NL2016207B1

NL2016207B1 - Selective cell penetrating peptides.

Info

Publication number: NL2016207B1
Application number: NL2016207A
Authority: NL
Inventors: Annerie Bode Saskia; Dolstra Harmen; Wilhelmus Petrus Maria Löwik Dennis; Cornelis Maria Van Hest Jan
Original assignee: Stichting Katholieke Univ
Priority date: 2016-02-02
Filing date: 2016-02-02
Publication date: 2017-08-21
Also published as: NL2016207A; WO2017134148A1

Abstract

The present invention founded on the finding that the cellular uptake capacity of the cell penetrating peptides (CPPs) can be modulated, by introducing at least one modified positively charged amino acid residue, which 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. This finding has been put to practice in the various aspects of the present invention. A first aspect concerns a method for selectively targeting a cell of interest, comprising (i) providing a modified CPP according to the invention, and (ii) contacting the modified CPP with a cell of interest. A second aspect concerns a method for screening for selective uptake of modified CPPs by a cell of interest, comprising (i) providing a modified CPP according to the invention, (ii) contacting the modified CPP with a sample comprising the cell of interest and at least one additional cell, (iii) determining the uptake of the modified CPP by the cell of interest and the at least one additional cell, (iv) comparing the uptake by the cell of interest with the uptake by the at least one additional cell. Furthermore, the modified CPPs are disclosed, as well as compositions comprising the modified CPP and their use for the treatment, prevention, delay, diagnosis, or detection of cancer.

Description

Selective cell penetrating peptides FIELD OF THE INVENTION

The present invention relates to the field of medicine, preferably to the field of cancer treatment.

BACKGROUND OF THE INVENTION

Since the rise of cancer as major cause of death, research has strived for effective medications to treat cancer. One of the major challenge therein is to create selectivity of a (potential) anti-cancer or chemotherapeutic agent for cancer cells, such that healthy cells are hardly affected by the treatment. Lack of such selectivity leads to the killing of random healthy cells and causes many side-effects that are associated with cancer treatment. Targeted drug delivery and methods to induce cellular uptake of drugs only by diseased cells and especially cancer cells, is of great importance. The major advantage of such methods is that healthy tissue is not affected by the pharmaceutical substance, and only diseased cells take up the drug, such that the many possible side-effects associated with chemotherapy are avoided as much as possible. As human cells are surrounded by a plasma membrane that with exception to specific molecules represents an impermeable banier, successful transport of chemotherapeutic agents into cells is dependent on the use of delivery vectors. Cell-penetrating peptides (CPPs), sometimes known as protein transduction domains (PTDs) represent a class of peptides that have the ability to penetrate the plasma membrane without induction of cytotoxicity.[1] In addition, 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.

In the art, several attempts have been made to overcome this limitation of CPPs.[3] In one approach, 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. An example of such active targeting system employs folate homing device for the delivery CPP decorated liposomes.[4] Cell-penetrating homing peptides that were identified by mRNA phage display technology are known from Kondo et al.[5] These peptides were able to bind to tumour cell lines, and intemalize into these cells afterwards. The specifïcity for tumour cells is encoded in the sequence of the homing peptide. The use of such dedicated targeting peptides to obtain selectivity is also disclosed in US 2014/038281 and US 2011/212028. Antibodies covalently attached to CPPs are disclosed in US5135736. The use of homing peptides for tumour-targeting approaches has some limitations. First of all, the specifïcity and affïnity of homing devices should be further improved, because binding to healthy tissue, either in an aspecifïc manner or via the presence of some tumour antigens, poses a major limitation of the methodology.[6] Secondly, the intrinsic activity of the CPPs to cross cell membranes causes undesired untargeted uptake in healthy cells.[7]

Another approach towards selectivity in CPPs is to activate them in the presence of the target tissue, by the use of extemal stimuli, such as a change in pH,[8,9] by an enzymatic reaction,110·'1 or by a UV-light trigger.[12] These so-called activatable CPPs (or ACPPs) are blocked and thus inactive, until they reach a specifïc tissue in which the environment triggers the release of the activated CPP. Because most CPPs are positively charged, a useful method to block their activity is by interaction with anions. In US 2014/038886, cargos are selectively released from a CPP in liver cells using a liver protease cleavable linker. Furthermore, poly(ethylene glycol) (PEG) chains have frequently been used to shield CPPs.[13] In these approaches the PEG shield is cleaved from the delivery vehicle by an cnzyme that is upregulated in tumour tissue, such as matrix metalloproteases (MMPs), thereby exposing the CPP, which is capable of penetrating any cell in the vicinity. Other blocking moieties, such as anionic peptides, have been used for similar strategies, which have been applied in tumour detection[14] and imaging.[11] In an altemative method for the development of ACPPs, Liu et al. made use of a legumain cleavable linker.[15] This enzyme is upregulated in various solid tumours. Herein, 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 specifïc enzymcs or a decrease in pH, to function. Furthermore, sometimes large alterations or additions in the CPP structure need to be included to induce an activatable and specifïc system, such as the incorporation of PEG chains, or enzyme cleavable linkers and shielding groups. As the micro-environment of tumour tissue may not always be that different from healthy tissue, ACPP based targeted drug delivery may also suffer reduced selectivity, causing undesirable side-effects. US2011009336 discloses chemically modifïed TAT peptides for use in the treatment of cancer. These modifïcations mainly concern amino acid substitutions, which would lead to enhanced immunostimulating characteristics.

There remains a need in the art for means to increase the selectivity of CPPs towards tumour cells, in order to provide cancer treatment wherein anti-cancer agents selectively target tumour cells and leave healthy cells unharmed such that undesirable side-effects of cancer treatment, in particular chemotherapy, are avoided to a greater extent. The present invention provides in this need, by modifïcation of at least one positively charged amino acid of a CPP. Such modifïed CPPs are known from Bode et al,[10] wherein modifïed Tat-peptides were used for cnzymatic 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. However, by co-incubation with enzymes that have the ability to cleave off the side-chain modifïcation, cellular uptake was restored. However, no uptake selectivity of one cell type over another of the modifïed Tat-peptide was disclosed by Bode et al.[10] US 2009/0099066 describes a screening method for identifying tissue-selective peptides, wherein libraries of modifïed CPPs are tested. The modifïed CPPs contain at least one elongated amino acid, wherein two or more carbon atoms are present between the N atom and the C=0 moiety of the peptide chain, such as beta-alanine. The screening occurs in vivo, by administration to a mammalian subject.

Further disclosures of similar modifïed Tat-peptides can be found in US 8524659, US 2009/062178, WO 2008/043366 and WO 2008/043366. US 8524659 discloses RNA virus-derived peptides having modifïed 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 fïve 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.

SUMMARY OF THE INVENTION

The inventors have surprisingly found that the cellular uptake capacity of the CPPs can be modulated, by introducing specifïc Chemical modifïcations, such that the modifïed CPPs are capable of targeting a specifïc cell of interest, such as a leukaemia cell. This fïnding has been put to practice in the various aspects of the present invention.

In a fïrst aspect, the present invention provides a method for selectively targeting a cell of interest, comprising: (i) providing a modifïed CPP, wherein at least one positively charged amino acid residue is modifïed 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 modifïed linking moiety, and (ii) contacting the modifïed CPP with a cell of interest.

In a second aspect, the present invention provides a method for screening for selective uptake of modifïed CPPs by a cell of interest, comprising: (i) providing a modifïed CPP, wherein at least one positively charged amino acid residue is modifïed 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 modifïed linking moiety, (ii) contacting the modifïed CPP with a sample comprising the cell of interest and at least one additional cell, (iii) determining the uptake of the modifïed CPP by the cell of interest and the at least one additional cell, (iv) comparing the uptake by the cell of interest with the uptake by the at least one additional cell.

In a third aspect, the present invention provides a modifïed CPP, wherein at least one positively charged amino acid residue is modifïed in that the positive charge is (a) absent, (b) located at least one atom more distant ffom the backbone of the CPP, (c) located at least one atom less distant ffom the backbone of the CPP, or (d) linked to the backbone of the CPP via a modifïed linking moiety.

In a fourth aspect, 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.

In a further aspects of the invention, a cell penetrating peptide according to the invention for use as a medicament, a composition comprising the cell penetrating peptide according to the invention for use as a medicament, a cell penetrating peptide according to the invention for use as a medicament for the treatment, prevention, delay, diagnosis, or detection of cancer, and 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.

LIST OF PREFERRED EMBODIMENTS 1. Method for selectively targeting a cell of interest, comprising: (i) providing a modifïed cell penetrating peptide (CPP), wherein at least one positively charged amino acid residue is modifïed 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 ffom the backbone of the CPP, or (d) linked to the backbone of the CPP via a modifïed linking moiety, and (ii) contacting the modifïed CPP with a cell of interest. 2. The method according to embodiment 1, wherein each of the at least one positively charged amino acid residues is lysine and/or arginine, preferably at least one is lysine. 3. The method according to embodiment 1 or 2, wherein the modifïed CPP comprises one or two positively charged amino acid residues that are modifïed. 4. The method according to any one of embodiments 1 to 3, wherein the cell of interest is comprised in a sample that further comprises additional cells, wherein uptake of the modifïed CPP by the cell of interest is selective for the cell of interest. 5. The method according to any one of embodiments 1 to 4, wherein the modifïed CPP is loaded with a cargo, preferably a detectable label or a pharmaceutically active substance. 6. The method according to any one of embodiments 1 to 5, wherein the modifïed CPP comprises RX3X4RRQRRR (SEQ ID NO: 29), wherein at least one of X3 and X4 is the modifïed positively charged amino acid residue and the other one of X3 and X4 is either lysine or a modifïed positively charged amino acid residue. 7. The method according to any one of embodiments 1 to 6, wherein the cell of interest is a cancer cell, preferably a leukaemia cell. 8. The method according to any one of embodiments 1 to 7, wherein the modifïed amino acid residue contains a side chain that contains a positive charge located at least one atom more distant from the backbone. 9. The method according to any one of embodiments 1 to 8, wherein the modifïed CPP has an increased uptake by the cell of interest as compared to the uptake by additional cells. 10. Method for screening for selective uptake of modifïed cell penetrating peptides (CPPs) by a cell of interest, comprising: (i) providing a modifïed CPP, wherein at least one positively charged amino acid residue is modifïed 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 modifïed linking moiety, (ii) contacting the modifïed CPP with a sample comprising the cell of interest and at least one additional cell, (iii) determining the uptake of the modifïed CPP by the cell of interest and the at least one additional cell, (iv) comparing the uptake by the cell of interest with the uptake by the at least one additional cell. 11. Method for screening according to embodiment 10, wherein the cell of interest is a cell that suffers from a condition. 12. Method for screening according to embodiment 11, wherein said cell of interest is a cancer cell, preferably a leukemia cell. 13. Method according to any of embodiments 1-9, wherein the modifïed cell penetrating peptide is identified by the method according to any one of embodiments 10-12. 14. A modifïed cell penetrating peptide (CPP), wherein at least one positively charged amino acid residue is modifïed 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 modifïed linking moiety. 15. The cell penetrating peptide according to embodiment 14, which is loaded with a cargo selected from the group consisting of a pharmaceutically active substance, a radioactive tracer, a specifïc isotope, a diagnostic marker, a hapten, Tokyo green, ASP, rhodamine, Cy3, Cy5, an Atto dye, an Alexa dye, calcein and an IR dye. 16. The cell penetrating peptide according to embodiment 14 or 15, which is represented with general formula (I): wherein

