Cell-penetrating polypeptides (CPPs) and their use in human therapy

Abstract

The present invention relates to a compound represented by general formula (I): X 0 GX 1 X 2 GX 3 X 4 X 5 GX 6 X 7 X 8 GX 9 X 10 X 11 X 12 X 13 X 14 , The present invention discloses a compound comprising or consisting of a polypeptide of the formula: (In the formula, G is glycine; X 0 is present or absent, and if present is an amino acid linker; X 1 is N or S, where N is asparagine and S is serine; X 2 is S or T, where S is serine and T is threonine; X 3 is present or absent and, if present, is S, where S is serine; X 4 is present or absent and, if present, is G, where G is glycine; X 5 is a basic polypeptide stretch consisting of 5 basic amino acid residues selected from R and K, where R is arginine, K is lysine and preferably X 5 contains three or more K amino acid residues, especially X 5 is KKKKK or KRKKK; X 6 is a basic amino acid residue selected from R and K, where R is arginine and K is lysine, preferably X 6 is K; X 7 is S or L, where S is serine and L is leucine; X 8is present or absent, and if present is a polypeptide stretch consisting of the amino acid sequence GLGS, where G is glycine, L is leucine, and S is serine; X 9 is a basic polypeptide stretch consisting of three basic amino acid residues selected from R and K, where R is arginine, K is lysine and preferably X 9 contains two or more K amino acid residues, especially X 9 is KKK or KKR; X 10 is present or absent and, when present, is L or a polypeptide stretch consisting of the amino acid sequence DPL or DPC, where L is leucine, D is aspartic acid, P is proline and C is cysteine; X 11 is present or absent and, if present, is a polypeptide stretch consisting of the amino acid sequence LR or GSGL, where L is leucine, R is arginine, G is glycine, and S is serine; X 12 is present or absent, and if present is a basic polypeptide stretch having 20% ​​or more basic amino acid residues selected from K and R and at least P residues, preferably X 12 KYKPKL, KYKPKLGT, or GX 3 X 4 X 5 GX 6 X 7 X 8 GX 9 X 10 where K, R, P, L, G, T, X 3 , X 4 , X 5 , GX 6 , X 7 , X 8 , GX 9 , and X 10 is defined as above, and Y is tyrosine; X 13is present or absent and, if present, is a polypeptide stretch consisting of the amino acid sequence GS, GST, GST, GSG, GSTG, or GSGL, where G, S, T, and L are defined as above; X 14 is present or absent, and if present is an amino acid linker; The polypeptide has a length of 20 to 75 amino acid residues, preferably 24 to 65 amino acid residues, and particularly 25 to 60 amino acid residues.

Description

[Technical field]

[0001] The present invention relates to the field of cell membrane permeable polypeptides (CPPs) and their use in human therapy, in particular cancer therapy. [Background technology]

[0002] The use of small peptides across cell membranes, called CPPs or protein transduction domains (PTDs), can enable cellular drug delivery of larger molecules and facilitate drug delivery of small molecule compounds. In therapy, small molecule compounds can cross cell membranes to some extent by diffusion, but the therapeutic use of macromolecules is limited by their poor penetration in tissues and their inability to cross cell membranes without an appropriate drug delivery system. Thus, CPP-directed transport of macromolecules across biological membranes allows for the development of therapeutic drug delivery vehicles for various applications. Multiple clinical trials are testing the first generation of CPP-mediated delivery of macromolecular CPP conjugates in various indications. CPP carrier peptides are a class of short peptide sequences, also known as protein transduction domains (PTDs), cell membrane-penetrating polypeptides (CPPs), or membrane translocation sequences (MTSs). Their ability to carry much larger molecules into cells provides an ideal tool for transporting polypeptides and other molecules into living cells for both research purposes and therapeutic applications. For example, CPPs have been reported to deliver therapeutic polypeptides, antisense oligonucleotides, liposomes, plasmids, nanoparticles, phages, and viruses into mammalian cells. Although CPPs are structurally very diverse (many of them are highly cationic and rich in arginine or lysine, but in addition, they show little sequence homology to each other), they are known to those skilled in the art as a functional class of polypeptides. Several examples are known (recently summarized, for example, in WO 2020 / 214846, especially in Table 1). Two specific cell membrane penetrating peptides, GKRKKKGKLGKKRDP and GKRKKKGKGLGKKRDPCLRKYK, have been disclosed in de Coupade et al. (Biochem. J. 390 (2005):407-418), WO 01 / 64738, and Lee et al. (BBRC 419 (2012):597-604). Both peptides are derived from the heparin-binding domain of epidermal growth factor-like growth factor.WO 2004 / 092194 teaches the fusion of a single-chain PvuII restriction endonuclease to a cell membrane-permeable peptide. Summary of the Invention [Problem to be solved by the invention]

[0003] Many currently known CPPs have major limitations such as poor tissue selectivity accompanied by off-target effects, toxicological constraints, moderate potency requiring high therapeutic concentrations, and wide tissue biodistribution at effective doses. Moreover, these molecules are often highly vulnerable to proteases and therefore present stability problems under manufacturing and storage conditions, drawbacks for implementation in the context of pharmaceutical formulations, limiting the druggability of the respective CPP conjugates. As a result, these CPPs exhibit unfavorable pharmacokinetic parameters as carriers of a given payload drug substance. The estimated therapeutic activity concentrations are relatively high, in the mid-to-low μM range. Given the beneficial principles for active intracellular drug delivery contained in the CPP technology, however the druggability is rather low due to the constraints, improvement of these parameters would be beneficial. Despite the promising results obtained with some CPP-linked polymer therapeutics, stability and / or delivery problems impair the effectiveness of many such therapeutics. Polymer-linked payloads often accumulate in late endosomes, are released to the desired compartment inefficiently, and require high concentrations to achieve therapeutic efficacy without significant general toxicity.

[0004] Therefore, there is a need for the development of improved CPPs that improve upon these limitations.The prior art still reflects the need for new alternative CPPs, particularly polymer-containing CPP-ligand conjugates and polymeric CPP compounds, that have the ability to efficiently translocate into biological membranes and provide cell-type-selective and / or subcellular compartment-selective molecular delivery, and thus have utility as therapeutic and / or research tools.Furthermore, there is a need in the art for CPP-linked polymeric therapeutics with significantly lower toxicity but useful therapeutic efficiency, or delivery systems that provide cell-type-selective and / or subcellular compartment-selective delivery of a wide range of therapeutic molecules, including biological macromolecules, small molecule drugs, or combinations of biological macromolecules and small molecule drugs. [Means for solving the problem]

[0005] It is an object of the present invention to provide improved CPPs with improved transport efficiency through cell membranes to enable successful delivery of payload molecules to the intracellular environment. A further object is to provide efficient compounds comprising such CPPs suitable for treating medical disorders and diseases, preferably treating various types of cancer patients, particularly for treating cancer patients already with metastasis. More specifically, it is another object to provide CPP-delivered therapeutics with significantly lower toxicity but useful therapeutic efficiency, or delivery systems that provide cell-type-selective and / or subcellular compartment-selective delivery of a wide range of therapeutic molecules, including biological macromolecules, small molecule drugs, or combinations of biological macromolecules and small molecule drugs. It is another object of the present invention to provide CPPs that have the ability to efficiently translocate into biological membranes and provide cell-type-selective and / or subcellular compartment-selective delivery of molecules, and thus have utility as therapeutic and / or research tools.

[0006] Thus, the present invention relates to a compound of general formula (I): X0GX1X2GX3X4X5GX6X7X8GX9X 10 X 11 X 12 X 13 X 14 , The present invention provides a compound comprising or consisting of a polypeptide of the above formula (I). (In the formula, G is glycine; X0 is present or absent, and if present is an amino acid linker; X1 is N or S, where N is asparagine and S is serine; X2 is S or T, where S is serine and T is threonine; X3 is present or absent and, if present, is S, where S is serine; X4 is present or absent and, if present, is G, where G is glycine; X5 is a basic polypeptide stretch consisting of 5 basic amino acid residues selected from R and K, where R is arginine and K is lysine, preferably X5 comprises 3 or more K amino acid residues, in particular X5 is KKKKK or KRKKK; X6 is a basic amino acid residue selected from R and K, where R is arginine and K is lysine, preferably, X6 is K; X7 is S or L, where S is serine and L is leucine; X8 is present or absent, and if present is a polypeptide stretch consisting of the amino acid sequence GLGS, where G is glycine, L is leucine, and S is serine; X9 is a basic polypeptide stretch consisting of three basic amino acid residues selected from R and K, where R is arginine and K is lysine, preferably X9 comprises two or more K amino acid residues, in particular X9 is KKK or KKR; X 10 is present or absent and, when present, is L or a polypeptide stretch consisting of the amino acid sequence DPL or DPC, where L is leucine, D is aspartic acid, P is proline and C is cysteine; X 11is present or absent and, if present, is a polypeptide stretch consisting of the amino acid sequence LR or GSGL, where L is leucine, R is arginine, G is glycine, and S is serine; X 12 is present or absent, and if present is a basic polypeptide stretch having 20% ​​or more basic amino acid residues selected from K and R and at least P residues, preferably X 12 KYKPKL, KYKPKLGT, or GX3X4X5GX6X7X8GX9X 10 where K, R, P, L, G, T, X3, X4, X5, GX6, X7, X8, GX9, and X 10 is defined as above, and Y is tyrosine; X 13 is present or absent and, if present, is a polypeptide stretch consisting of the amino acid sequence GS, GST, GST, GSG, GSTG, or GSGL, where G, S, T, and L are defined as above; X 14 is present or absent, and if present is an amino acid linker; The polypeptide has a length of 20 to 75 amino acid residues, preferably 24 to 65 amino acid residues, and particularly 25 to 60 amino acid residues.

[0007] The compounds of the present invention comprise or consist of a new class of CPPs with unexpected cell membrane permeability and transmembrane transport properties that allow efficient transport of payload molecules (often also referred to as "targets", "targeted molecules", "effective molecules", "active molecules", "active drugs", "active biologics", etc.). The CPPs of the present invention also have improved properties for combating tumor cells, especially when covalently linked to molecules effective in killing tumor cells. The suitability of using the CPPs of the present invention to treat tumor patients lies in the efficient cell membrane permeability function of the polypeptides of the present invention.

[0008] The compounds provided by the present invention with improved CPP moieties show better efficiency in membrane crossing and intracellular sorting processes. The CPPs of the present invention are particularly suitable as platforms for transporting various payloads for various pharmaceutical applications. In addition, the efficient CPPs of the present invention also show improved solubility in recombinant expression systems and robust stability in the production process. This allows for the subsequent transfer and upscaling of the CPP conjugates, which are the compounds of the present invention, to API manufacturing processes, resulting in pharma- ceutically acceptable stability of the respective formulations. The advantages in the manufacturing process of the compounds of the present invention are particularly useful when the payload is a polypeptide. Most studies that have examined PTD-payload fusion polypeptides or CPP-payload fusion polypeptides in bacteria have resulted in problems such as polypeptide solubility, formation of inclusion bodies, and lack of eukaryotic post-translational modifications important for selective applications. These problems are not observed with the CPPs of the present invention or are at least significantly reduced with known CPPs. These differences in the performance and efficiency of the CPPs of the present invention also apply to the peptides GKRKKKGKLGKKRDP and GKRKKKGKGLGKKRDPCLRKYK.

[0009] The present invention also allows for modifying the payload in selected CPPs for better pharmacokinetic (PK) and pharmacodynamic (PD) properties. The compounds of the present invention are useful in solving one or more (or all) of the objectives set out above.

[0010] The CPP conjugates of the present invention allow for increased in vivo half-life; reduced immunogenicity, reduced toxicity, and reduced selective clearance rate; successful transport through cell membranes; protection against proteolysis; modification of electroosmotic flow; increased pH and thermal stability; small volume distribution and sustained adsorption from the injection site; and improved formulation properties of polypeptides. These superior properties can increase effective efficacy, improve response to drugs, increase patient tolerance, reduce side effects, and reduce overall dosage. For example, many biological macromolecules, including proteins, peptides, polynucleotides, and nucleic acids, have been found to be useful in the treatment of various health problems.

[0011] According to a preferred embodiment, the CPP moiety in the compound of the present invention is a compound of general formula (I), in which X1 is N, X2 is S, X3 is S, X4 is G, X5 is KKKKK or KRKKK, X6 is K, X9 is KKK or KKR, and X 10 is L or a polypeptide stretch consisting of the amino acid sequence DPL or DPC (where N, S, G, R, L, D, P, and C are defined as above).

[0012] According to a preferred embodiment, the number of arginine or cysteine ​​residues in the CPP moiety of general formula (I). Preferably, the compounds of the invention have a general formula (I) that contains a single arginine and / or a single cysteine, or that contains no more than two arginine and / or no more than two cysteine ​​residues. According to a particularly preferred embodiment, the CPP has a general formula (I) that is devoid of arginine and / or cysteine ​​residues, in particular that the general formula (I) is devoid of both arginine and cysteine ​​residues.

[0013] Substitution of arginine residues and / or replacement of arginine residues with lysine residues has been shown to improve the stability of the compounds of the invention, both by preventing protease degradation in vivo (i.e., when the compounds of the invention are applied to human patients) and by preventing degradation during the manufacturing process. Replacement of arginine with another amino acid, particularly with lysine, can, for example, block a protease site.

[0014] This lack of cysteine ​​residues in the CPP moiety is, of course, independent of the use of cysteine ​​residues in the linking moiety that links the CPP to the payload molecule (see below). Moreover, cysteine ​​residues may also be used as preferred sites of PEGylation to provide the PEGylated compounds of the invention.

[0015] According to another preferred embodiment, the compound of the invention comprises two or more polypeptides of general formula (I), preferably the CPP consists of two polypeptides of general formula (I).

[0016] A particularly preferred embodiment of the invention is a compound wherein the CPP is selected from the group consisting of GNSGSGGKKKKKGKGSGLGSGKKKDPL (DDM34) and GNSGSGGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKKDPLGST (DDM36).

[0017] Preferred embodiments of the CPP of the present invention include GNSGSGKRKKKGKGLGKKRDPCLRKYKPKLGTGSTG(HHHHHH)(DDM11), GNSGKRKKKGKGSGLGSGKKRDPCLRKYKPKLGSTG(HHHHHH)(DDM18), GNSGSGKRKKKGKGLGKKRDPCLRKYKPKLGTGSTG(HHHHHH)(DDM21), GNSGSGKRKKKGKGSGLGSGKKRDPLGSTG(HHHHHH)(DDM26), GNSG(HHHHHH)GSTGGKRKKKGKGSGLGSGKKRDPL(DDM27), GNSGSGKRKKKGKGSGLGSGKKKDPLGSTG(HHHHHH)(DDM28), GNSG(HHHHHH)GSTGGKRKKKGKGSGLGSGKKKDPL(DDM29), GNSGSGKKKKKGKGSGLGSGKKRDPLGSTG(HHHHHH)(DDM30), GNSG(HHHHHH)GSTGGKKKKKGKGSGLGSGKKRDPL(DDM31), GNSGSGKKKKKGKGSGLGSGKKKDPLGSTG(HHHHHH)(DDM32), GNSG(HHHHHH)GSTGGKKKKKGKGSGLGSGKKKDPL(DDM33), GNSGSGKKKKKGKGSGLGSGKKKDPL(DDM34), GNSGSGKKKKKGKGSGLGSGKKKLGSTG(HHHHHH)(DDM35), GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST(DDM36), and GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGSTG(HHHHHH)GST(DDM44), where (HHHHHH; histidine tag) may be present or absent; Preferably, GNSGSGKRKKKGKGLGKKRDPCLRKYKPKLGTGSTG(DDM11), GNSGKRKKKGKGSGLGSGKKRDPCLRKYKPKLGSTG(DDM18), GNSGSGKRKKKGKGLGKKRDPCLRKYKPKLGTGSTG(DDM21), GNSGSGKRKKKGKGSGLGSGKKRDPLGSTG(DDM26), GNSGGSTGGKRKKKGKGSGLGSGKKRDPL(DDM27), GNSGSGKRKKKGKGSGLGSGKKKDPLGSTG(DDM28), GNSGGSTGGKRKKKGKGSGLGSGKKKDPL(DDM29), GNSGSGKKKKKGKGSGLGSGKKRDPLGSTG(DDM30), GNSGGSTGGKKKKKGKGSGLGSGKKRDPL(DDM31), GNSGSGKKKKKGKGSGLGSGKKKDPLGSTG(DDM32), GNSGSGKKKKKGKGSGLGSGKKKDPL(DDM34), and GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST(DDM36), GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGSTGGST(DDM44), especially, GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST(DDM36), and GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGSTGGST(DDM44).

[0018] According to a preferred embodiment, the polypeptide of general formula (I) in the compound of the invention is a payload molecule to be delivered into a biological cell, preferably (CROMOC01) SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKI- YGNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKKKKGGSGLGSGKKKKDPLGST、 (CROMOC02) SHPDNLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERWYSDGHKDINPKIPVKYVMEHGTKI- YGNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKKKKGGSGLGSGKKKKDPLGST、 (CROMOC36) SHPDNLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERWYSDGHKDINPKIPVKYVMEHGTKIYGSGGGSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG-NDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI-YRLEPKDLEFYYDKWERWYSDGHKDINPKIPVKYVMEHGTKI- YGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKKKGKSGLGSGKKKDPLGST、 (CROMOC60) SHPDNLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGGSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKI-YGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST、 (CROMOC62) SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG-NDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST、 (CROMOC63) SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST、 (CROMOC70) SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKI-YGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST、 (CROMOC87) SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST, (CROMOC88) SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST, and (CROMOC89) SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST is covalently attached to the C-terminus of a compound selected from the group consisting of:

[0019] The term "coupled" or "covalently coupled" refers to the binding of two parts of the compound of the invention, preferably via a covalent peptidic bond, e.g. a peptidic bridge, thereby forming a polypeptide. Of course, other bonds (e.g. via S-S bonds or other chemical (covalent) bonds) may also be used to link different parts of the compound. However, peptidic bonds are the preferred method of (covalently) binding parts of the compound (e.g. the polypeptide of general formula (I), the payload molecule, the linker, etc.) to each other to form a polypeptide with a single amino acid chain.

