Designed ankyrin repeat proteins binding p53, and uses thereof

Abstract

The invention is based on designed ankyrin repeat proteins (DARPins) specifically binding to a DNA binding domain of human p53. The DARPins of the invention are capable of forming a binding interface with the DNA binding domain of p53, and upon binding facilitate p53 protein stability. The DARPins of the invention are for use in the treatment of p53 related diseases such as cancer. Further, the invention pertains to nucleic acid constructs encoding the DARPin of the invention, methods for their production and cells comprising the DARPins or nucleic acid constructs of the invention.

Description

[0001] DESIGNED ANKYRIN REPEAT PROTEINS BINDING P53, AND USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] [1] The invention is based on designed ankyrin repeat proteins (DARPins) specifically binding to a DNA binding domain of human p53. The DARPins of the invention are capable of forming a binding interface with the DNA binding domain of p53, and upon binding facilitate p53 protein stability. The DARPins of the invention are for use in the treatment of p53 related diseases such as cancer. Further, the invention pertains to nucleic acid constructs encoding the DARPin of the invention, methods for their production and cells comprising the DARPins or nucleic acid constructs of the invention.

[0004] DESCRIPTION

[0005] [2] Several subtypes of the human papillomavirus (HPV) cause anogenital carcinomas. In particular, cervical cancer has been associated with HPV infection, with the two high-risk HPV16 and HPV18 subtypes being responsible for more than 70% of human cervical carcinoma, which rises to 90% when all HPV subtypes are included. In addition to anogenital cancers, an HPV infection can also cause squamous cell carcinoma of the head and neck (HNSCC), in particular HNSCC located in the oral cavity (oropharyngeal squamous cell carcinoma). The molecular mechanism of the HPV-induced tumorigenesis is well studied and is based on the inactivation of important cellular factors by viral proteins, especially by the early expressed proteins E6 and E7. Mechanistic investigations have revealed that the E7 protein binds to the phosphorylated tumor suppressor Rb and thus releases the transcription factor E2F1 , which initiates the E2F1- dependent transcription of genes involved in DNA synthesis and reentry into the cell cycle. The second viral protein that is responsible for the transformation of cells is the E6 protein. It consists of two small Zn-binding domains that are flexibly linked by a helix, and it can bind leucine-rich peptides in the cleft between both domains. One important interaction is the binding to the cellular E3 ligase E6AP. The E6AP leucine-rich peptide structures the cleft between both E6 domains and renders the complex capable of binding to the DNA-binding domain (DBD) of p53. This interaction recruits the E6AP E3 ligase to the vicinity of p53, which results in ubiquitination and proteasomal degradation of p53, thereby eliminating p53’s tumor suppressor function. Long-term infections with high-risk HPV viruses can thus transform cells. The development of vaccination against high-risk HPV strains has become a powerful weapon against virus-induced tumorigenesis, however, vaccination is not available in many countries, especially developing countries where HPV-induced cancers continue to be a severe health problem. For instance, of the worldwide -470,000 new cases of HPV-induced cervical cancer per year, more than two thirds are diagnosed in less developed countries in South-Eastern Asia, Latin America and sub-Saharan Africa. In addition to the expansion of vaccination programs, the development of an anti-HPV therapy would be important to provide treatment options for these cancer cases. One avenue towards such a therapy is the reactivation of p53 in HPV-infected cells. In principle, the cleft between the two Zn-binding domains of E6 could be druggable and corresponding drug screens have already been performed. However, this cleft has not emerged as a druggable binding site for small molecules, typically requiring a larger peptide for strong binding. That inhibition of the degradation function of the E6 protein is possible was demonstrated using a construct in which a leucine-rich peptide was fused to a PDZ domain. This protein binds both to the cleft between the two Zn-binding domains and to the C-terminal peptide of E6 with nM affinity. In cell culture experiments, this bivalent ligand stabilized p53 and induced apoptosis in HPV-positive cells.