- Rn is the N-terminal end of the CPP containing at least one N-terminal amino acid residue; - Rc is the C-terminal end of the CPP containing at least three C-terminal amino acid residues; wherein the backbone of the CPP comprises at least eight amino acids; - R1 is a fïrst amino acid side chain represented by -L1-N+(R3)3, wherein each R3 is individually selected from H and Ci-4-alkyl and L1 is a linker containing 1-20 optionally substituted backbone atoms selected from C, N, O and S; and - R2 is a second amino acid side chain represented by -L2-N+(R4)3, wherein each R4 is individually selected from H and Ci-4-alkyl and L2 is a linker containing 1-20 optionally substituted backbone atoms selected from C, N, O and S, wherein at least one of R1 and R2 is not -(CH2)4N+H3. 17. The cell penetrating peptide according to embodiment 16, wherein RN is XnR and Rc is RRQRRRXm, wherein each X is individually an amino acid residue and n and m are each individually integers in the range of 0 - 10. 18. The cell penetrating peptide according to embodiment 16 or 17, wherein at least one of R1 and R2 is -(CIU^NHR5, wherein R5 is a peptide containing 1-5 amino acid residues, preferably 1 or 2 amino acids. 19. The cell penetrating peptide according to embodiment 18, wherein R5 is Ala- or GlyPro-. 20. The cell penetrating peptide according to any one of embodiments 14-19, which is represented by RX3X4RRQRRR (SEQ ID NO: 29), wherein at most one of X3 and X4 is a lysine residue and at least one of X3 and X4 is a modifïed lysine residue, wherein the modifïed lysine residue is modifïed in that the positive charge is located at least one atom more distant from the backbone of the CPP. 21. A composition, preferably a pharmaceutical composition, comprising a cell penetrating peptide according to any one of embodiments 14-20 and a pharmaceutically acceptable carrier. 22. The cell penetrating peptide according to any one of embodiments 14 - 20 or the composition according to embodiment 21 for use as a medicament. 23. The cell penetrating peptide according to any one of embodiments 14 - 20 or the composition according to embodiment 21 for use in the treatment, prevention, delay, diagnosis or detection of cancer, preferably leukaemia. 24. The cell penetrating peptide or the composition for use according to embodiment 23, wherein the cell penetrating peptide is represented by RX3X4RRQRRR (SEQ ID NO: 29), wherein at most one of X3 and X4 is a lysine residue and at least one of X3 and X4 is a modifïed lysine residue, wherein the modifïed lysine residue is modifïed in that the positive charge is located at least one atom more distant from the backbone of the CPP. 25. Use of a modifïed cell penetrating peptide (CPP) according to any one of embodiments 14 -20 or of a composition according to embodiment 21, for the in vitro or in vivo targeting of a cell of interest by contacting the cell of interest with the modifïed CPP or with the composition. 26. The use according to embodiment 25, wherein said use is for the detection of said cell of interest, preferably in a diagnostic application. 27. The use according to embodiment 25, wherein said use is for treatment, preferably for treatment of cancer.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, it has now been demonstrated that cell penetrating peptides (CPPs) that are selectively taken up by specifïc cells or cell types can be provided. Such selective cell penetrating peptides according to the invention, and methods and uses according to the invention, are preferred over methods or uses for achieving selective CPPs known in the art. In the art, 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. There is no actual preference for uptake by a specific cell of interest, such that untargeted cells are often still penetrated by the CPP, which can lead to undesirable side effects in therapeutic applications such as cell death of the untargeted yet penetrated cell. To avoid such side-effects, there is a need in the art for CPPs that have inherent selectivity. To date, no means have been found to modulate the selectivity of CPPs such that uptake selectivity by a certain type of cells is achieved.

The modifïed CPPs according to the invention overcome this problem. They are not taken up by any cell, but only by a specifïc kind of cell. The CPPs according to the invention require no activation or in situ modifïcation to be taken up by this target cell. As such, 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. In preferred embodiments of the invention, CPPs according to the invention are not activated in a method or during use.

Without being bound to a theory, it is believed that the cellular uptake of the modifïed CPP is slightly reduced compared to known CPPs by modulating the distribution of positive charges around the CPP. The modifïed CPPs according to the invention are only slightly modifïed compared to known CPPs, wherein the modifïcation slightly changes the positive charge distribution of the CPP, as such slightly reducing the uptake capacity of the CPP. As such, the modifïed CPP is no longer readily taken up by any cell, but only by a specifïc cell of interest.

This fïnding by the inventors that the cellular uptake selectivity of CPPs can be modulated or tuned towards uptake by a specific cell of interest can be put to practice in several applications. Hence, the present invention relates in a fïrst aspect to a method for selective targeting a cell of interest. In a second aspect, the present invention relates to a method for screening modifïed cell penetrating peptides (CPP) for selective uptake by a cell of interest. In a third aspect, the present invention relates a modifïed cell penetrating peptide. In a fourth aspect, the present invention relates to a composition comprising the modifïed cell penetrating peptide according to the invention. In further aspects, the invention relates to the use of the modifïed 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.

To put these aspects in the right perspective, some general terms common to all aspects are fïrst further defïned. A cell of interest, as defïned herein, is a cell that is the intended target for the modifïed CPP. In other words, the cell of interest takes up the modifïed CPP, while cells of a different kind do not or to a lesser extent take up the modifïed 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 identifïed as being of a different kind ffom 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. Altematively, the difference between the cell of interest and the other cells may reside in the state of the cell. For example, the cell of interest may be in a diseased state, i.e. it suffers ffom a condition, while other cells, form the same type or not, do not suffer ffom that condition. An example of such a condition is cancer, such as leukaemia. Thus, in one embodiment of the present invention, the cell of interest is a leukemie cell, such as a leukemie lymphoblast, while other cells include non-leukemie cells, including healthy lymphoblasts, neuronal cells and bone cells. In this embodiment, the modifïed CPPs are taken up by leukemie 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. Preferably, the cell of interest is a human cell. In a preferred embodiment, the cell of interest suffers from a condition and differentiates from other cells in that the latter do not suffer from said condition. In this context, cancer is a preferred condition, most preferably the condition is leukaemia. Preferred forms of leukaemia in the context of the present invention are acute myeloid leukaemia (AML) and B-cell lymphocytic leukaemia (ΒΑΤΕ). 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.

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. In preferred embodiments, obtaining the sample is not part of the method according to the invention. Also 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. Herein, 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, as defined herein, 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. Thus, 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. As a non-limiting example, when a heterogeneous population of cells is present such as in a mammal, 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. As non-limiting example, 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 unmodifïed Tat). It is preferred that the uptake of a modifïed 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 defïned herein, uptake is the uptake of a substance by a cell, in particular of a (modifïed) 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 (hypcr)nuclcation zones, direct translocation and cytosolic delivery through nucleation zones. In the context of the invention, entering a cell through endocytosis or via direct penetration is also considered uptake. In this application, uptake is explicitly differentiated from association, which can involve binding of a substance to the outside of a cell. 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 α-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. Herein, 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. Typically, 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 confïguration, preferably L. However, the amine can also be more distant from the carboxylic acid. As known to a person skilled in the art, 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 omithine (Om), 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. 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). In this context, a terminal amino acid is the last or the first amino acid of a sequence of amino acids or of a peptide. When the terminal amino acid is linked to its sloe neighbouring residue via its carboxylic acid moiety, then that terminus is an N-terminus. When 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, in particular the modified CPPs as defined herein, may be conjugated to a further moiety, such as a cargo, optionally via a linker moiety. In case such conjugation is present in a peptide, any 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). Exemplary is 6-aminohexanoic acid (ahx), which is also known as 6-aminocaproic acid (acp). Ahx or acp is a linker moiety which is capable to connect two moieties together.

Peptide sequences as defïned herein are to be interpreted as defïning (part of) the chain of amino acids only, and such peptides may possibly feature various end group modifïcations, unless such moieties are expressly represented. In the context of the present invention, when a sequence is recited without explicitly mentioned end group( modifïcation)s or terminal moieties, 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 modifïcations may be designated in between parentheses behind the three letter code of the residue. For example, Lys(Boc) represents a lysine residue wherein a Boe group is attached to its side chain amine. Preferably, within the peptides according to the invention, at least one of the amino acid residues of the peptide is a natural amino acid, preferably an L-amino acid. In a preferred embodiment of the invention, at least two of said residues are natural amino acids. In a more preferred embodiment of the invention, each of the residues, except for the modified residues as further defïned below, are natural amino acids. The amino acid residues may be either L-amino acids and/or D-amino acids. In one embodiment, the amino acid residues are all L-amino acids. In one embodiment, 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.

Whenever a peptide is provided, this can be bought, synthesized or obtained by isolating it ff om a natural product. Peptides can be obtained through isolation from a digest of a larger protein. Preferably, 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, semi-automatic synthesizers, or automatic synthesizers. SPPS strategies known in the art allow both N-terminal and C-terminal modifïcation, such as alkylation, amidation, or labelling.

Cell penetrating peptides (CPPs) are known in the art, e.g. from Miletti, Drug Disc Today, 2012, 17(15-16), 850-860, and refer to a specifrc type of peptide that can independently enter cells, e.g. through membrane translocation, through mediated translocation, or through translocation through the formation of a transitory structure, such as a pore. Modified CPPs in the context of the present invention, are modifred with respect to known CPPs, such as those defïned here. In the context of the present invention, CPPs are (highly) cationic (often referred to as “oligocationic” or “polycationic”). Thus, the CPP is typically an oligocationic or a polycationic CPP, more preferably a polycationic CPP. Although the terms “(oligo- or poly)cationic CPP” are wel 1-known in the art and are used interchangeably, they 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 ff om naturally occurring polypeptides or they are synthetic. Such derived CPPs are typically derived ffom 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„, wherein n is an integer in the range 4-17, preferably in the range 7 -10 (e.g. R9 (SEQ ID NO: 1, nona-arginine), Rs (SEQ ID NO: 2, octa-arginine), R7 (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), GRRRRRRRRRPPQ (SEQ ID NO: 8, also known as R9-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-II Rex-(4-16)); KCFQWQRNMRKVRGPPVSCIKR (SEQ ID NO: 16 which is also referred to as hLF or human lactoferrin); RQIKIWFQNRRMKWKK (SEQ ID NO: 17, which is also referred to as penetratin or antennapedia); (RAbu)óR (Abu = 2-aminobutyric acid, SEQ ID NO: 18); (RAca)f,R (Aca = 2-aminocaproic acid, SEQ ID NO: 19); (RG)óR (SEQ ID NO: 20); (RM)6R (SEQ ID NO: 21); (RT)6R (SEQ ID NO: 22); (RS)6R (SEQ ID NO: 23); and (RA)6R (SEQ ID NO: 24). To avoid doubt, 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.

