPP exhibits selective uptake by the cell of interest when selective uptake as defined above by the cell of interest over the additional cell is observed.
  • the modified CPP according to the invention is further defined below.
  • Selective uptake is preferably expressed as a parameter selected from the group consisting of:
  • the ratio of uptake by the cell of interest relative to uptake by other cells than the cell of interest which is greater than 1, preferably greater than 10;
  • the modified CPP exhibits selective uptake by the cell of interest when its uptake by the cell of interest is larger than the corresponding uptake by the additional cell.
  • this uptake may be the only detectable uptake, thus the ratio of uptake by the cell of interest over the average uptake by additional cell(s) in the sample is (close to) 1 and (close to) 100 % of the detected uptake is by the cell of interest.
  • At least 10% of the total detectable uptake is by the cell of interest for the uptake to be classified as selective uptake.
  • Selective uptake thus also includes preferential uptake of the modified CPP according to the invention by the cell of interest over the additional cells, wherein uptake of the modified CPP is 1% more prevalent than uptake by the additional cells, preferably 5% more prevalent, more preferably 10%>, 15%, 20%>, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% or more percent more prevalent than uptake by the additional cells.
  • modified CPPs that are selective for a specific kind of cell of interest can be identified. These identified modified CPPs may be suitably used in the method according to the first aspect.
  • the method according to the second aspect preferably involves the screening of a library of distinct modified CPPs. As such, steps (ii) - (iv) are repeated for each member in the library and step (i) involves providing a library of modified CPPs as defined above.
  • the library of modified CPPs preferably comprises at least 2 distinct modified CPPs according to the present invention, more preferably at least 5 distinct modified CPPs according to the present invention, most preferably at least 10 distinct modified CPPs according to the present invention. Although it is generally preferred that the number of distinct modified CPPs according to the present invention that is present in the library is as high as possible, the upper limit is typically determined from a practical point of view. In one embodiment the library comprises up to 1000 distinct modified CPPs according to the present invention.
  • the method for screening as described above wherein the cell of interest is a cell that suffers from a condition.
  • the method for screening as described above is provided, wherein the cell of interest is a cancer cell, preferably a leukaemia cell. Preferred embodiments thereof are further defined hereinabove.
  • the cell of interest is not a HEK cell or a pancreatic cancer cell (AsPC-1).
  • the method for screening is an in vitro method, wherein the sample is not a living subject, such as a human, although the sample may have been taken from a subject prior to the method according to this aspect is executed.
  • the sample contains the cell of interest and at least one other type of cell, the additional cell(s). Both the cell of interest and the additional cell(s) may be a multiplicity of such cells.
  • the cell of interest thus forms a subpopulation within a larger population of the sample.
  • the cell of interest is of a different type as the additional cell(s) comprised in the sample. In one embodiment, the cell of interest is different from the additional cell(s) in that it suffers from a condition while the additional cell(s) are healthy cells.
  • the modified CPP(s) is/are modified CPP(s) according to the third aspect of the present invention.
  • the modified CPP(s) is/are linked to a cargo, preferably a detectable label or a pharmaceutically active substance, as further defined above for the first aspect.
  • a detectable label such as a fluorescent label is especially preferred in the context of the present aspect, for easy detection of cellular uptake.
  • the possible modifications that may be introduced in the modified CPPs that are to be screened is typically very diverging, such that the chances to identify suitable candidate(s) to specifically target the cell of interest are maximized.
  • the scope of modification between which the skilled person can choose, within the framework of the modifications of types (a) - (d), is in principle unlimited, and further options, definitions and preferred embodiments are described here below.
  • a peptide is provided that is a modified cell penetrating peptide (CPP), wherein at least one positively charged amino acid residue is modified in that that positive charge is (a) absent, (b) located at least one atom more distant from the backbone of the CPP, (c) located at least one atom less distant from the backbone of the CPP, or (d) linked to the backbone of the CPP via a modified linking moiety.
  • CPP modified cell penetrating peptide
  • the modified enhanced cell penetrating peptide according to the invention as defined herein is preferable used in the method for selectively targeting a cell of interest according to the first aspect of the invention.
  • the modified enhanced cell penetrating peptide according to the invention as defined herein is preferable used in the method for screening for selective uptake according to the second aspect of the invention.
  • the modified enhanced cell penetrating peptide according to the invention as defined herein is preferable comprised in the composition according to the invention as defined further below.
  • the modified enhanced cell penetrating peptide according to the invention as defined herein is preferable used in any of the uses defined further below.
  • the modified CPP is linked to a cargo, as specified above.
  • the cell penetrating peptide does not comprise a Pro-Ser-Gln-Ser-Lys-Ser-Lys-Ala amino acid sequence. In one embodiment of this aspect, the cell penetrating peptide does not comprise an N-myristoyl-Pro-Ser-Gln-Ser- Lys-Ser-Lys-Ala-OH motif.
  • the cell penetrating peptide is not N-myristoyl-Pro-Ser-Gln-Ser-Lys-Ser-Lys-Ala-OH, wherein both lysine residues are modified according to modification (a) defined below, wherein a parabromobenzoyl residue is condensed to the primary amino moiety of the lysine residues.
  • the cell penetrating peptide does not comprise an Ac-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-ahx-Cys-NH2 motif.
  • the cell penetrating peptide of the invention is not linked to fluorescein diacetate 5-maleimide. In one embodiment, the cell penetrating peptide according to the invention is not Ac-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-ahx-Cys-NH2, linked to fluorescein diacetate 5-maleimide to form a conjugate.
  • the cell penetrating peptide according to the invention is not Ac-Gly-Arg-Lys-Lys-Arg-Arg-Gln- Arg-Arg-Arg-ahx-Cys-NH 2 , wherein one or both of the lysine residues are modified according to modification (b) defined below, wherein an alanine residue or a GlyPro dipeptide is condensed to the primary amino moiety of one or both lysine residues.
  • the cell penetrating peptide according to the invention is not Ac-Gly- Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-ahx-Cys-NH 2 , linked to fluorescein diacetate 5-maleimide to form a conjugate, wherein one or both of the lysine residues are modified according to modification (b) defined below, wherein an alanine residue or a GlyPro dipeptide is condensed to the primary amino moiety of one or both lysine residues.
  • Preferred cell penetrating peptides according to the invention are not linked to fluorescein diacetate 5-maleimide.
  • Preferred cargos are pharmaceutically active substances, or fluorescent labels selected from Tokyo green, ASP (preferably 4-(4- (didecylamino)styryl)-N-methylpyridinium iodide), rhodamine, Cy3, Cy5, Atto dyes, Alexa dyes, calcein and IR dyes (e.g. CW8000).
  • the cell penetrating peptide according to the invention does not contain a homing peptide, preferably not a homing device (homing peptide or antibody), as cargo.
  • the cargo is not fluorescein diacetate 5-maleimide, preferably not a fluorescein moiety.
  • the cell penetrating peptide according to the invention does not contain an N-terminal myristate, preferably no N-terminal lipidation or even no lipidation in general, as cargo.
  • the modified CPPs according to the invention are modified compared to known CPPs, such as modified compared to one of the CPPs according to SEQ ID NO 1 - 25.
  • the modification may also be referred to as a chemical modification.
  • CPPs are known, the skilled person appreciates when a CPP classifies as "modified CPP".
  • the modification leads to an altered distribution of positive charge around the CPP, when compared to the known CPP.
  • the modification resides in that at least one positively charged amino acid residue of the modified CPP modified such that the positive charge that would be present in the corresponding amino acid residue of the known CPP is (a) absent, (b) located at least one atom more distant from the backbone of the CPP, (c) located at least one atom less distant from the backbone of the CPP, or (d) linked to the backbone of the CPP via a modified linking moiety.
  • the modification is of type (a). In one embodiment, the modification is of type (b). In one embodiment, the modification is of type (c). In one embodiment, the modification is of type (d).
  • the modification is of type (a), (b) or (c), more preferably of type (a) or (b), most preferably of type (b).