[0006] [3] An alternative to peptide-based reagents (which might not be stable in the intracellular environment) are designed ankyrin repeat proteins (DARPins). DARPins consist of multiple stacked helical hairpin units. Certain positions in the loops and in the helices are randomized to result in a contiguous interaction surface and can be used for in vitro selection of tight binders for a folded target domain. The advantages of DARPins relative to other selective binders such as antibodies are their small size (14-18 kDa), high stability, and the absence of stabilizing disulfide bonds, which makes them ideally suited as inhibitors in the reducing cellular cytoplasm. Recently, DARPins were developed against all folded domains of p63 as well as DARPins that selectively target the hetero-tetramer consisting of a p63 dimer and a p73 dimer. With this tool, one could show that such hetero-complexes of both proteins exist in primary mouse and human tissues.

[0007] [4] The present invention seeks to provide p53-binding proteins that have characteristics suitable for use in therapeutic applications, in particular in the treatment of proliferative disorders.

[0008] [5] The present invention is based on the characterization of a DARPin that binds to the same site as the HPV E6 protein, thereby preventing its interaction with p53. Surprisingly, in HPV-infected cells, this DARPin stabilized p53 and reactivates its transcriptional activity, including the initiation of apoptosis. Thus, the developed DARPin provides a tool for studying E6-mediated tumorigenesis and for translational studies that target HPV-dependent cancers.

[0009] BRIEF DESCRIPTION OF THE INVENTION

[0010] [6] Generally, and by way of brief description, the main aspects of the present invention can be described as follows:

[0011] [7] In a first aspect, the invention pertains to a p53 binding protein, comprising a designed ankyrin repeat protein (DARPin), wherein the DARPin is capable of forming a binding interface with a DNA binding domain (DBD) of (human) p53, preferably wherein said binding interface comprises amino acid position Trp146 of hp53.

[0012] [8] In a second aspect, the invention pertains to a nucleic acid construct (NAC), comprising a nucleic acid sequence encoding a p53 binding protein, wherein the p53 binding protein comprises a designed ankyrin repeat protein (DARPin), wherein the DARPin is capable of forming a binding interface with a DNA binding domain (DBD) of (human) p53, preferably wherein said binding interface comprises amino acid position Trp146 of hp53.

[0013] [9] In a third aspect, the invention pertains to a recombinant cell comprising a p53 binding protein or a NAC of the invention.

[0014]

[0010] In a fourth aspect, the invention pertains to a pharmaceutical composition, comprising a p53 binding protein, a NAC, or a cell according to the previous aspects, optionally together with a pharmaceutically acceptable carrier or excipient

[0015]

[0011] In a fifth aspect, the invention pertains to a medicinal product for use in the treatment of a disease, such as a proliferative disorder, wherein the medicinal product comprises a p53 binding protein, a NAC, a cell, or a pharmaceutical composition according to the previous aspects.

[0016] DETAILED DESCRIPTION OF THE INVENTION

[0017]

[0012] In the following, the elements of the invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine two or more of the explicitly described embodiments or which combine the one or more of the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0018]

[0013] In a first aspect, the invention pertains to a p53 binding protein, comprising a designed ankyrin repeat protein (DARPin), wherein the DARPin is capable of forming a binding interface with a DNA binding domain (DBD) of (human) p53, preferably wherein said binding interface comprises amino acid position Trp146 of hp53.

[0019]

[0014] Cellular tumor antigen p53 is encoded by the TP53 gene and its sequence and coding gene sequences can be derived via the UniProt database in the version of July 11 , 2024, under the accession No. P04637 (preferably the canonical sequence P04637-1). A DNA binding domain (DBD) of p53 is well known, however, a preferred region is localized between amino acids 94-294 of human p53, wherein the start and endpoint in some embodiments may be localized + / - 5 amino acid positions around amino acid 94, or 294 respectively.