[0020] According to another preferred embodiment, the polypeptide of general formula (I) is covalently attached to the C-terminus of a payload molecule to be delivered into a biological cell which is a class II restriction endonuclease, preferably PvuII or a subunit thereof, the payload molecule being optionally attached at its C-terminus to a linker molecule comprising 8 to 12 amino acids comprising or consisting of glycine, serine and / or cysteine ​​residues, the polypeptide of general formula (I) is covalently attached to the C-terminus of the payload molecule or the linker molecule, and the compound comprises one or two payload molecules, optionally separated by a linker molecule comprising 8 to 12 amino acids comprising or consisting of glycine, serine and / or cysteine ​​residues.

[0021] Preferably, the polypeptide of general formula (I) is covalently attached to the C-terminus of a payload molecule to be delivered into a biological cell, the compound comprising a first payload molecule, which is a first subunit of PvuII linked at its C-terminus to a linker molecule comprising 8-12 amino acids comprising or consisting of glycine, serine and / or cysteine ​​residues, the linker molecule being attached at its C-terminus to a second subunit of PvuII, the polypeptide of general formula (I) being covalently attached to the C-terminus of the second subunit of PvuII. An example of such a compound of the invention may have the structure (unless otherwise stated: always from N-terminus to C-terminus): first subunit of PvuII-DDM. Another example is a compound having the structure of first subunit of PvuII-8-12 amino acid linker comprising glycine, serine and / or cysteine ​​residues-second subunit of PvuII-DDM.

[0022] The PvuII restriction endonuclease is 157 amino acid residues long and naturally has a homodimeric form. The homodimer can be easily converted to a single chain polypeptide (sc PvuII). The two subunits can be linked in tandem via a short peptide linker (see above). The configuration of single chain PvuII (sc PvuII) can be (2-157)-linker-(2-157), where (2-157) represents the amino acid residues of the enzyme subunit. PvuII endonuclease activity as a sc enzyme can be expressed at high levels as a soluble protein. The purified enzyme was shown to have the expected molecular mass for the designed sc protein. The cleavage specificity of sc PvuII is indistinguishable from that of the wild-type (wt) enzyme.

[0023] These preferred CPP moieties of the invention ("drug delivery module" or "DDM" sequences) have been found to significantly increase membrane crossing activity and the desired intracellular sorting activity, increase stability against proteolysis, increase the solubility of a given payload (particularly a polypeptide payload) or the expression of a polypeptide (as a payload) encoded by a polynucleotide molecule, and simplify payload purification.

[0024] According to a preferred embodiment, the compound of the present invention further comprises a payload molecule to be delivered into the biological cell covalently linked to the polypeptide of general formula (I), said payload molecule being preferably a chemotherapeutic molecule, a cytotoxic molecule, a DNA damaging molecule, an antimetabolite molecule, a therapeutic molecule, a small molecule having a therapeutic effect in the biological cell, a DNA molecule, an RNA molecule, an antibody molecule or an antibody derivative having an antibody-like function, a restriction endonuclease, a nicking enzyme or a DNA- or RNA-dependent endonuclease exhibiting double-stranded / single-stranded cleavage activity or no nuclease activity, more preferably the payload molecule is a class II restriction endonuclease such as PvuII, EcoRV, PvuII, HinfI, or a sc homodimer, subunit or functional fragment thereof, in particular the payload molecule is (CP01) SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERWYSDGHGDINNPKIPVKYVMEHGTKIY、 (CP02) SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERWYSDGHGDINNPKIPVKYVMEHGTKIY、 (CP36) SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINPKIPVKYVMEHGTKIYGSGGGSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG-NDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY、 (CP60) SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGGSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY、 (CP62) SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY、 (CP63) SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG-NDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY、 (CP70) SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY、 (CP87) SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY, (CP88) SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY, and (CP89) SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY is selected from the group consisting of:

[0025] According to a preferred embodiment, the compound of the present invention further comprises a homopolymer moiety covalently attached to the polypeptide of general formula (I). Although such homopolymer-containing CPP compounds have been identified in the prior art as being advantageous in principle for enhancing CPP function in druggable situations (e.g., WO 2020 / 214846 and other documents cited in the search report of this international application), this presents challenges due to the size of the polymer and the resulting molecules due to modification by endosomal trafficking. Furthermore, it has been reported that the use of polymeric therapeutic agents is hindered because cell membrane barriers often impede their intracellular delivery.

[0026] The CPP moiety in the compounds of the present invention has been found to be very potent in delivering payloads through cell barriers, and the addition of polymers, particularly homopolymers, can be predicted in the compounds of the present invention such that the art-accepted advantages of such polymers attached to CPPs, including the ability of a particular compound to be tailored for the pharmacokinetic (PK) and pharmacodynamic (PD) properties of a given payload, are present for the compounds of the present invention without the disadvantages of size expansion, low cell type selectivity, and overall equal tissue distribution. Preferably, the homopolymer of the compounds of the present invention is selected from the group consisting of polyethylene glycol (PEG), particularly PEG with a MW of 5 kDa to 30 kDa; dextran, polysialic acid, hyaluronic acid, dextrin, hydroxyethyl starch, poly(2-ethyl 2-oxazoline), etc. (Pasut, Polymers 6 (2014), 160-178; Grigoletto et al., Nanomed. Nanobiotechnol. 2020, e1689). Alternatively, polypeptide techniques such as protein conjugation with XTEN peptides or PASylation can be used.

[0027] Virtually all polymer therapeutics used in relevant therapeutic applications are water-soluble polymers. One of the most commonly used polymers in polymer therapeutics is PEG, which is particularly preferred for the present invention. PEG has been approved for human administration by various routes of administration, e.g., oral, injection, or skin application. The structure of linear PEG is HO-(CH2-CH2-O) n -H, and n indicates the number of repeating ethylene oxide units in the PEG. PEG is a linear or branched neutral polyether, commercially available in a variety of molecular masses. The polymerization can be controlled to narrow the molecular mass distribution. PEG derivatives can have a variety of sizes, typically with molecular weights ranging from hundreds to thousands of Da. The PEG molecules used in the polymer molecules are selectively highly purified molecules with little or no diol impurities. The preferred PEG moieties used in the present invention are monodisperse or exhibit only a narrow molecular mass range difference (±5% or ±3% is better). Many PEG derivatives are commercially available or can be prepared by methods known in the art.

[0028] Therefore, the present invention is preferably related to PEGylated compounds. Many of the advantages of PEGylated therapeutics lie in the properties of PEG. PEG is a neutral hydrophilic polymer that is soluble in water and various organic solvents. In addition, PEG is inert, non-toxic, has low immunogenicity, and the polymer is easily cleared from the body, mainly via the kidney for molecules with a molecular mass of less than 20 kDa, or via a combination of the kidney and liver for molecules with a molecular mass of more than 20 kDa. To date, the maximum PEG molecular mass used in the preparation of polymeric therapeutics is 40 kDa. PEGylation plays an important role in stabilizing drugs, reducing their antigenicity, and reducing drug doses, in addition to enhancing the biodistribution capacity by binding biologics to their surface. PEG has the most suitable qualities for the preparation of bioactive drugs and biologics. A variety of functional groups are available, allowing the introduction of PEG chains to drugs, enzymes, phospholipids, and other biologics. Covalent conjugation of hydrophobic macromolecules with activated PEG leads to the formation of polymeric micelles, which allow the homogeneous dispersion of hydrophobic drugs in aqueous media.

[0029] The conjugation of PEG or a PEG derivative to a polypeptide (such as a polypeptide of general formula (I)) can be achieved by conjugation of a PEG-NHS derivative to a polypeptide amine (e.g., an epsilon group of a lysine) (PEG-NHS+polypeptide-NH2), a PEG-aldehyde derivative to an NH2 group of a polypeptide (including a secondary amine, such as the N-terminus of a polypeptide, a frequently used aldehyde linker being methoxy-PEG-CO(CH2)nCOO-NHS, where n represents an integer between 1 and 3) (PEG-Ald+polypeptide-NH2); or a PEG-maleimide derivative to an SH group of a polypeptide (PEG-maleimide+polypeptide-SH), for example to an exposed cysteine ​​contained within the polypeptide or engineered into the polypeptide by recombinant techniques, or a PEG-NH2 derivative to a COOH group of a polypeptide (e.g., a surface-exposed glutamic acid of a polypeptide) (PEG-NH2+polypeptide-COOH), or a PEG-p-nitrophenyloxycarbonyl derivative to an NH2 group of a polypeptide (PEG-NP+polypeptide-NH2).

[0030] PEG derivatives include monofunctional linear PEGs, such as NHS active esters / carbonates, p-nitrophenyl carbonate PEGs, aldehyde PEGs, aminopropyl PEGs, aminoethyl PEGs, thiol PEGs, maleimide PEGs, aminoxy PEGs, hydrazide PEGs, iodoacetamide PEGs; bifunctional PEGs, such as NHS-PEGs, amine PEGs, thiol PEGs, maleimide PEGs, or multi-arm PEGs, such as 4-arm-PEGs or 8-arm-PEGs; branched PEGs, such as 2-arm branched PEGs, 3-arm branched PEGs, 4-arm branched PEGs, or lysine branched PEGs; heterofunctional PEGs, such as Boc-protected amino-PEG-carboxylic acid, 9-fluorenyl-methyloxycarbonyl-protected amino-PEG-carboxylic acid, maleimide-PEG-carboxylic acid, maleimide-PEG-NHS esters / carbonates, amino-PEG-carboxylic acids, Boc-protected aminoPEG-NHS carbonates, protected mercapto-PEGs. The PEG derivatives can be modified as NHS esters, 9-fluorenylmethyloxycarbonyl protected amino PEG-NHS esters, azido-PEG-NHS-esters / carbonates, azido-PEG-amines, biotin-PEG-NHS esters / carbonates, biotin-PEG-maleimide, biotin-PEG-amines; or branched PEGs, e.g., NHS-PEG, amine PEG, or maleimide PEG. For such applications, various PEG derivatives have been developed. Most polymer-macromolecules can be chemically synthesized and attached to virtually all known classes of therapeutically suitable molecules, such as nucleic acids, PNAs, lipids, small peptides, or proteins, or mixtures thereof, using linker technologies known in the art. Most of these molecules, especially those with extended molecular masses >1000 Da, are at least partially synthesized using molecular recombination methods, and it would be advantageous to have at hand polymer-macromolecules that can be produced recombinantly, including polymers that can be produced as covalent fusions that allow for single-step expression. Overall, the covalent conjugation of polymers (e.g., PEG) with small molecule drugs and / or biological macromolecules (e.g., polypeptides or polynucleotides) is a promising approach for pharmaceutical applications.Because such conjugates exhibit altered (improved) pharmacokinetic properties, including longer in vivo half-life, reduced immunogenicity, reduced toxicity, protection against proteolysis, improved water solubility, and increased pH and thermal stability, while the biological activity of the small molecule drug and / or biological macromolecule is generally retained in those conjugates.

[0031] The compounds of the present invention do not exhibit undesirable off-target effects and frequently do not require high therapeutic doses, as reported for prior art polymer-CPP compounds.

[0032] In fact, the compounds of the present invention generally show sufficient and often advantageous target specificity to select the target-selective activity principle. The compounds of the present invention are useful for increasing local tissue distribution and obtaining selective cell type specificity. Thus, the compounds of the present invention provide better target specificity for therapeutic agents, especially polymeric therapeutic agents. Such polymeric therapeutic agents include polymer-polymer conjugates, drug-polymer conjugates, and supramolecular drug delivery systems. In addition to favorable biodistribution, these polymeric therapeutic agents can achieve several additional objectives to optimize CPP activity and tailor the pharmacokinetic profile of the polymer.

[0033] The combination of the novel CPP moiety and polymer-modified macromolecular single activity profile compound or the combination of the CPP and polymer-modified macromolecular multiple activity profile compound provided by the present invention can be engineered to sort into specific locations (specific biodistribution) after systemic treatment in vivo. Moreover, such compounds (even with the polymer moiety attached) show efficient membrane penetration by receptor-mediated energy-dependent endocytosis pathway. In order to design the compounds of the present invention, it has been found that rather than linking the CPP to the payload molecule via the polymer moiety, it is advantageous (often compulsory for the effective transport of many payload molecules) to link the polymer moiety and the payload molecule to different amino acid residues of the CPP moiety of the compounds of the present invention.

[0034] According to a preferred embodiment, the compound of the present invention further comprises a restriction endonuclease as a payload molecule to be delivered into the biological cell covalently linked to the polypeptide of general formula (I), said restriction endonuclease being preferably a class II restriction endonuclease, in particular a PvuII restriction endonuclease or a derivative thereof, said derivative being a single-chain PvuII restriction endonuclease, e.g. SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY, SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGG, SGGSSHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY, SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSG、 MSHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGC、 CSGGSSHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY、 MSHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY, MSHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY, MSHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGSGGS, SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGSGGS, or GSGGSGGSSHPDLNKLLELWPHIQEYQDLALKHGIN- DIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIA- KYRQVPWIFAIYRGIAIEAIYRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVME- HGTKIY It is a derivative having the amino acid sequence of

[0035] Thus, a particularly preferred embodiment of the compound of the invention comprises as payload two monomeric subunits of a type II restriction endonuclease or a part thereof. Preferably, the two monomeric alpha-helical subunits of the type II restriction endonuclease may be covalently linked by a linker. More preferably, the two monomeric alpha-helical subunits may be subunits of PvuII. Specifically, compounds comprising two monomeric subunits of the type II restriction endonuclease PvuII or a part thereof covalently linked by a linker to the CPP portion of the compound of the invention are preferred. The linker is preferably an amino acid linker of 1 to 20 amino acid residues in length, preferably 1 to 15 amino acid residues in length, more preferably 7 to 13 amino acid residues in length, in particular 8 to 12 amino acid residues in length. Preferred amino acid linkers are linkers which comprise or consist of a cysteine, serine or glycine residue (or 2, 3, 4 or 5 consecutive cysteine ​​or glycine residues), in particular one or two adjacent cysteine ​​residues; or an amino acid linker which comprises a glycine residue and a serine residue, or which comprises or consists of the amino acid sequence GSG, SGG, GGC, GCS, CSG, GGS, GSGG, SGGS, GSGGC, CSGGS, GSGGSGGS, GSGGCCSGGS, GSGCCCSGGGS, or GSGGCSGGS.

[0036] Type II restriction endonucleases have been reviewed, for example, by Pingoud et al. (NAR 29 (2001), 3705-3727; and NAR 42 (2014), 7489-7527); thus, the general structure and function of type II restriction endonucleases is well available to the skilled artisan. A "derivative" of a restriction endonuclease is a polypeptide that is modified compared to a wild-type restriction endonuclease but still contains the main function of the wild-type restriction endonuclease, i.e., specific cleavage properties, i.e., the ability to cleave a nucleic acid molecule at a sequence-specific site. In the preferred example of PvuII, this means that a PvuII derivative is a derivative of the wild-type PvuII polypeptide and is modified compared to the wt PvuII polypeptide, but still contains the function of wild-type PvuII to specifically recognize the double-stranded DNA sequence 5'-CAGCTG-3' and cleave after G-3 (Cheng et al., EMBO J. 13 (1994), 3927-3935).

[0037] According to a preferred embodiment, the polypeptide of general formula (I) in the compound of the present invention is covalently linked by a linker to another moiety, which is preferably a payload molecule, a labeling group and / or a homopolymer to be delivered into a biological cell.

[0038] A preferred embodiment is a compound of the invention further comprising a capping group, preferably an alkoxy group, in particular a methoxy, ethoxy, propoxy, or butoxy group; a halogen atom; or a tosylate; an isocyanate, hydrazine hydrate, maleimide, orthopyridyl disulfide, N-succinimidyloxy, sulfo-N-succinimidyloxy, 1-benzotriazole, 1-imidazolyloxy, p-nitrophenyloxy, or an aldehyde.

[0039] Another aspect of the present invention relates to a compound of the present invention for use in the treatment of a tumor patient, preferably a tumor patient suffering from neuroblastoma, colon cancer, hepatocellular carcinoma, small cell lung cancer, ovarian cancer, lung cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, placental cancer, breast cancer, kidney cancer, liver cancer, pancreatic cancer, muscle cancer, skin cancer, connective tissue cancer, bone cancer, and any of these cancers in which the patient has already developed metastases, in particular for the treatment of patients with neuroblastoma, colon cancer, hepatocellular (liver) cancer, small cell lung cancer, lung cancer, breast cancer, pancreatic cancer, and any of these cancers in which the patient has already developed metastases.

[0040] Another aspect of the present invention relates to a method for treating a tumor patient, wherein an effective amount of a compound according to the present invention is administered to a patient in need thereof, preferably the tumor patient is suffering from neuroblastoma, colon cancer, hepatocellular carcinoma, small cell lung cancer, ovarian cancer, lung cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, placental cancer, breast cancer, kidney cancer, liver cancer, pancreatic cancer, muscle cancer, skin cancer, connective tissue cancer, bone cancer and any of these cancers in which the patient has already developed metastases, in particular for the treatment of patients with neuroblastoma, colon cancer, hepatocellular (liver) cancer, small cell lung cancer, lung cancer, breast cancer, pancreatic cancer and any of these cancers in which the patient has already developed metastases.

[0041] Another aspect of the present invention relates to the use of a compound according to the present invention for the preparation of a medicament, preferably for the treatment of tumor patients, more preferably for the treatment of patients with neuroblastoma, colon cancer, hepatocellular carcinoma, small cell lung cancer, ovarian cancer, lung cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, placental cancer, breast cancer, kidney cancer, liver cancer, pancreatic cancer, muscle cancer, skin cancer, connective tissue cancer, bone cancer and any of these cancers in which the patient has already developed metastases, in particular for the treatment of patients with neuroblastoma, colon cancer, hepatocellular (liver) cancer, small cell lung cancer, lung cancer, breast cancer, pancreatic cancer and any of these cancers in which the patient has already developed metastases.

[0042] As used herein, "CPP" refers to an amino acid sequence or a polynucleotide encoding said amino acid sequence that facilitates active transport of biological macromolecules through biological membranes or physiological barriers. Transduction through biological membranes or physiological barriers can be determined by various processes, for example, by a cell membrane permeability test, which has a first incubation step with a PTD conjugated to a marker in the presence of cultured cells, followed by a fixation step, and then detection of the presence of the marked peptide in the cells. Preferably, the CPP activity of a given molecule is tested according to the model system disclosed in the Examples section, using the CROMOC molecule as a payload model. Alternatively, detection can be achieved by incubating the CPP in the presence of a labeled antibody against the CPP, and then detecting an immunological reaction between the amino acid sequence of the CPP and the labeled antibody in the cytoplasm or in the immediate vicinity of or within the cell nucleus. Cell membrane permeability tests are well known to those skilled in the art.