  • screening for uptake selectivity may provide modified CPPs wherein more amino acid residues are modified, it is preferred that at most 5, more preferably at most 4, even more preferably at most 3 and most preferably at most 2 amino acids residues are modified, in order not to reduce the uptake capacity of the CPP too much such that cellular uptake is completely blocked. So, most preferably, one or two positively charged amino acid residues are modified.
  • Each of the modified amino acid residues may have the same type of modification (a), (b), (c) or (d), or a different type of modification, although it is preferred that each modified amino acid residue has the same type of modification (a), (b), (c) or (d).
  • Each of the at least one positively charged amino acid residue that is modified is preferably selected from arginine, lysine and histidine, more preferably from arginine and lysine, most preferably one or more lysine residues are modified.
  • the CPPs according to the invention are modified compared to any of the Tat variant, preferably modified compared to one of the CPPs according to SEQ ID NO 5 - 8 and 25, more preferably according to any one of SEQ ID NO 5 - 7, most preferably according to SEQ ID NO 6.
  • at least the lysine residue closest to the N-terminus of the Tat peptide is modified, and the other lysine moiety may or may not be modified. Most preferably both lysine moieties are modified.
  • said modification is of type (b), more preferably by condensing an alanine residue to the side-chain of the lysine residue(s).
  • the modification of type (a) may take the form of the replacement of a positively charged amino acid residue with a neutral amino acid residue.
  • a lysine or arginine residue may be replaced by an alanine, valine, leucine or iso leucine residue.
  • the cationic primary amino moiety of the lysine or arginine side chain is replaced by a secondary or tertiary amino moiety, which are not cationic under physiological conditions, preferably by a secondary amino moiety.
  • the -NH 3 + moiety is replaced by a -NHR 6 moiety, wherein R 6 is a shielding moiety as known in the art, such as acetyl (Ac), t-butyl carbamate (tBoc), 9-fluorenylmethyl carbamate (Fmoc), benzyl carbamate (Z), Ci-4-alkyl.
  • R 6 is a shielding moiety as known in the art, such as acetyl (Ac), t-butyl carbamate (tBoc), 9-fluorenylmethyl carbamate (Fmoc), benzyl carbamate (Z), Ci-4-alkyl.
  • Non-limiting examples of modified Tat peptides according to the invention, wherein the positive charge that is present in the corresponding amino acid residue of Tat is absent are: (1) H-Ala-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-OH, and (2) H-Arg- Lys-Lys(Ac)-Arg-Arg-Gln-Arg-Arg-OH.
  • modified Tat peptide 1 the positive charge that is present at the N-terminal arginine of Tat is replaced by alanine.
  • modified Tat peptide 2 the positive charge that is present at the side chain of the second lysine residue is blocked by an acetyl moiety
  • the positive charge that is naturally present in the side chain of a positively charged amino acid reside e.g. in lysine or arginine side chains, is relocated to a position at least one atom further away from the backbone of the CPP (the peptide main chain).
  • the positive charge may be located 1 - 20 atoms further away, preferably 2 - 10 atoms further away, more preferably 3 - 6 atoms further away from the backbone, even more preferably three, four, or six atoms more distant from the backbone. Most preferably, the charge is three or six atoms more distant.
  • Such elongation of the linking moiety between the backbone of the peptide and the positive charge is typically accomplished by condensing at least one, such as 1 - 7, preferably 1 - 5, most preferably 1 - 2 amino acids to the primary amino group.
  • the primary amino group that originates from the side chain of the positively charged amino acid is connected to the carboxylic acid moiety of a further amino acid or to the C-terminus of a small peptide containing 2 - 7, preferably 2 - 5, most preferably 2 amino acids residues, such that an amide bond exists.
  • a dipeptide is condensed to the primary amino acid group.
  • the amine moiety of the further amino acid or the N-terminus of the small peptide than bears the positive charge, such that the positive charge is located more distant from the backbone of the CPP. Condensing one naturally occurring amino acid residue to the cationic primary amino group thus relocates the positive charge three atoms more distant from the backbone of the CPP, while condensation of two naturally occurring amino acid residues leads to a shift of six atoms.
  • the amino acid residues that are condensed in this embodiment are preferably all naturally occurring. Preferably, at most one, more preferably none of these amino acid residues is cationic. Thus, preferably no Arg, Leu or His is used as amino acid residue in this respect.
  • the one or more amino acid residues that are condensed to the primary amino group are selected from the group consisting of Ala, Asp, Leu, He, Val, Gly, Pro, lysine that is acetylated at its ⁇ -amine, and norleucine.
  • one or two amino acid residues selected from Ala, Gly and Pro are condensed to the primary amino group.
  • one alanine residue is condensed to the primary amino group.
  • a GlyPro dipeptide is condensed to the primary amino group.
  • modified Tat peptides according to the invention that are modified in that at least one positively charged amino acid residue has the positive charge at least one atom more distant from the backbone of the CPP are: (3) H-Arg-Lys(Gly)-Lys-Arg- Arg-Gln-Arg-Arg-Arg-OH and (4) H-Arg-Arg-Lys-Arg-Arg-Gln-Arg-Arg-OH.
  • the positive charge of the first lysine residue is three atoms more distant from the backbone of the CPP, because amidation with glycine neutralises the charge on the ⁇ -amine of lysine, yet introduces a new positive charge through glycine's own amine at its N-terminus.
  • the ⁇ -amine and the two carbon atoms of glycine contribute a total of three atoms to the distance of the charge from the backbone.
  • the positive charge of the first lysine residue of the Tat peptide is one atom more distant from the backbone in the modified CPP, because arginine features a side chain that has a length of more atoms than the side chain of lysine.
  • the positive charge may be located any number of atom closer to the peptide backbone as possible.
  • the positively charged amino acid moiety is lysine, having four carbon atoms between the positively charged nitrogen atom and the peptide backbone
  • the positive charge is located 1 - 4 atoms closer to the backbone, preferably 1 - 2 atoms closer.
  • the positive charge is located 1 - 5 atoms closer to the backbone, preferably 1 - 2 atoms closer.
  • histidine the positive charge is equally distributed over both nitrogen atoms of the imidazole ring.
  • the farthest nitrogen atom has three carbon atoms between the positively charged nitrogen atom and the peptide backbone, and the positive charge is thus located 1 - 3 atoms closer to the backbone, preferably 1 - 2 atoms closer.
  • the positive charge is located 1 - 2 atoms closer to the peptide backbone.
  • the positive charge is located 1 atom closer to the peptide backbone.
  • the positive charge is located 2 atoms closer to the peptide backbone.
  • Examples of a modified Tat peptide according to the invention that is modified in that at least one positively charged amino acid residue has the positive charge at least one atom less distant from the backbone of the CPP is: (5) H-Arg-Orn-Lys-Arg-Arg- Gln-Arg-Arg-OH.
  • the positive charge at the first lysine residue of the Tat peptide is one atom closer to the backbone, since ornithine comprises one fewer methylene moieties than lysine.
  • linker moiety represents the linker between the positive charge (the positively charged atom) and the peptide backbone, such as -C4H8- for lysine.
  • linker moiety represents the linker between the positive charge (the positively charged atom) and the peptide backbone, such as -C4H8- for lysine.
  • the number of atoms between the positive charge and the peptide backbone remains the same.
  • the moiety typically a CH 2 moiety, directly adjacent to the positive charge is replaced, is this is expected to have the greatest influence on the positive charge distribution of the CPP.
  • Examples of a modified Tat peptide according to the invention that is modified in that at least one positively charged amino acid residue is connected via a modified linker moiety are:(6): H-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Orn(Gly)-OH and (7): H-Arg- Dap(Gly)-Lys-Arg-Arg-Gln-Arg-Arg-OH.
  • the positive charge of the last amino acid residue is spaced an equal amount of atoms distant from the backbone as in Arg (the naturally occurring amino acid in Tat), but the linker moiety is modified to the one of Orn(Gly).