In one embodiment, all Tat variants are together represented by the general sequence X1X2RX3X4RRX5RRRX6X7 (SEQ ID NO: 25), wherein X1 = absent or optionally Tyr (Y) if X2 = Gly (G), X2 = absent or Gly (G), X3 = Lys (K) or Arg (R), X4 = Lys (K) or Arg (R), X5 = Gin (Q) or Arg (R), X6 = absent or (Pro)p ((P)p), wherein p = 1 or 2, and X7 = absent or optionally Gin (Q) if X6 = (Pro)p. X1 = absent or optionally Y if X2 = G. So, if X2 is absent, X1 is also absent. Preferably, X1 = absent. X2 = absent or G, preferably X2 = absent. X3 = K or R, preferably X3 = K. X4 = K or R, preferably X4 = K. X5 = Q or R, preferably X5 = Q. X6 = absent or (P)p, wherein p = 1 or 2, preferably p = 1. Most preferably X6 = absent. X7 = absent or optionally Q if X6 = (P)p. So, if X6 is absent, X7 is also absent. Preferably, X7 = absent.

Within the context of the present invention, the sequence of SEQ ID NO: 25 can be incorporated in a larger peptide, which is preferably represented by X„X1X2RX3X4RRX5RRRX6X7Xm, wherein each of X1, X2, X3, X4, X5, X6 and X7 are as defïned 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 Tat variants are presented by X1X2RX3X4RRX5RRRX6X7 (SEQ ID NO: 25) wherein X3 = X4 = K, i.e. they are represented by X1X2RKKRRX5RRRX6X7 (SEQ ID NO: 26). Preferred variants of Tat are GRKKRRQRRRPQ (SEQ ID NO: 5), RKKRRQRRR (SEQ ID NO: 6), YGRKKRRQRRRPQ (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. In this respect, 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. In the context of the present invention, 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”. When the CPP is loaded with a cargo, 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. As a result, 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.

All said here about peptides in general and CPPs in general specifically applies to the modifïed CPPs according to the invention. As they typically contain one or more non-natural amino acids, they are still classifïed as peptides. Also in conjugated form, i.e. when conjugated to a cargo, the modifïed CPP according to the invention classifïes as a peptide as defïned herein.

Whenever a parameter of a substance is discussed in the context of this invention, it is assumed that unless otherwise specifïed, the parameter is determined, measured, or manifested under physiological conditions. 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 (ffom 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.

Whenever reference is made to 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. Thus, as the skilled person will appreciate, the primary amino moiety of the side chain of lysine and the guanidino moiety in arginine is positively charged at physiological conditions. Also the side chain of histidine, having a \>KA of about 6, is considered positively charged in the context of the present invention.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items foliowing the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

Method for selective targeting

Accordingly, in a first aspect 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 (ii) contacting the modified CPP with a cell of interest.

This method may also be referred to as a method for improving or enhancing the uptake selectivity of a CPP.

In this context, 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” In this context, the CPP that is contacted in step (ii) can also be a composition comprising the CPP. As such, 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. When 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 modified CPP is selectively taken up by the cell of interest. As such, when the cell of interest is comprised in the sample where other cells are also present, the modified CPP is taken up by the cell of interest exclusively when said sample is contacted with the modified CPP. Altematively, only the cell of interest is present in a sample. In such cases, all cells are penetrated by the modified CPP. This can fïnd 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 fïnd use in applications where detection of the cell of interest is desirable.

In a preferred embodiment of this aspect, a method as described above is provided, 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. Typically, 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. Preferably, at least 10% of the total detectable uptake, more preferably 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% 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. In one embodiment, 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 modifïed CPP. Herein the uptake selectivity of the modifïed 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. However, uptake selectivity for the modified CPP can also be provided when the uptake of the modifïed 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 modifïed CPP for additional cells is even further reduced compared to the uptake in additional cells by the template CPP.

The modifïed CPP according to the invention, which is to be used in the method of this fïrst aspect, is further defined below. Herein, it is especially preferred that each of the at least one positively charged amino acid residue that is modifïed is lysine or arginine. Thus, the modifïed amino acid residues can be referred to as a modifïed lysine or a modifïed arginine. When defined in relation to a template CPP, the amino acid residue(s) in question in the template CPP are lysine and/or arginine. In an especially preferred embodiment, at least one of the positively charged amino acid residues is lysine, most preferably all are lysine. Herein, it is especially preferred that 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. As such, the modified CPP differs from a known CPP in that one or two positively charged amino acid residues are modified. In a preferred embodiment, the modified CPP comprises one amino acid residue is modified. In a further preferred embodiment, the modified CPP comprises two amino acid residues are modified.

In a further preferred embodiment of this aspect, the modified CPP is loaded with a cargo, preferably wherein the cargo is a detectable label or a pharmaceutically active substance. In one embodiment, 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). In one embodiment, 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). In preferred embodiments, the cargo does not comprise fluorescein diacetate 5-maleimide. In one embodiment, the 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 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. Preferably, 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. Examples of 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). Most preferably anti-cancer agent are present as cargo, such as daunorubicin and doxorubicin. In the context of the preferred embodiment wherein the cancer is leukaemia, 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.

In a preferred embodiment of this aspect, a method as described above is provided, wherein the modifïed CPP is a modifïed CPP according to the third aspect of the present invention. In a preferred embodiment of this aspect, a method as described above is provided, wherein the modifïed 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) modifïcation, which are further defïned below. In preferred embodiments of this aspect have the positive charge located three, four, or six atoms more distant from the backbone. Most preferably, the charge is three or six atoms more distant.

In a preferred embodiment of this aspect a method as described above is provided, wherein the modifïed CPP comprises SEQ ID NO: 27 or 28 or 29. SEQ ID NO: 27 refers to X1X2RX3X4RRX5RRRX6X7, wherein X1, X2, X5, X6 and X7 are as defïned for Tat (see SEQ ID NO: 25) and at least one of X3 and X4 is not lysine (K), preferably not lysine (K) or arginine (R), most preferably at least one of X3 and X4 is a lysine residue that is modifïed according to any one of modifïcations (a), (b), (c) or (d) as defined below. SEQ ID NO: 28 refers to RX3X4RRX5RRR, wherein X5 is as defïned for Tat (see SEQ ID NO: 25) and at least one of X3 and X4 is not K, preferably not K or R, most preferably at least one of X3 and X4 is a lysine residue that is modifïed according to any one of modifïcations (a) , (b), (c) or (d) as defïned below. SEQ ID NO: 29 refers to RX3X4RRQRRR, wherein at least one of X3 and X4 is not K, preferably not K or R, most preferably at least one of X3 and X4 is a lysine residue that is modifïed according to any one of modifïcations (a), (b) , (c) or (d) as defïned below. The modification over Tat thus resides in at least one of X3 and X4 not being lysine, preferably not being lysine or arginine. In one embodiment, the second one of X3 and X4 is lysine or arginine, preferably lysine. Modifïed CPPs according to the present embodiment are further defïned below, and preferred candidates are Tat-A(ll), Tat-A(Ol), Tat-A(lO), Tat-GP(ll), Tat-GP(Ol), Tat-GP(lO), Tat-G(ll), Tat-G(lO), Tat-G(Ol), Tat-V(lO), Tat-L(lO), Tat-bA(ll), Tat-bA(lO), Tat-bA(Ol), Tat-AA(10), Tat-ObA(ll), Tat-ObA(lO), Tat-ObA(Ol), Tat-OA(ll), Tat-OA(IO), Tat-OA(Ol), Tat-F(ll), Tat-F(lO), Tat-F(Ol), Tat-DpbA(ll), Tat-DpbA(lO), Tat-DpbA(Ol), Tat-DbbA(ll), Tat-DbbA(lO), Tat-DbbA(Ol), Tat-DpA(ll), Tat-DpA(lO), Tat-DpA(Ol), Tat-DbA(ll), Tat-DbA(lO) and Tat-DbA(Ol). Among these modifïed CPPs, Tat-A(Ol), Tat-A(lO), Tat-A(ll), Tat-GP(Ol), Tat-GP(lO), Tat-GP(ll), Tat-G(Ol), Tat-G(10) and Tat-G(l 1) are most preferred.

In a preferred embodiment of the method according to the fïrst aspect, the modifïed CPP that is selective for a particular cell of interest is identifïed by the method according to the second aspect.

In a preferred embodiment of this aspect, a method is provided as described above, wherein the cell of interest suffers ffom a condition. In this embodiment, cells that are of the same type as the cell of interest yet that do not suffer ffom the same condition are considered cells of a different kind and thus not cells of interest. In a preferred embodiment 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.

In this context, 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 ffom the bladder; brain; breast; colon; esophagus; gastrointestine; head; kidney; liver; lung; nasopharynx; neck; ovary; prostate; skin; stomach; testis; tongue; neuron or uterus. In addition, the cancer may specifïcally 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 adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duet carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; ovarian stromal tumour, malignant; and roblastoma, malignant; Sertoli cell carcinoma. A cancer may be neuroblastoma.

In this context 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.

In this context a lymphoma includes any of: Small lymphocytic lymphoma, Lymphoplasmacytic lymphoma (such as Waldenström macroglobulinemia), 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, Enteropathy-type T cell lymphoma, Hepatosplenic T cell lymphoma, Blastic NK cell lymphoma, Mycosis fungoides / Sezary syndrome, Primary cutaneous CD30-positive T cell lymphoproliferative disorders, Primary cutaneous anaplastic large cell lymphoma, Lymphomatoid papulosis, Angioimmunoblastic T cell lymphoma, Peripheral T cell lymphoma, unspecifïedAnaplastic large cell lymphoma, Hodgkin lymphoma, Immunodefïciency-associated lymphoproliferative disorders.

In a preferred embodiment of this aspect a method as described above is provided, wherein the modifïed CPP has an increased uptake selectivity by the cell of interest as compared to the uptake selectivity of a template CPP. In this context, said uptake selectivity is preferably expressed as a parameter selected from the group consisting of: 1. 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; 2. the ratio of uptake of the modifïed CPP according to the invention by the cell of interest relative to uptake of a template CPP by the cell of interest, which is greater than 1.

Method for screening

In a second aspect of the invention, a method for screening is provided. This method is for screening modifïed cell penetrating peptides (CPPs) for selective uptake by 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 modifïed linking moiety, (ii) contacting the modifïed CPP with a sample comprising the cell of interest and at least one additional cell, (iii) determining the uptake of the modifïed CPP by the cell of interest and the at least one additional cell, (iv) comparing the uptake by the cell of interest with the uptake by the additional cell.