[0020]

[0015] The described p53 binding proteins, or binding domains thereof, comprising designed ankyrin repeat motifs or modules are also referred herein as DARPin® proteins (see Stumpp et al., Curr Opin Drug Discov Devel. 10(2): 153-9 (2007); and Binz et al., Nature Biotech. 22(5): 575-582 (2004)). DARPin® proteins can be considered as antibody mimetics with high specificity and high binding affinity to a target protein, in general, a DARPin© protein comprises at least one ankyrin repeat domain, and may comprise 2, 3, 4, 5, or more ankyrin repeat domains. Designed ankyrin repeat proteins are a class of binding molecules which have the potential to overcome limitations of monoclonal antibodies, hence allowing novel therapeutic approaches. Such ankyrin repeat proteins may comprise a single designed ankyrin repeat domain or may comprise a combination of two or more designed ankyrin repeat domains with the same or different target specificities (Stumpp et al, Drug Discov. Today 13, 695-701 , 2008; U.S. Patent No. 9,458,211). Ankyrin repeat proteins comprising only a single designed ankyrin repeat domain are small proteins (14 kDa) which can be selected to bind a given target protein with high affinity and specificity. These characteristics, and the possibility of combining two or more designed ankyrin repeat domains in one protein, make designed ankyrin repeat proteins ideal agonistic, antagonistic and / or inhibitory drug candidates. Furthermore, such ankyrin repeat proteins can be engineered to carry various effector functions, e.g. cytotoxic agents or half-life extending agents, enabling completely new drug formats.

[0021]

[0016] The ankyrin repeat domains described herein generally comprise a core scaffold that provides structure, and target binding residues that bind to a target. The structural core includes conserved amino acid residues, and the target binding surface includes amino acid residues that differ depending on the target.

[0022]

[0017] Designed ankyrin repeat proteins may also target epitopes which are not readily accessible with monoclonal antibodies. Further advantages of the described designed ankyrin repeat proteins are that they generally have low immunogenic potential and no or insignificant off-target effects. DARPin® candidates also display favorable development properties including rapid, low-cost and high-yield manufacturing and up to several years of shelf-life at 4°C. Taken together, designed ankyrin repeat proteins are an example of the next generation of protein therapeutics with the potential to surpass existing antibody drugs.

[0023]

[0018] The term "protein" refers to a polypeptide, wherein at least part of the polypeptide has, or is able to acquire a defined three-dimensional arrangement by forming secondary, tertiary, or quaternary structures within and / or between its polypeptide chain(s). If a protein comprises two or more polypeptides, the individual polypeptide chains may be linked non-covalently or covalently, e.g. by a disulfide bond between two polypeptides. A part of a protein, which individually has, or is able to acquire a defined three-dimensional arrangement by forming secondary or tertiary structures, is termed "protein domain." Such protein domains are well known to the practitioner skilled in the art.

[0024]

[0019] The term "recombinant" as used in recombinant protein, recombinant protein domain and the like, means that the polypeptides are produced by the use of recombinant DNA technologies well known by the practitioner skilled in the relevant art. For example, a recombinant DNA molecule (e.g. produced by gene synthesis) encoding a polypeptide can be cloned into a bacterial expression plasmid (e.g. pQE30, Qiagen). When such a constructed recombinant expression plasmid is inserted into a bacteria (e.g. E. coli), this bacteria can produce the polypeptide encoded by this recombinant DNA. The correspondingly produced polypeptide is called a recombinant polypeptide.

[0025]

[0020] The term "polypeptide tag" refers to an amino acid sequence attached to a polypeptide / protein, wherein the amino acid sequence is useful for the purification, detection, or targeting of the polypeptide / protein, or wherein the amino acid sequence improves the physicochemical behavior of the polypeptide / protein, or wherein the amino acid sequence possesses an effector function. The individual polypeptide tags, moieties and / or domains of a binding protein may be connected to each other directly or via polypeptide linkers. These polypeptide tags are all well known in the art and are fully available to the person skilled in the art. Examples of polypeptide tags are small polypeptide sequences, for example, His, myc, FLAG, or Strep-tags or moieties such as enzymes (for example enzymes like alkaline phosphatase), which allow the detection of the polypeptide / protein, or moieties which can be used for targeting (such as immunoglobulins or fragments thereof) and / or as effector molecules.

[0026]

[0021] The p53 binding proteins of the invention in some embodiments may be functionalized, for example covalently or non-covalently fused to another chemical entity, such as a another protein, nucleic acid, sugar or fatty acid. For example, if the p53 binding proteins of the invention are fused to one or more proteins, polypeptide linkers may be employed to covalently fuse such moiety to the p53 binding protein of the invention.