  • peptide 7 the positive charge of the first lysine residue of Tat is equidistant from the backbone, yet a - CH 2 CH 2 - moiety (the ⁇ and ⁇ carbons in the side chain) is replaced by a -C(0)NH- moiety.
  • the modified CPPs according to the invention are defined as being modified with respect to a template CPP.
  • a "template CPP” refers to a CPP that is known in the art. The modifications as defined above are modifications of these known template CPPs, or in other words differences with respect to these known template CPPs.
  • a modified CPP according to the invention contains thus at least one modified positively charged amino acid compared to a template CPP, which is modified in that positive charge is (a) absent, (b) located at least one atom more distant from the backbone of the CPP, (c) located at least one atom less distant from the backbone of the CPP, or (d) linked to the backbone of the CPP via a modified linking moiety.
  • Preferred template CPPs are discussed above and represented by SEQ ID Nos: 1 - 6. Template CPPs of SEQ ID NO: 5 and 6 are especially preferred and amino acid sequence of SEQ ID NO: 6 is most preferred as template CPP.
  • each of the at least one positively charged amino acid residue that is modified in the modified CPP according to the invention is a lysine or arginine residue.
  • the modified amino acid residues can be referred to as a modified lysine or a modified arginine.
  • the amino acid residue(s) in question in the template CPP are lysine and/or arginine.
  • at least one of the positively charged amino acid residues is lysine, most preferably all are lysine.
  • the modified CPP comprises one or two modified amino acid residues, which are each modified according to one of the modifications (a), (b), (c) and (d) as defined below.
  • the modified CPP differs from a known CPP in that one or two positively charged amino acid residues are modified.
  • the modified CPP comprises one amino acid residue is modified.
  • the modified CPP is loaded with a cargo, preferably wherein the cargo is a detectable label or a pharmaceutically active substance.
  • a detectable label is a preferred cargo.
  • a label is understood to be any moiety that facilitates detection using a method for detection, whereby such a label is a fluorophore, a chromophore, a radioactive tracer, a specific isotope, a diagnostic marker, or a hapten, wherein a hapten is preferably biotin.
  • Preferred labels are defined hereinabove.
  • the compound is radioactively labelled, preferably by having incorporated a radioactively labelled amino acid (e.g. 3 H or 14 C), whereby more preferably the radioactively labelled amino acid is a tritium-labelled amino acid.
  • a label is a diagnostic marker, it may be a fluorogenic substrate to detect the activity of a pathologically relevant protease, for example a caspase involved in the initiation and execution of apoptosis in a cell.
  • detectable labels are substances that are radio labelled or fluorescently or phosphorescently labelled, more preferably the label is a fluorescent label.
  • Fluorescent labels are preferably selected from the group consisting of fluorescein and its derivatives such as FITC or Tokyo green, ASP (preferably 4-(4-(didecylamino)styryl)-N-methylpyridinium iodide), rhodamine, Cy3, Cy5, Atto dyes, Alexa dyes, calcein and IR dyes (e.g. CW800).
  • the cargo does not comprise fluorescein diacetate 5-maleimide.
  • Appropriate labels differ for each individual application and the person skilled in the art is capable of selecting a proper label.
  • An alternative preferred cargo is a pharmaceutically active substance, such as a drug, pro-drug or a drug candidate.
  • a pharmaceutically active substance can be a small molecule, a peptide, a depsipeptide, an acyldepsipeptide, an antibiotic, an antimicrobiotic, a polypeptide, a protein, a protein fragment, an oligooxopiperazine, a nucleic acid, a nucleic acid analogue, or parts thereof, a monosaccharide, oligosaccharide or polysaccharide, a chemotherapeutic, a nanoparticle, a polymeric drug carrier, a dendritic drug carrier, a liposome, a decoy molecule, or any other entity or combination thereof.
  • a nucleic acid can be selected from the group comprising DNA molecules, RNA molecules, PNA molecules, oligonucleotides, siRNA molecules, antisense molecules, ribozymes, aptamers, and spiegelmers.
  • a drug is understood to be any entity that can assert a therapeutic effect, which includes vaccination and diagnosis.
  • the cargo is a selected from the group of anti-cancer agents, cytotoxins, antiviral agents, antibacterials agents, peptides, oligonucleotides and nucleoic acids, in particular siRNA.
  • cytotoxins examples include colchicine, vinca alkaloids, anthracyclines, camptothecins, doxorubicin, daunorubicin, taxanes, calicheamycins, tubulysins, irinotecans, an inhibitory peptide, siRNA, amanitin, deBouganin, duocarmycins, maytansines, auristatins or pyrrolobenzodiazepines (PBDs).
  • anti-cancer agent are present as cargo, such as daunorubicin and doxorubicin.
  • the anti-cancer agent is preferably an anti- leukaemia agent, such as chlorambucil, cyclophosphamide, fludarabine, pentostatin, tretinoin, cladribine, prednisone, vincristine, anthracycline, imatinib, cyclophosphamide, doxorubicin, etoposide and bleomycin. All of these agents are used in the treatment of leukaemia.
  • an anti- leukaemia agent such as chlorambucil, cyclophosphamide, fludarabine, pentostatin, tretinoin, cladribine, prednisone, vincristine, anthracycline, imatinib, cyclophosphamide, doxorubicin, etoposide and bleomycin. All of these agents are used in the treatment of leukaemia.
  • a peptide as described above wherein each of the at least one positively charged amino acid residue that is modified is lysine and/or arginine.
  • the modified amino acid residues can be referred to as a modified lysine and/or a modified arginine.
  • the amino acid residue(s) in question in the template CPP are lysine and/or arginine.
  • at least one of the positively charged amino acid residues is lysine, most preferably all are lysine.
  • a peptide as described above is provided, wherein the modified CPP comprises one or two modified amino acid residues, which are each modified according to one of the modifications (a), (b), (c) and (d).
  • the modified CPP differs from a known CPP in that one or two positively charged amino acid residues are modified.
  • the modified CPP comprises one amino acid residue is modified.
  • the modified CPP comprises two amino acid residues are modified.
  • a cell penetrating peptide as described above is provided, which is represented by general formula (I):
  • each Y is individually selected from NH, N(Ci_4)alkyl and O, preferably each Y is NH;
  • R N is the N-terminal end of the CPP containing at least one N-terminal amino acid residue
  • R c is the C-terminal end of the CPP containing at least three C-terminal amino acid residues
  • backbone of the CPP comprises at least eight amino acids
  • R 1 is a first amino acid side chain represented by -L 1 -N + (R 3 ) 3 , wherein each R 3 is individually selected from H and (Ci-4)alkyl and L 1 is a linker containing 1 - 20 optionally substituted backbone atoms selected from C, N and O; and
  • R 2 is a second amino acid side chain represented by -L 2 -N + (R 4 ) 3 , wherein each R 4 is individually selected from H and (Ci-4)alkyl and L 2 is a linker containing 1 - 20 optionally substituted backbone atoms selected from C, N, O and S.
  • R 1 and R 2 are not -(CH 2 )4N + H 3 , i.e. not a lysine side-chain.
  • R 1 is not -(CH 2 ) 4 N + H 3 and R 2 is or is not -(CH 2 ) 4 N + H 3 .
  • both R 1 and R 2 are not -(CH 2 ) 4 N + H 3 .
  • Alkyl groups as recited herein may be linear or branched, saturated or unsaturated, cyclic or linear. Preferably, they are linear or branched, saturated and linear. Examples of alkyl groups include methyl, ethyl, propyl, 2-propyl and t-butyl.
  • both R 1 and R 2 contain a cationic moiety. It is however also encompassed by the present invention that the neutral amine moieties are provided, which are in situ, converted to cationic moieties.
  • - N + (R 4 ) 3 can also be represented by -N(R 4 ) 2 , wherein the at least one R 4 that represents H is absent; this applies to R 3 mutatis mutandis.
  • the cell penetrating peptide of general formula (I) is preferably loaded with a cargo as further specified above.