[0043] The term "capping group" as used herein means any suitable chemical group, which is non-reactive or reactive with other chemical moieties as desired. Thus, the capping group is selected to provide monofunctionality, i.e., a terminal aldehyde group, or bifunctionality, i.e., an aldehyde group at one end and a different functional moiety at the opposite end. If the capping group is non-reactive with other chemical moieties, the resulting polymeric aldehyde derivative structure is monofunctional and thus can be covalently linked to only one chemical moiety of interest. In other words, if the capping group is non-reactive, the terminal aldehyde group of the compound of the present invention allows for easy covalent linkage to the chemical moiety of interest, for example, to the α-amino group of a polypeptide. Suitable capping groups are generally known in the art and are disclosed, for example, in WO 2004 / 013205. Suitable non-reactive capping groups include, for example, alkoxy, such as methoxy, ethoxy, propoxy, or butoxy; halogen atoms (i.e., fluorine, chlorine, bromine, or iodine); or tosylate; and suitable reactive capping groups include, for example, isocyanate, hydrazine hydrate, maleimide, orthopyridyl disulfide, N-succinimidyloxy, sulfo-N-succinimidyloxy, 1-benzotriazole, 1-imidazolyloxy, p-nitrophenyloxy, or aldehyde.

[0044] The terms "group," "functional group," "moiety," "active moiety," "active compound," "effective compound," "target," "target molecule," "radical," and the like, are somewhat synonymous in the chemical arts and are used in the art and herein to refer to distinct, definable portions or units of the compounds of the invention and units that perform some function or activity and are reactive with other molecules or portions of molecules. In this sense, small molecule drugs or polypeptide residues, when attached to the compounds of the invention, can be considered functional groups or moieties.

[0045] The term "small molecule drug" or "small molecule" as used herein preferably refers to a medicinal organic compound having a molecular mass of less than 1000 Da, typically between 300 Da and 1000 Da, preferably between 300 Da and 700 Da. Small molecule drugs activate or inhibit the function of a biomolecule, thereby providing a therapeutic benefit to a patient, e.g., a mammal, preferably a human. This is because small molecule drugs usually bind with high affinity to a biomolecule, such as a polypeptide, nucleic acid, or polysaccharide, and alter the activity or function of the biomolecule. Small molecule drugs can be natural compounds (e.g., alkaloids, terpenoids, steroids, glycosides, natural phenols, phenazines, polyketides, fatty acid synthase products, non-ribosomal peptides, macrolactones, and secondary metabolites including polyphenols, etc.) or their artificial counterparts.

[0046] Preferred small molecule drugs for use in the present invention are cytotoxic agents, particularly those used in cancer therapy. Such drugs generally include DNA damaging agents, antimetabolites, natural products, and analogs thereof.

[0047] Preferred classes of cytotoxic agents include, for example, natural or synthetic tubulysins; natural or synthetic epothilones; duocarmycins; auristatins; maytansinoids; calicheamicins; mesothelin; anthracyclines; dihydrofolate reductase inhibitors; and thymidylate synthase inhibitors; DNA intercalators; DNA cleaving agents; topoisomerase inhibitors; vinca drugs; mitomycins; bleomycins; cytotoxic nucleosides; the pteridine family of drugs; diynenes; podophyllotoxins; differentiation inducers; and taxanes. Useful members of these classes include, for example, erlotinib (TARCEVAR®, Genentech / OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diammine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ), trastuzumab (HERCEPTIN®, Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide, CAS number 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethanamine, NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1 / 2, HPPD, and rapamycin. Also useful cytotoxic agents include oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.),Sutent (SUNITINIB®, SU1 1248, Pfizer), Letrozole (FEMARA®, Novartis), Imatinib Mesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007 / 044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1 126 (PI3K inhibitor, Semaphores), Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787 / ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™ (Cremophor-free), vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chlorambucil, AG1478, AG1571 (SU5271; Sugen), temsirolimus (TEZ-1000; Sigma-Aldrich), tetanib ... Limus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), camposamide (TELCYTA®, Telik), thiotepa, and cyclophosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, metholedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide,Ethylenimines and methylamelanamines, including triethylenethiophosphoramide, and trimethylomelanine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatins; kallistatins; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analog KW-2189 and the synthetic analog CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongiostatins; nitrogen mustards such as chlorambucil, chromafazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trophosphamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calciamine, calciamine gamma 1, calciamine omega 11 (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186), dynemicin, dynemicin A; bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromoprotein enediine antibiotic chromophores), aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detrevicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, queramycin, rodorubicin, streptomycin, streptozocin, tubercidin, ubenistatin, zinostatin,zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmoful, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenergics such as aminoglutethimide, mitotane, trilostane ;Folic acid supplements such as floric acid;Aceglatone;Aldophosphamide glycosides;Aminolevulinic acid;Eniluracil;Amsacrine;Vestravcil;Bisantrene;Edatraxate;Defofamine;Demecolcine;Diazicon;Elfomitin;Elliptinium acetate;Epothilone;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidynin;Maytansinoids such as maytansine and ansamitocin;Mitoguazone;Mitoxantrone;Mopidanmol;Nitrelin;Pentostatin;Phenamet;Pirarubicin;Rosoxantrone;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); Razoxane; Rhizoxin; Sizofiran; Spirogermanium; Tenuazonic acid; Triazicon; 2,2',2"-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, veraculin A, roridin A, and anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitrol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C ");cyclophosphamide;thiotepa;6-thioguanine;mercaptopurine;methotrexate;platinum analogues such as cisplatin and carboplatin;vinblastine;etoposide (VP-16);ifosfamide;mitoxantrone;vincristine;vinorelbine (NAVELBINE®);novantrone;teniposide;edatrexate;daunomycin;aminopterin;capecitabine (XELODA®,Roche); ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharma- ceutically acceptable salts, acids, and known functional derivatives thereof.

[0048] Also included within the definition of "chemotherapeutic agent" are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxyphene, ketoxifene, LY117018, onapristone, and FARESTON® (toremifene citrate); (ii) anti-inflammatory drugs, such as, for example, 4(5)-imidazole, aminoglutethimide, MEGASE ( (iii) aromatase inhibitors, which inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as AZIMILAR ® (megestrol acetate), AROMASIN ® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR ® (vorozole), FEMARA ® (letrozole; Novartis), and ARIMIDEX ® (anastrozole; AstraZeneca); (iv) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analogues; (iv) protein kinase inhibitors, such as MEK inhibitors (WO 2007 / 044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, and H-Ras, e.g., oblimersen (GENASENSE®, Genta Inc.);(vii) ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME®), and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, such as m-RNA vaccines, ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitors such as LURTOTECAN®; ABARELIX® rmRH;(ix) antiangiogenic agents such as bevacizumab (AVASTIN®, Genentech); leurosine, carminomycin, tallysomycin, podophyllotoxin, retinoic acid, butyric acid, N. 8 -Acetylspermidine, as well as pharma- ceutically acceptable salts, acids, and any known functional analogues thereof.

[0049] The term "labeling group" as used herein refers to a label, tag, tracer, or marker, i.e., a readily recognizable chemical moiety, that allows tracking of the translocation and / or chemical or biological transformation of a moiety of interest, for example, a conjugate formed with a compound of the present invention. Suitable labeling groups include, for example, affinity labels, including antibodies and antibody fragments, radiolabels, and fluorophores. Particularly useful examples of labeling groups include fluorescein, Cy5.5, and related cyanines that emit in the near UV.

[0050] As used herein, the term "biological macromolecule" refers to any macromolecule produced by an organism, including large polymeric molecules such as antibodies, enzymes, polypeptides involved in cell signaling and signal transduction processes, membrane polypeptides that act as receptors, polysaccharides, lipids, nucleic acids, and polynucleotides, as well as polypeptides (which include the term "protein"), including primary metabolites, secondary metabolites, and natural products. Preferably, a "biological macromolecule" is a polypeptide or a polynucleotide.

[0051] As used herein, the terms "polypeptide," "protein," and "peptide" define an organic compound consisting of two or more amino acid residues arranged in a linear chain, in which the individual amino acids are linked by peptide bonds, i.e., amide bonds formed between adjacent amino acid residues. By convention, the primary structure of a polypeptide is reported starting from the amino-terminus (N)-terminus to the carboxyl-terminus (C)-terminus.

[0052] The term "antibody" herein is used in the broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. Antibodies may be murine, human, humanized, chimeric, or derived from other species. Antibodies are polypeptides generated by the immune system capable of recognizing and binding to a specific antigen. A target antigen generally has multiple binding sites, also called epitopes, that are recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules, or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to an antigen of interest or a portion thereof, such targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulin can be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. The immunoglobulin can be from any species, including from human, murine, or rabbit origin.

[0053] An "antibody fragment" comprises a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; bispecific antibodies; linear antibodies; fragments produced by a Fab expression library; anti-idiotypic (anti-Id) antibodies, CDRs (complementarity determining regions), and epitope-binding fragments of any of the above that immunospecifically bind to a cancer cell antigen, a viral antigen, or a microbial antigen; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0054] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for the possibility of naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by hybridoma methods or by recombinant DNA methods. Monoclonal antibodies may also be isolated from phage antibody libraries using established techniques. Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chains are identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. Chimeric antibodies include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey or Ape) and human constant region sequences.

[0055] An "intact antibody" herein comprises a VL domain and a VH domain, as well as a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. An intact antibody may have one or more "effector functions", which refer to the biological activities attributable to the Fc region of an antibody (native sequence Fc region or amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down-regulation of cell surface receptors such as B cell receptor and BCR. Depending on the amino acid sequence of the constant domain of the heavy chain, intact antibodies can be assigned to different "classes". There are five major classes of complete antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0056] Particularly useful antibodies include alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech / Biogen), and rituximab (RITUXAN®, Genentech / Biogen). Idec), pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), as well as the humanized monoclonal antibodies alemtuzumab, apolizumab, aselizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labbetu tuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, norobizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pexituzumab, pectuzumab, pertuzumab, pexelizumab, ralibizumab, ranibizumab, reslivizumab, reslizumab, resi Therapeutic antibodies such as vizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab celmoleukin, tucituzumab, umavizumab, urtoxazumab, and visilizumab.

[0057] The term "amino acid" as used herein refers to any organic acid containing one or more amino substituents of an aliphatic carboxylic acid, such as an α-amino derivative, a β-amino derivative or a γ-amino derivative. Preferably, the term "amino acid" refers to the 22 most common naturally occurring L-amino acids selected from the group consisting of glycine, leucine, isoleucine, valine, alanine, phenylalanine, tyrosine, tryptophan, aspartic acid, asparagine, glutamic acid, glutamine, cysteine, methionine, arginine, lysine, proline, serine, threonine, histidine, selenocysteine, and pyrrolysine. Of these, the 20 amino acids encoded by the universal genetic code may also be referred to herein by their conventional three-letter or one-letter abbreviations, which abbreviations follow conventional usage. The remaining two, selenocysteine ​​and pyrrolysine, are incorporated into polypeptides by their own synthetic mechanisms. Unless otherwise specified herein, all references to "amino acid" or "amino acid residue" are intended to refer to any of the 20 naturally occurring L-amino acids encoded by the genetic code.

[0058] The term "amino acid side chain" includes groups found in (i) naturally occurring amino acids, such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; (ii) minor amino acids, such as ornithine and citrulline; and (iii) non-natural amino acids, β-amino acids, synthetic analogs, and derivatives of naturally occurring amino acids.

[0059] The terms "nucleic acid", "nucleic acid sequence", "oligonucleotide", "nucleotide", or "polynucleotide" as used herein refer to any DNA- or RNA-like material of natural or synthetic origin, including DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA, gRNA, crRNA, traceRNA), peptide nucleic acid (PNA), or, for example, iRNA, ribonucleoproteins (e.g., double-stranded iRNA, e.g., iRNP), of genomic or synthetic origin, which may be single-stranded or double-stranded and may represent the sense or antisense strand. These terms encompass natural and synthetic nucleic acids, including known analogs of natural nucleotides. The term also encompasses nucleic acid-like structures with synthetic backbones. "Oligonucleotide" includes either a single-stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands, which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and therefore will not ligate to another oligonucleotide unless a phosphate is added with an ATP in the presence of a kinase. Synthetic oligonucleotides can be ligated to fragments that have not been dephosphorylated. A "coding sequence" or "nucleotide sequence encoding" a particular polypeptide is a nucleic acid sequence that is transcribed and translated into a polypeptide when placed under the control of appropriate regulatory sequences. Nucleic acids used to practice the invention may be isolated from a variety of sources, genetically engineered, amplified, and / or expressed / produced recombinantly. Techniques for the manipulation of nucleic acids, such as subcloning, labeling probing (e.g., random primer labeling with Klenow polymerase, nick translation, amplification), sequencing, hybridization, and the like, are well described in the scientific and patent literature. Nucleic acids encoding a polypeptide of the invention are assembled in appropriate phase with a leader sequence capable of directing secretion of the translated polypeptide or fragment thereof.

[0060] The compounds referred to herein are meant and claimed as "isolated" and / or "purified" and / or "synthetic" compounds. As used herein, the term "isolated" means that the compound is removed from any natural environment, e.g., if the compound (or a portion thereof) occurs in nature. The term "purified" does not require absolute purity. Rather, it is intended as a relative definition. As used herein, the term "synthetic" means that the compound does not occur in nature, but is synthesized in vitro by well-known chemical or biological (recombinant) synthesis techniques. The term "recombinant" or "chimeric" means that two or more functional moieties that are not linked, combined, or do not exist in a natural environment are linked together, thereby providing a novel combination of the functional moieties that does not occur in nature, e.g., a polypeptide domain from one protein is linked to a polypeptide domain from a second, different, unrelated protein. Recombinant polypeptides are produced from nucleic acids encoding the recombinant polypeptides by expression in a suitable expression system, which can be further purified from the expression system, and further individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect, or plant cell expression systems.

[0061] The compound preferably further comprises a restriction endonuclease or a nicking enzyme as a payload.

[0062] Preferred restriction endonucleases are type II restriction endonucleases, especially AatII, AbaSI, Acc65I, AccI, AciI, AclI, AcuI, AfeI, AflII, AflIII, AgeI, AhdI, AleI, AluI, AlwI, AlwNI, ApaI, ApaLI, ApeKI, ApoI, AscI, AseI, AsiSI, AvaI, AvaII, BaeGI, BaeI, BamHI, BanI, BanII, BbsI, BbvCI, BbvI, BccI, BceAI, BcgI, BciVI, BclI, BcoDI, BfaI, BfuAI, BfuCI, BglI, BglII, BlpI, BmgBI, BmrI, BmtI, BpmI, Bpu10I, BpuEI, BsaAI, BsaBI, BsaHI, BsaI, BsaJI, BsaWI, BsaXI, BseRI, BseYI, BsgI, BsiEI, BsiHKAI, BsiWI, BslI, BsmAI, BsmBI, BsmFI, BsmI, BsoBI, Bsp1286I, BspCNI, BspDI, BspEI, BspHI, BspMI, BspQI, BsrBI, BsrDI, BsrFI, BsrGI, BsrI, BssHII, BssKI, BssSαI, BstAPI, BstBI, BstEII, Bst, BstUI, BstXI, BstYI, BstZ17I, Bsu36I, BtgI, BtgZI, BtsαI, BtsCI, Cac8I, ClaI, CspCI, CviAII, CviKI-1, CviQI, DdeI, DpnI, DpnII, DraI, DraIII, DrdI, EaeI, EagI, EarI, EciI, Eco53kI, EcoNI, EcoO109I, EcoP15I, EcoRI, EcoRV, FatI, FauI, Fnu4HI, FokI, FseI, FspEI, FspI, HaeIII, HgaI, HhaI, HincII, HindIII, HinfI, HinP1I, HpaI, HpaII, HphI, Hpy166II, Hpy188I, Hpy188III, Hpy99I, HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V, I-CeuI, I-SceI, KasI, KpnI, LpnPI, MboI, MboII, MfeI, MluCI, MluI, MlyI, MmeI, MnlI, MscI, MseI, MslI, MspA1I,MspI, MspJI, MwoI, NaeI, NarI, Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BtsI, NciI, NcoI, NdeI, Ng oMIV, NheI, NlaIII, NlaIV, NmeAIII, NotI, NruI, NsiI, NspI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI. Nt.BspQI, Nt.BstNBI, Nt.CviPII, PacI, PaeR7I, PciI, PflFI, PflMI, PI-PspI, PI-SceI, Ple I, PluTI, PmeI, PmlI, PpuMI, PshAI, PsiI, PspGI, PspOMI, PspXI, PstI, PvuI, PvuII, RsaI, Rs rII, Sac, SacII, SalI, SapI, Sau3AI, Sau96I, SbfI, ScaI, ScaI, ScrFI, SexAI, SfaNI, SfcI, S fiI, SfoI, SgrAI, SmaI, SmlI, SnaBI, SpeI, SphI, SspI, StuI, StyD4I, StyI, SwaI, TaqαI, Tfi I, TseI, Tsp45I, TspMI, TspRI, Tth111I, XbaI, XcmI, XhoI, XhoI, XmaI, XmnI, and ZraI were strong; In this case, there are a number of snowflakes Machine (Free Enrichment of DNA Shuang-yong Xu,Biomolecular Concepts,De Gruyter August 7,2015).