[0027]

[0022] The term "polypeptide linker" refers to an amino acid sequence, which is able to link, for example, two protein domains, a polypeptide tag and a protein domain, a protein domain and a non-polypeptide moiety such as polyethylene glycol or two sequence tags. Such additional domains, tags, non-polypeptide moieties and linkers are known to the person skilled in the relevant art. A list of example is provided in U.S. Patent No. 7,417,130 and U.S. Patent No. 8,1 10,653. Examples of such linkers are glycine-serine-linkers and proline-threonine-linkers of variable lengths. In one embodiment, the linkers have a length of between 2 and 24 amino acids; in another embodiment, the linkers have a length of between 2 and 16 amino acids.

[0023] The term "polypeptide" relates to a molecule consisting of one or more chains of multiple, i.e. two or more, amino acids linked via peptide bonds. In one embodiment, a polypeptide consists of more than eight amino acids linked via peptide bonds.

[0028]

[0024] Examples of non-proteinaceous polymer moieties fused to a p53 binding protein of the invention are hydroxyethyl starch (HES), polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene. The term "PEGylated" means that a PEG moiety is covalently attached to, for example, a polypeptide of the invention. Examples of repeat proteins containing a polypeptide linker between the repeat domain and a C-terminal Cys residue useful for binding a non- proteinaceous polymer moiety are SEQ ID NO:2, 3, 5, 6 and 7.

[0029]

[0025] The term "binding protein" refers to a protein comprising one or more binding domains and, in one embodiment, one or more polymer moieties as further explained below. In one embodiment, the binding protein comprises up to four binding domains. In one embodiment, the binding protein comprises up to two binding domains. In another embodiment, the binding protein has only one binding domain. Furthermore, any such binding protein may comprise additional protein domains that are not binding domains, multimerization moieties, polypeptide tags, polypeptide linkers and / or a single Cys residue. Examples of multimerization moieties are immunoglobulin heavy chain constant regions which pair to provide functional immunoglobulin Fc domains, and leucine zippers or polypeptides comprising a free thiol which forms an intermolecular disulfide bond between two such polypeptides. The single Cys residue may be used for conjugating other moieties to the polypeptide, for example, by using the maleimide chemistry well known to the person skilled in the art.

[0030]

[0026] The term "binding interface" refers to an interface created using binding agents comprising a first protein and a second protein which have a capability to bind to each other via one or more amino acid residues in each of the proteins. The amino acids of both proteins that are involved in the binding between the two proteins shall define a binding interface according to the invention. Accordingly if for example an amino acid of the first protein is comprised within said binding interface, then the term specifies that said amino acid is involved in the binding interaction between the two proteins. Preferably such amino acid is essential for said binding, and if substituted or removed, the binding interface would be altered, and the binding affinity is reduced.

[0031]

[0027] In this context it should be understood that the term “capable of forming” shall mean that there is a possibility of forming a binding interface in the event that the binding partners of the interface are in proximity to each other.

[0032]

[0028] In one embodiment, said DARPin comprises an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 1 to 4 are optionally missing, and wherein the second last position and / or the last position of SEQ ID NOs: 1 to 4 are optionally substituted. Thus, in one embodiment, said ankyrin repeat domain comprises an amino acid sequence with at least 90% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4. In another embodiment, said ankyrin repeat domain comprises an amino acid sequence with at least 93% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4; and in a further embodiment, said ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4. In one embodiment, said ankyrin repeat domain comprises an amino acid sequence with at least 98% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4; and in one embodiment, said ankyrin repeat domain comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 4. Thus, in one embodiment, said p53 binding protein comprises an ankyrin repeat domain having binding specificity for p53, in particular to a DBD of p53, wherein said DARPin comprises an amino acid sequence selected from SEQ ID NOs: 1 to 4, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 1 to 4 are optionally missing, and wherein L at the second last position and / or N at the last position of SEQ ID NOs: 1 to 4 are optionally substituted.

[0033]

[0029] In some embodiments of the invention the p53 binding protein is preferred, wherein the DARPin has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, or preferably 95% sequence identity to any one of SEQ ID NO: 1 to 4.