  • each R 3 is individually H, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl, more preferably each R 3 is individually H or methyl. It is preferred that all instances of R 3 are the same, most preferably all instances of R 3 are H.
  • each R 4 is individually H, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl, more preferably individually R 4 is individually H or methyl. It is preferred that all instances of R 4 are the same, most preferably all instances of R 4 are H.
  • Linkers L 1 and L 2 contain a backbone, i.e. the shortest chain of atoms between the a-carbon of the amino acid and the cationic nitrogen atom.
  • the backbones of L 1 and L 2 contain 1 - 20 backbone atoms that define this shortest chain.
  • Each of these backbone atoms is selected from C, N, O and S, and may optionally be substituted.
  • the backbone atoms are selected from C, N and O, more preferably from C and N. In one embodiment, all backbone are C.
  • the number of backbone heteroatoms is 0 - 2, preferably 0 or 1.
  • Optional substitutions are typically selected from (Ci-i 2 )-alkyl groups, (C 2 -i 2 )-alkenyl groups, (C 2 -i 2 )-alkynyl groups, (C 3 -i 2 )-cycloalkyl groups, (C5-12)- cycloalkenyl groups, (C8-i 2 )-cycloalkynyl groups, (Ci-i 2 )-alkoxy groups, (C2-12)- alkenyloxy groups, (C 2 -i 2 )-alkynyloxy groups, (C 3 _i 2 )-cycloalkyloxy groups, halogens, amino groups, oxo and silyl groups, wherein the silyl groups can be represented by the formula (R 2 ) 3 Si-, wherein R 2 is independently selected from the group consisting of (Ci_ i 2 )-groups, (C 2 -i 2 )-alkenyl groups, (C 2 -
  • the alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, alkoxy groups, alkenyloxy groups, alkynyloxy groups and cycloalkyloxy groups are optionally substituted, the alkyl groups, the alkoxy groups, the cycloalkyl groups and the cycloalkoxy groups being optionally interrupted by one of more hetero-atoms selected from the group consisting of O, N and S.
  • Preferred optional substitutions include halogen, an hydroxyl moiety, an amino moiety, a methoxy moiety, an azide an oxo moiety, a thiol moiety, or a thioxo moiety.
  • L 1 and L 2 linkers are -(CH 2 )n-NH-C(0)-(CH 2 ) m -, -(CH 2 )n-C(0)-N-(CH 2 ) m -, and -(CH 2 ) 0 - wherein n and m are integers from 0 to 18, provided that the n + m is in the range of 0 - 18, and o is an integer from 1 to 20.
  • Integers n, m and o are preferably each individually in the range of 1 - 10, more preferably 1 - 5. Most preferably, m is 1 or 2. Most preferably, n is 4. Most preferably, o is 4.
  • L 1 and L 2 linkers are -(CH 2 ) 5 -, - (CH 2 ) 4 - -(CH 2 ) 3 - -(CH 2 ) 2 -,-(CH 2 )-, -(CH 2 ) 4 -NH-C(0)-CHR 4 - and -(CH 2 ) 4 -NH- C(0)-CHR 4 -NH-C(0)-CHR 4 - (wherein each R 4 is individually H or an optional substituent as defined above).
  • a most preferred L 1 linker is -(CH 2 ) 4 -.
  • a most preferred L 2 linker is -(CH 2 ) 4 -.
  • the cell penetrating peptide according to the invention is represented by general formula (I) as described above, wherein R N is X n Arg- and R c is -ArgArgGlnArgArgArgX m , wherein each X is individually an amino acid and n and m are each individually integers between 0 and 10.
  • each X is individually a naturally occurring amino acid and 0 - (n + m), preferably 0 - 2, of the occurrences of X is an amino acid that may function as linker, such as Ahx.
  • This linker may further be conjugated to a cargo.
  • R N is preferably cargo-ahx-Gly-Arg-, wherein the cargo is as defined above.
  • R N is FITC-ahx-Gly-Arg-.
  • R c is -Arg-Arg-Gln-Arg- Arg-Arg-ahx-Cys-NH 2 .
  • R c is -Arg-Arg-Gln-Arg-Arg-NH 2 .
  • X n is not Gly.
  • X m is not ahx-Cys-cargo.
  • the cell penetrating peptide according to the invention is represented by general formula (I) as described above, wherein at least one of R 1 and R 2 is -(CH 2 ) 4 NHR 5 , wherein R 5 is a peptide containing 1 - 5 amino acid residues.
  • the peptide is thus condensed to the lysine side-chain(s) of the cell penetrating peptide according to the invention.
  • R 5 is a peptide that is connected to the NH in (CH 2 ) 4 NHR 5 through an amide bond involving the C-terminus of the peptide R 5 .
  • R 5 is a peptide the C-terminus of which is conjugated to the primary amino moiety of a lysine residue.
  • the N-terminus of peptide R 5 forms the - N + (R 3 ) 3 and/or the N + (R 4 ) 3 moiety as present in R 1 and R 2 respectively.
  • R 5 is selected from Ala-, or GlyPro-, or Phe- or H- Ala-, wherein the n- terminus is as defined above.
  • R 5 is Ala- or GlyPro-.
  • R 1 is -(CH 2 ) 4 NHR 5 and R 2 is not, preferably R 2 is -(CH 2 ) 4 NH 3 + .
  • R 2 is -(CH 2 ) 4 NHR 5 and R 1 is not, preferably R 1 is -(CH 2 ) 4 NH 3 + . In one embodiment, R 1 and R 2 are both -(CH 2 ) 4 NHR 5 .
  • the modified cell penetrating peptide according to the invention comprises the sequence of SEQ ID NO: 27, 28 or 29, preferably of SEQ ID NO: 28 or 29, most preferably of SEQ ID NO: 29, wherein at least one of X 3 and X 4 is not lysine (K), preferably not lysine (K) or arginine (R), most preferably at least one of X 3 and X 4 is a lysine residue that is modified according to any one of modifications (a), (b), (c) or (d) as defined above.
  • the sequence of SEQ ID NO: 27, 28 or 29 can be incorporated in a larger peptide.
  • such a larger peptide is preferably represented by X tractX 1 X 2 RX 3 X 4 RRX 5 RRRX 6 X 7 X m , wherein each of X 1 , X 2 , X 3 , X 4 , X 5 , X 6 and X 7 are as defined above for SEQ ID NO: 27, and each X is individually an amino acid residue and n and m are each individually integers in the range of 0 - 10, preferably in the range of 0 - 3.
  • such a larger peptide is preferably represented by X encompassRX 3 X 4 RRX 5 RRRX m , wherein each of X 3 , X 4 and X 5 are as defined above for SEQ ID NO: 28, and each X is individually an amino acid residue and n and m are each individually integers in the range of 0 - 10, preferably in the range of 0 - 3.
  • such a larger peptide is preferably represented by XnRX 3 X 4 RRQRRRXm, wherein each of X 3 and X 4 are as defined above for SEQ ID NO: 29, and each X is individually an amino acid residue and n and m are each individually integers in the range of 0 - 10, preferably in the range of 0 - 3.
  • the modified cell penetrating peptide according to the invention consist of the sequence of SEQ ID NO: 27, 28 or 29, preferably of SEQ ID NO: 28 or 29, most preferably of SEQ ID NO: 29, meaning that no amino acids are present at the C- or N-termini of the sequences of SEQ ID NO: 27, 28 or 29, but other moieties such as capping groups as known in the art may be present.
  • RX 3 X 4 RRQRRR when X 3 X 4 is K , the sequence is RK RRQRRR, which is a sequence that can be referred to as Tat (SEQ ID NO: 6).
  • Each of X 3 and X 4 is preferably selected from at most one lysine and at least one modified lysine, wherein the modified lysine is modified according to any of the modifications (a) - (d) as defined above, preferably by modification (b).