Herein, the modified CPP exhibits selective uptake by the cell of interest when selective uptake as defïned above by the cell of interest over the additional cell is observed. The modifïed CPP according to the invention is further defïned below. Selective uptake is preferably expressed as a parameter selected from the group consisting of: 1. 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; or 2. the ratio of uptake of the modifïed CPP according to the invention by the cell of interest relative to uptake of a template CPP by the cell of interest, which is greater than 1.

More in particular, the modifïed 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. In case selective uptake of the modifïed CPP by the cell of interest is observed, 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. Preferably, at least 10% of the total detectable uptake, more preferably 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the total detectable uptake is by the cell of interest for the uptake to be classifïed as selective uptake. Selective uptake thus also includes preferential uptake of the modifïed CPP according to the invention by the cell of interest over the additional cells, wherein uptake of the modifïed 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 underlying mechanism for the method according to the second aspect is the same as for the method according to the fïrst aspect. With the method according to the second aspect, modifïed CPPs that are selective for a specifïc kind of cell of interest can be identifïed. These identifïed modifïed CPPs may be suitably used in the method according to the fïrst aspect.

The method according to the second aspect preferably involves the screening of a library of distinct modifïed CPPs. As such, steps (ii) - (iv) are repeated for each member in the library and step (i) involves providing a library of modifïed CPPs as defïned above. The library of modifïed CPPs preferably comprises at least 2 distinct modifïed CPPs according to the present invention, more preferably at least 5 distinct modifïed CPPs according to the present invention, most preferably at least 10 distinct modifïed CPPs according to the present invention. Although it is generally preferred that the number of distinct modifïed 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 modifïed CPPs according to the present invention.

In an embodiment of this aspect, the method for screening as described above is provided, wherein the cell of interest is a cell that suffers from a condition. In an embodiment of this aspect, 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 defïned hereinabove. In one embodiment, 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.

In a preferred embodiment of this aspect, a method as described above is provided, wherein the modifïed CPP(s) is/are modified CPP(s) according to the third aspect of the present invention. In preferred embodiments within this aspect, the modified CPP(s) is/are linked to a cargo, preferably a detectable label or a pharmaceutically active substance, as fiirther 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 modifïcations 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. Hence, in the present aspect, it is not crucial that every member of the library of modified CPPs is capable of being intemalized by the cell of interest, but merely that specifïc modified CPPs that are capable to do so can be identifïed. Even if no suitable modified CPP could be identifïed, the method according to the second aspect provides valuable Information. The scope of modifïcation between which the skilled person can choose, within the framework of the modifïcations of types (a) - (d), is in principle unlimited, and further options, definitions and preferred embodiments are described here below.

Peptide product

In a third aspect of the invention, a peptide is provided that is a modifïed cell penetrating peptide (CPP), wherein at least one positively charged amino acid residue is modifïed 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 modifïed linking moiety. Such an enhanced cell penetrating peptide is referred to herein as a CPP according to the invention. The modifïed enhanced cell penetrating peptide according to the invention as defïned herein is preferable used in the method for selectively targeting a cell of interest according to the fïrst aspect of the invention. The modifïed enhanced cell penetrating peptide according to the invention as defïned herein is preferable used in the method for screening for selective uptake according to the second aspect of the invention. The modifïed enhanced cell penetrating peptide according to the invention as defïned herein is preferable comprised in the composition according to the invention as defïned further below. The modifïed enhanced cell penetrating peptide according to the invention as defïned herein is preferable used in any of the uses defïned further below. In preferred embodiments of this aspect, the modifïed CPP is linked to a cargo, as specifïed above.

In one embodiment of this aspect, the cell penetrating peptide does not comprise an Ac-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-ahx-Cys-Ntk motif. In one embodiment, 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-Nth, linked to fluorescein diacetate 5-maleimide to form a conjugate. 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-Nfh, wherein one or both of the lysine residues are modifïed according to modifïcation (b) defïned below, wherein an alanine residue or a GlyPro dipeptide is condensed to the primary amino moiety of one or both lysine residues. 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, wherein one or both of the lysine residues are modifïed according to modifïcation (b) defïned 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, in this context, 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). In one embodiment, 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. In one embodiment, the cargo is not fluorescein diacetate 5-maleimide, preferably not a fluorescein moiety.

The modifïed CPPs according to the invention are modifïed compared to known CPPs, such as modifïed compared to one of the CPPs according to SEQ ID NO 1-25. The modifïcation may also be referred to as a Chemical modifïcation. As CPPs are known, the skilled person appreciates when a CPP classifïes as “modifïed CPP”. In the context of the present invention, the modifïcation leads to an altered distribution of positive charge around the CPP, when compared to the known CPP. The modifïcation resides in that at least one positively charged amino acid residue of the modifïed CPP modifïed 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 ffom the backbone of the CPP, (c) located at least one atom less distant ffom the backbone of the CPP, or (d) linked to the backbone of the CPP via a modifïed linking moiety. In one embodiment, the modifïcation is of type (a). In one embodiment, the modifïcation is of type (b). In one embodiment, the modifïcation is of type (c). In one embodiment, the modifïcation is of type (d). Preferably, the modifïcation is of type (a), (b) or (c), more preferably of type (a) or (b), most preferably of type (b). Although screening for uptake selectivity may provide modifïed CPPs wherein more amino acid residues are modifïed, 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 modifïed, 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 modifïed. Each of the modifïed amino acid residues may have the same type of modifïcation (a), (b), (c) or (d), or a different type of modifïcation, although it is preferred that each modifïed amino acid residue has the same type of modifïcation (a), (b), (c) or (d). Each of the at least one positively charged amino acid residue that is modifïed is preferably selected ffom arginine, lysine and histidine, more preferably from arginine and lysine, most preferably one or more lysine residues are modifïed.

In an especially preferred embodiment, the CPPs according to the invention are modifïed compared to any of the Tat variant, preferably modifïed compared to one of the CPPs according to SEQ ID NO 5-8 and 25, more preferably according to any one of SEQID NO 5-7, most preferably according to SEQID NO 6. Tat according to general sequence SEQ ID NO: 25, wherein X3 = X4 = Lys, comprises two lysine residues, and one or two of those lysine residues is modified and none of the further residues is modified. Preferably, 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. Preferably, 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. For example, a lysine or arginine residue may be replaced by an alanine, valine, leucine or isoleucine residue. Altematively, 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. In other words, the -Nfh moiety is replaced by a -NHR6 moiety, wherein R6 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^-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-Arg-OH. For modified Tat peptide 1, the positive charge that is present at the N-terminal arginine of Tat is replaced by alanine. For 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

For modifications of type (b), 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 fürther 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. Most preferably, 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 trom 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. In an especially preferred embodiment, 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, Ile, Val, Gly, Pro, lysine that is acetylated at its ε-amine, and norleucine. Most preferably, one or two amino acid residues selected from Ala, Gly and Pro are condensed to the primary amino group. In an especially preferred embodiment, one alanine residue is condensed to the primary amino group. In an especially preferred embodiment, a GlyPro dipeptide is condensed to the primary amino group.

Examples of modifïed Tat peptides according to the invention that are modifïed 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-Arg-OH. For modified Tat peptide 3, the positive charge of the fïrst lysine residue is three atoms more distant from the backbone of the CPP, because amidation with glycine neutralises the charge on the ε-amine of lysine, yet introducés a new positive charge through glycine’s own amine at its N-terminus. The ε-amine and the two carbon atoms of glycine contribute a total of three atoms to the distance of the charge from the backbone. For modifïed Tat peptide 4, the positive charge of the fïrst lysine residue of the Tat peptide is one atom more distant from the backbone in the modifïed CPP, because arginine features a side chain that has a length of more atoms than the side chain of lysine.

For modifications of type (c), 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 closer to the backbone of the CPP (the peptide main chain). The positive charge may be located any number of atom closer to the peptide backbone as possible. Thus, in case 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. For arginine, having 5 atoms (four carbon and 1 nitrogen) between the positively charged nitrogen atom and the peptide backbone, the positive charge is located 1-5 atoms closer to the backbone, preferably 1-2 atoms closer. For 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. In a preferred embodiment, the positive charge is located 1-2 atoms closer to the peptide backbone. In one embodiment, the positive charge is located 1 atom closer to the peptide backbone. In one embodiment, the positive charge is located 2 atoms closer to the peptide backbone.

Examples of a modifïed 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-Om-Lys-Arg-Arg-Gln-Arg-Arg-Arg-OH. The positive charge at the first lysine residue of the Tat peptide is one atom closer to the backbone, since omithine comprises one fewer methylene moieties than lysine.

For modifications of type (d), 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 connected to the backbone of the modified CPP via a modified linker moiety. Herein, “linker moiety” represents the linker between the positive charge and the peptide backbone, such as -C4H8- for lysine. In this embodiment, the number of atoms between the positive charge and the peptide backbone remains the same. Preferred modified linkers have at least one, preferably 1-3, CRH (R = H or NH2) and/or NH moieties replaced by a moiety selected from CH2, C(O), NH, C(NH), O, S(O) or S(0)2 (excluding the replacement of CH2 for CH2 and the replacement of NH for NH). Most preferably, the moiety, typically a CH2 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. In one possible modification, a -XCH2- moiety, wherein X = Cth or NH is replaced by a -C(0)NH- moiety. In one possible modification, a -CH2NHCHR- moiety, wherein R = H or NH2, preferably R = NH2, is replaced by a -C(0)NHCH2- moiety.

Examples of a modifïed Tat peptide according to the invention that is modifïed in that at least one positively charged amino acid residue is connected via a modifïed linker moiety are:(6): H-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Om(Gly)-OH and (7): H-Arg-Dap(Gly)-Lys-Arg-Arg-Gln-Arg-Arg-Arg-OH. In peptide 6, 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 modifïed to the one of Om(Gly). In peptide 7, the positive charge of the fïrst lysine residue of Tat is equidistant from the backbone, yet a - CH2CH2- moiety (the β and γ carbons in the side chain) is replaced by a -C(0)NH- moiety.

In one embodiment, the modifïed CPPs according to the invention are defïned as being modifïed with respect to a template CPP. A “template CPP” refers to a CPP that is known in the art. The modifïcations as defïned above are modifïcations of these known template CPPs, or in other words differences with respect to these known template CPPs. A modifïed CPP according to the invention contains thus at least one modifïed positively charged amino acid compared to a template CPP, which is modifïed 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 modifïed 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.

It is especially preferred that each of the at least one positively charged amino acid residue that is modifïed in the modifïed CPP according to the invention is a lysine or arginine residue. Thus, the modifïed amino acid residues can be referred to as a modifïed lysine or a modifïed arginine. When defïned in relation to a template CPP, the amino acid residue(s) in question in the template CPP are lysine and/or arginine. In an especially preferred embodiment, at least one of the positively charged amino acid residues is lysine, most preferably all are lysine. Herein, it is especially preferred that the modifïed CPP comprises one or two modifïed amino acid residues, which are each modifïed according to one of the modifïcations (a), (b), (c) and (d) as defïned below. As such, the modifïed CPP differs ffom a known CPP in that one or two positively charged amino acid residues are modifïed. In a preferred embodiment, the modifïed CPP comprises one amino acid residue is modifïed.