[0034]

[0030] Preferably, when referring herein to a DARPIN sequence shown in any one of SEQ ID NO: 1 to 4, in any of the specific aspects or embodiments disclosed, the G at position 1 and / or S at position 2 of said ankyrin repeat domains are optionally missing; and A at the second last position and / or A at the last position of said ankyrin repeat domains of SEQ ID NOs: 1 to 4, are optionally substituted by another amino acid, such as L at the second last position and / or N at the last position. Most preferably the second last position and the last position remain A.

[0035]

[0031] In some embodiments of the invention the p53 binding protein is preferred, wherein the DARPin has at least 95%, 96%, 97%, 98%, or preferably 99% sequence identity to SEQ ID NO: 1 (C10).

[0036]

[0032] In some embodiments of the invention the p53 binding protein is preferred, wherein the DARPin consists of, or consists essentially of, SEQ ID NO: 1 , 2, 3 or 4; preferably of SEQ ID NO: 1 or 4; optionally with not more than 10, preferably not more than 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acid substitution, deletion, or addition compared to these sequences.

[0037]

[0033] In one embodiment, said DARPin comprises an amino acid sequence with at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with any one of SEQ ID NOs: 1 to 10 (preferably any one of SEQ ID NOs: 9 and 10), wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 1 to 10 are optionally missing, and wherein the second last position and / or the last position of SEQ ID NOs: 1 to 10 are optionally substituted. Thus, in one embodiment, said ankyrin repeat domain comprises an amino acid sequence with at least 90% amino acid sequence identity with any one of SEQ ID NOs: 1 to 10. In another embodiment, said ankyrin repeat domain comprises an amino acid sequence with at least 93% amino acid sequence identity with any one of SEQ ID NOs: 1 to 10; and in a further embodiment, said ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with any one of SEQ ID NOs: 1 to 10. In one embodiment, said ankyrin repeat domain comprises an amino acid sequence with at least 98% amino acid sequence identity with any one of SEQ ID NOs: 1 to 10; and in one embodiment, said ankyrin repeat domain comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 10. Thus, in one embodiment, said p53 binding protein comprises an ankyrin repeat domain having binding specificity for p53, in particular to a DBD of p53, wherein said DARPin comprises an amino acid sequence selected from SEQ ID NOs: 1 to 10, wherein G at position 1 and / or S at position 2 of SEQ ID NOs: 1 to 10 are optionally missing, and wherein L at the second last position and / or N at the last position of SEQ ID NOs: 1 to 10 are optionally substituted.

[0038]

[0034] In some embodiments of the invention the p53 binding protein is preferred, wherein the DARPin has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, or preferably 95% sequence identity to any one of SEQ ID NO: 1 to 10 (preferably to any one of SEQ ID NOs: 9 and 10).

[0039]

[0035] Preferably, when referring herein to a DARPIN sequence shown in any one of SEQ ID NO: 1 to 10, in any of the specific aspects or embodiments disclosed, the G at position 1 and / or S at position 2 of said ankyrin repeat domains are optionally missing; and A at the second last position and / or A at the last position of said ankyrin repeat domains of SEQ ID NOs: 5 to 10, are optionally substituted by another amino acid, such as L at the second last position and / or N at the last position. Most preferably the second last position and the last position remain A.

[0040]

[0036] In some embodiments of the invention the p53 binding protein is preferred, wherein the DARPin consists of, or consists essentially of, SEQ ID NO: 5, 6, 7, 8, 9 or 10; preferably of SEQ ID NO: 9 or 10; optionally with not more than 10, preferably not more than 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acid substitution, deletion, or addition compared to these sequences. More preferably, in some embodiments, said amino acid substitution or deletion is not at positions 16, 18, 19 and 82 when aligned to SEQ ID NO: 1.

[0041]

[0037] The term "identity" refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e. , an "algorithm"). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, Fl. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991 , New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0042]

[0038] In calculating percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences. One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wl). The computer algorithm GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid or nucleotide (the "matched span", as determined by the algorithm). A gap opening penalty (which is calculated as 3x the average diagonal, wherein the "average diagonal" is the average of the diagonal of the comparison matrix being used; the "diagonal" is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1 / 10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm.