[0063] Further preferred payloads, particularly polypeptidic payloads, are zinc finger nucleases (ZFNs), zinc finger transcriptional activators (ZFAs), zinc finger transcriptional repressors (ZFRs), and zinc finger methylases (ZFMs); enzymes in diseases for enzyme replacement therapy, such as glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (G6PD), 21-hydroxylase (EC 1.14.99.10), steroid 11β-monooxygenase (EC 1.14.15.4), 3α-hydroxysteroid-3-dehydrogenase (EC 1.1.1.50), steroid 17α-monooxygenase (EC 1.14.99.9), cholesterol monooxygenase (EC 1.14.15.6), glucosylceramidase (EC 3.2.1.45), galactosylceramidase (EC 3.2.1.46), pyruvate kinase (EC 2.7.1.40), α-galactosidase (EC 3.2.1.22), β-galactosidase (EC 3.2.1.23), acid α-glucosidase (GAA), α-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2.), collagenase (EC 3.4.24.7), glutaminase (EC 3.5.1.2), asparaginase (EC 3.5.1.1), lysozyme (EC 3.2.1.17), bilirubin oxidase (EC 1.3.3.5), thiosulfate sulfur transferase (EC 2.8.1.1), β-lactamase (EC 3.5.2.6), streptokinase, urokinase (EC 3.4.21.73), hyaluronate glucosaminidase (EC 3.2.1.35), hyaluronate glucuronidase (EC 3.2.1.36), hyaluronate lyase (EC 4.2.2.1), superoxide dismutase (EC 1.15.1.1), urease (EC 3.5.1.5), arginase (EC 3.5.3.1), glutamyl-transferase (EC 2.3.2.2.), glutathione-S-transferase (EC 6.3.1.13), creatine kinase (EC 2.7.3.2), aldolase (EC 4.1.2.13), lipase (EC 3.1.1.79), hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34), xanthine oxidase, PRPP synthase (EC 2.7.6.1), hypoxanthine-guanine-phosphoribosyltransferase (EC 2.4.2.8), DNAse (EC 3.1.22.1), RNAse (EC 3.1.27.5), branched-chain ketoacid dehydrogenase (EC 1.2.4.4), adenosine deaminase (EC 3.5.4.4), hexoseaminidase (EC 3.2.1.52), β-galactosidase (EC 3.2.1.23), sphingomyelinase (EC 3.1.4.12), ceramidase (EC 3.5.1.23), homogenate oxidase, β-glucosidase (EC 3.2.1.21), lactase (EC 3.2.1.108), HIV-protease (EC 3.4.23.16), dihydrofolate reductase (EC 1.5.1.3), alcohol dehydrogenase (EC 1.1.1.2), iduronate-2-sulfatase (EC 3.1.6.13), or sphingomyelin phosphodiesterase (EC 3.1.4.12).

[0064] ZFN is achieved by linking several zinc finger (ZF) motifs in tandem to form ZFP (zinc finger protein). For the designed ZFP, other functionalities such as a non-specific FokI cleavage domain (N, nuclease), a transcription activator domain (A), a transcription repressor domain (R), and a methylase (M) are fused to the ZFP to form ZFN, respectively.

[0065] The compounds of the present invention can also include editing nucleases, CRISPR nucleases, and hybrids thereof. The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA-carrying sequences that are complementary to the 20-nucleotide segment in the guide RNA. Cas9 and its orthologues have emerged as versatile molecular tools for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. The HNH motif (His-Asn-His) nuclease-like domain in Cas9 cleaves the DNA strand that is complementary to the RNA guide, and the RuvC endonuclease-like domain initiates the cleavage of the non-complementary DNA strand to the guide RNA, resulting in a blunt-ended DNA double-strand break between the +3 and +4 phosphates upstream of the PAM sequence, respectively. This clearly separable molecular feature is used in the art to engineer single-stranded cutters (or nickases), or catalytically inactive enzymes that still exhibit guide RNA-dependent binding selectivity. Along these lines, single point mutations at H840A, N863A, N854A, or D839A, or combinations thereof, result in inactive HNH nuclease cleavage, and mutations at any of the catalytic amino acids H983A, D986A, D10A, or E762A result in loss of function of RuvC nuclease-like activity, thus producing a Cas9 enzyme that can be used as a single-stranded cutter or nickase. Loss of Cas9 nuclease function can result from a combination of HNH nuclease null mutations and RuvC nuclease null mutations in a single Cas9 polypeptide, e.g., D10A, D839A, H840A, N863A. These variations of CRISPR nucleases can be produced as polypeptides fused to a polypeptide of general formula (I) in a similar manner as illustrated for HNH mutant nickase, RuvC mutant nickase, or Cas9-NLS-DDM, which are examples of expression constructs for Cas9 null nuclease. This is also applicable to non-natural editing enzymes. In the present invention, "Cas9" refers toIt refers to any type of functional Cas9 polypeptide nuclease enzyme with or without one or more guide RNAs (gRNAs), including Cas9 enzymes with nicking activity or with impaired nuclease but DNA binding activity; sequences for designing Cas9 molecules include those derived from, for example, Acetobacter, Acholeplasma, Acidaminococcus, Acidovorax, Actinobacillus, us), Afipia, Alicycliphilus, Alistipes, Aminomonas, Anaerococcus, Arthrobacter, Atopobium, Bacillus, Bacteroidales, Bacteroides, Bacteroidetes, Barnesiella la), Bdellovibrio, Belliella, Bergeyella, Bibersteinia, Bifidobacterium, Blastopirellula, Bordetella, Bradyrhizobium, Brevibacillus, Brochothrix, Burkholderiales deriales, Butyrivibrio, Campylobacte, Capnocytophaga, Catellicoccus, Catenibacterium, Chryseobacterium, Clostridiales Clostridium, Collinsella, Coprobacillus,Coriobacteriaceae, Corynebacterium, Cryptobacterium, Cryptococcus, Cyclobacteriaceae, Defluviimonas, Dialister, Dinoroseobacter, Dolosigranulum, Dorea, Eggerthella, Elizabethanth Elizabethkingia, Elusimicrobium, Enterococcus, Escherichia, Eubacterium, Facklamia, Fibrobacter, Filifactor, Finegoldia, Firmicutes, Flavobacterium, Fluviicola taphensis taffensis, Francisella, Francisella, Fusobacterium, Galbibacter, Gemella, Geobacillus, Gluconacetobacter, Gordonibacter, Gracilibacillus, Granulibacter bacter, Haemophilus, Halobacillus, Helcococcus, Helicobacter, Hyphomonas, Ignavibacterium, Ilyobacter, Indibacter, Joostella, Kingella, Kordia,Lachnospiraceae, Lactobacillus, Legionella, Leptotrichia, Leuconostoc, Listeria, Mannheimia, Maritimibacter, Marmosops, Megasphaera, Mollicutes, icutes, Mucilaginibacter, Mucinivorans, Mucispirillum, Mycoplasma, Myroides, Narcissus, Negativicoccus, Neisseria, Niabella, Nitratifractor, Nitrobacter, Nitroso Nitrosomonas, Novosphingobium, Oceanobacillus, Odoribacter, Oenococcus, Oligella, Olsenella, Oribacterium, Ornithobacterium, Parabacteroides, Parasutterel la), Parvibaculum, Parvimonas, Pasteurella, Pediococcus, Pedobacter, Peptoniphilus, Peptostreptococcaceae, Phascolarctobacterium, Planococcus,Porphyromonadaceae, Porphyromonas, Prevotella, Proteobacterium, Pseudoramibacter, Psychroflexus, Puniceispirillum, Ralstonia, Rhodopseudomonas, Rhodospirillum illum, Rhodovulum, Riemerella, Roseburia, Ruminococcus, Saccharibacter, Salinispira, Scardovia, Simonsiella, Solobacterium, Sphaerochaeta, Sphingobacterium, Sphingobacterium, Sphingobium, Sphingomonas, Spiroplasma, Sporocytophaga, Staphylococcus, Streptobacillus, Streptococcus, Subdoligranulum, Succinatimonas, Sulfitobacter, Sulfurimonas, Sulfuritalea, Sulfurospirillum, Sulfurovum, Sutterella, Tannerella, Tistrella, Treponema, Veillonella, Verminephrobacter, Weeksella, Wolinella,The genera include the type II CRISPR system from Zunongwangia. Based on the published Cas9 sequences from these genera (UNIprot-Expasy), the corresponding DNA can be easily synthesized by synthetic DNA techniques well described in the art or by PCR cloning from the respective hosts. Cas9 also has RNA binding and targeting functions used to form Cas9-like molecules, and nucleases have nucleic acid modifying enzyme activities, , methylases, acetylases, deacetylases, polymerases, reverse transcriptases, RNases, Rnase H; helicases, recA-like activity, topoisomerases, transcription factors, activators, transcription repressors, ubiquitin, and chimeras supplemented or replaced by other enzymatic functions such as smoligases, debuchitinases, etc. By "gRNA" we mean the mature crRNA:tracrRNA complex, or truncated RNA chimeras containing these engineered hairpins. These RNAs can ideally be obtained by chemical synthesis or in vitro transcription protocols well known in the art. Chemical synthesis further offers the possibility of using gRNAs containing non-natural RNA nucleotides, which provides an additional advantage for using Cas9-CPP polypeptide-RNA complexes as medicines. Examples of modified nucleotides are partial or complete use of chemically modified RNA, such as phosphorothioate (PS), locked nucleic acid (LNA) 2'-O-methoxyethyl (MOE), 2'-O-methyl (OMe), or 2'-fluoro (2'-F) modified nucleotides. These compounds of the present invention can be applied to in vitro, ex vivo, and in vivo editing of genetic information. For this, the compounds of the present invention are applied directly to cell culture medium or cell culture buffer for in vitro or ex vivo application, or directly injected by applying specific Cas9-CPP compounds into the bloodstream or subcutaneously to the whole body for in vivo application. These compounds of the present invention can be used in particular for gain-of-function editing by homologous recombination using a nucleic acid-containing compound as a template for recombination and Cas9 nuclease to introduce a site-specific double-strand break near the mutation. This mixture can be used to introduce site-specific genetic changes useful for introducing site-specific mutations, including repair of gene defects.

[0066] The Cas9-CPP polypeptides and Cas9-CPP:RNA complexes of the invention can be obtained by a) recombinant techniques expressing any Cas9-CPP molecule, followed by purification of this molecule by a chromatographic step, optional PEGylation / purification, followed by loading of the RNA compound required for function. Alternatively, b) Cas9-CPP protein moieties can be co-expressed with the required RNA compound, either encoded from a plasmid or integrated into the genomic locus of the host cell, preparing extracts, purifying the Cas9-CPP:RNA complex by a chromatographic step under conditions that maintain the Cas9-CPP:RNA complex, and then PEGylating and purifying these isolates to homogeneity as an optional final step. The resulting polypeptides can be lyophilized or frozen for storage in the absence or presence of stabilizers such as glucose or trehalose.

[0067] According to a further aspect, the present invention also relates to a pharmaceutical formulation comprising a compound according to the invention and a protein kinase inhibitor (PKI), preferably a DNA-dependent PKI, more preferably a non-homologous end joining (NHEJ) and V(D)J repair factor DNA-dependent PKI, in particular 7-methyl-2-[(7-methyl[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one (AZD7648).

[0068] Therefore, the present invention discloses the following preferred embodiments.

[0069] 1.General formula (I): X0GX1X2GX3X4X5GX6X7X8GX9X 10 X 11 X 12 X 13 X 14 , A compound comprising or consisting of a polypeptide of the above formula (1). (In the formula, G is glycine; X0 is present or absent, and if present is an amino acid linker; X1 is N or S, where N is asparagine and S is serine; X2 is S or T, where S is serine and T is threonine; X3 is present or absent and, if present, is S, where S is serine; X4 is present or absent and, if present, is G, where G is glycine; X5 is a basic polypeptide stretch consisting of 5 basic amino acid residues selected from R and K, where R is arginine and K is lysine, preferably X5 comprises 3 or more K amino acid residues, in particular X5 is KKKKK or KRKKK; X6 is a basic amino acid residue selected from R and K, where R is arginine and K is lysine, preferably, X6 is K; X7 is S or L, where S is serine and L is leucine; X8 is present or absent, and if present is a polypeptide stretch consisting of the amino acid sequence GLGS, where G is glycine, L is leucine, and S is serine; X9 is a basic polypeptide stretch consisting of three basic amino acid residues selected from R and K, where R is arginine and K is lysine, preferably X9 comprises two or more K amino acid residues, in particular X9 is KKK or KKR; X 10 is present or absent and, when present, is L or a polypeptide stretch consisting of the amino acid sequence DPL or DPC, where L is leucine, D is aspartic acid, P is proline and C is cysteine; X 11 is present or absent and, if present, is a polypeptide stretch consisting of the amino acid sequence LR or GSGL, where L is leucine, R is arginine, G is glycine, and S is serine; X12 is present or absent, and if present is a basic polypeptide stretch having 20% ​​or more basic amino acid residues selected from K and R and at least P residues, preferably X 12 KYKPKL, KYKPKLGT, or GX3X4X5GX6X7X8GX9X 10 where K, R, P, L, G, T, X3, X4, X5, GX6, X7, X8, GX9, and X 10 is defined as above, and Y is tyrosine; X 13 is present or absent and, if present, is a polypeptide stretch consisting of the amino acid sequence GS, GST, GST, GSG, GSTG, or GSGL, where G, S, T, and L are defined as above; X 14 is present or absent, and if present is an amino acid linker; The polypeptide has a length of 20 to 75 amino acid residues, preferably 24 to 65 amino acid residues, and particularly 25 to 60 amino acid residues.

[0070] 2. X1 is N, X2 is S, X3 is S, X4 is G, X5 is KKKKK or KRKKK, X6 is K, X9 is KKK or KKR, and X 10 is L or a polypeptide stretch consisting of the amino acid sequence DPL or DPC, where N, S, G, R, L, D, P, and C are defined as above.

[0071] 3. The compound is GNSGSGKRKKKGKGLGKKRDPCLRKYKPKLGTGSTG(HHHHHH)(DDM11), GNSGKRKKKGKGSGLGSGKKRDPCLRKYKPKLGSTG(HHHHHH)(DDM18), GNSGSGKRKKKGKGLGKKRDPCLRKYKPKLGTGSTG(HHHHHH)(DDM21), GNSGSGKRKKKGKGSGLGSGKKRDPLGSTG(HHHHHH)(DDM26), GNSG(HHHHHH)GSTGGKRKKKGKGSGLGSGKKRDPL(DDM27), GNSGSGKRKKKGKGSGLGSGKKKDPLGSTG(HHHHHH)(DDM28), GNSG(HHHHHH)GSTGGKRKKKGKGSGLGSGKKKDPL(DDM29), GNSGSGKKKKKGKGSGLGSGKKRDPLGSTG(HHHHHH)(DDM30), GNSG(HHHHHH)GSTGGKKKKKGKGSGLGSGKKRDPL(DDM31), GNSGSGKKKKKGKGSGLGSGKKKDPLGSTG(HHHHHH)(DDM32), GNSG(HHHHHH)GSTGGKKKKKGKGSGLGSGKKKDPL(DDM33), GNSGSGKKKKKGKGSGLGSGKKKDPL(DDM34), GNSGSGKKKKKGKGSGLGSGKKKLGSTG(HHHHHH)(DDM35), GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST(DDM36), and GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGSTG(HHHHHH)GST(DDM44) is selected from the group consisting of where (HHHHHH; histidine tag) may be present or absent; Preferably, GNSGSGKRKKKGKGLGKKRDPCLRKYKPKLGTGSTG(DDM11), GNSGKRKKKGKGSGLGSGKKRDPCLRKYKPKLGSTG(DDM18), GNSGSGKRKKKGKGLGKKRDPCLRKYKPKLGTGSTG(DDM21), GNSGSGKRKKKGKGSGLGSGKKRDPLGSTG(DDM26), GNSGGSTGGKRKKKGKGSGLGSGKKRDPL(DDM27), GNSGSGKRKKKGKGSGLGSGKKKDPLGSTG(DDM28), GNSGGSTGGKRKKKGKGSGLGSGKKKDPL(DDM29), GNSGSGKKKKKGKGSGLGSGKKRDPLGSTG(DDM30), GNSGGSTGGKKKKKGKGSGLGSGKKRDPL(DDM31), GNSGSGKKKKKGKGSGLGSGKKKDPLGSTG(DDM32), GNSGSGKKKKKGKGSGLGSGKKKDPL(DDM34), GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST(DDM36), and GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGSTGGST(DDM44), is selected from the group consisting of especially, GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST(DDM36), and GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGSTGGST(DDM44) Selected from the group consisting of: A compound according to embodiment 1 or embodiment 2.

[0072] 4. The compound further comprises a payload molecule to be delivered into the biological cell covalently linked to the polypeptide of general formula (I), the payload molecule is preferably a chemotherapeutic molecule, a cytotoxic molecule, a DNA damaging molecule, an antimetabolite molecule, a therapeutic molecule, a small molecule having a therapeutic effect in the biological cell, a DNA molecule, an RNA molecule, an antibody molecule or an antibody derivative having an antibody-like function, a restriction endonuclease, a nicking enzyme, or a DNA-dependent endonuclease exhibiting double-stranded / single-stranded cleavage activity or no nuclease activity, more preferably the payload molecule is a class II restriction endonuclease, such as PvuII, EcoRV, PvuII, HinfI, or a sc homodimer, subunit, or functional fragment thereof, in particular the payload molecule is SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY, SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY, SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGGSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINPKIPVKYVMEHGTKIYGSGGGSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG-NDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINPKIPVKYVMEHGTKIYGSGGCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHCDINNPKIPVKYVMEHGTKIYGSGGCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGRE-NDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINPKIPVKYVMEHGTKIYGSGGCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY, SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY, and SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG-NDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY The compound according to any one of embodiments 1 to 3, which is selected from the group consisting of:

[0073] 5. The compound according to any one of the preceding claims, wherein the compound further comprises a homopolymer moiety covalently attached to the polypeptide of general formula (I), preferably the homopolymer being selected from the group consisting of polyethylene glycol (PEG), in particular PEG with a MW between 5 kDa and 30 kDa; dextran, polysialic acid, hyaluronic acid, dextrin, hydroxyethyl starch, or poly(2-ethyl 2-oxazoline).

[0074] 6. The compound further comprises a restriction endonuclease as a payload molecule to be delivered into the biological cell covalently linked to the polypeptide of general formula (I), said restriction endonuclease being preferably a class II restriction endonuclease, in particular a PvuII restriction endonuclease or a derivative thereof, wherein said derivative is a single-chain PvuII restriction endonuclease, e.g. SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY, SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGG、 SGGSSHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSG、 MSHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGC、 CSGGSSHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY、 MSHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY、 MSHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY、 MSHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGSGGS, SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGSGGS, or GSGGSGGSSHPDLNKLLELWPHIQEYQDLALKHGIN- DIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIA- KYRQVPWIFAIYRGIAIEAIYRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVME- HGTKIY The compound according to any one of embodiments 1 to 5, which is a derivative having the amino acid sequence of

[0075] 7. The compound according to any one of the preceding embodiments, wherein the polypeptide of general formula (I) is covalently linked by a linker to another moiety, which is preferably a payload molecule, a labelling group and / or a homopolymer to be delivered into a biological cell.

[0076] 8. The compound according to any one of the preceding embodiments, wherein the compound further comprises a capping group, preferably an alkoxy group, in particular a methoxy, ethoxy, propoxy, or butoxy group; a halogen atom; or a tosylate; an isocyanate, hydrazine hydrate, maleimide, orthopyridyl disulfide, N-succinimidyloxy, sulfo-N-succinimidyloxy, 1-benzotriazole, 1-imidazolyloxy, p-nitrophenyloxy, or an aldehyde.