In a further preferred embodiment of this aspect, the modifïed 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 defïned hereinabove. In a preferred embodiment, the compound is radioactively labelled, preferably by having incorporated a radioactively labelled amino acid (e.g. 3H or 14C), whereby more preferably the radioactively labelled amino acid is a tritium-labelled amino acid. If 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. Further preferred detectable labels are substances that are radiolabelled or fluorescently or phosphorescently labelled, more preferably the label is a fluorescent label. Fluorescent labels are preferably selected fromthe 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). In preferred embodiments, 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 altemative 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. Preferably, 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. Examples of 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). Most preferably anti-cancer agent are present as cargo, such as daunorubicin and doxorubicin. In the context of the preferred embodiment wherein the cancer is leukaemia, 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.

In a preferred embodiment of this aspect, a peptide as described above is provided wherein each of the at least one positively charged amino acid residue that is modifïed is lysine and/or arginine. Thus, the modifïed amino acid residues can be referred to as a modifïed lysine and/or a modifïed arginine. When defïned in related to a template CPP, the amino acid residue(s) in question in the template CPP are lysine and/or arginine. In an especially preferred embodiment, at least one of the positively charged amino acid residues is lysine, most preferably all are lysine.

In a preferred embodiment of this aspect, a peptide as described above is provided, wherein the modifïed CPP comprises one or two modifïed amino acid residues, which are each modifïed according to one of the modifïcations (a), (b), (c) and (d). As such, the modifïed CPP differs from a known CPP in that one or two positively charged amino acid residues are modifïed. In a preferred embodiment, the modifïed CPP comprises one amino acid residue is modifïed. In a further preferred embodiment, the modifïed CPP comprises two amino acid residues are modifïed.

In a preferred embodiment of this aspect, a cell penetrating peptide as described above is provided, which is represented by general formula (I):

wherein - each Y is individually selected ffomNH, N(Ci-4)alkyl and O, preferably each Y is NH; - Rn is the N-terminal end of the CPP containing at least one N-terminal amino acid residue; - Rc is the C-terminal end of the CPP containing at least three C-terminal amino acid residues; wherein the backbone of the CPP comprises at least eight amino acids; - R1 is a fïrst amino acid side chain represented by -L'-N+(R3)3, wherein each R3 is individually selected ff om H and (Ci^t)alkyl and L1 is a linker containing 1-20 optionally substituted backbone atoms selected from C, N and O; and - R2 is a second amino acid side chain represented by -L2-N+(R4)3, wherein each R4 is individually selected from H and (Ci^)alkyl and L2 is a linker containing 1-20 optionally substituted backbone atoms selected from C, N, O and S.

Herein, at least one of R1 and R2 is not -(CH2)4N+H3, i.e. not a lysine side-chain. Preferably, R1 is not -(CH2)4N+H3 and R2 is or is not -(CH2)4N+H3. Most preferably, both R1 and R2 are not -(CH2)4N+H3.

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.

Thus, both R1 and R2 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. Whenever at least one of R4 is H, it is understood that -N+(R4)3 can also be represented by -N(R4)2, wherein the at least one R4 that represents H is absent; this applies to R3 mutatis mutandis. The cell penetrating peptide of general formula (I) is preferably loaded with a cargo as further specifïed above.

In preferred embodiments of this aspect, each R3 is individually H, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl, more preferably each R3 is individually H or methyl. It is preferred that all instances of R3 are the same, most preferably all instances of R3 are H. In related preferred embodiments of this aspect, each R4 is individually H, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl, more preferably individually R4 is individually H or methyl. It is preferred that all instances of R4 are the same, most preferably all instances of R4 are H.

Linkers L1 and L2 contain a backbone, i.e. the shortest chain of atoms between the α-carbon of the amino acid and the cationic nitrogen atom. The backbones of L1 and L2 contain 1-20 backbone atoms that defïne this shortest chain. Each of these backbone atoms is selected from C, N, O and S, and may optionally be substituted. Preferably, the backbone atoms are selected from C, N and O, more preferably from C and N. In one embodiment, all backbone are C. Typically, the number of backbone heteroatoms is 0 -2, preferably 0 or 1. Optional substitutions are typically selected from (Ci i2)-alkyl groups, (C2-i2)-alkenyl groups, (C2-i2)-alkynyl groups, (C3-i2)-cycloalkyl groups, (C5-12)-cycloalkenyl groups, (C8-i2)-cycloalkynyl groups, (Ci_i2)-alkoxy groups, (C2-12)-alkenyloxy groups, (C2-i2)-alkynyloxy groups, (C3-i2)-cycloalkyloxy groups, halogens, amino groups, oxo and silyl groups, wherein the silyl groups can be represented by the formula (R2)3Si-, wherein R2 is independently selected from the group consisting of (Ci-i2)-groups, (C2-i2)-alkenyl groups, (C2-i2)-alkynyl groups, (C3-i2)-cycloalkyl groups, (Ci-i2)-alkoxy groups, (C2-i2)-alkenyloxy groups, (C2-i2)-alkynyloxy groups and (C3-12)-cycloalkyloxy groups. Herein, 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. Preferred L1 and L2 linkers are -(CH2)n-NH-C(0)-(CH2)m-, -(CH2)n-C(0)-N-(CH2)m-, and -(CH2)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. Particularly preferred L1 and L2 linkers are -(CH2)5-, -(CH2)4-, -(CH2)3-, -(CH2)2-,-(CH2)-, -(CH2)4-NH-C(0)-CHR4- and -(CH2)4-NH-C(0)-CHR4-NH-C(0)-CHR4- (wherein each R4 is individually H or an optional substituent as defïned above). A most preferred L1 linker is -(CH2)4- A most preferred L2 linker is -(CH2)4-,

In a preferred embodiment of this aspect, the cell penetrating peptide according to the invention is represented by general formula (I) as described above, wherein RN is XnArg- and Rc is -ArgArgGlnArgArgArgXm, wherein each X is individually an amino acid and n and m are each individually integers between 0 and 10. In preferred embodiments, 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. For example, RN is preferably cargo-ahx-Gly-Arg-, wherein the cargo is as defïned above. In one embodiment, RN is FITC-ahx-Gly-Arg- In one embodiment, Rc is -Arg-Arg-Gln-Arg-Arg-Arg-ahx-Cys-NH2. In one embodiment, Rc is -Arg-Arg-Gln-Arg-Arg-Arg-NH2. In one embodiment, X„ is not Gly. In one embodiment, Xm is not ahx-Cys-cargo.

In a preferred embodiment of this aspect, the cell penetrating peptide according to the invention is represented by general formula (I) as described above, wherein at least one of R1 and R2 is -(CH2)4NHR5, wherein R5 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. Typically, R5 is a peptide that is connected to the NH in (CH2)4NHR5 through an amide bond involving the C-terminus of the peptide R5. In other words, R5 is a peptide the C-terminus of which is conjugated to the primary amino moiety of a lysine residue. The N-terminus of peptide R5 forms the - N+(R3)3 and/or the N+(R4)3 moiety as present in R1 and R2 respectively. In an especially preferred embodiment, R5 is selected ffom Ala-, or GlyPro-, or Phe- or H-pAla-, wherein the n-terminus is as defïned above. In the most preferred embodiments, R5 is Ala- or GlyPro-. In one embodiment, R1 is -(CH2)4NHR5 and R2 is not, preferably R2 is -(CH2)4NH3+. In one embodiment, R2 is -(CH2)4NHR5 and R1 is not, preferably R1 is -(CH2)4NH3+. In one embodiment, R1 and R2 are both -(CH2)4NHR5.

In a preferred embodiment of this aspect, the modifïed 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 X3 and X4 is not lysine (K), preferably not lysine (K) or arginine (R), most preferably at least one of X3 and X4 is a lysine residue that is modifïed according to any one of modifïcations (a), (b), (c) or (d) as defïned above. Within the context of the present invention, the sequence of SEQ ID NO: 27, 28 or 29 can be incorporated in a larger peptide. For SEQ ID NO: 27, such a larger peptide is preferably represented by X„X1X2RX3X4RRX5RRRX6X7Xm, wherein each of X1, X2, X3, X4, X5, X6 and X7 are as defïned 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. For SEQ ID NO: 28, such a larger peptide is preferably represented by XnRX3X4RRX5RRRXm, wherein each of X3, X4 and X5 are as defïned 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. For SEQ ID NO: 29, such a larger peptide is preferably represented by XnRX3X4RRQRRRXm, wherein each of X3 and X4 are as defïned 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.

In one embodiment, the modifïed 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. In RX3X4RRQRRR, when X3X4 is KK, the sequence is RKKRRQRRR, which is a sequence that can be referred to as Tat (SEQ ID NO: 6). Each of X3 and X4 is preferably selected from at most one lysine and at least one modifïed lysine, wherein the modifïed lysine is modifïed according to any of the modifïcations (a) - (d) as defïned above, preferably by modifïcation (b). Preferred modifïed CPPs according to the invention comprise a sequence selected from the group consisting of:

Tat-A(l 1): Arg-Lys(H-Ala)-Lys(H-Ala)-Arg-Arg-Gln-Arg-Arg-Arg T at- A(01): Arg-Lys-Lys(H-Ala)-Arg-Arg-Gln-Arg-Arg-Arg T at-Α( 10): Arg-Lys(H- Ala)-Lys- Arg-Arg-Gln- Arg- Arg-Arg T at-GP( 11): Arg-Lys(H-Gly-Pro)-Lys(H-Gly-Pro)-Arg-Arg-Gln-Arg-Arg-Arg T at-GP(01): Arg-Lys-Lys(H-Gly-Pro)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-GP(lO): Arg-Lys(H-Gly-Pro)-Lys-Arg-Arg-Gln-Arg-Arg-Arg

Tat-G( 11): Arg-Lys(H-Gly)-Lys(H-Gly)-Arg-Arg-Gln-Arg-Arg-Arg T at-G( 10): Arg-Lys(H-Gly)-Lys- Arg-Arg-Gln- Arg- Arg-Arg T at-G(01): Arg-Lys-Lys(H-Gly)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-bA(l 1): Arg-LysiH-β Ala)-LysfH-β Ala)-Arg-Arg-Gln-Arg-Arg-Arg T at-bA( 10): Arg-Lys(H-P Ala)-Lys- Arg- Arg-Gln- Arg-Arg- Arg

Tat-bA(Ol): Arg-Lys-Lys(H-PAla)-Arg- Arg-Gln- Arg-Arg- Arg T at-ObA( 11): Arg-Om(H-pAla)-Om(H-pAla)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-ObA(lO): Arg-Om(H-PAla)-Lys- Arg- Arg-Gln- Arg-Arg-Arg T at-ObA(01): Arg-Lys-Orn(H-PAla)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-OA(l 1): Arg-Om(H-Ala)-Om(H-Ala)-Arg-Arg-Gln- Arg- Arg-Arg