  • Preferred modified CPPs according to the invention comprise a sequence selected from the group consisting of: Tat-A(l l) Arg-Lys(H-Ala)-Lys(H-Ala)-Arg-Arg-Gln-Arg-Arg-Arg Tat-A(Ol) Arg-Lys-Lys(H-Ala)-Arg-Arg-Gln-Arg-Arg-Arg-Arg
  • Tat- AA(01 ) Arg-Lys-Lys(H- Ala- Ala)- Arg- Arg-Gln- Arg- Arg- Arg
  • Tat-G( 10) Arg-Lys(H-Gly)-Lys- Arg- Arg-Gln- Arg- Arg- Arg- Arg
  • Tat-G(01 ) Arg-Lys-Lys(H-Gly)- Arg- Arg-Gln- Arg- Arg- Arg- Arg
  • Tat- V(01 ) Arg-Lys(H- Val)-Lys- Arg- Arg-Gln- Arg- Arg- Arg
  • Tat- V( 10) Arg-Lys-Lys(H-Val)- Arg- Arg-Gln- Arg- Arg- Arg
  • Tat- VFmoc(01 ) Arg-Lys(H-Val-Fmoc)-Lys- Arg- Arg-Gln- Arg- Arg- Arg
  • Tat-G AB A( 10) Arg-Lys(H-G AB A)-Lys- Arg- Arg-Gln- Arg- Arg- Arg- Arg
  • Tat-[20,3K] Arg-Orn-Lys- Arg- Arg-Gln- Arg- Arg- Arg
  • Tat-[2,30] Arg-Orn-Orn-Arg- Arg-Gln- Arg- Arg- Arg
  • Tat-[2K,30G] Arg-Lys-Orn(H-Gly)-Arg- Arg-Gln- Arg- Arg- Arg
  • Tat-[20A,3K] Arg-Orn(H-Ala)-Lys- Arg- Arg-Gln- Arg- Arg- Arg Tat-[2 ,30A] : Arg-Lys-Orn(H-Ala)- Arg-Arg-Gln- Arg-Arg-Arg-Arg
  • Tat-[2,30A] Arg-Orn(H-Ala)-Orn(H-Ala)-Arg-Arg-Gln- Arg-Arg-Arg
  • Tat-[20bA,3K] Arg-Orn(H-pAla)-Lys- Arg-Arg-Gln- Arg-Arg-Arg
  • Tat-[2 ,30bA] Arg-Lys-Orn(H-p Ala)-Arg- Arg-Gln- Arg-Arg-Arg
  • Tat-[2Db,3K] Arg-Dab-Lys- Arg-Arg-Gln- Arg-Arg-Arg
  • Tat-[2K,3Db] Arg-Lys-Dab- Arg-Arg-Gln- Arg-Arg-Arg
  • Tat-[2,3Db] Arg-Dab-Dab- Arg-Arg-Gln- Arg-Arg-Arg
  • Tat-[2DbA,3K] Arg-Dab(H-Ala)-Lys- Arg-Arg-Gln- Arg-Arg-Arg
  • Tat-[2K,3DbA] Arg-Lys-Dab(H-Ala)-Arg-Arg-Gln- Arg-Arg-Arg
  • Tat-[2DbAA,3 ] Arg-Dab(H-Ala-Ala)-Lys- Arg-Arg-Gln- Arg-Arg-Arg
  • Tat-[2K,3DbAA] Arg-Lys-Dab(H- Ala- Ala)-Arg-Arg-Gln- Arg-Arg-Arg
  • Tat-[2DbGABA,3 ] Arg-Dab(H-GABA)-Lys- Arg-Arg-Gln- Arg-Arg-Arg
  • Tat-[2K,3DbGABA] Arg-Lys-Dab(H-GABA)- Arg-Arg-Gln- Arg-Arg-Arg
  • Tat-[2Dp,3K] Arg-Orn-Lys- Arg-Arg-Gln- Arg-Arg-Arg
  • Tat-[2K,3Dp] Arg-Lys-Orn- Arg-Arg-Gln- Arg-Arg-Arg
  • Tat-[2,3Dp] Arg-Orn-Orn- Arg-Arg-Gln- Arg-Arg-Arg
  • Tat-[2K,3DpG] Arg-Lys-Dap(H-Gly)-Arg-Arg-Gln- Arg-Arg-Arg
  • Tat-[2,3DpG] Arg-Dap(H-Gly-Dap(H-Gly)- Arg-Arg-Gln- Arg-Arg-Arg-Arg
  • Tat-[2DpA,3K] Arg-Dap(H-Ala)-Lys- Arg-Arg-Gln- Arg-Arg-Arg
  • Tat-[2K,3DpA] Arg-Lys-Dap(H-Ala)-Arg-Arg-Gln- Arg-Arg-Arg
  • Tat-[2,3DpA] Arg-Dap(H-Ala-Dap(H-Ala)-Arg-Arg-Gln- Arg-Arg-Arg
  • Tat-[2DpbA,3K] Arg-Dap(H-pAla)-Lys- Arg-Arg-Gln- Arg-Arg-Arg
  • GABA is gamma-aminobutyric acid.
  • Tat-A(Ol), Tat-A(lO), Tat-A(l l), Tat-GP(Ol), Tat-GP(lO), Tat- GP(l l), Tat-G(Ol), Tat-G(lO) and Tat-G(l l) are most preferred.
  • the modified CPPs according to the invention do not contain any further amino acid residues in addition to those mentioned in the sequences listed directly above.
  • the modified CPPs according to the invention consist of the sequences listed directly above.
  • the cell penetrating peptide according to the invention is not
  • the cell penetrating peptide according to the invention does not comprise the sequence of Tat-A(Ol), Tat-A(lO), Tat-A(l l), Tat-GP(Ol), Tat-GP(lO) or Tat- GP(l l).
  • a composition comprising a cell penetrating peptide according to the invention, and a pharmaceutically acceptable carrier or excipient.
  • this composition consists of said cell penetrating peptide and one or more pharmaceutically acceptable carriers or excipients.
  • Such compositions are suitable for use as a medicament, in particular in the treatment, prevention, delay, diagnosis, or detection of a disorder, preferably wherein the disorder is cancer.
  • a preferred cancer is leukaemia.
  • Such compositions are also suitably used in the methods and uses according to the invention.
  • the cell penetrating peptide according to the invention is present in an effective dose.
  • Effective dose refers to the dose of a compound or composition that can assert a desired effect, such as improving a symptom of a disorder, or changing a parameter associated with a disorder. Such a dose may also be referred to as a therapeutically effective dose or effective amount. More specifically, a therapeutically effective amount refers to an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is within the capability of those skilled in the art.
  • 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. 1 p. 1).
  • the amount of compound or composition administered will, of course, depend on many factors, as appreciated by the skilled person, including the subject being treated, the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
  • composition that comprises a cell penetrating peptide according to the invention, a pharmaceutically acceptable carrier, and that further comprises a further therapeutic compound.
  • the composition is for use in the treatment, delay, prevention, cure or stabilization of a disorder, preferably cancer, of a subject in need thereof, said use comprising administration to the subject of an effective dose of the composition.
  • a pharmaceutical composition that comprises a cell penetrating peptide according to the invention in combination with a further therapeutic compound can be supplied such that the compound and one or more of the composition components, and the further therapeutic compound are in the same container, e.g. in solution, in suspension, or in powder form.
  • the cell penetrating peptide according to the invention can also be provided separately from one or more of the further components of the composition according to the invention, such as the further therapeutic compound.
  • the cell penetrating peptide is mixed with the separate component(s) prior to administration.
  • a combination therapy wherein a composition according the invention is administered together with an effective dose of a further therapeutic compound or composition.
  • a composition according to this invention is administered simultaneously with the administration of a further therapeutic compound or composition, which is referred to as simultaneous administration.
  • the administration of a further therapeutic compound or composition can take place either before or after the administration of the composition according to this invention, which is referred to as separate administration.
  • Separate administration may involve administration events that are separated by a minimal amount of e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, or more minutes, or by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more hours, or by 1, 2, 3, 4, 5, or more days, or by a week or by a month or longer.