[0077] 9. The compound according to any one of the preceding embodiments, wherein the compound further comprises a small molecule as a payload, the small molecule being a medicinal organic compound having a molecular mass of less than 1000 Da, preferably between 300 Da and 1000 Da, in particular between 300 Da and 700 Da.

[0078] 10. The compound according to any one of the preceding embodiments, wherein the compound further comprises as a payload a secondary metabolite, including alkaloids, terpenoids, steroids, glycosides, natural phenols, phenazines, polyketides, fatty acid synthase products, non-ribosomal peptides, macrolactones, and polyphenols.

[0079] 11. Compounds may be used as payloads, such as natural or synthetic tubulysins; natural or synthetic epothilones; duocarmycins; auristatins; maytansinoids; calicheamicins; mesothelin; anthracyclines; dihydrofolate reductase inhibitors; and thymidylate synthase inhibitors; DNA intercalators; DNA cleaving agents; topoisomerase inhibitors; vinca drugs; mitomycins; bleomycins; cytotoxic nucleosides; pteridine family drugs; diynenes; podophyllotoxins; differentiation inducers; and taxanes; erlotinib (TARCEVAR®, Genentech / OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS number 51-21-8), gemcitabine (GEMZAR®), Lilly), PD-0325901 (CAS number 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum(II), CAS number 15663-27-1), carboplatin (CAS number 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb 11. The compound of any one of embodiments 1 to 10, further comprising a cytotoxic agent comprising: trastuzumab (HERCEPTIN®, Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide, CAS number 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethanamine, NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1 / 2, HPPD, and rapamycin. Also useful cytotoxic agents include oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®,Millennium Pharm.), Sutent (SUNITINIB®, SU1 1248, Pfizer), Letrozole (FEMARA®, Novartis), Imatinib Mesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007 / 044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1 126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787 / ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH 66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™ (Cremophor-free), vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chlorambucil, AG1478, AG1571 (SU5271 ; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), camposamide (TELCYTA®, Telik), thiotepa, and cyclophosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, metholedopa, and uredopa; altretamine,Ethylenimines and methylamelanamines, including triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatins; kallistatins; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobi pancratistatin; sarcodictin; spongiostatin; nitrogen mustards such as chlorambucil, chromafazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trophosphamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calciamine, calciamine gamma 1, calciamine omega 11 (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186), dynemicin, dynemicin A; bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromoprotein enediine antibiotic chromophores), aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunol. bicine, detrebicine, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, queramycin, rodorubicin, streptomycin, streptozocin,tubercidin, ubenistatin, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmoful, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenergics such as aminoglutethimide, Mitotane, trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diazicon; elfomitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitrerin; pentostatin; phenamet; pirarubicin; rosoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Oreg.); Razoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triazicon; 2,2',2"-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, veraculin A, roridin A,and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche); ibandronate; CPT-11; topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharma- ceutically acceptable salts, acids, and known functional derivatives thereof;

[0080] Antihormonal agents that act to regulate or block hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs) (including tamoxifen (NOLVADEX®; including tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxyphene, ketoxifene, LY117018, onapristone, and FARESTON® (toremifene citrate); 4(5)-imidazole, aminoglutethimide, MEGASE® (megestrol acetate) aromatase inhibitors, which block the enzyme aromatase that regulates estrogen production in the adrenal glands, such as bromocriptine (exemestane; Pfizer), bromocriptine (formestanie), fadrozole, RIVISOR (vorozole), FEMARA (letrozole; Novartis), and ARIMIDEX (anastrozole; AstraZeneca); antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analogs; protein kinase inhibitors, such as MEK inhibitors; lipid kinase inhibitors; antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, and H-Ras, such as oblimersen (GENASENSE®, Genta Inc.); ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME®), and HER2 expression inhibitors; gene therapy vaccines, m-RNA vaccines, such as ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitors, such as LURTOTECAN®; ABARELIX® rmRH; antiangiogenic agents, such as bevacizumab (AVASTIN®, Genentech); leurosine, carminomycin, tallysomycin, podophyllotoxin, retinoic acid, butyric acid, N 8-acetylspermidine, as well as pharma- ceutically acceptable salts, acids, and any known functional analogs thereof.

[0081] 12. Compounds that contain as payloads alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech / Biogen Idec), pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), as well as the humanized monoclonal antibodies alemtuzumab, apolizumab, acelizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab, tuzumab-mertansine, cantuzumab-mertansine, cedelizumab, certolizumab pegol, cidofusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab-ozogamicin, inotuzumab-ozogamicin, ipilimumab, labetuzumab, lintuzumab, matsutake zumab, mepolizumab, motavizumab, motavizumab, natalizumab, nimotuzumab, norobizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pexituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resivizumab, rovelizumab, ruplizumab, sibromo 12. The compound of any one of embodiments 1 to 11, further comprising a therapeutic antibody, such as tuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab celmoleukin, tuxituzumab, umavizumab, urtoxazumab, and visilizumab.

[0082] 13. The compound has, as a payload, a restriction endonuclease, preferably a type II restriction endonuclease, in particular AatII, AbaSI, Acc65I, AccI, AciI, AclI, AcuI, AfeI, AflII, AflIII, AgeI, AhdI, AleI, AluI, AlwI, AlwNI, ApaI, ApaLI, ApeKI, ApoI, AscI, AseI, AsiSI, AvaI, AvaII, BaeGI, BaeI, BamHI, BanI, BanII, BbsI, BbvCI, BbvI, BccI, BceAI, BcgI, BciVI, BclI, BcoDI, BfaI, BfuAI, BfuCI, BglI, BglII, BlpI, BmgBI, BmrI, BmtI, BpmI, Bpu10I, BpuEI, BsaAI, BsaBI, BsaHI, BsaI, BsaJI, BsaWI, BsaXI, BseRI, BseYI, BsgI, BsiEI, BsiHKAI, BsiWI, BslI, BsmAI, BsmBI, BsmFI, BsmI, BsoBI, Bsp1286I, BspCNI, BspDI, BspEI, BspHI, BspMI, BspQI, BsrBI, BsrDI, BsrFI, BsrGI, BsrI, BssHII, BssKI, BssSαI, BstAPI, BstBI, BstEII, Bst, BstUI, BstXI, BstYI, BstZ17I, Bsu36I, BtgI, BtgZI, BtsαI, BtsCI, Cac8I, ClaI, CspCI, CviAII, CviKI-1, CviQI, DdeI, DpnI, DpnII, DraI, DraIII, DrdI, EaeI, EagI, EarI, EciI, Eco53kI, EcoNI, EcoO109I, EcoP15I, EcoRI, EcoRV, FatI, FauI, Fnu4HI, FokI, FseI, FspEI, FspI, HaeIII, HgaI, HhaI, HincII, HindIII, HinfI, HinP1I, HpaI, HpaII, HphI, Hpy166II, Hpy188I, Hpy188III, Hpy99I, HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V, I-CeuI, I-SceI, KasI, KpnI, LpnPI, MboI, MboII, MfeI, MluCI, MluI, MlyI, MmeI, MnlI, MscI, MseI,MslI、MspA1I、MspI、MspJI、MwoI、NaeI、NarI、Nb.BbvCI、Nb.BsmI、Nb.BsrDI、Nb.BtsI、Nc iI、NcoI、NdeI、NgoMIV、NheI、NlaIII、NlaIV、NmeAIII、NotI、NruI、NsiI、NspI、Nt.AlwI、N t.BbvCI、Nt.BsmAI、Nt.BspQI、Nt.BstNBI、Nt.CviPII、PacI、PaeR7I、PciI、PflFI、PflMI、 PI-PspI, PI-SceI, PleI, PluTI, PmeI, PmlI, PpuMI, PshAI, PsiI, PspGI, PspOMI, PspXI, Ps tI, PvuI, PvuII, RsaI, RsrII, Sac, SacII, SalI, SapI, Sau3AI, Sau96I, SbfI, ScaI, ScaI ScrFI, SexAI, SfaNI, SfcI, SfiI, SfoI, SgrAI, SmaI, SmlI, SnaBI, SpeI, SphI, SspI, StuI StyD4I, StyI, SwaI, TaqαI, TfiI, TseI, Tsp45I, TspMI, TspRI, Tth111I, XbaI, XcmI, XhoI XhoI, XmaI, XmnI, ZraI, II 1000000000000000000000000000120120101200101000000000000000000000000000050050001 and 1000

[0083] 14. The compound has, as a payload, a nicking enzyme, preferably AatII, Acc65I, AccI, AciI, AclI, AcuI, AflII, AgeI, AhdI, AluI, AlwNI, ApaI, ApaLI, ApeKI, ApoI, AscI, AseI, AvaI, AvaII, AvrII, BaeGI, BaeI, BamHI, BbsI, BbvI, BciVI, BclI, BcoDI, BfuAI, BfuCI, BglI, BglII, BlpI, BmgBI, BpmI, BpuEI, BsaAI, BsaHI, BsaWI, BsaXI, BseRI, BsgI, BsiEI, BsiWI, BslI, BsmAI, BsmBI, BsmI, BsoBI, Bsp1286I, BspCNI, BspEI, BspHI, BspQI, BsrBI, BsrDI, BsrGI, BsrI, BssHII, BssKI, BstBI, BstEII, BstNI, BstUI, BstXI, BstYI, BstZ17I, Bsu36I, BtgI, BtsCI, Cac8I, ClaI, CspCI, CviAII, CviQI, DdeI, DpnI, DpnII, DraI, DraIII-HF (registered trademark), DrdI, EagI, EarI, Eco53kI, EcoNI, EcoO109I, EcoP15I, EcoRI, EcoRV, Fnu4HI, FseI, FspEI, FspI, HaeIII, HhaI, HincII, HinfI, HinP1I, HpaII, HphI, Hpy166II, HpyAV, HpyCH4IV, HpyCH4V, I-CeuI, I-SceI, KpnI, LpnPI, MboI, McrBC, MluCI, MluI, MlyI, MmeI, MnlI, MseI, MslI, MspA1I, MspI, MspJI, MwoI, Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BtsI, NciI, NcoI, NdeI, NgoMIV, NheI, NlaIII, NotI, NruI, NsiI, NspI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nt.The compound according to any one of the preceding embodiments, further comprising a nicking enzyme selected from CviPII, PacI, PaeR7I, PflFI, PflMI, PI-PspI, PI-SceI, PmeI, PmlI, PpuMI, PshAI, PstI, PvuI, PvuII, RsaI, SacI, SacII, SalI, SapI, SbfI, SfiI, SfoI, SmaI, SpeI, StuI, StyD4I, StyI, SwaI, TaqαI, TfiI, TseI, TspMI, TspRI, Tth111I, XbaI, XhoI, XmaI, and XmnI.

[0084] 15. Compounds that are used as payloads include zinc finger nucleases (ZFNs), zinc finger transcription activators (ZFAs), zinc finger transcription repressors (ZFRs), and zinc finger methylases (ZFMs); enzymes in diseases for enzyme replacement therapy, such as glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (G6PD), 21-hydroxylase (EC 1.14.99.10), steroid 11β-monooxygenase (EC 1.14.15.4), 3α-hydroxysteroid-3-dehydrogenase (EC 1.1.1.50), steroid 17α-monooxygenase (EC 1.14.99.9), cholesterol monooxygenase (EC 1.14.15.6), glucosylceramidase (EC 3.2.1.45), galactosylceramidase (EC 3.2.1.46), pyruvate kinase (EC 2.7.1.40), α-galactosidase (EC 3.2.1.22), β-galactosidase (EC 3.2.1.23), acid α-glucosidase (GAA), α-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2.), collagenase (EC 3.4.24.7), glutaminase (EC 3.5.1.2), asparaginase (EC 3.5.1.1), lysozyme (EC 3.2.1.17), bilirubin oxidase (EC 1.3.3.5), thiosulfate sulfur transferase (EC 2.8.1.1), β-lactamase (EC 3.5.2.6), streptokinase, urokinase (EC 3.4.21.73), hyaluronate glucosaminidase (EC 3.2.1.35), hyaluronate glucuronidase (EC 3.2.1.36), hyaluronate lyase (EC 4.2.2.1), superoxide dismutase (EC 1.15.1.1), urease (EC 3.5.1.5), arginase (EC 3.5.3.1), glutamyl-transferase (EC 2.3.2.2.), glutathione-S-transferase (EC 6.3.1.13), creatine kinase (EC 2.7.3.2), aldolase (EC 4.1.2.13), lipase (EC 3.1.1.79), hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34), xanthine oxidase, PRPP synthase (EC 2.7.6.1), hypoxanthine-guanine-phosphoribosyltransferase (EC 2.4.2.8), DNAse (EC 3.1.22.1), RNAse (EC 3.1.27.5), branched-chain ketoacid dehydrogenase (EC 1.2.4.4), adenosine deaminase (EC 3.5.4.4), hexoseaminidase (EC 3.2.1.52), β-galactosidase (EC 3.2.1.23), sphingomyelinase (EC 3.1.4.12), ceramidase (EC 3.5.1.23), homogenate oxidase, β-glucosidase (EC 3.2.1.21), lactase (EC 3.2.1.108), HIV-protease (EC 3.4.23.16), dihydrofolate reductase (EC 1.5.1.3), alcohol dehydrogenase (EC 1.1.1.2), iduronate-2-sulfatase (EC 3.1.6.13), or sphingomyelin phosphodiesterase (EC 3.1.4.12).

[0085] 16. The compound according to any one of the preceding embodiments, wherein the compound further comprises as payload two monomeric subunits of a type II restriction endonuclease or a part thereof, preferably two monomeric alpha-helical subunits of a type II restriction endonuclease covalently linked to the polypeptide of general formula (I) by a linker.

[0086] 17. The compound according to any one of the preceding embodiments, wherein the compound further comprises two monomeric alpha-helical subunits of PvuII as payload, preferably wherein the two monomeric subunits of the type II restriction endonuclease PvuII or a part thereof are covalently linked to the polypeptide of general formula (I) by a linker.

[0087] 18. The compound according to any one of embodiments 1 to 17, wherein the compound further comprises a linker, the linker being an amino acid linker of 1 to 20 amino acid residues in length, preferably 1 to 15 amino acid residues in length, more preferably 7 to 13 amino acid residues in length, in particular 8 to 12 amino acid residues in length.

[0088] 19. The compound according to any one of embodiments 1 to 18, wherein the compound further comprises a linker, which is an amino acid linker selected from a linker comprising or consisting of a cysteine, serine or glycine residue, preferably one or two adjacent cysteine ​​residues; an amino acid sequence comprising a glycine and a serine residue, or an amino acid sequence comprising or consisting of the amino acid sequence GSG, SGG, GGC, GCS, CSG, GGS, GSGG, SGGS, GSGGC, CSGGS, GSGGSGGS, GSGGCCSGGS, GSGCCCSGGGS, or GSGGCSGGS.

[0089] 20. The compound according to any one of the preceding embodiments, wherein the compound further comprises an editing nuclease, a CRISPR nuclease, and / or a hybrid thereof as a payload.

[0090] 21. The compound according to any one of the preceding embodiments, wherein general formula (I) contains a single arginine and / or a single cysteine ​​or contains up to two arginine residues and / or up to two cysteine ​​residues.

[0091] 22. The compound according to any one of the preceding embodiments, wherein general formula (I) lacks arginine and / or cysteine, preferably general formula (I) lacks arginine and cysteine.

[0092] 23. A compound according to any one of embodiments 1 to 22, comprising two or more polypeptides of general formula (I).

[0093] 24. The compound is GNSGSGKKKKKGKGSGLGSGKKKDPL (DDM34) and The compound of any one of embodiments 1 to 23, wherein the compound is selected from the group consisting of: GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST (DDM36).

[0094] 25. The polypeptide of general formula (I) is used as a payload molecule to be delivered into biological cells, preferably SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKI- YGNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST, SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKI- YGNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST, SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGGSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGGSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST, and SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST The compound of any one of embodiments 1 to 24, wherein the compound is covalently attached to the C-terminus of a compound selected from the group consisting of:

[0095] 26. The compound according to any one of embodiments 1 to 25, wherein the polypeptide of general formula (I) is covalently attached to the C-terminus of a payload molecule to be delivered into a biological cell, which is a class II restriction endonuclease, preferably PvuII or a subunit thereof, and the payload molecule is optionally attached at its C-terminus to a linker molecule comprising 8 to 12 amino acids comprising or consisting of glycine, serine and / or cysteine ​​residues, and the polypeptide of general formula (I) is covalently attached to the C-terminus of the payload molecule or of the linker molecule, and the compound comprises one or two payload molecules, optionally separated by a linker molecule comprising 8 to 12 amino acids comprising or consisting of glycine, serine and / or cysteine ​​residues.

[0096] 27. The compound according to any one of embodiments 1 to 26, wherein the polypeptide of general formula (I) is covalently attached to the C-terminus of a payload molecule to be delivered into a biological cell, the compound comprising a first payload molecule which is a first subunit of PvuII linked at its C-terminus to a linker molecule comprising 8 to 12 amino acids comprising or consisting of glycine, serine and / or cysteine ​​residues, the linker molecule being attached at its C-terminus to a second subunit of PvuII, and the polypeptide of general formula (I) is covalently attached to the C-terminus of the second subunit of PvuII.

[0097] 28. A compound according to any one of embodiments 1 to 27 for use in the treatment of a tumor patient, preferably the tumor patient is suffering from neuroblastoma, colon cancer, hepatocellular carcinoma, small cell lung cancer, ovarian cancer, lung cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, placental cancer, breast cancer, kidney cancer, liver cancer, pancreatic cancer, muscle cancer, skin cancer, connective tissue cancer, bone cancer and any of these cancers in which the patient has already developed metastases, in particular for the treatment of patients with neuroblastoma, colon cancer, hepatocellular (liver) cancer, small cell lung cancer, lung cancer, breast cancer, pancreatic cancer and any of these cancers in which the patient has already developed metastases.

[0098] 29. A method for treating a tumor patient, comprising administering an effective amount of a compound according to any one of embodiments 1 to 27 to a patient in need thereof, preferably the tumor patient suffering from neuroblastoma, colon cancer, hepatocellular (liver) cancer, small cell lung cancer, ovarian cancer, lung cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, placental cancer, breast cancer, kidney cancer, liver cancer, pancreatic cancer, muscle cancer, skin cancer, connective tissue cancer, bone cancer, and any of these cancers in which the patient has already developed metastases, in particular for the treatment of patients with neuroblastoma, colon cancer, hepatocellular (liver) cancer, small cell lung cancer, lung cancer, breast cancer, pancreatic cancer, and any of these cancers in which the patient has already developed metastases.