Tat-OA(IO): Arg-Om(H-Ala)-Lys- Arg-Arg-Gln- Arg- Arg-Arg T at-0 A(01): Arg-Lys-Om(H-Ala)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-F(l 1): Arg-Lys(H-Phe)-Lys(H-Phe)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-F(lO): Arg-Lys(H-Phe)-Lys-Arg-Arg-Gln-Arg-Arg-Arg T at-F (01): Arg-Lys-Lys(H-Phe)-Arg-Arg-Gln- Arg- Arg-Arg T at-DpbA( 11): Arg-Dap(H-P Ala)-Dap(H-P Ala)-Arg-Arg-Gln- Arg-Arg-Arg T at-DpbA( 10): Arg-Dap(H-P Ala)-Lys- Arg-Arg-Gln- Arg- Arg-Arg T at-DpbA(01): Arg-Lys-Dap(H-P Ala)-Arg-Arg-Gln- Arg- Arg-Arg T at-DbbA( 11): Arg-Dab(H-P Ala)-Dab(H-P Ala)-Arg-Arg-Gln- Arg-Arg-Arg T at-DbbA( 10): Arg-Dab(H-P Ala)-Lys- Arg-Arg-Gln- Arg- Arg-Arg T at-DbbA(01): Arg-Lys-Dab(H-P Ala)-Arg-Arg-Gln- Arg- Arg-Arg Tat-DpA(l 1): Arg-Dap(H-Ala)-Dap(H-Ala)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-DpA(lO): Arg-Dap(H-Ala)-Lys-Arg-Arg-Gln-Arg-Arg-Arg T at-Dp A(01): Arg-Lys-Dap(H-Ala)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-DbA(l 1): Arg-Dab(H-Ala)-Dab(H-Ala)-Arg-Arg-Gln-Arg-Arg-Arg T at-DbA( 10): Arg-Dab(H-Ala)-Lys- Arg- Arg-Gln- Arg-Arg- Arg T at-DbA(01): Arg-Lys-Dab(H-Ala)-Arg-Arg-Gln-Arg-Arg-Arg T at-AA( 10): Arg-Lys(H-Ala-Ala)-Lys- Arg-Arg-Gln- Arg- Arg-Arg T at-AA(01): Arg-Lys-Lys(H-Ala-Ala)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-AA(ll): Arg-Lys(H-Ala-Ala)-Lys(H-Ala-Ala)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-AAA( 10): Arg-Lys(H- Ala- Ala- Ala)-Lys- Arg-Arg-Gln- Arg-Arg-Arg

Tat- AAA(01): Arg-Lys-Lys(H-Ala- Ala- Ala)-Arg-Arg-Gln- Arg-Arg-Arg

Tat-AAA( 11): Arg-Lys(H- Ala- Ala- Ala)-Lys(H-Ala- Ala- Ala)-Arg-Arg-Gln- Arg-

Arg-Arg

Tat-G( 10): Arg-Lys(H-Gly)-Lys- Arg-Arg-Gln- Arg- Arg-Arg

Tat-G(01): Arg-Lys-Lys(H-Gly)-Arg-Arg-Gln- Arg- Arg-Arg

Tat-G(l 1): Arg-Lys(H-Gly)-Lys(H-Gly)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-V (01): Arg-Lys(H-V al)-Lys-Arg-Arg-Gln-Arg-Arg-Arg

Tat-Y (10): Arg-Lys-Lys(H-V al)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-V(l 1): Arg-Lys(H-Val)-Lys(H-Val)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-VFmoc(Ol): Arg-Lys(H-Val-Fmoc)-Lys-Arg-Arg-Gln-Arg-Arg-Arg Tat-VFmoc(lO): Arg-Lys-Lys(H-Val-Fmoc)-Arg-Arg-Gln-Arg-Arg-Arg Tat-VFmoc(ll): Arg-Lys(H-Val-Fmoc)-Lys(H-Val-Fmoc)-Arg-Arg-Gln-Arg-Arg-Arg Tat-L(01): Arg-Lys(H-Leu)-Lys- Arg-Arg-Gln- Arg-Arg- Arg

Tat-L( 10): Arg-Lys-Lys(H-Leu)-Arg-Arg-Gln- Arg-Arg- Arg T at-L( 11): Arg-Lys(H-Leu)-Lys(H-Leu)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-LFmoc(01): Arg-Lys(H-Leu-Fmoc)-Lys-Arg-Arg-Gln-Arg-Arg-Arg Tat-LFmoc(lO): Arg-Lys-Lys(H-Leu-Fmoc)-Arg-Arg-Gln-Arg-Arg-Arg Tat-LFmoc(l 1): Arg-Lys(H-Leu-Fmoc)-Lys(H-Leu-Fmoc)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-GABA( 10): Arg-Lys(H-GABA)-Lys-Arg-Arg-Gln-Arg-Arg-Arg Tat-GABA(01): Arg-Lys-Lys(H-GABA)-Arg-Arg-Gln-Arg-Arg-Arg Tat-GABA( 11): Arg-Lys(H-GABA)-Lys(H-GABA)-Arg-Arg-Gln-Arg-Arg-Arg Tat-[20,3K]; Arg-Om-Lys-Arg- Arg-Gln- Arg-Arg- Arg Tat-[2K,30]: Arg-Lys-Orn-Arg-Arg-Gln-Arg-Arg-Arg

Tat-[2,30]: Arg-Om-Orn-Arg-Arg-Gln-Arg-Arg-Arg

Tat-[20G,3K]: Arg-Om(H-Gly)-Lys-Arg-Arg-Gln-Arg-Arg-Arg Tat-[2K,30G]: Arg-Lys-Om(H-Gly)-Arg-Arg-Gln-Arg-Arg-Arg Tat-[2,30G]: Arg-0m(H-Gly)-0m(H-Gly)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-[20A,3K]: Arg-Om(H-Ala)-Lys-Arg-Arg-Gln-Arg-Arg-Arg Tat-[2K,30A]: Arg-Lys-Om(H-Ala)-Arg-Arg-Gln-Arg-Arg-Arg Tat-[2,30A]: Arg-0m(H-Ala)-0m(H-Ala)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-[20bA,3K]: Arg-Om(H-PAla)-Lys-Arg-Arg-Gln-Arg-Arg-Arg Tat-[2K,30bA]: Arg-Lys-Orn(H-PAla)-Arg-Arg-Gln-Arg-Arg-Arg Tat-[2,30bA]: Arg-0m(H-PAla)-0m(H-PAla)-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-[2,3DbA]: Arg-Dab(H-Ala)-Dab(H-Ala)-Arg-Arg-Gln-Arg-Arg-Arg Tat-[2DbAA,3K]: 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-[2,3DbAA]: Arg-Dab(H-Ala-Ala)-Dab(H-Ala-Ala)-Arg-Arg-Gln-Arg-Arg-

Arg

Tat-[2DbGABA,3K]: 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-[2,3DbGABA]: Arg-Dab(H-GABA)-Dab(H-GABA)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-[2Dp,3K]: Arg-Om-Lys-Arg-Arg-Gln-Arg-Arg-Arg

Tat-[2K,3Dp]: Arg-Lys-Orn-Arg-Arg-Gln-Arg-Arg-Arg

Tat-[2,3Dp]: Arg-Om-Orn-Arg-Arg-Gln-Arg-Arg-Arg

Tat-[2DpG,3K]: Arg-Dap(H-Gly)-Lys-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

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

Tat-[2K,3DpbA]:Arg-Lys-Dap(H-pAla)-Arg-Arg-Gln-Arg-Arg-Arg

Tat-[2,3DpbA]: Arg-Dap(H-PAla-Dap(H-PAla)-Arg-Arg-Gln-Arg-Arg-Arg

Herein, GABA is gamma-aminobutyric acid. Among the modified CPPs mentioned above, Tat-A(Ol), Tat-A(lO), Tat-A(ll), Tat-GP(Ol), Tat-GP(lO), Tat-GP(ll), Tat-G(Ol), Tat-G(lO) and Tat-G(ll) are most preferred. Preferably, the modified CPPs according to the invention do not contain any fiirther amino acid residues in addition to those mentioned in the sequences listed directly above. In one embodiment, the modifïed CPPs according to the invention consist of the sequences listed directly above.

In one embodiment, the cell penetrating peptide according to the invention is not Tat-A(Ol), Tat-A(lO), Tat-A(ll), Tat-GP(Ol), Tat-GP(lO) or Tat-GP(ll). In one embodiment, the cell penetrating peptide according to the invention does not comprise the sequence of Tat-A(Ol), Tat-A(lO), Tat-A(ll), Tat-GP(Ol), Tat-GP(lO) or Tat-GP(ll).

Composition

In a fourth aspect of the invention, a composition is provided comprising a cell penetrating peptide according to the invention, and a pharmaceutically acceptable carrier or excipient. In a preferred embodiment of this aspect, 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. In this context, a preferred cancer is leukaemia. Such compositions are also suitably used in the methods and uses according to the invention.

As will be understood, the cell penetrating peptide according to the invention, as well as any further therapeutic compound that may be present in the composition 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 specifïcally, 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 patiënt'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.

Preferred in this aspect is a composition that comprises a cell penetrating peptide according to the invention, a pharmaceutically acceptable carrier, and that further comprises a further therapeutic compound. In an embodiment of this aspect of the invention, 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 ffom one or more of the further components of the composition according to the invention, such as the further therapeutic compound. Typically, the cell penetrating peptide is mixed with the separate component(s) prior to administration. Also encompassed in this aspect is a combination therapy, wherein a composition according the invention is administered together with an effective dose of a further therapeutic compound or composition. Encompassed by this aspect are embodiments wherein 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. Also encompassed by this aspect are embodiments where 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 peptide conjugates. These examples are also preferred pharmaceutically active substances that can be loaded as cargo to a cell penetrating peptide according to the invention.

Various packaging options are possible and known in the art, depending, among others, on the route and mechanism of administration. For example, where the cell penetrating peptide according to the invention is supplied separately ffom one or more of the further therapeutic compounds, 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. Altematively, 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. In particular, 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 effïciently 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.

Use

In a further aspect of the invention, a cell penetrating peptide according to the invention for use as a medicament is provided. In a further aspect of the invention, 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.

In a further aspect of the invention, 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. In a further aspect of the invention, 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. 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, prevention, delay, diagnosis, or detection of cancer. These 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. Within these aspects, said cancer is preferably leukaemia. The cell penetrating peptide according to the invention can also be for the curing or stabilizing of a condition.

In the context of this invention, 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. Within the embodiments of the invention, a preferred disorder is cancer.

In preferred embodiments of this aspect, a cell penetrating peptide is provided as defïned 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. In the context of the invention, 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 patiënt 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.

Within the embodiments of the invention, 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. In certain embodiments, 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. The detection of 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-[18F]-fluor-2-deoxy-D-glucose (FDG-PET) or [18F]-'3-fluoro-'3-deoxy-L-thymidine PET. An ex vivo altemative may be staining of a tumour biopsy with Ki67.

Within the embodiments of the invention, a subject can be any living entity. Preferably, the subject is a mammal, more preferably a human.