[0043]

[0039] A standard comparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) may also be used by the algorithm.

[0044]

[0040] Examples of parameters that can be employed in determining percent identity for polypeptides or nucleotide sequences using the GAP program are the following: (i) Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453; (ii) Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra,- (iii) Gap Penalty: 12 (but with no penalty for end gaps); (iv) Gap Length Penalty: 4; (v) Threshold of Similarity: 0.

[0045]

[0041] In some embodiments the DARPin has a sequence that when aligned with SEQ ID NO: 1 comprises a variant amino acid at a corresponding position of SEQ ID NO: 1 selected from the group of Q16, D18, E19 and H82.

[0042] In some embodiments the DARPin has a sequence that when aligned with SEQ ID NO: 1 comprises variant amino acids at two or three corresponding positions of SEQ ID NO: 1 selected from the group of Q16, D18, E19 and H82.

[0046]

[0043] In some embodiments the DARPin has a sequence that when aligned with SEQ ID NO: 1 comprises variant amino acids at corresponding positions Q16, D18 and H82 in SEQ ID NO: 1, or (preferably) comprises variant amino acids at corresponding positions Q16, E19 and H82 in SEQ ID NO: 1.

[0047]

[0044] In some embodiments the DARPin has a sequence that when aligned with SEQ ID NO: 1 does not comprise variant amino acids at corresponding positions Trp13 and Trp46 of SEQ ID NO: 1

[0048]

[0045] In some embodiments the DARPin has a sequence that when aligned with SEQ ID NO:

[0049] 1 comprises a tryptophan residue at corresponding positions E19 of SEQ ID NO: 1.

[0050]

[0046] In some embodiments the DARPin has an amino acid sequence selected from SEQ ID:

[0051] 2 (H82R), 3 (Q16E, D18N, H82R) or 4 (Q16E, E19R, H82R).

[0052]

[0047] In some embodiments the DARPin has an amino acid sequence selected from SEQ ID: 5 (Q16E), 6 (Q16E, D18N), 7 (Q16E, E19R), 8 (Q16E, N18R, H82R), 9 (Q16E, R19W, H82R) or 10 (Q16E, D18N, R19W, H82R).

[0053]

[0048] In preferred embodiments, the DARPin has an amino acid sequence shown in SEQ ID: 4.

[0054]

[0049] In preferred embodiments, the DARPin has an amino acid sequence shown in SEQ ID: 9 or 10.

[0055]

[0050] Yet another embodiment pertains to a p53 binding protein, wherein the binding protein binds to p53 protein with a KD of less than 250nM, more preferably less than 100nM, more preferably of less than 80nM, most preferably of less than 50nM, and preferably of about 72nM, of about 42nM or of about 29nM. In preferred embodiments, the binding affinity is determined by isothermal titration calorimetry (ITC), preferably as described in Example 2. For example the affinity is determined with the isolated p53 DBD expressed in E. coli. The examples of this disclosure show the respective conditions used to determine DARPin binding affinity (see also materials and methods). Buffer conditions are preferably: 50 mM HEPES pH 7.4, 150 mM NaCI, 0.5 mM TCEP). Preferably, measurements were performed at 15°C or 25°C; most preferably the measurements are performed at 25°C.

[0056]

[0051] Yet another embodiment pertains to a p53 binding protein, wherein the binding protein binds to p53 protein with a KD of about 3 nM. Preferably, measurements were performed at 15°C, 25°C, 30°C, or 37°C; most preferably the measurements are performed at 25°C or 30°C.

[0052] In an embodiment of the invention a p53 binding protein, in particular when bound to p53, does not inhibit DNA binding (capability) of p53.

[0057]

[0053] In another embodiment, the p53 binding protein reduces E6-mediated protein degradation of p53.

[0058]

[0054] The present invention is based on the surprising finding that the DARPins when bound to p53 reduce or inhibit their E6 mediated degradation and thereby increase protein stability.

[0059]

[0055] In a second aspect, the invention pertains to a nucleic acid construct (NAC), comprising a nucleic acid sequence encoding a p53 binding protein, wherein the p53 binding protein comprises a designed ankyrin repeat protein (DARPin), wherein the DARPin is capable of forming a binding interface with a DNA binding domain (DBD) of (human) p53, preferably wherein said binding interface comprises amino acid position Trp146 of hp53.