[0099] 30. The use of a compound according to any one of embodiments 1 to 27 for the manufacture of a medicament, preferably for the treatment of tumor patients, more preferably for the treatment of patients with neuroblastoma, colon cancer, hepatocellular carcinoma, small cell lung cancer, ovarian cancer, lung cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, placental cancer, breast cancer, kidney cancer, liver cancer, pancreatic cancer, muscle cancer, skin cancer, connective tissue cancer, bone cancer and any of these cancers in which the patient has already developed metastases, in particular for the treatment of patients with neuroblastoma, colon cancer, hepatocellular (liver) cancer, small cell lung cancer, lung cancer, breast cancer, pancreatic cancer and any of these cancers in which the patient has already developed metastases.

[0100] 31. A pharmaceutical formulation comprising a compound according to any one of embodiments 1 to 27 and a protein kinase inhibitor (PKI), preferably a DNA-dependent PKI, more preferably a non-homologous end joining (NHEJ) and V(D)J repair factor DNA-dependent PKI, in particular 7-methyl-2-[(7-methyl[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one (AZD7648; Fok et al., Nat. Comm. 10 (2019), 5056).

[0101] The present invention is further illustrated by the following examples and figures, without however being limited thereto. [Brief description of the drawings]

[0102] [Figure 1A] SDS gel profile of CROMOC36 purification, 3-column downstream processing of drug intermediate. SDS gel profile of CROMOC36 purification. Lanes from left to right: molecular mass marker, total extract, peak fraction affinity chromatography, anion chromatography flow-through, flow-through cation chromatography, low salt elution cation chromatography, medium salt elution cation chromatography, first peak fraction (I) "preCROMOC", high salt elution cation chromatography, peak fraction (II) high salt elution cation chromatography. The gel shows that the described processing allows to reach highly concentrated proteins of the invention. [Figure 1B] SDS gel profile of CROMOC36 by PEGylation of the peak fraction in Figure 1A and the subsequent purified final product: right lane. From left to right: lane 1, molecular mass markers, lane 2) peak fraction (I) from cation chromatography, lanes 3-7 are PEG reaction intermediates. Lane 8 is the final product of the purified PEGylated molecule "CROMOC36" used in the examples. The gel shows that the described treatment allows to reach a highly concentrated protein of the invention.

[0103] [Diagram 2] CROMOC36 exhibits antiproliferative activity at nM concentrations in tumor cells representing a variety of tumor indications (55 / 80 cell lines IC50 ≦100 nM). [Diagram 3] CROMOC36 exhibits antiproliferative activity at nM concentrations and is 40- to 100-fold less cytotoxic to immortalized non-tumor cells than to cancer cells. [Figure 4]In vitro activity of CROMOC36. Human pancreatic cancer cell lines. IC50 was assessed by propidium iodide monolayer assay for 96 hours. CROMOC36 showed selective and concentration-dependent antitumor activity against human pancreatic cell lines tested. CROMOC36 was most sensitive against PATU8902, PATU8988T, PAXF1657L, and PAXF546L cell lines. Results show resistant cells with IC50 in the low μM range and highly sensitive, 1000-fold more sensitive cell lines in the low nM range.

[0104] [Diagram 5] Comparison of uptake kinetics of PEGylated CROMOC36-AF680 (CROMOC36-AF680) versus non-PEGylated preCROMOC-AF680. Fluorescence intensity was measured by flow cytometry. U2OS cells were incubated for the indicated time points and surface-bound molecules were washed extensively with ice-cold PBS before cells were fixed. [Figure 6] CROMOC endocytosis inhibitor studies to evaluate the pathways important for CROMOC membrane crossing. These studies show that uptake of CROMOC36 is ATP-dependent, via clathrin- and caveolin-dependent endocytosis for DDM-targeted transport, and not micropinocytosis. Conditions tested for CROMOC36 were (1) at 4°C or (2, CD) in the presence of cytocollisin D; (3, CPZ) in the presence of chlorpromazine; (4, MβCD) in the presence of methyl-β-cyclodextrin; (5, CPZ+MβCD) in the presence of chlorpromazine and methyl-β-cyclodextrin; (5, HP) in the presence of heparin, and cellular uptake was measured using flow cytometry.

[0105] [Figure 7]HCT116 p53- / - human colon cancer xenograft. Treatment for advanced tumors (volume >50mm3). Dose: CROMOC36 4mg / kg; CROMOC60 4mg / kg. Treatment: sc on days 0, 1, 2, 3, 6, 7, 8, 9, 10, 20, 22, 24. [Figure 8] Immunohistochemistry (IHC) analysis of paraffin-embedded liver specimens from male Sprague Dawley rats after a single IV treatment with CROMOC36. Samples were collected 13-15 days later and IHC was used to assess histone surrogate markers of DNA double strand breaks phospho-H2AX and phospho-H3 to analyze the persistence of DNA double strand breaks (DSBs) at very high CROMOC concentrations in tumors compared to the KI67 marker. This demonstrates that DSBs are induced by CROMOC molecules and that the DDM sequence can guide the payload into the nucleus of cells in vivo.

[0106] [Figure 9] In vivo antitumor and antimetastatic activity of CROMOC36 in combination with cyclophosphamide against murine Lewis lung carcinoma. [Figure 10] CROMOC36 antitumor efficacy against HCT116 (p53- / -) human colon cancer xenograft model as a single agent and in combination with cisplatin or vincristine. [Figure 11] Treatment of xenografted mice bearing IMR32 sc (implanted) advanced tumors (volume >50 mm3). Variation in tumor volume expressed as a percentage of the initial volume (day 0; designated as 100%). Final measurements for the vehicle (PBS) and doxorubicin groups were taken on day 42. For the remaining groups, final measurements were taken on day 63, the end of the experiment. Decreased proliferation compared to controls on day 42 (p<0.01). [Figure 12]CROMOC36 has strong antimetastatic activity in liver M5076 allografts, iv route (M5076). (A) Blue: CROMOC 16mg / kg (q7dx3), Red: Doxorubicin 6mg / kg (q4dx3). (Left) Metastasis inhibition (%), (Right) ILS (%). (B) Liver weight.

[0107] [Figure 13] In vivo images: Biodistribution of CROMOC04. [Figure 14] In vivo images: Biodistribution of CROMOC05. [Figure 15] In vivo images: Biodistribution of CROMOC06. [Figure 16] In vivo images: Biodistribution of CROMOC36. [Figure 17] In vivo images: Biodistribution of proCROMOC36. [Figure 18] In vivo organ distribution of CROMOC36 molecules in healthy mice. [Figure 19] In vivo organ distribution of CROMOC36-AF680 molecules in tumor-bearing mice.

[0108] [Figure 20] IC50 comparison using CLUSTAL W(1.83) multiple sequence alignment and endonuclease assay of various DDM sequence alignments of the DDM-CPP subfamily. [Figure 21] Comparison of DDM36 with TAT, pentratin, and transportan. [Figure 22] UPB buffer. [Figure 23] UPB buffer.

[0109] [Figure 24] IC50 values ​​of CROMOC36 and sorafenib against human hepatocellular carcinoma cell lines. [Diagram 25] SRB buffer. [Figure 26A]The concentration-dependent response to CROMOC62 in the presence of various concentrations of the DNA-PK inhibitor AZD7648 was determined for U2-OS by SRB assay. [Figure 26B] Concentration-dependent responses to CROMOC62 in the presence of various concentrations of the DNA-PK inhibitor AZD7648 were determined by SRB assay in b) A549 cells. [Figure 26C] The calculated EC50 values ​​are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0110] Example 1 To develop a new family of CPPs that show efficiency at concentrations in the low nM range, are more stable in vitro and in vivo, do not interfere with solubility, and allow the development of drug-like properties, synthetic CPPs were developed as drug delivery modules (DDMs). To this end, different DDM sequences were tested for the IC of nuclease payloads in cell-based assays as a primary screening assay. 50 The structure-activity relationship was analyzed using the β-linked ...

[0111] Example 2 Monolayer assay for in vitro testing of IC50 activity of CROMOC compounds. The following cells were tested in the monolayer assay. The results are shown in Figures 2, 3, 4, and 24:

[0112] 22RV1, 5637, 786O, A204, A375, A431, A549, A673, ACHN, ASPC1, BT20, BXPC3, CACO2, CAKI1, CALU6, CLS439, COLO205, COLO678, DLD1, DU145, EFO21, EJ28, HCT15 , HEK293, CASKI, C33A, HELA, SAOS2, SKHEP1, LIXF575L, HEPG2, HEP3B, HS578T, HS729, HT1080, HT29, IGROV1, IMR90, J82, JAR, JEG3, JIMT1, LOVO, MCF7, MDAMB23 1, MDAMB435, MDAMB436, MDAMB468, U2OS, MG63, MHHES1, MIAPACA2, MT3, NCIH292, NCIH358M, NCIH460, NCIH82, A2780, OVCAR3, OVCAR4, PANC1, PANC1005, PC3, PLC-PRF-5, RD, RDES, SF268, SF295, SKBR3, SKLMS1, SKMEL28, SKMEL5, SKNAS, SKNSH, SKOV3, SNB75, SW620, T24, TE671, U87MG, UMUC3, UO31, and human peripheral blood mononuclear cells (PBMCs). BEN;COLO-699;FU-OV-1;GI-LI-N;H-1339;HCC-15;HCC-44;HCC-827;HCT-116 p53+ / +;HCT-116 p53- / -;HCT-116 p21- / -;primary hepatocytes;IMR-32;LAN-5;LCLC-97TM1;LCLC-103H;ONCO-DG-1;SH-SY-5Y;STA-NB3;STA-NB10;U2-OS;WI-38.

[0113] Example 2A. Monolayer assay using sulforhodamine B (SRB) staining. Cells were harvested from exponential phase cultures and 10,000-30,000 cells were seeded into 96-well flat-bottom microtiter plates depending on the cell line. After a 24-h recovery period to allow cells to resume exponential growth, the supernatant was discarded and 100 μl of culture medium (8 control wells / plate) or culture medium containing test compound was added by a liquid handling robotic system (Microlab® Starlet, Hamilton) and treatment was continued for 72 h. Compounds were applied at nine concentrations to eight wells per concentration. At least three independent experiments were performed. For SRB staining, cells were fixed with SRB01 for 1 h at 4 °C (final concentration of TCA = 10%) and washed 5 times with HO using a plate washer (Hydroflex, Tecan ©); stained with 100 μl of SRB02 for 10 min at room temperature, washed 4 times with SRB03 and finally washed with 100 μl of developer SRB04 for 5 min with shaking. Optical density was evaluated at 540 nm using a plate reader (Sunrise, Tecan ©). The concentration required for 50% inhibition of cell proliferation (IC 50 ) was calculated by nonlinear regression (log[inhibitor concentration] (%T / C) vs. response) using the analytical software GraphPad Prism®, Prism 5 (for windows, version 5.01) (GraphPad Software Inc., CA) (SRB buffer, FIG. 25).

[0114] Example 2B Monolayer assay Propidium iodide (PI). Cells were harvested from exponential phase cultures, counted, and 3,000 to 20,000 cells were seeded into 96-well flat-bottom microtiter plates depending on the cell line. After a 24-h recovery period to allow cells to resume exponential growth, 10 μl of culture medium (4 control wells / plate) or culture medium containing test compound was added by a liquid handling robotic system and treatments were continued for 4 days. Compounds were applied in duplicate at 10 concentrations in half-log increments. Three independent experiments were performed. Cells were then washed with 200 μl of PBS to remove dead cells, followed by the addition of 200 μl of a solution containing 7 μg / ml propidium iodide (PI) and 0.1% (v / v) Triton X-100. After a 1-2 h incubation period at room temperature, the amount of attached viable cells was quantified by measuring fluorescence (FU) using a Cytofluor 4000 microplate reader (excitation λ = 530 nm, emission λ = 620 nm). The concentration required for 50% inhibition of cell proliferation (IC 50 ) was calculated by nonlinear regression (log [inhibitor concentration] vs. response (% T / C)) using the analytical software GraphPad Prism®, Prism 5 (for Windows, version 5.01) (GraphPad Software Inc., CA).

[0115] Example 3 Expression of the proteins of the invention. Protein expression was obtained from E. coli strains using a two-plasmid vector system as described in Kuhne C., et al. (NAR 31 (2003), 7227-7237). Cell growth for protein expression was performed constitutively in 2 L Erlenmeyer flasks with a culture volume of 400 ml and without antibiotics. Culture medium was incubated at 4°C for an optical cell density OD of 0.008. 600The cultures were inoculated with freshly prepared precultures previously grown under kanamycin and ampicillin antibiotic selection at 100 rpm (UPB-001, UPB-004, UPB-005). Growth medium consisted of Terrific Broth (TB, UPB-002) supplemented with UPB-003. Growth was at 210 RPM, 30°C, for 16 hours in a Certomat BS-1 shaking incubator (Sartorious). After 16 hours of growth, the OD of 6.7 (+ / - 0.2) was reached. 600 The pH reached 7.2 (+ / -0.05) and the E. coli culture was centrifuged at 3557g, +4°C for 30 minutes. The pellet was then incubated on ice with 25ml wash buffer (UPB-006 / 400ml growth pellet for 30 minutes), resuspended and centrifuged again as above. The supernatant was discarded and the wet biomass was measured. Under these conditions, a 400ml culture yields a wet weight pellet of 5g (+ / -0.4g). The wet pellet is stored at -80°C (UPB buffers are listed in Figure 22).

[0116] Example 4 Purification and PEG modification of the protein of the invention. The following steps were performed: Cell pellet disruption: Frozen cell pellets (example) stored at -80°C were thawed in a water bath at 30°C for 10 minutes. The pellets were then resuspended in 7.5 ml of ice-cold buffer DPB-013 per gram of cell pellet and then incubated on ice for 30 minutes. Homogenization: A homogenizer GEA NS 1001L-2K (GEA Niro Soavi) was pre-equilibrated in ice-cold buffer DPB-013; cells were disrupted by 4 cycles at 1100 bar while keeping the temperature at 4°C. Centrifugation: Clarification of the homogenate was performed by centrifugation at 25,000 x g and 4°C for 1 hour using a 3K30 centrifuge (Sigma). Sterile filtration: The clarified supernatant was sterile filtered using a Stericup PVDF Durapore® 0.22 μm filtration device (Millipore). Affinity chromatography: Sample loading was performed at 79 cm / h (20 ml / min) and the remaining chromatography was performed at 159 cm / h (40 ml / min); AKTA Purifier UPC 100 (GE Healthcare Bio-Sciences). Sterile filtered samples were loaded onto a pre-equilibrated (in buffer DPB-014) IMAC Sepharose 6 Fast Flow 50 ml (GE Healthcare Bio-Sciences AB). Unbound proteins were washed off with 10 CV of DPB-014, then washed with DPB-015 to reduce salt concentration and eluted with one step 100% DPB-016, 3 CV. Anion exchange chromatography: All runs were performed at 150 cm / h (20 ml / min); AKTA Purifier UPC 100 (GE Healthcare Bio-Sciences AB). The peak fractions from the affinity chromatography elution were loaded directly onto a pre-equilibrated (in buffer DPB-015) Fractogel EMD TMAE Medcap(M) 30 ml (Merck KGaA-Life Science Products-Darmstadt, Germany) and the flow-through was collected (fraction not binding to the column).Wash with DPB-015 for 10 column volumes (CV) and elute with one step of 100% DPB-014, 3 CV. (I) Cation exchange chromatography: all runs were performed at 197 cm / h (50 ml / min). AKTA Purifier UPC 100 (GE Healthcare Bio-Sciences AB). The flow-through fraction from the anion exchange chromatography was loaded onto 40 ml of pre-equilibrated (in buffer DPB-004) Fractogel EMD SO3 (M) (Merck KGaA-Life Science Products-Darmstadt, Germany) and eluted for 10 CV. The mixture was washed with DPB-004 and eluted with a three-step gradient, 25% buffer DPB-005 (C-terminally truncated SUB36 protein (theoretical mass 37928 / 37930 (amino acids 2-331), molecular mass 40113 / 40115 (aa2-355)) 40%); buffer DPB-005 (native full-length SUB36 protein-compound, peak fraction 43404,4 / 43406,8 (aa2-387)); 100% buffer DPB-005 (non-native isomeric form). The protein peak fractions were frozen at -80 °C in sterile tubes. For post-expression modification of CROMOC molecules with PEG, the protein from -80 was thawed on ice (3 h). The precipitate was removed by centrifugation for 30 min at 25.000xg and 4°C in a 3K30 centrifuge (Sigma). Protein pools were prepared. N-terminal PEGylation reaction: Native full-length SUB36 protein compound was diluted to 1 mg / ml in 0.1 M NaH2PO4 / Na2HPO4 (pH 5.0) and diluted with a 10-fold molar excess of mPEG-Butyraldehyde 30 kDa (Laysan The mixture was mixed with 1000-fold molar excess of NaBH3CN (Sigma Aldrich) and incubated at 25°C and 125 rpm in a CERTOMAT BS-1 incubator (Sartorius) for 16 hours. The reaction was quenched by adding a 30-fold molar excess of glycine solution. (II) Cation exchange chromatography: All runs were performed at 157 cm / hr (40 ml / min).AKTA Purifier UPC 100, (GE Healthcare Bio-Sciences AB, Bjorkgatan 30, 75184 Uppsala, Sweden). The clarified supernatant is diluted 1:2,5 with buffer DPB-004. The sample is loaded onto 40 ml pre-equilibrated (in buffer DPB-004) Fractogel EMD SO3(M) (Merck KGaA-Life Science Products-Darmstadt, Germany), washed with 10 CV DPB-004 and the protein is eluted with a 3-step gradient: 20% buffer DPB-005 (poly-PEGylated protein), 30% buffer DPB-005 (mono-PEGylated full-length SUB36 protein-compound), 100% buffer DPB-005 (non-PEGylated protein). Microfiltration: Mono-PEGylated full-length SUB36 protein compound was concentrated 1:10 by centrifugation and microfiltration (Vivaspin 15R, MWCO 30000, Sartorius) followed by 3 cycles of washing with PBS. Stored at -80°C. UPB buffer is listed in Figure 23. An example of SDS-gel of the different purification steps is shown in Figure 1.