Administration of a cell penetrating peptide according to the invention can be achieved by any method known in the art, as further defïned later in this text.

In one embodiment, 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.

In a further aspect of this 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.

As defïned hereinabove, 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. In embodiments of the invention, 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. Herein, at least one other cell than said cell of interest is present in the sample.

In a preferred embodiment of this aspect, the cell penetrating peptide according to the invention is loaded with a cargo as further defïned above. In preferred embodiments within this aspect, this substance of interest is not fluorescein diacetate 5-maleimide.

In a preferred embodiment, the use or method for targeting a cell of interest is for the detection of the cell of interest. Such 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. For instance, when the cell of interest is a cancer cell, and when the cell of interest is detected in a subject, cancer can be diagnosed with the use or method according to this aspect of the invention. Altematively, a diagnosis can be made when the cell of interest is not detected. For instance, when a cancer cell is the cell of interest, and the cell is not detected in a subject, the absence of cancer can be diagnosed. In preferred embodiments of this aspect, the use or method is for the aid in diagnosis, or to assist in diagnosis, and not for diagnosis itself. In one embodiment, 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 defïned hereinabove.

In an altemative preferred embodiment, the use or method for targeting a cell of interest is for treatment. In one embodiment, the treatment is of a human subject. In one embodiment, the treatment is not of a human subject. Herein, treatment refers to the treatment of a subject that suffers from a condition. In an especially preferred embodiment, the treatment is the treatment of cancer, most preferably of leukaemia. In this embodiment, it is preferred that the cell penetrating peptide according to the invention is loaded with a cargo being a pharmaceutically active substance as defïned hereinabove, preferably an anti-cancer agent.

Preferred modifïed cell penetrating peptide according to the invention to be used in the treatment, prevention, delay, diagnosis, or detection of cancer have been identifïed 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). Each occurrence of X is preferably selected from at most one lysine and at least one modifïed lysine, wherein the modifïed lysine is modifïed according to any of the modifïcations (a) - (d) as defïned above, preferably by modifïcation (b). CPPs of SEQID NO: 7 are further defïned below, and preferred candidates are Tat-A(ll), Tat-A(Ol), Tat-A(lO), Tat-GP(ll), Tat-GP(Ol), Tat-GP(lO), Tat-G(ll), Tat-G(lO), Tat-G(Ol), Tat-bA(ll), Tat-bA(10), Tat-bA(Ol), Tat-ObA(ll), Tat-ObA(lO), Tat-ObA(Ol), Tat-OA(ll), Tat-OA(IO), Tat-OA(Ol), Tat-F(ll), Tat-F(lO), Tat-F(Ol), Tat-DpbA(ll), Tat-DpbA(lO), Tat-DpbA(Ol), Tat-DbbA(l 1), Tat-DbbA(lO), Tat-DbbA(Ol), Tat-DpA(ll), Tat-DpA(lO), Tat-DpA(Ol), Tat-DbA(ll), Tat-DbA(lO) and Tat-DbA(Ol). Among these modifïed CPPs, Tat-A(Ol), Tat-A(lO), Tat-A(l 1), Tat-GP(Ol), Tat-GP(lO), Tat-GP(l 1), Tat-G(Ol), Tat-G(lO) and Tat-G(l 1) are most preferred.

In the context of this aspect, 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. In addition, the cancer may specifïcally 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 adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infïltrating duet carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; ovarian stromal tumour, malignant; and roblastoma, malignant; Sertoli cell carcinoma. A cancer may be neuroblastoma.

In this context 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.

Table 1. Sequences

1] Abu = 2-aminobutyric acid; Aca = 2-aminocaproic acid; [2] X1 = absent or optionally Tyr (Y) if X2 = Gly (G), X2 = absent or Gly (G), X3 = Lys (K) or Arg (R), X4 = Lys (K) or Arg (R), X5 = Gin (Q) or Arg (R), X6 = absent or (Pro)p ((P)p), wherein p = 1 or 2, and X7 = absent or optionally Gin (Q) if X6 = (Pro)p; [3] X1 = absent or optionally Tyr (Y) if X2 = Gly (G), X2 = absent or Gly (G), X5 = Gin (Q) or Arg (R), X6 = absent or (Pro)p ((P)p), wherein p = 1 or 2, and X7 = absent or optionally Gin (Q) if X6 = (Pro)p; [4] X1 = absent or optionally Tyr (Y) if X2 = Gly (G), X2 = absent or Gly (G), X5 = Gin (Q) or Arg (R), X6 = absent or (Pro)p ((P)p), wherein p = 1 or 2, and X7 = absent or optionally Gin (Q) if X6 = (Pro)p, and at least one of X3 and X4 is not Lys (K); [5] X5 = Gin (Q) or Arg (R), and at least one of X3 and X4 is not Lys (K); [6] at least one of X3 and X4 is not Lys (K).

EXAMPLES

The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

Unless stated otherwise, the practice of the invention will employ Standard conventional methods of molecular biology, virology, microbiology or biochemistry. Such techniques are described in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; in Volumes 1 and 2 of Ausubel et al. (1994)

Current Protocols in Molecular Biology, Current Protocols, USA; and in Volumes I and II ofBrown (1998) Molecular Biology LabFax, Second Edition, Academie Press (UK); Oligonucleotide Synthesis (N. Gait editor); Nucleic Acid Hybridization (Hames and Higgins, eds.).

Experimental

Peptide synthesis and purifïcation: The following peptides were used in examples 1 and 2: T at: Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg T at-a(01): Arg-Lys-Lys(H-Ala*)-Arg-Arg-Gln-Arg-Arg-Arg T at-A( 11): Arg-Lys(H-Ala)-Lys(H-Ala)-Arg- Arg-Gln- Arg-Arg- Arg T at- A(01): Arg-Lys-Lys(H-Ala)-Arg-Arg-Gln-Arg-Arg-Arg T at-A( 10): Arg-Lys(H-Ala)-Lys-Arg-Arg-Gln-Arg-Arg-Arg T at-GP( 11): Arg-Lys(H-Gly-Pro)-Lys(H-Gly-Pro)-Arg-Arg-Gln- Arg-Arg- Arg T at-GP(01): Arg-Lys-Lys(H-Gly-Pro)-Arg-Arg-Gln-Arg-Arg-Arg T at-GP( 10): Arg-Lys(H-Gly-Pro)-Lys-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. 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.

Cell culture: 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 humidifïed atmosphere of 7.5% CO2. 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). HL-60, THP-1, KGla, BV-173 and REH cells were maintained in sterile conditions in RPMI medium 1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells were maintained on tissue culture plastic and kept at 37 °C in a humidifïed atmosphere of 5% CO2. 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). CLSM analvsis of uptake in HEK and HeLa cells: 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 μΜ 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. Fluorescein was excited 488 nm and emission was collected between 500 and 550 nm. For the HEK/mCherry HeLa co-culture experiments, 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. Fluorescein was excited at 488 nm and emission was collected between 500 and 550 nm, mCherry was excited by 561 nm laser and emission was collected between 600 and 650 nm.

Flow cvtometrv experiments with HEK and HeLa cells: HEK or HeLa cells were seeded in 24-well plates (Sarstedt, Numbrecht, Germany) one (60,000 cells/well) ortwo (30,000 cells/well) days prior to the experiment. On the day of the experiment, cells were incubated with the peptide Solutions (5 μΜ) 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 MgCk, 1.8 mM CaCk), cells were detached by trypsinisation for 5 minutes, spun down and resuspended in 200 pL RPMI + 10% FCS. Afterwards, the cells were spun down again and resuspended in HBS buffer supplemented with 0.1% bovine serum albumin (BSA). 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. For analysis of the HEK/mCherry HeLa co-culture, 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 (Beekman Coulter Ine.) and the data was analyzed using the Summit Software. Results were based on 10,000 gated cells.

Flow cvtometrv experiments with normal WBCs and RBCs: For uptake experiments in WBCs, 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 NH4CI; 5 mM KHCO3; 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 supematant was discarded. The pellet was washed with PBS twice, and afterwards the amount of WBCs was counted. 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 μΜ) 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 pL 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 (Beekman Coulter Ine. Miami) and the data was analysed using the Kaluza software (Beekman Coulter). Results were based on 10,000 gated cells. For uptake experiments in RBCs, peripheral blood was drawn from a healthy individual on the day of the experiment. The cells were spun down at 1500 rpm for 10 minutes and the pellet was resuspended in PBS. The amount of RBCs was counted and dilutions were made so that there were 150,000 RBCs per sample. Subsequently, the cells were treated as described above, only the trypsin/EDTA was excluded from the procedure.

Flow cytometry experiments with leukemie cells: On the day of the experiment, HL-60, THP-1, KGla, 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 μΜ) 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 pL 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 (Beekman Coulter Ine. Miami) and the data was analyzed using the Kaluza software (Beckmam Coulter). Results were based on 10,000 gated cells.

Example 1. Selective uptake by HeLa cells

The uptake of Tat and the modifïed Tat peptides was studied in HEK, obtained trom healthy tissue, and tumorous HeLa cells. The uptake of the peptides was visualized using confocal laser scanning microscopy (CLSM) and quantifïed by flow cytometry. For this purpose, adherent cells were treated with 5 μΜ 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. As a positive control, unmodifïed Tat was included in the uptake experiments, and it was observed that this peptide was taken up by both HeLa and HEK cells. For all modifïed Tat peptides, no fluorescence was observed in HEK cells, meaning the peptides were unable to enter these cells. However, in HeLa cells, bright punctate spots were observed, similar to cells treated with unmodifïed Tat. This demonstrated that the HeLa cells were able to take up the modifïed Tat peptides. The experiment was repeated for analysis by flow cytometry. We again observed no uptake by HEK cells, but in contrast, the HeLa cells did take up all modifïed peptides with varying effïcacy (Table 2).

Table 2. Uptake efficacies *

* Summary of flow cytometry data for uptake of Tat and the modifïed Tat peptides by HEK293 and HeLa cells. The median fluorescence of Tat is set to 100% for both cell lines, to allow for comparison of uptake efficacies of the modifïed Tat peptides. Standard deviation are given in brackets.

To confirm that the observed selective uptake by HeLa cells was indeed an inherent feature of the modifïed Tat-peptides and not by an enzymatic reaction, 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. It was observed that 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.

To further investigate the possibility that HeLa cells express enzymes that may cleave off the side-chain modifïcation of the peptides, 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. On the day of the experiment, the cells were treated in the same way as described above and were analyzed by both CLSM and flow cytometry. Erom the CLSM micrographs it could be concluded that uptake by endocytosis took only place for the mCherry HeLa cells, while the HEK cells remained without fluorescent signal. Using flow cytometry, four cell populations could potentially be observed: unlabeled cells which correspond to HEK cells that did not take up any peptide, LITC-labeled cells which correspond to HEK cells in which peptide was intemalized, mCherry labeled cells that correspond to HeLa cells not having taken up any peptide and doublé labeled cells that represent HeLa cells that have taken up peptide. A sample treated with the positive control, LITC-labeled Tat, showed equal uptake by both HEK and HeLa cells. Lor the Tat-A series, hardly any uptake by HEK cells was observed, as in agreement with the flow cytometry experiments described above for this cell line. The HeLa cells were able to take up the three peptides from this series. Even though the Tat-GP series showed to be less selective than the Tat-A series, as slight uptake by HEK cells was observed, uptake by mCherry HeLa cells remained predominant (Table 3).