[0060]

[0056] A NAC of the invention can comprise one or more additional features permitting the expression of the encoded p53 binding protein or component of said p53 binding protein (eg the DARPin) in a cell (such as in a host cell). Examples of NACs of the invention include, but are not limited to, plasmid vectors, viral vectors, mRNA, non-episomal mammalian vectors and expression vectors, for example, recombinant expression vectors. The nucleic acid constructs of the invention can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a cell, such as a host cell, (see below). The nucleic acid constructs of the invention will be, typically, recombinant nucleic acids, and / or may be isolated and / or substantially pure. Recombinant nucleic acids will, typically, be non-natural; particularly if they comprise portions that are derived from different species and / or synthetic, in-vitro or mutagenic methods.

[0061]

[0057] The NAC of the invention is preferably a nucleic acid is selected from DNA, RNA or a DNA / RNA hybrid. However, such NAC may comprise one or more nonstandard nucleic bases. For example, the NAC can be an mRNA construct suitable for cellular delivery and translation of the encoded DARPin, or is a DNA construct comprising one or more sequence elements, such as a promoter, enhancer etc., for cellular expression of the DARPin.

[0062]

[0058] Delivery of NAC of the invention, for example for the herein disclosed medical applications, may involve known methods of nucleic acid delivery into human organisms. Particular preferred is that the NAC is RNA based and delivered using lipid nanoparticles (LNPs). Herein, the term “lipid nanoparticles” is defined as molecules that are spherical in shape and comprise a solid lipid core stabilized by a surfactant. The core lipids can be steroids, fatty acids, acylglycerols, waxes, and combinations of them. Surfactants may be biological membrane lipids such as phospholipids, sphingomyelins and bile salts (e.g., sodium taurocholate). All of these may be utilized as stabilizers in the lipid nanoparticles used for pharmaceutical compositions of the invention. The use of LNP for drug delivery is well known to the skilled artisan.

[0063]

[0059] In a third aspect, the invention pertains to a recombinant cell comprising a p53 binding protein or a NAC of the invention. A cell according to the invention may comprise an expression cell suitable for the expression of DARPin or alternatively a target cell

[0064]

[0060] In a fourth aspect, the invention pertains to a pharmaceutical composition, comprising a p53 binding protein, a NAC, or a cell according to the previous aspects, optionally together with a pharmaceutically acceptable carrier or excipient.

[0065]

[0061] A pharmaceutical composition of the invention may for example contain LNPs as described above.

[0066]

[0062] Pharmaceutically acceptable carriers and / or diluents are known to the person skilled in the art and are explained in more detail below. Even further, a diagnostic composition is provided comprising one or more of the above mentioned recombinant binding proteins and / or designed ankyrin repeat domains, and / or nucleic acids, in particular recombinant binding proteins and / or nucleic acids of the present invention.

[0067]

[0063] A pharmaceutical composition comprises a p53 binding protein, and / or a designed ankyrin repeat domain, and / or a nucleic acid, preferably a recombinant binding protein and / or a nucleic acid, as described herein and a pharmaceutically acceptable carrier, excipient or stabilizer, for example as described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980.

[0068]

[0064] Suitable carriers, excipients or stabilizers known to one of skill in the art include, for example, saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers and preservatives. Other suitable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids and amino acid copolymers. A pharmaceutical composition may also be a combination formulation, comprising an additional active agent, such as an anti-cancer agent or an anti-angiogenic agent, or an additional bioactive compound.

[0069]

[0065] The formulations to be used for in vivo administration must be aseptic or sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0070]

[0066] In a fifth aspect, the invention pertains to a medicinal product for use in the treatment of a disease, such as a proliferative disorder, wherein the medicinal product comprises a p53 binding protein, a NAC, a cell, or a pharmaceutical composition according to the previous aspects.