[0117] Example 5 In vitro activity of CROMOC36. CROMOC36 and sorafenib in human hepatocellular carcinoma cell lines IC 50 Values, 96 h treatment. CROMOC36 had a geometric mean IC 50 It showed concentration-dependent activity against all cell lines tested. The most sensitive cell line was HEP3B (IC 50 = 0.0122 μM), followed by HEPG2 (IC 50 =0.0137 μM) and LIXF 575L (IC 50 = 0.017 μM). Individual results for CROMOC36 are shown in Figure 24. The reference compound, sorafenib, had a geometric mean IC of 4.68 μM for these three cell lines. 50 The most sensitive cell line to sorafenib was HEP3B (IC 50= 2.83 μM). HEPG2 (IC 50 = 5.97 μM) and LIXF 575L (IC 50 Near-identical sensitivity was detected for sorafenib (IC = 6.07 μM). Individual results for sorafenib are included in (Figure 24). The study showed that the three HCC cell lines tested had a geometric mean IC of 0.0141 μM. 50 We show that these cell lines are sensitive to CROMOC36 in vitro. CROMOC36 was more than 300-fold more potent than the reference compound sorafenib against these cell lines. These results suggest that CROMOC36 has potent anticancer activity against HCC in vitro.

[0118] Example 6 In Vitro Activity of CROMOC36. Human pancreatic cancer cell lines. The aim of this study was to investigate the antitumor activity of CROMOC36 in vitro against a panel of 13 human pancreatic tumor cell lines. A propidium iodide-based monolayer proliferation assay was used to evaluate antitumor activity after 4 days of CROMOC36 treatment. Ten concentrations of CROMOC36 were tested in half-log increments up to 1 μM, with up to four experiments performed for each cell line. Taxol, an antitumor agent commonly used to treat pancreatic cancer, was used as a reference compound and was tested in half-log steps up to 0.3 μM. CROMOC36 had an overall geometric mean IC of 45.9 nM. 50 PATU8902 (IC 50 =2.33nM), PATU8988T(IC 50 = 2.79 nM), and PAXF1657L (IC 50 = 4.73 nM), followed by PAXF546L (IC 50 = 6.49 nM). In addition, MIAPACA2, HUPT3, and PANC1 also showed above-average sensitivity (IC 50 CROMOC36 showed no or only slight activity against five of the cell lines tested. The reference compound, taxol, had a geometric mean IC of 1.99 nM. 50CROMOC36 showed concentration-dependent activity against all cell lines tested at 100% CI, with the highest concentration-dependent activity against human pancreatic cell lines tested. CROMOC36 was most sensitive against the PATU8902, PATU8988T, PAXF1657L, and PAXF546L cell lines (Figure 4).

[0119] Example 7 To analyze the cytostatic activity in different tumor cell lines, the in vitro activity of CROMOC36 was analyzed in 80 different human tumor cell lines representing Mayor's tumor indications by a 72-hour monolayer sulforhodamine B (SRB) staining assay. CROMOC36 shows antiproliferative activity at nM concentrations in tumor cells from different indications (55 / 80 cell lines IC 50 <100 nM), indicating broad cytostatic antitumor activity in a variety of indications (Figure 2). CROMOC36 exhibits antiproliferative activity at nM concentrations.

[0120] I C 50 Range: <0.5 nM - 19 nM. Notably, CROMOC36 is 40-100 times less cytotoxic to immortalized non-tumor cells than to cancer cells. IC 50 Compared to >10 μM non-tumor primary hepatocytes.

[0121] Example 9 A comparison of the uptake kinetics of PEGylated CROMOC36-AF680 (CROMOC36-AF680) versus non-PEGylated preCROMOC-AF680 was performed. Fluorescence intensity was measured by flow cytometry. U2OS cells were incubated for the indicated time points, and the surface-bound molecules were washed extensively with ice-cold PBS before cells were fixed. As shown in Figure 4, the cellular uptake of PEGylated CROMOC36-AF680 was 6 times slower than that of non-PEGylated (preCROMOC36-AF680).

[0122] Example 10 CROMOC endocytosis inhibitors test to evaluate the pathways important for CROMOC membrane crossing. U2OS cells were incubated with fluorescein-labeled CROMOC36 at (1) 4°C or in the presence of (2, CD) Cytocollacin D; (3, CPZ) Chlorpromazine; (4, MβCD) Methyl-β-cyclodextrin; (5, CPZ+MβCD) Chlorpromazine and Methyl-β-cyclodextrin; (5, HP) Heparin, and cellular uptake was measured using flow cytometry. CD inhibits macropinoptosis; CPZ inhibits clathrin-dependent endocytosis; MβCD inhibitor of caveolin-dependent endocytosis competes with heparin receptors, and 4°C inhibits energy-dependent endocytosis but not macropinocytosis or phagocytosis. X-axis shows CROMOC uptake inhibition (%). This example confirms that DDM CPP uptake is energy dependent, i.e. ineffective at 4°C and inhibited by CPZ and MβCD, and demonstrates clathrin- and caveolin-dependent endocytosis, but not micropinocytosis, for DDM targeted transport, as summarized in Figure 5. Similar results were obtained in various cell lines, including colon cancer cells HCT116 or lung adenocarcinoma cell line A549.

[0123] Example 11 A study was conducted to test the antitumor efficacy of CROMOC36 against advanced human colon cancer using a null p53 (HCT116 p53- / -) xenograft model and compare it to vehicle or nuclease impaired CROMOC60 treatment groups. CROMOC60 is no longer catalytically active as a nuclease but can be transported to the nucleus of the cell. This study was also designed to prove that the active principle of CROMOC's tumor growth inhibitory activity is its nuclease activity in vivo. Both CROMOC36 and CROMOC60 were administered subcutaneously (sc) at a dose of 4 mg / kg on days 0, 1, 2, 3, 6, 7, 8, 9, 10, 20, 22, and 24. This study showed that the progression of HCT116 p53- / - human colon cancer was significantly inhibited by CROMOC36 but not by CROMOC60 compared to vehicle control. This demonstrates that nuclease activity is responsible for the consistent antitumor effects seen in mouse xenograft experiments, and also demonstrates that CROMOC36 can function as an effective DNA DSB reagent. A summary graph is shown in Figure 7.

[0124] Example 12 Immunohistochemistry (IHC) analysis of paraffin-embedded liver specimens of male Sprague Dawley rats after a single IV treatment with CROMOC36 as proof of nuclease activity in vivo. Samples were collected 13-15 days later and IHC was used to evaluate histone surrogate markers of DNA double strand breaks phospho-H2AX and phospho-H3 to analyze the persistence of DNA double strand breaks (DSBs) at very high CROMOC concentrations in tumors compared to the KI67 marker. This proves that DSBs are induced by CROMOC molecules and that the DDM sequence is able to guide the payload into the nucleus of cells in vivo. A representative example is shown in Figure 8.

[0125] Example 13 The efficacy of CROMOC36 on the Lewis Lung Carcinoma (LLC) model was chosen to test its activity against primary tumor growth and metastasis formation. CROMOC36 was tested as a single agent and in combination with cyclophosphamide (CYPP). Furthermore, by using single agent CYPP as a reference compound, it was possible to compare the efficacy of CROMOC36 with commonly used antitumor agents (summarized in Figure 9). This study showed that mouse LLC primary tumor growth was not inhibited after repeated subcutaneous treatments with CROMOC36 [five treatments every two days (q2d x 5)] [15.2% tumor growth inhibition (TI)]. Conversely, treatment with CYPP inhibited primary tumor growth in this model (78.6% TI). The simultaneous combination of CROMOC36 with CYPP demonstrated a statistically significant (p = 3 x 10) inhibition of primary tumor growth compared to treatment with either compound as a single agent. -8 ) increase (85.6% TI). In contrast to the primary tumors, CROMOC36 showed a strong anti-metastatic effect: compared to the control, CROMOC36 (4 mg / kg, q2d x 5) reduced the number of small, medium, and large metastases by 61.2%, 49.2%, and 69.1%, respectively. Similar results were observed after treatment with 180 mg / kg CYPP. The combination of the two treatments further reduced the number of metastases, with a reduction of 81.1% for small, 66.7% for medium, and 89.2% for large metastases.

[0126] Overall, CROMOC36 at 4 mg / kg and 0.4 mg / kg inhibited the total number of metastases by 66% and 24%, respectively; CYPP inhibited the number of metastases by 62%, and the combination of CROMOC36 (4 mg / kg) and CYPP (180 mg / kg) inhibited the number of metastases by 81%. Comparable inhibition of metastasis volume was observed in all treatment groups.

[0127] No major issues were identified regarding the tolerability of CROMOC36. The combination of CROMOC36 and CYPP reduced body weight (BW) during the treatment schedule more than treatment with each agent as a single agent. However, BW recovered towards the end of the experiment, with a final reduction in BW of 3.7% (compared to the beginning of the experiment), which was comparable to the other treatment groups. This model clearly shows that CROMOC36 is highly active in an orthotopic metastasis model, but does not interfere with the primary tumor injected intramuscularly. This is in contrast to CYPP, which is active in the primary tumor, and the effect seen in metastasis comes from the fact that the primary tumor cannot escape to the metastatic site.

[0128] Example 14 A study was conducted in female athymic nude mice to evaluate the efficacy and tolerability of CROMOC36 against the HCT116 human colon cancer xenograft model with null p53 (HCT116 p53- / -). CROMOC36 was administered subcutaneously (sc) as a single agent three times at two-week intervals (q14d x 3) or in combination with 4 mg / kg cisplatin (CDDP) or 2 mg / kg vincristine (VCR), both administered five times at one-week intervals (q7d x 5). CROMOC36 was tested at 32 mg / kg or with reduced doses of 32-16-16 mg / kg. CDDP and VCR were also administered as single agents to allow for the comparison of the efficacy of CROMOC36 with commonly used anticancer drugs. The doses and schedules of CDDP and VCR treatments remained the same when tested as single agents or in combination with CROMOC36. This study demonstrated that progression of HCT116 p53- / - human colon cancer was inhibited by CROMOC36 at both the repeated concentration of 32 mg / kg (82.9% tumor growth inhibition; p<0.005 vs. untreated control) and the reduced concentration of 32-16-16 mg / kg (91.6% tumor growth inhibition; p<0.005 vs. untreated control), demonstrating better efficacy than single agent CDDP (47.2% tumor growth inhibition) or VCR (45.4% tumor growth inhibition). The tumor growth inhibition observed when 32-16-16 mg / kg CROMOC36 was administered in combination with CDDP or VCR was not significantly altered with treatment with single agent CROMOC36. The inhibitory effect observed in the groups treated with 3632-16-16 mg / kg CROMOC, either as a single agent or in combination with CDDP or VCR, persisted until day 50 (i.e., 3 weeks after the last treatment), with no significant reduction in tumor inhibition calculated at day 31. Efficacy was also assessed by measuring tumor growth delay (TGD) and number of relapsing responses. The control group had achieved tumor growth of 1000 mm by day 23. 3 The study endpoint (TTE), defined as a tumor volume of 1000 mm3 or greater, was reached in all groups treated with CROMOC36, either as a single agent or in combination with CDDP or VCR. 3The TTE in these groups was >50 days, corresponding to >117% TGD (%). In the CDDP and VCR groups, the TTE was 34 days (48% TGD) and 35 days (52% TGD), respectively. Furthermore, treatment with 32-16-16 mg / kg CROMOC36 resulted in one partial regression response and one tumor-free survivor. The graph is shown in Figure 10.

[0129] Example 15 The purpose of this study was to evaluate the in vivo efficacy of CROMOC36 on IMR32 human neuroblastoma xenograft model. CROMOC36 was administered at two different doses (32mg / Kg) at 2-week intervals (q14d×2) subcutaneously (sc) and in combination with doxorubicin administered intravenously (iv) (32 / kg CROMOC36+4+4mg / Kg doxorubicin, sc and iv, q14d×2). Doxorubicin was also used alone as a reference compound (8mg / kg, iv, q14d×2). Vehicle was PBS. This study showed that the growth of IMR32 human neuroblastoma was dramatically inhibited by CROMOC36 (32mg / Kg) both alone and in combination with doxorubicin, but in contrast, the efficacy of treatment with doxorubicin alone showed much lower efficiency (TGI 10%) compared to vehicle control. Treatment with CROMOC36 and CROMOC36 plus doxorubicin resulted in 82% and 89% tumor growth inhibition, respectively, compared to controls.

[0130] In conclusion, these results show that CROMOC36 has strong antitumor activity as a single agent and in combination with doxorubicin at tolerated doses against the human IMR32 neuroblastoma model. These findings further support the in vivo efficacy of CROMOC36 against experimental tumor models after repeated sc treatment. Doxorubicin is accepted as the first-line treatment in late-stage neuroblastoma, and the superiority of CROMOC36 has important implications for the therapeutic use of neuroblastoma. A summary graph is shown in FIG.

[0131] Example 16 The M5076 ovarian reticulosarcoma tumor cell line model was designed to test anti-metastatic activity in liver allografts, iv route (M5076). M5076 cells were maintained in vivo as ascites tumors (by ip injection) in syngeneic female C57bl / 6 mice. To obtain artificial liver metastases, cells were harvested under sterile conditions from the peritoneal ascites, washed with PBS and injected into the lateral tail vein (5 × 10 cells in 200 μl PBS per mouse). 4 The mice were injected into the 100-well plate (1000 x 100 live cells) at 4 °C for 12 min. Cell viability was assessed by trypan blue dye. Randomization criteria were as follows: all cell injections were by the same person, mice were caged for 7 days before cell injection, and then randomly remixed into new cages. Mice were treated iv with 16 mg / kg CROMOC36 or PBS as vehicle, 3 times (q7d x 3) once a week, starting 2 days after tumor cell injection. Each treatment group consisted of 8 animals.

[0132] Antitumor activity was measured at the time of sacrifice (day 20) and mice from groups G1 (vehicle PBS), G2 (doxorubicin 6 mg / kg (q4d x 3), and G3 (CROMOC36 16 mg / kg (q7d x 3)) were killed. Livers were harvested and fixed in Bouin's fixative (SIGMA-Aldrich). Surface metastatic nodules were assessed by macroscopic observation under magnification. Determination of metastatic volume was performed by weighting each liver (Figure 12B).

[0133] The efficacy of CROMOC36 and doxorubicin treatment in reducing liver metastasis formation was reported as the percentage of inhibition comparing the number of metastases and liver weight in all treatment groups to the control group (100% T / C).

[0134] In the second experiment, the mean survival time was evaluated. Three groups treated under the same conditions as the animals used in the efficacy test against metastasis were not sacrificed and were used to evaluate the mean survival time (MST). Mice were examined daily and severely moribund mice were culled to prevent unwanted suffering to the animals.

[0135] The survival time of each mouse was calculated as the number of days elapsed from tumor cell injection to death or euthanasia.

[0136] Treatment efficacy was evaluated by comparing the MST in treated mice with the control group and expressed as ILS (%).

[0137] Treatments were considered significant if ILS (%)>20%. As can be seen from the left panel ILS in Figure 12, CROMOC (68%) showed significantly higher survival time than the vehicle control (calculated as 0%) as well as doxorubicin (32%) at the MTD, which is considered the gold standard for this tumor model.

[0138] Example 16a In vivo imaging was performed to compare the biodistribution in mice of CROMOC-DDM sequences with first generation CPPs engineered into CROMOC payloads. For this, 8 mg of CROMOC molecules of fluorescently labeled CROMOC-AF680 (Alexafluor 680 (AF680)) containing various CPPs (TAT CROMOC04, Penetratin CROMOC 05, Transportan CROMOC06, and DDM36 CROMOC 36) were injected iv into athymic nude nu / nu (from balb / c wild type) female mice. The in vivo localization of CROMOC-AF680 in mice was monitored over time by bioluminescence analysis. Animals were anesthetized and the bladder was excluded from image recording. Fluorescence emitted from CROMOC-AF680 fused to the described CPPs was detected using a cooled charge-coupled device camera mounted in a light-tight specimen box (IVIS Lumina-II imaging system; Perkin-Elmer). Regions of interest from the displayed images were quantified as total photon counts or photons / second using living image® software (Perkin Elmer). Measurements were taken 2, 4, 6, and 24 hours after injection. Biodistribution varied significantly between the various CPPs. CROMOC36 was exclusively localized to the liver, whereas all other molecules tested show distinct localization characteristics with more general overall staining during life imaging. CROMOC04 shows whole body staining, with signal also in the head region and strong liver fluorescence. CROMOC05 shows strong staining in the liver reminiscent of CROMOC36, but in addition, distinct spots in the limbs and upper intestine can be observed. With CROMOC006, whole body staining was observed, with only slight signal from the liver, but strong signal from the intestine, head region, and limbs. All CROMOC proteins evaluated above were PEGylated. The individual CPP localization properties were less pronounced in the non-PEGylated "pre-CROMOC" versions, indicating that post-expression modifications result in a more defined biodistribution and longer half-life of the molecules.

[0139] An example of CROMOC05 is shown in FIG. 13, an example of CROMOC06 is shown in FIG. 14, an example of CROMOC06 is shown in FIG. 15, an example of CROMOC36 is shown in FIG. 16, and an example of preCROMOC36 is shown in FIG.

[0140] Example 17 In vivo organ distribution of CROMOC36 molecules in healthy mice. Four hours after iv injection of 8 mg / kg of CROMOC molecules fluorescently labeled with AF680 dye, organs (liver, kidney, spleen, intestine, heart, gallbladder, lung) were isolated and fluorescence intensity was evaluated ex vivo (total photon flux shown on x-axis logarithmic scale). Figure 18 shows the average fluorescence intensity of organs from three animals, as shown on the x-axis. These experiments show that in healthy animals, CROMOC36-AF680 is distributed mainly in the liver, which has 3 times more CROMOC than intestine and 6 times more CROMOC than gallbladder, kidney, lung, and spleen. In agreement with the observations in the toxicology experiments, only residual signals were detected in the heart, and no cardiotoxic effects were observed in the various tumor models. The latter also confirmed the exclusion of CROMOC36 from cardiac and skeletal muscle cells observed from the Lewis lung allograft metastasis experiment in Figure 9.

[0141] Example 18 In vivo organ distribution of CROMOC36-AF680 molecules in tumor-bearing mice. The first orthotopic 4T1 breast tumor lung metastasis mouse model was analyzed for CROMOC-AF680 distribution in vivo (Figure 19A). Four hours after iv injection of 8 mg / kg of CROMOC molecules labeled with AF680 fluorescent dye, lungs containing 4T1 cell metastases were isolated and fluorescence intensity was evaluated ex vivo (total photon flux shown on X-axis logarithmic scale). Tumor-free animals do not show significant retention of CROMOC36 molecules, but more than 10.000-fold enrichment of CROMOC36 can be detected in breast tumor metastasis-bearing animals (Figure 19A).