  • Non-limiting preferred examples of preferred further therapeutic compounds or compositions include Actinomycin, All-trans retinoic acid, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, Oxaliplatin, Paclitaxel (Taxol), Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, or Vinorelbine, or their peptid
  • the compositions may, if desired, be presented in a pack having more than one chamber, and in which a barrier can be ruptured, ripped, or melted to provide mixing of the compound or composition according to the invention with the further therapeutic compound.
  • two separately provided elements can be mixed in a separate container, optionally with the addition to one or more other carriers, solutions, etc.
  • One or more unit dosage forms containing the further therapeutic compound can be provided in a pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • compositions comprising a cell penetrating peptide according to the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labelled for treatment of an indicated condition.
  • Suitable conditions indicated on the label may include any disease which may be treated or prevented or diagnosed using the compositions according to the invention.
  • the invention is ideally suited for cancer therapy or chemotherapy or cancer diagnostics.
  • compositions and pharmaceutical compositions according to the invention may be manufactured by processes well known in the art; e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes, which may result in liposomal formulations, coacervates, oil-in-water emulsions, nanoparticulate/microparticulate powders, or any other shape or form.
  • Compositions for use in accordance with the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Preferred modes of formulation is dependent on the route of administration chosen.
  • the compound or composition according to the invention may for example be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. As such, particular organs, tissues, tumor sites, sites of inflammation, etc, are more efficiently targetted.
  • Formulations for infection may be presented in unit dosage form, e.g., in ampoules or in multi-dose container, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions or pharmaceutical compositions for parenteral administration include aqueous solutions of the compositions in water soluble form. Additionally, suspensions of the compositions may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compositions to allow for the preparation of highly concentrated solutions.
  • a cell penetrating peptide according to the invention for use as a medicament is provided.
  • a composition comprising the cell penetrating peptide according to the invention for use as a medicament is provided.
  • These aspects of the invention may also be worded as the use of a cell penetrating peptide according to the invention or a composition comprising the same for the manufacture of a medicament for use in the treatment or prevention of a disorder.
  • These aspects of the invention may also be worded as a method for the treatment or prevention of a disorder, comprising the administration of a cell penetrating peptide according to the invention or a composition comprising the same to a subject in need thereof.
  • a cell penetrating peptide according to the invention for use as a medicament for the treatment, prevention, delay, diagnosis, or detection of cancer is provided.
  • a composition comprising the cell penetrating peptide according to the invention for use as a medicament for the treatment, prevention, delay, diagnosis, or detection of cancer is provided.
  • aspects of the invention may also be worded as a method for the treatment, prevention, delay, diagnosis, or detection of cancer, comprising the administration of a cell penetrating peptide according to the invention or a composition comprising the same to a subject in need thereof.
  • said cancer is preferably leukaemia.
  • the cell penetrating peptide according to the invention can also be for the curing or stabilizing of a condition.
  • a disorder can be any type of disorder with which an aberrant cell is associated and/or in which an aberrant cell might hinder treatment.
  • a preferred disorder is cancer.
  • a cell penetrating peptide is provided as defined above, for use in the treatment, delay, prevention, cure, detection, diagnosis, or stabilization of a cancer of a subject in need thereof, comprising administration to the subject of an effective dose of the cell penetrating peptide.
  • the treatment of a cancer may also be the inhibition of tumour cell proliferation, the induction or increased induction of tumour cell death, the prevention or delay of the occurrence of metastases, the prevention or delay of tumour cell migration, an inhibition or prevention or delay of the increase of a tumour weight or growth, and/or a prolongation of patient survival of at least one month, several months or more (compared to those not treated or treated with a control or compared with the subject at the onset of the treatment) and/or improvement of the quality of life and observed pain relief.
  • inhibition of the proliferation of tumour cells is preferably at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%), or more.
  • Proliferation of cells may be assessed using known techniques, such as FACS or resazurin assays or by MRI, PET, SPECT, or CT, or by otherwise determining changes in tumour volume or metabolic activity.
  • the proliferation and the status of tumour cells may be assessed through biopsies and (immuno-)histological characterization of the tumour and its surrounding tissue.
  • An induction of tumour cell death may be at least 1%, 5%, 10%>, 15%, 20%>, 25%, or 25 more.
  • Tumour growth may be inhibited at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more.
  • Tumour cell death may be assessed using techniques known to the skilled person. Tumour cell death may be assessed using MRI, PET, SPECT, or CT, or by otherwise determining changes in tumour volume or metabolic activity. The death or decrease in activity of tumour cells may be assessed through biopsies and (immuno-) histological characterization of the tumour and its surrounding tissue.
  • tumour weight increase or tumour growth may be inhibited at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. Tumour weight or tumour growth may be assessed using techniques known to the skilled person.
  • tumour growth or the detection of the proliferation of tumour cells may be assessed in vivo by measuring changes in glucose utilization by positron emission tomography with the glucose analogue 2-[ 18 F]-fluor-2-deoxy-D-glucose (FDG-PET) or [ 18 F]-'3-fluoro-'3- deoxy-L-thymidine PET.
  • FDG-PET glucose analogue 2-[ 18 F]-fluor-2-deoxy-D-glucose
  • An ex vivo alternative may be staining of a tumour biopsy with Ki67.
  • a subject can be any living entity.
  • the subject is a mammal, more preferably a human.
  • a cell penetrating peptide according to the invention can be achieved by any method known in the art, as further defined later in this text.
  • the cell penetrating peptide according to the invention are for the use in a method according to this invention, or for a use according to the invention.
  • the use of a cell penetrating peptide according to the invention or of a composition according to the invention is provided, wherein said use is for the in vitro or in vivo targeting of a cell of interest by contacting the cell of interest with the cell penetrating peptide or the composition.
  • This aspect may also be worded as the method for targeting a cell of interest, wherein the cell penetrating peptide according to the invention or of a composition according to the invention is contacted with a sample or subject comprising the cell of interest.
  • the use or method may be in vitro, ex vivo or in vivo. In a preferred embodiment of this aspect, the use or method is in vitro or ex vivo.
  • a cell of interest can be comprised in a sample or in a subject.
  • a sample or a subject can comprise multiple cells, and often contains additional cells in addition to the cell of interest.
  • the cell of interest forms a subpopulation within a larger population of the sample or subject.
  • the cell penetrating peptide or composition according to the invention may thus be used to selectively target a cell of interest within a larger population of cells comprised in a sample or subject.
  • at least one other cell than said cell of interest is present in the sample.
  • the cell penetrating peptide according to the invention is loaded with a cargo as further defined above.
  • this substance of interest is not fluorescein diacetate 5-maleimide.
  • the use or method for targeting a cell of interest is for the detection of the cell of interest.
  • detection is preferably used in a diagnostic application, wherein a diagnosis is typically made when the cell of interest is detected, or, more exceptionally, not detected.
  • a diagnosis can be made when the cell of interest is not detected.
  • the use or method is for the aid in diagnosis, or to assist in diagnosis, and not for diagnosis itself.
  • the diagnosis is of a human subject. In one embodiment, the diagnosis is not of a human subject. In a preferred embodiment, the diagnosis is executed in vitro or ex vivo. In this embodiment, it is preferred that the cell penetrating peptide according to the invention is loaded with a cargo being a detectable label as defined hereinabove.
  • the use or method for targeting a cell of interest is for treatment.
  • the treatment is of a human subject.
  • the treatment is not of a human subject.
  • treatment refers to the treatment of a subject that suffers from a condition.
  • the treatment is the treatment of cancer, most preferably of leukaemia.
  • the cell penetrating peptide according to the invention is loaded with a cargo being a pharmaceutically active substance as defined hereinabove, preferably an anti-cancer agent.
  • Preferred modified cell penetrating peptide according to the invention to be used in the treatment, prevention, delay, diagnosis, or detection of cancer have been identified by the inventors, and are represented by SEQ ID NO: 7, which is RXXRRQRRR (Arg- Xaa-Xaa-Arg-Arg-Gln-Arg-Arg-Arg), wherein at least one of X is not lysine (K).