Table 3. Uptake efficacies *

* Summary of flow cytometry data for uptake of Tat and the modifïed Tat peptides by a co-culture of HEK293 and mCherry HeLa cells. The median fluorescence of Tat is set to 100%, to allow for comparison of uptake efficacies of the modifïed Tat peptides. The median fluorescence for each population was calculated as relative population ffaction. [no upt. = no uptake, no FITC label observed; upt. = uptake is observed, FITC label was intemalized]

Thus, the modifïed 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 HeEa cells are present, the modifïed Tats are taken up by the HeEa cells only.

Example 2. Selective uptake by leukemie cells

White blood cells (WBCs) were obtained from peripheral blood of a healthy human donor. Erythrocytes (red blood cells, RBCs) were lysed from the population, because these cells are much more abundant in blood making the analysis of WBCs challenging. Furthermore, the membrane of RBCs can be very sticky, which may lead to peptides being entrapped on the RBC membrane. The remaining WBCs (lymphocytes, monocytes and granulocytes) were incubated with 5 μΜ 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 modifïed Tat peptides according to the invention was analysed by flow cytometry. Only unmodified Tat was intemalized into WBCs, and that the lymphocytes took up the peptide most effïciently. The modifïed Tat peptides were not able to effïciently enter WBCs, with their uptake reduced to maximally 15% compared to Tat (Table 4).

Table 4. Uptake efficacies *

* Flow cytometry results of uptake of Tat and the modifïed Tat peptides by normal WBCs. The average median fluorescence of Tat was set to 100%.

Leukemie cells were obtained trom three cell lines derived from patients with acute myeloid leukemia (AML) and two cell lines derived ffom 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 μΜ 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.

For the HL60 cell line, which corresponds to early AML, it was observed that the peptides were taken up by these cells with varying uptake efficiency. The Tat-A(ll) peptide was taken up almost equally well as Tat itself. Interestingly, for the Tat-GP series, also the doublé modifïed Tat-GP(ll) peptide was intemalized more effïciently than Tat-GP(lO). Notably, Tat-A(lO) and Tat-GP(Ol) were hardly taken up by these cells. For the THP-1 cells, which are known to be more phagocytic than the HL60 cells, the Tat-A series were all taken up with high efficiency, while for the peptides of the Tat-GP series, the doublé modifïed Tat-GP(l 1) peptide was taken up most effïciently. KG la cells are derived from a patiënt with erythroleukemia 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 efïlciently than Tat, while the other peptides Tat-A(lO), Tat-GP(lO) and Tat-GP(11) were all intemalized with an efficacy of ± 50% compared to Tat.

Table 5. Uptake efficacies *

* Summary of flow cytometry results of uptake of Tat and modifïed Tat peptides by HL-60, THP-1 and KG la cells. For each cell line, the average median fluorescence of Tat was setto 100%. BV-173 cells, which are B cell precursor B-ALL cells, were obtained from the peripheral blood of a patiënt 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 efficiënt intemalization 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. For the second B-ALL cell line, B-cell precursor leukemia cell line REH were established ffom a peripheral blood sample from a patiënt with acute lymphocytic leukemia at first relapse. Tat-A(l 1) was taken up remarkably efficiently, even more than Tat, while the single modifïed peptides Tat-A(01) and Tat-A(lO) were hardly taken up. For the Tat-GP series a similar, but less pronounced uptake preference for the doublé modifïed Tat-GP(l 1) was observed. A summary of the flow cytometry data for the BV-173 and REH cells can be found in Table 6. In this graph, the uptake of the peptides by lymphocytes derived from the peripheral blood of a healthy donor is also included, to allow for comparison of intemalization of the peptides in leukemie lymphocytes and normal lymphocytes. From this, it can be observed that none of the modifïed peptides were taken up by healthy lymphocytes, but that for the leukemie lymphocytes at least one modifïed Tat peptide induced efficiënt uptake.

Table 6. Uptake efficacies *

* Summary of flow cytometry results of uptake of Tat and modified Tat peptides by REH and BV 173 cells and normal lymphocytes. For each cell line, the average median fluorescence of Tat was set to 100%. A summary of the uptake efficacies of Tat and the various modified Tat peptides in the different cell lines tested in this study is given in Table 7. Notably, for each cancer cell line at least one modified Tat peptide showed promising uptake selectivity for the leukemia over healthy cells.

Table 7. Summary of uptake efficacies

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Claims

CHANGED CONCLUSIONS (annotated)

A method for selectively targeting a cell of interest, comprising: (i) providing a modified cell-penetrating peptide (CPP), wherein at least one positively charged amino acid residue has been modified such that the positive charge (a) is absent, ( b) at least one atom further away from the backbone of the CPP, (c) at least one atom less far away from the backbone of the CPP, or (d) connected to the backbone of the CPP via a modified linker and (ii) contacting the modified CPP with the cell of interest wherein the cell of interest is included in a sample which further comprises additional cells wherein uptake of the modified CPP by the cell of interest is selective for the cell of interest.

The method of claim 1, wherein each of the at least one positively charged amino acid residues is lysine and / or arginine, preferably at least one lysine.

The method of claim 1 or 2, wherein the modified CPP comprises one or two modified positively charged amino acid residues.

The method of any one of claims 1 to 3, wherein the cell of interest is contained in a sample taken from a subject, preferably a human subject, which further comprises additional cells including recording of —The modified CPP by the cell of interest is selective for the cell of interest.

The method of any one of claims 1 to 4, wherein the modified CPP is loaded with a charge, preferably a detectable label or a pharmaceutically active substance.

The method of any one of claims 1 to 5, wherein the modified CPP comprises RX3X4RRQRRR (SEQ ID NO: 29), wherein at least one of X3 and X4 is the modified positively charged amino acid residue and the other of X3 and X4 or a lysine residue or a modified positively charged amino acid residue.

The method of any one of claims 1 to 6, wherein the cell of interest is a cancer cell, preferably a leukemia cell.

The method of any one of claims 1 to 7, wherein the modified amino acid residue contains a side chain that contains a positive charge that is at least one atom further from the backbone of the CPP.

The method of any one of claims 1 to 8, wherein the modified CPP has an increased uptake by the cell of interest compared to uptake by additional cells.

A method for screening for uptake selectivity by a cell of interest of modified cell penetrating peptides (CPP), comprising: (i) providing a modified CPP, wherein at least one positively charged amino acid residue is modified such that the positive charge (a) is absent, (b) at least one atom is further away from the backbone of the CPP, (c) is at least one atom less far away from the backbone of the CPP, or (d) via a modified linker with the backbone of the CPP is connected, (ii) contacting the modified CPP with a sample comprising the cell of interest and at least one additional cell, (iii) determining the uptake of the modified CPP by the cell of interest and the at least one additional cell, and (iv) comparing the uptake by the cell of interest with the uptake by the at least one additional cell.

The method of claim 10, wherein the cell of interest suffers from a disorder.

The method of claim 11, wherein the cell of interest is a cancer cell, preferably a leukemia cell.

The method of any one of claims 1 to 9, wherein the modified CPP is identified by the method of any one of claims 10 to 12.

14. Modified cell-penetrating peptide (CPP), wherein at least one positively charged amino acid residue has been modified such that the positive charge (a) is absent, (b) is at least one atom further from the baekbone of the CPP, (c) at least one atom less distant from the baekbone of the CPP, or (d) is connected to the baekbone of the CPP via a modified linker. 15. The cell-penetrating peptide according to claim 14, loaded with a charge selected from a pharmaceutically active substance, a radioactive tracer, a specific isotope, a diagnostic marker, a hapten, Tokyo green, ASP, rhodamine, Cv3, Cy5, an Atto dye , an Alexa dyc, calcein and an IR dye.

The cell-penetrating peptide according to claim 14 or 15, which is represented by structure (I):

wherein - R N is the N-terminal end of the CPP, which comprises at least one N-terminal amino acid residue; - Rc is the C-terminal end of the CPP, which comprises at least three C-terminal amino acid residue; wherein the baekbone of the CPP comprises at least eight amino acid residues; - R 1 is a first amino acid side chain represented by -L 1 -N + (R 3): wherein each R 3 is independently selected from H and C 1-10 alkyl and L 1 is a linker which has 1-20 optionally substituted backbone atoms selected from C, N, O and S; and - R 2 is a second amino acid side chain represented by -L 2 -N + (R 4) 3, wherein each R 4 is independently selected from H and C 1-4 alkyl and L 2 is a linker which is 1-20 optionally substituted backbone atoms selected from C, N, O and S, wherein at least one of R 1 and R 2 is not (CH 1 -1 NH).

The cell penetrating peptide of claim 16, wherein R N is X n R - and R 0 is -RRQRR R X m, wherein each X is independently an amino acid residue and n and m are each independently integers in the range 0-10.

The cell penetrating peptide according to claim 16 or 17, wherein at least one of R1 and R2 is - (CH2) 4 NHR5, wherein R5 is a peptide comprising 1-5 amino acid residues, preferably 1 or 2 amino acid residues.

The cell-penetrating peptide of claim 18, wherein R 5 is Ala or GlyPro.

The cell-penetrating peptide according to any of claims 14-19, which is represented by RX3X4RRQRRR (SEQ ID NO: 29), wherein at least one of X3 and X4 is a lysine residue and at least one of X3 and X4 is a modified lysine residue, wherein the modified lysine residue is modified such that the positive is at least one atom further from the backbone of the CPP.

A composition, preferably a pharmaceutical composition, which comprises a cell-penetrating peptide according to any one of claims 14 to 20 and a pharmaceutically acceptable carrier.

The cell-penetrating peptide of any one of claims 14 to 20 or the composition of claim 21 for use as a medicament.

The cell-penetrating peptide according to any of claims 14 to 20 or the composition according to claim 21 for use in the treatment, prevention, delay, diagnosis or detection of cancer, preferably leukemia.

The cell-penetrating peptide or composition for use according to claim 23, wherein the cell-penetrating peptide is represented by RX3X4RRQRRR (SEQ ID NO: 29), wherein most of one of X3 and X4 is a lysine residue and at least one of X 'and X4 is a modified lysine residue, wherein the modified lysine residue is modified such that the positive at least one atom is further from the backbone of the CPP.

Use of a modified cell-penetrating peptide (CPP) according to any one of claims 14 to 20 or of a composition according to claim 21, for targeting a cell of interest in vitro or in vivo by contacting the cell of interest with the modified CPP or with the composition.

The use according to 25, wherein the use for the detection of the cell is important, preferably in a diagnostic application.

The use according to 25, wherein the use is pre-treatment, preferably for cancer treatment.