[0142] Tumor cell enrichment of CROMOC was further confirmed by experiments in the liver, where CROMOC enrichment in orthotopic M5076 breast tumor metastases was strongly enriched compared to the surrounding liver tissue. Figure 19B shows an example in which mice bearing M5076 liver metastases were intravenously injected with 8 mg / kg CROMOC36-AF680, and the fluorescence intensity was measured in the metastatic liver tumor (T) and compared to the surrounding liver tissue (L) (Figure 19B). Quantitative evaluation for three mice showed that the total photon flux in the tumor tissue was 2-3 orders of magnitude higher than in normal liver tissue, proving the enrichment of CROMOC in the metastatic sites of the liver.

[0143] Example 19: In vitro activity of non-homologous end joining (NHEJ) and V(D)J repair factors in osteosarcoma U2OS of CROMOC62 and lung cancer cell line A549 in combination with the DNA protein kinase inhibitor (DNA-PKi) AZD7648 (7-methyl-2-[(7-methyl[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one). Concentration-dependent responses to CROMOC62 in the presence of different concentrations of the DNA-PK inhibitor AZD7648 were determined by SRB assay for a) U2-OS (Figure 26A) and b) A549 cells (Figure 26B). The calculated EC50 values ​​are shown in Figure 26C.

[0144] 2000 U2-OS (osteosarcoma cell line grown in RPMI) or A549 cells (lung cancer cell line grown in DMEM) were seeded the day before treatment in 100 μl medium supplemented with 10% FCS and penicillin / streptomycin per 96-well. The indicated substances (CROMOC62 and AZD7648 at the given concentrations) were added in 50 μl of medium, medium only served as a negative control and 250 nM CROMOC62 served as a positive control. After 70 h, cells were fixed for 1 h at 4° C. after adding 37.5 μl of 50% trichloroacetic acid. Fixed cells were washed 5 times with water and 100 μl of 0.06% sulforhodamine B in 1% acetic acid was added and incubated for 10 min at room temperature, followed by 4 washing steps with 1% acetic acid. 100 μl of 10 mM TRIS was added per well, shaken for 5 min, and absorbance was measured with a 560 / 20 nm filter, excluding border wells. Normalization was performed using positive and negative controls, and EC50 calculations were performed via Graphpad Prism 6 (Graph Pad Software, San Diego, CA) fitted to the formula: (Y=100 / (1+10^((LogEC50-X)*HillSlope))). Sample size n=3, error bars indicate standard deviation.

[0145] Sequence Listing [ka]

[0146] [ka]

[0147] [ka]

[0148] [ka]

[0149]

change

[0150]

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Claims

1. General formula (I): X ０ GX １ X ２ GX ３ X ４ X ５ GX ６ X ７ X ８ GX ９ X １０ X １１ X １２ X １３ X １４ 、 A compound comprising or consisting of a polypeptide of the above formula (1). (In the formula, G is glycine; X ０ is present or absent and, if present, is an amino acid linker; X １ is N or S, where N is asparagine and S is serine; X ２ is S or T, where S is serine and T is threonine; X ３ is present or absent and, if present, is S, where S is serine; X ４ is present or absent and, if present, is G, where G is glycine; X ５ is a basic polypeptide stretch consisting of 5 basic amino acid residues selected from R and K, where R is arginine, K is lysine and preferably X ５ contains three or more K amino acid residues, particularly X ５ is KKKKK or KRKKK; X ６ is a basic amino acid residue selected from R and K, where R is arginine and K is lysine, preferably X ６ is K; X ７ is S or L, where S is serine and L is leucine; X ８ is present or absent, and if present is a polypeptide stretch consisting of the amino acid sequence GLGS, where G is glycine, L is leucine, and S is serine; X ９ is a basic polypeptide stretch consisting of three basic amino acid residues selected from R and K, where R is arginine, K is lysine and preferably X ９ contains two or more K amino acid residues, in particular X ９ is KKK or KKR; X １０ is present or absent and, when present, is L or a polypeptide stretch consisting of the amino acid sequence DPL or DPC, where L is leucine, D is aspartic acid, P is proline and C is cysteine; X １１ is present or absent and, if present, is a polypeptide stretch consisting of the amino acid sequence LR or GSGL, where L is leucine, R is arginine, G is glycine, and S is serine; X １２ is present or absent, and if present is a basic polypeptide stretch having 20% ​​or more basic amino acid residues selected from K and R and at least P residues, preferably １２ KYKPKL, KYKPKLGT, or GX ３ X ４ X ５ GX ６ X ７ X ８ GX ９ X １０ where K, R, P, L, G, T, X ３ , X ４ , X ５ , G.X. ６ , X ７ , X ８ , G.X. ９ , and X １０ is defined as above, and Y is tyrosine; X １３ is present or absent, and if present is a polypeptide stretch consisting of the amino acid sequence GS, GST, GST, GSG, GSTG, or GSGL, where G, S, T, and L are defined as above; X １４ is present or absent and, if present, is an amino acid linker; The polypeptide has a length of 20 to 75 amino acid residues, preferably 24 to 65 amino acid residues, particularly 25 to 60 amino acid residues.

2. X １ is N and X ２ is S and X ３ is S and X ４ is G and X ５ is KKKKK or KRKKK, and X ６ is K and X ９ is KKK or KKR, and X １０ is L or a polypeptide stretch consisting of the amino acid sequence DPL or DPC, where N, S, G, R, L, D, P, and C are defined as above.

3. The compound is GNSGSGKRKKKGKGLGKKRDPCLRKYKPKLGTGSTG (HHHHHH) (DDM11), GNSGKRKKKGKGSGLGSGKKRDPCLRKYKPKLGSTG (HHHHHH) (DDM18), GNSGSGKRKKKGKGLGKKRDPCLRKYKPKLGTGSTG (HHHHHH) (DDM21), GNSGSGKRKKKGKGSGLGSGKKRDPLGSTG (HHHHHH) (DDM26), GNSG (HHHHHH) GSTGGKRKKKGKGSGLGSGKKRDPL (DDM27), GNSGSGKRKKKGKGSGLGSGKKKDPLGSTG (HHHHHH) (DDM28), GNSG (HHHHHH) GSTGGKRKKKGKGSGLGSGKKKDPL (DDM29), GNSGSGKKKKKGKGSGLGSGKKRDPLGSTG (HHHHHH) (DDM30), GNSG (HHHHHH) GSTGGKKKKKGKGSGLGSGKKRDPL (DDM31), GNSGSGKKKKKGKGSGLGSGKKKDPLGSTG (HHHHHH) (DDM32), GNSG (HHHHHH) GSTGGKKKKKGKGSGLGSGKKKDPL (DDM33), GNSGSGKKKKKGKGSGLGSGKKKDPL (DDM34), GNSGSGKKKKKGKGSGLGSGKKKLGSTG (HHHHHH) (DDM35), GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST (DDM36), and GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGSTG (HHHHHH) GST (DDM44) is selected from the group consisting of where (HHHHHH; histidine tag) may be present or absent; Preferably, GNSGSGKRKKKGKGLGKKRDPCLRKYKPKLGTGSTG (DDM11), GNSGKRKKKGKGSGLGSGKKRDPCLRKYKPKLGSTG (DDM18), GNSGSGKRKKKGKGLGKKRDPCLRKYKPKLGTGSTG (DDM21), GNSGSGKRKKKGKGSGLGSGKKRDPLGSTG (DDM26), GNSGGSTGGKRKKKGKGSGLGSGKKRDPL (DDM27), GNSGSGKRKKKGKGSGLGSGKKKDPLGSTG (DDM28), GNSGGSTGGKRKKKGKGSGLGSGKKKDPL (DDM29), GNSGSGKKKKKGKGSGLGSGKKRDPLGSTG (DDM30), GNSGGSTGGKKKKKGKGSGLGSGKKRDPL (DDM31), GNSGSGKKKKKGKGSGLGSGKKKDPLGSTG (DDM32), GNSGSGKKKKKGKGSGLGSGKKKDPL (DDM34), GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST (DDM36), and GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGSTGGST (DDM44), is selected from the group consisting of especially, GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST (DDM36), and GNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGSTGGST (DDM44) Selected from the group consisting of: A compound according to claim 1 or claim 2.

4. Said compound further comprises a payload molecule to be delivered into the biological cell covalently linked to said polypeptide of general formula (I), said payload molecule being preferably a chemotherapeutic molecule, a cytotoxic molecule, a DNA damaging molecule, an antimetabolite molecule, a therapeutic molecule, a small molecule having a therapeutic effect in the biological cell, a DNA molecule, an RNA molecule, an antibody molecule or an antibody derivative having an antibody-like function, a restriction endonuclease, a nicking enzyme, or a DNA- or RNA-dependent endonuclease exhibiting double-stranded / single-stranded cleavage activity or no nuclease activity, more preferably said payload molecule is a class II restriction endonuclease, such as PvuII, EcoRV, PvuII, HinfI, or a sc homodimer, subunit or functional fragment thereof, in particular said payload molecule being SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY, SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY, SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGGSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- The SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLEM- - - - - The SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLEM- - - PDLNKLLELWPHIEYEEEETE - The SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLEM- - - - - The SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLEM- - - PDLNKLLELWPHIEYEEEETE - The SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLEM- - - PDLNKLLELWPHIEYEEEETE - The SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLEM- - - PDLNKLLELWPHIEYEEEETE - YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY, and SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG-NDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY The compound according to any one of claims 1 to 3, selected from the group consisting of:

5. The compound according to any one of claims 1 to 4, wherein the compound further comprises a homopolymer moiety covalently attached to the polypeptide of general formula (I), preferably the homopolymer is selected from the group consisting of polyethylene glycol (PEG), in particular PEG with a MW of 5 kDa to 30 kDa; dextran, polysialic acid, hyaluronic acid, dextrin, hydroxyethyl starch, or poly(2-ethyl 2-oxazoline).

6. The compound further comprises a restriction endonuclease as a payload molecule to be delivered into the biological cell covalently linked to the polypeptide of general formula (I), the restriction endonuclease being preferably a class II restriction endonuclease, in particular a PvuII restriction endonuclease or a derivative thereof, the derivative being a single-chain PvuII restriction endonuclease, e.g. SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY, SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLEM- - * SGFSHPDLNKLLELWPHIEYEETLTNKHFEFTNGEL - - SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLEM- - * MSHPDLNKLLELWPHIEYEETTTLLTPHIEETTLLM - - SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLEM- - * CSGGSIHPDLNKLLELWPHIEYETTTLPHIEYETTLMEMEMM - - MSHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY, MSHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIY, MSHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGSGGS, SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGSGGS, or GSGGSGGSSHPDLNKLLELWPHIQEYQDLALKHGIN- DIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQEYELKSINIDLTKGFSTHHHHMNPVIIA- KYRQVPWIFAIYRGIAIEAIYRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVME- HGTKIY The compound according to any one of claims 1 to 5, which is a derivative comprising the amino acid sequence of

7. The compound according to any one of claims 1 to 6, wherein said polypeptide of general formula (I) is covalently linked by a linker to another moiety, said another moiety being preferably a payload molecule, a labelling group and / or a homopolymer to be delivered into a biological cell.

8. 8. The compound according to any one of claims 1 to 7, wherein the compound further comprises a capping group, preferably an alkoxy group, in particular a methoxy, ethoxy, propoxy or butoxy group; a halogen atom; or a tosylate; an isocyanate, hydrazine hydrate, maleimide, orthopyridyl disulfide, N-succinimidyloxy, sulfo-N-succinimidyloxy, 1-benzotriazole, 1-imidazolyloxy, p-nitrophenyloxy, or an aldehyde.

9. 9. The compound according to any one of claims 1 to 8, wherein the compound further comprises a linker, said linker being an amino acid linker of 1 to 20 amino acid residues in length, preferably 1 to 15 amino acid residues in length, more preferably 7 to 13 amino acid residues in length, in particular 8 to 12 amino acid residues in length.

10. 10. The compound according to any one of claims 1 to 9, wherein the compound further comprises a linker, which is an amino acid linker selected from a linker comprising or consisting of a cysteine, serine or glycine residue, preferably one or two adjacent cysteine ​​residues; an amino acid sequence comprising a glycine and a serine residue, or an amino acid sequence comprising or consisting of the amino acid sequences GSG, SGG, GGC, GCS, CSG, GGS, GSGG, SGGS, GSGGC, CSGGS, GSGGSGGS, GSGGCCSGGS, GSGGCCSGGS, or GSGGCSGGS.

11. The compound according to any one of claims 1 to 10, wherein said compound further comprises as payload two monomeric subunits of a type II restriction endonuclease or parts thereof, preferably two monomeric alpha-helical subunits of a type II restriction endonuclease, covalently linked to said polypeptide of general formula (I) by a linker.

12. The compound according to any one of claims 1 to 11, wherein said compound further comprises two monomeric alpha-helical subunits of PvuII as payload, preferably said two monomeric subunits of the type II restriction endonuclease PvuII or parts thereof are covalently linked to said polypeptide of general formula (I) by a linker.

13. 13. A compound according to any one of claims 1 to 12, wherein said general formula (I) comprises a single arginine and / or a single cysteine ​​or comprises up to two arginine residues and / or up to two cysteine ​​residues.

14. 14. The compound according to any one of claims 1 to 13, wherein said general formula (I) is devoid of arginine and / or cysteine, preferably said general formula (I) is devoid of arginine and cysteine.

15. The compound according to any one of claims 1 to 14, comprising two or more polypeptides of general formula (I).

16. The compound is GNSGSGKKKKKKGKGSGLGSGKKKDPL (DDM34) and 16. The compound of any one of claims 1 to 15, selected from the group consisting of: GNSGSGKKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST (DDM36).

17. The polypeptide of general formula (I) is preferably a payload molecule to be delivered into a biological cell, SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKI- YGNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST, SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKI- YGNSGSGKKKKKGKGSGLGSGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGGSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGGSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST、 SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST, SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST, SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELKSINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKKGKGSGLGSGKKKDPLGST, and SHPDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREG- NDAVDNAGQEYELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAIYRLEPKD- LEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGSGGCCCSGGSSH- PDLNKLLELWPHIQEYQDLALKHGINDIFQDNGGKLLQVLLITGLTVLPGREGNDAVDNAGQE- YELASINIDLTKGFSTHHHMNPVIIAKYRQVPWIFAIYRGIAIEAI- YRLEPKDLEFYYDKWERKWYSDGHKDINNPKIPVKYVMEHGTKIYGNSGSGKKKKKGKGSGLG- SGKKKDPLGSGLGSGKKKKKGKGSGLGSGKKKDPLGST The compound of any one of claims 1 to 16, wherein the compound is covalently attached to the C-terminus of a compound selected from the group consisting of:

18. 18. The compound according to any one of claims 1 to 17, wherein the polypeptide of general formula (I) is covalently attached to the C-terminus of a payload molecule to be delivered into a biological cell which is a class II restriction endonuclease, preferably PvuII or a subunit thereof, and the payload molecule is optionally attached at its C-terminus to a linker molecule comprising 8 to 12 amino acids comprising or consisting of glycine, serine and / or cysteine ​​residues, and the polypeptide of general formula (I) is covalently attached to the C-terminus of the payload molecule or of the linker molecule, and the compound comprises one or two payload molecules, optionally separated by the linker molecule comprising 8 to 12 amino acids comprising or consisting of glycine, serine and / or cysteine ​​residues.

19. 19. The compound according to any one of claims 1 to 18, wherein the polypeptide of general formula (I) is covalently attached to the C-terminus of a payload molecule to be delivered into a biological cell, the compound comprising a first payload molecule which is a first subunit of PvuII linked at its C-terminus to a linker molecule comprising 8 to 12 amino acids comprising or consisting of glycine, serine and / or cysteine ​​residues, the linker molecule being attached at its C-terminus to a second subunit of PvuII, the polypeptide of general formula (I) being covalently attached to the C-terminus of the second subunit of PvuII.

20. 20. A compound according to any one of claims 1 to 19 for use in the treatment of a tumor patient, preferably said tumor patient suffering from neuroblastoma, colon cancer, hepatocellular carcinoma, small cell lung cancer, ovarian cancer, lung cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, placental cancer, breast cancer, kidney cancer, liver cancer, pancreatic cancer, muscle cancer, skin cancer, connective tissue cancer, bone cancer and any of these cancers in which said patient has already developed metastases, in particular for the treatment of patients with neuroblastoma, colon cancer, hepatocellular (liver) cancer, small cell lung cancer, lung cancer, breast cancer, pancreatic cancer and any of these cancers in which said patient has already developed metastases.

21. A method for treating a tumor patient, comprising administering an effective amount of a compound according to any one of claims 1 to 19 to a patient in need thereof, preferably said tumor patient suffering from neuroblastoma, colon cancer, hepatocellular (liver) cancer, small cell lung cancer, ovarian cancer, lung cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, placental cancer, breast cancer, kidney cancer, liver cancer, pancreatic cancer, muscle cancer, skin cancer, connective tissue cancer, bone cancer and any of these cancers in which said patient has already developed metastases, in particular for the treatment of patients with neuroblastoma, colon cancer, hepatocellular (liver) cancer, small cell lung cancer, lung cancer, breast cancer, pancreatic cancer and any of these cancers in which said patient has already developed metastases.

22. Use of a compound according to any one of claims 1 to 19 for the preparation of a medicament, preferably for the treatment of tumor patients, more preferably for the treatment of patients with neuroblastoma, colon cancer, hepatocellular (liver) cancer, small cell lung cancer, ovarian cancer, lung cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, placental cancer, breast cancer, kidney cancer, liver cancer, pancreatic cancer, muscle cancer, skin cancer, connective tissue cancer, bone cancer and any of these cancers in which the patient has already developed metastases, in particular for the treatment of patients with neuroblastoma, colon cancer, hepatocellular (liver) cancer, small cell lung cancer, lung cancer, breast cancer, pancreatic cancer and any of these cancers in which the patient has already developed metastases.

23. 20. A pharmaceutical formulation comprising a compound according to any one of claims 1 to 19 and a protein kinase inhibitor (PKI), preferably a DNA dependent PKI, more preferably a non-homologous end joining (NHEJ) and V(D)J repair factor DNA dependent PKI, in particular 7-methyl-2-[(7-methyl[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one (AZD7648).

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