  • RXXRRQRRR Arg- Xaa-Xaa-Arg-Arg-Gln-Arg-Arg-Arg
  • Each occurrence of X is preferably selected from at most one lysine and at least one modified lysine, wherein the modified lysine is modified according to any of the modifications (a) - (d) as defined above, preferably by modification (b).
  • CPPs of SEQ ID NO: 7 are further defined below, and preferred candidates are Tat-A(l l), Tat-A(Ol), Tat-A(lO), Tat- GP(l l), Tat-GP(Ol), Tat-GP(lO), Tat-GP(lO), Tat-G(l l), Tat-G(lO), Tat-G(Ol), Tat-bA(l l), Tat- bA(10), Tat-bA(Ol), Tat-ObA(l l), Tat-ObA(lO), Tat-ObA(Ol), Tat-OA(l l), Tat- OA(10), Tat-OA(Ol), Tat-F(l l), Tat-F(lO), Tat-F(Ol), Tat-DpbA(l 1), Tat-DpbA(lO), Tat-DpbA(Ol), Tat-DbbA(l 1), Tat-DbbA(lO), Tat-DbbA(Ol), Tat-Dp
  • Tat-A(Ol), Tat-A(lO), Tat-A(l l), Tat-GP(Ol), Tat-GP(lO), Tat-GP(l l), Tat-G(Ol), Tat-G(lO) and Tat-G(l 1) are most preferred.
  • cancer includes any type of cancer, such as cancer of epithelial origin or neuronal origin or a carcinoma or a solid tumour or a sarcoma or a liquid tumour such a leukaemia or a lymphoma.
  • Cancer may be from the bladder; brain; breast; colon; esophagus; gastrointestine; head; kidney; liver; lung; nasopharynx; neck; ovary; prostate; skin; stomach; testis; tongue; neuron or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm; malignant; carcinoma; carcinoma undifferentiated; giant and spindle cell carcinoma; small cell cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma; malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma; familial polyposis coli; solid carcinoma; carcinoid tumour; malignant; branchiolo-alveolar carcinoma; papillary carcinoma; squamous cell carcinoma; basal adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic a
  • a leukaemia includes any of: Acute lymphoblastic leukaemia (ALL) such as precursor B acute lymphoblastic leukaemia, precursor T acute lymphoblastic leukaemia, Burkitt's leukaemia, and acute biphenotypic leukaemia, Chronic lymphocytic leukaemia (CLL) such as B-cell prolymphocytic leukaemia, a more aggressive disease, Acute myelogenous leukaemia (AML) such as acute promyelocytic leukaemia, acute myeloblastic leukaemia, and acute megakaryoblastic leukaemia, Chronic myelogenous leukaemia (CML) such as chronic monocytic leukaemia, Hairy cell leukaemia (HCL), Tcell prolymphocytic leukaemia (T-PLL), Large granular lymphocytic leukaemia and Adult T-cell leukaemia.
  • ALL Acute lymphoblastic leukaemia
  • X 5 Gin (Q) or Arg (R), and at least one of X 3 and X 4 is not Lys (K);
  • At least one of X 3 and X 4 is not Lys (K).
  • Tat-a(01 ) Arg-Lys-Lys(H- Ala*)-Arg- Arg-Gln- Arg- Arg-Arg-Arg
  • Tat- A(01 ) Arg-Lys-Lys(H- Ala)- Arg- Arg-Gln- Arg- Arg- Arg
  • Tat-GP(l 1) Arg-Lys(H-Gly-Pro)-Lys(H-Gly-Pro)- Arg- Arg-Gln- Arg- Arg- Arg- Arg
  • Tat-GP(01 ) Arg-Lys-Lys(H-Gly-Pro)- Arg- Arg-Gln- Arg- Arg- Arg
  • All amino acids herein are L-amino acids, except for Ala*, which refers to D-alanine. All peptides bear fluorescein isothiocyanate (FITC) as cargo for easy detection.
  • FITC fluorescein isothiocyanate
  • the Peptides were synthesized and purified as described by Bode et al. (Bioconj Chem, 2015). Tat-a(Ol) was synthesized using the same procedure as for Tat-A(Ol) described therein.
  • HEK 293, HeLa-CCL-2 and mCherry HeLa cells were maintained in sterile conditions in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells were maintained on tissue culture plastic and kept at 37 °C in a humidified atmosphere of 7.5% C0 2 . Cells were passaged every 2-3 days. Prior to cellular uptake studies, cells within a confluent layer were detached using trypsin/EDTA. Cells were then resuspended in FBS-supplemented DMEM and the number of cells was counted using a standard inverted microscope and a cell counting chamber (Fuchs-Rosenthal).
  • DMEM Dulbecco's Modified Eagle's medium
  • FBS heat-inactivated fetal bovine serum
  • HL-60, THP-1, KG la, BV-173 and REH cells were maintained in sterile conditions in RPMI medium 1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS).
  • FBS heat-inactivated fetal bovine serum
  • Cells were maintained on tissue culture plastic and kept at 37 °C in a humidified atmosphere of 5% C0 2 . Cells were passaged every 2- 3 days. Prior to cellular uptake experiments, cells were spun down for 5 minutes at 1500 rpm, thoroughly washed with cell culture medium and the number of cells was counted using a standard inverted microscope and a cell counting chamber (Fuchs-Rosenthal).
  • HEK or HeLa-CCL-2 cells were seeded in 8-well chambered coverslips (Nunc, Wiesbaden, Germany) at a density of 40,000 cells (one day) or 20,000 cells per well (two days prior) to the experiment. Cells were incubated with the 5 ⁇ peptide for 30 min at 37 °C. Cells were washed twice after incubation with DMEM + 10 % FCS and living cells were analysed immediately by confocal microscopy using a TCS SP2 confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with an HCX PL APO 63 x N. A. 1.2 oil immersion lens.
  • a mixture of 20,000 HEK and 20,000 mCherry HeLa cells were seeded one day prior to the experiment in each well of the above mentioned chambered coverslips.
  • the protocol for cellular uptake was than performed the same as for the HEK cells.
  • HEK or HeLa cells were seeded in 24-well plates (Sarstedt, Numbrecht, Germany) one (60,000 cells/well) or two (30,000 cells/well) days prior to the experiment. On the day of the experiment, cells were incubated with the peptide solutions (5 ⁇ ) for 30 min at 37 °C in RPMI + 10% FBS. After washing the cells with HBS buffer pH 7.4 (10 mM HEPES, 135 mM NaCl, 5 mM KC1, 5 mM MgCl 2 , 1.8 mM CaCl 2 ), cells were detached by trypsinisation for 5 minutes, spun down and resuspended in 200 RPMI + 10% FCS.
  • the cells were spun down again and resuspended in HBS buffer supplemented with 0.1 %> bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the fluorescence was measured using a FACSCalibur flow cytometer (BD Biosciences, Erembodegem, Belgium) and subsequently data was analysed with the Summit software (Fort Collins, U.S.A.). Results were based on 10,000 gated cells.
  • a mixture of 30,000 HEK cells and 30,000 mCherry HeLa cells were seeded in each well of a 24-well plate (Sarstedt, Numbrecht, Germany) one day prior to the uptake experiments.
  • the experimental procedure was the same as described above for HEK or HeLa cells.
  • the fluorescence was measured using a Cyan flow cytometer (Beckman Coulter Inc.) and the data was analyzed using the Summit Software. Results were based on 10,000 gated cells.
  • Flow cytometry experiments with normal WBCs and RBCs peripheral blood was drawn from a healthy individual on the day of the experiment. Subsequently, the RBCs were lysed from the blood using erythrocyte lysis buffer pH 7.4 (75 mM NH 4 C1; 5 mM KHC0 3 ; 0.05 mM EDTA-4Na) and incubation for 10 minutes at RT. Subsequently, the samples were spun down for 10 minutes at 1500 rpm and afterwards the supernatant was discarded. The pellet was washed with PBS twice, and afterwards the amount of WBCs was counted.
  • erythrocyte lysis buffer pH 7.4 75 mM NH 4 C1; 5 mM KHC0 3 ; 0.05 mM EDTA-4Na
  • the cells were resuspended in 10% FBS-containing IMDM so that there were 150,000 cells per sample. Subsequently, the cells were incubated with the peptide solutions (5 ⁇ ) for 30 min at 37 °C in RPMI + 10% FBS. After washing the cells with PBS buffer, the cells were treated with trypsin/EDTA for 5 minutes to remove membrane-bound peptide, spun down and resuspended in 200 IMDM + 10%> FCS. Afterwards, the cells were spun down again and resuspended in PBS buffer supplemented with 0.1 % bovine serum albumin (BSA). The fluorescence was measured using an FC500 flow cytometer (Beckman Coulter Inc.
  • HL-60, THP-1, KG la, BV-173 or REH cells were washed twice in 10% FCS-containing RPMI, counted and distributed in 24-well plates (Sarstedt, Numbrecht, Germany) so that there were 150,000 cells per well.
  • the cells were incubated with the peptide solutions (5 ⁇ ) for 30 min at 37 °C in RPMI + 10% FBS. After washing the cells with PBS buffer, the cells were treated with trypsin/EDTA for 5 minutes to remove membrane-bound peptide, spun down and resuspended in 200 ⁇ , IMDM + 10%> FCS.
  • the cells were spun down again and resuspended in PBS buffer supplemented with 0.1%> bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the fluorescence was measured using an FC500 flow cytometer (Beckman Coulter Inc. Miami) and the data was analyzed using the Kaluza software (Beckmam Coulter). Results were based on 10,000 gated cells.
  • the uptake of Tat and the modified Tat peptides was studied in HEK, obtained from healthy tissue, and tumorous HeLa cells.
  • the uptake of the peptides was visualized using confocal laser scanning microscopy (CLSM) and quantified by flow cytometry.
  • CLSM confocal laser scanning microscopy
  • adherent cells were treated with 5 ⁇ peptide in cell culture medium containing 10% fetal calf serum (FCS) (v/v) and incubated for 30 minutes at 37 °C. After this incubation period, the cells were washed and analysed by CLSM immediately.
  • FCS fetal calf serum
  • Tat-a(Ol) was synthesized, which is the same peptide as Tat-A(Ol) only the Ala residue is replaced by D-Ala, to hamper enzymatic cleavage of the alanine residue from the lysine side-chain.
  • Tat-a(Ol) behaved similarly as Tat-A(Ol) in both HeLa and HEK cells, demonstrating that the preferential uptake by HeLa cells is not caused by an enzymatic trigger.
  • co-culture experiments with HEK and HeLa cells were performed. To be able to differentiate between the two cell lines using CLSM and flow cytometry use was made of HeLa cells with mCherry labeled nuclei. HEK and mCherry HeLa cells were seeded in a 1 : 1 ratio one day prior to the experiment.
  • the modified Tat-peptides according to the invention do not show uptake selectivity caused by differences in micro-environment, as even in samples wherein both HEK and HeLa cells are present, the modified Tats are taken up by the HeLa cells only.
  • WBCs White blood cells
  • Erythrocytes red blood cells, RBCs
  • RBCs red blood cells
  • the remaining WBCs were incubated with 5 ⁇ peptide in FCS-containing cell culture medium for 30 minutes. Afterwards, a short trypsin treatment was performed to remove any membrane-bound peptide. Uptake by Tat and the modified Tat peptides according to the invention was analysed by flow cytometry.
  • Leukemic cells were obtained from three cell lines derived from patients with acute myeloid leukemia (AML) and two cell lines derived from patients with B-cell lymphocytic leukemia (B-ALL).
  • the AML cell lines correspond with different stages of the disease, with HL60 cells being the most immature cells, followed by THP-1 cells and KG la cells.
  • the experiments were conducted by incubating the selected cells with 5 ⁇ peptide in FCS-containing cell culture medium for 30 minutes. Membrane-bound peptide was subsequently removed by a short trypsin treatment. Analysis of uptake was performed using flow cytometry. For all cell lines, the uptake of Tat was used as positive control and set to 100%.
  • the results for the AML cells are depicted in Table 5.
  • Tat-A(l l) peptide was taken up almost equally well as Tat itself.
  • the double modified Tat-GP(l l) peptide was internalized more efficiently than Tat-GP(lO).
  • Tat-A(lO) and Tat-GP(Ol) were hardly taken up by these cells.
  • Tat- A series were all taken up with high efficiency, while for the peptides of the Tat- GP series, the double modified Tat-GP(l 1) peptide was taken up most efficiently.
  • KGla cells are derived from a patient with erythro leukemia that developed into acute myeloid leukemia.
  • Tat-A(Ol), Tat-A(l 1) and Tat-GP(Ol) showed to be taken up equally or even more efficiently than Tat, while the other peptides Tat-A(lO), Tat-GP(lO) and Tat- GP(11) were all internalized with an efficacy of ⁇ 50% compared to Tat.
  • BV-173 cells which are B cell precursor B-ALL cells, were obtained from the peripheral blood of a patient with chronic myeloid leukemia in a blast crisis.
  • the Tat-A series were taken up more efficiently by these cells than the Tat-GP peptides, with the most efficient internalization observed for Tat-A(l 1) (110% compared to Tat).
  • the peptides of the Tat- GP series were taken up around 60% compared to Tat.
  • B-cell precursor leukemia cell line REH were established from a peripheral blood sample from a patient with acute lymphocytic leukemia at first relapse.
  • Tat-A(l 1) was taken up remarkably efficiently, even more than Tat, while the single modified peptides Tat-A(Ol) and Tat-A(lO) were hardly taken up.
  • Tat-GP series a similar, but less pronounced uptake preference for the double modified Tat-GP(l 1) was observed.
  • Table 6 A summary of the flow cytometry data for the BV-173 and REH cells can be found in Table 6.
  • the uptake of the peptides by lymphocytes derived from the peripheral blood of a healthy donor is also included, to allow for comparison of internalization of the peptides in leukemic lymphocytes and normal lymphocytes. From this, it can be observed that none of the modified peptides were taken up by healthy lymphocytes, but that for the leukemic lymphocytes at least one modified Tat peptide induced efficient uptake. Table 6. Uptake efficacies

Abstract

La présente invention se fonde sur la découverte que la capacité d'absorption cellulaire des peptides pénétrant dans les cellules (CPP) peut être modulée, par l'introduction d'au moins un résidu d'acide aminé à charge positive modifiée, qui est modifiée en ce que cette charge positive est (a) absente, (b) située au moins un atome plus loin du squelette du CPP, (c) située au moins un atome moins loin du squelette du CPP, ou (d) liée au squelette du CPP par le biais d'une fraction de liaison modifiée. Cette découverte a été mise en œuvre dans les divers aspects de la présente invention. Un premier aspect concerne un procédé permettant de cibler sélectivement une cellule digne d'intérêt, consistant (i) à utiliser un CPP modifié selon l'invention, et (ii) à mettre en contact le CPP modifié avec une cellule digne d'intérêt. Un deuxième aspect concerne un procédé de criblage servant à l'absorption sélective des CPP modifiés par une cellule digne d'intérêt, consistant (i) à utiliser un CPP modifié selon l'invention, (ii) à mettre en contact le CPP modifié avec un échantillon comprenant la cellule digne d'intérêt et au moins une cellule supplémentaire, (iii) à déterminer l'absorption du CPP modifié par la cellule digne d'intérêt et la ou les cellules supplémentaires, (iv) à comparer l'absorption par la cellule digne d'intérêt avec l'absorption par la ou les cellules supplémentaires. En outre, les CPP modifiés sont décrits, ainsi que des compositions comprenant le CPP modifié et leur utilisation servant à traiter, prévenir, retarder, diagnostiquer ou détecter un cancer.
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