[0071]

[0067] In a preferred embodiment, the disease is a proliferative disorder, preferably cancer. Specifically preferred is that the proliferative disorder is characterized in that it involves p53. In some embodiments, the proliferative disorder of the invention is a cancer or tumor, such as a solid or liquid tumor. In context of the invention, a cancer or tumor is associated with a reduced stability of p53.

[0072]

[0068] The therapeutic application of the medicinal product of the invention comprises the administration of a therapeutically effective amount of the p53 binding protein to a subject suffering from the disease. The subject preferably is a mammalian, more preferably a human subject.

[0073]

[0069] The medical uses of the invention preferably comprise a method of treatment of a tumor or cancer in a subject, the method comprising the step of administering a therapeutically effective amount of a medicinal product as defined herein to the subject. Such therapy preferably thereby involves a stabilization of p53 protein in cells involved with the disease caused by administration of the medicinal product. Preferably, the stabilization is a reduced E6- mediated protein degradation of p53. As an example of a tumor or cancer disease, the present invention preferably relates to cervical cancer, such as a cervical cancer caused by HPV infection.

[0074]

[0070] The terms “of the [present] invention”, “in accordance with the invention”, “according to the invention” and the like, as used herein are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0075]

[0071] As used herein, the term “comprising” is to be construed as encompassing both “including” and “consisting of”, both meanings being specifically intended, and hence individually disclosed embodiments in accordance with the present invention. Where used herein, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. In the context of the present invention, the terms “about” and “approximately” denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±20%, ±15%, ±10%, and for example ±5%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. Where an indefinite or definite article is used when referring to a singular noun, e.g. "a", "an" or "the", this includes a plural of that noun unless something else is specifically stated.

[0076]

[0072] It is to be understood that application of the teachings of the present invention to a specific problem or environment, and the inclusion of variations of the present invention or additional features thereto (such as further aspects and embodiments), will be within the capabilities of one having ordinary skill in the art in light of the teachings contained herein.

[0077]

[0073] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0078]

[0074] All references, patents, and publications cited herein are hereby incorporated by reference in their entirety.

[0079]

[0075] In view of the above, it will be appreciated that the present invention also relates to the following itemised embodiments:

[0080] Item 1 : A p53 binding protein, comprising a designed ankyrin repeat protein (DARPin), wherein the DARPin is capable of forming a binding interface with a DNA binding domain (DBD) of (human) p53, wherein said binding interface comprises amino acid position Trp146 of hp53.

[0081] Item 2: The p53 binding protein of item 1 , wherein the DARPin comprises an amino acid sequence having not more than 90% sequence identity to the amino acid sequence shown in any one of SEQ ID NO: 1 to 4.

[0082] Item 3: The p53 binding protein of item 1 or 2, wherein the DARPin has a sequence that when aligned with SEQ ID NO: 1 comprises an amino acid substitution at a corresponding position of SEQ ID NO: 1 selected from the group of Q16, D18, E19 and H82; and I or wherein the DARPin has a sequence that when aligned with SEQ ID NO: 1 comprises variant amino acids at two or three corresponding positions of SEQ ID NO: 1 selected from the group of Q16, D18, E19 and H82; and / or wherein the DARPin has a sequence that when aligned with SEQ ID NO: 1 comprises variant amino acids at corresponding positions Q16, D18 and H82 in SEQ ID NO: 1 , or (preferably) comprises variant amino acids at corresponding positions Q16, E19 and H82 in SEQ ID NO: 1. Item 4: The p53 binding protein of item 1 to 3, wherein the DARPin has a sequence that when aligned with SEQ ID NO: 1 does not comprise variant amino acids at corresponding positions Trp13 and Trp46 of SEQ ID NO: 1

[0083] Item 5: The p53 binding protein of any one of items 1 to 4, wherein the DARPin has an amino acid sequence selected from SEQ ID: 2 (H82R), 3 (Q16E,D18N,H82R) or 4 (Q16E, E19R, H82R), optionally with not more than 9 amino acid substitutions, additions and / or deletions.

[0084] Item 6: The p53 binding protein of any one of items 1 to 5, wherein the binding protein binds to p53 protein with a KD of less than 250nM, more preferably less than 100nM, more preferably of less than 80nM, most preferably of less than 50nM, and preferably of about 72nM, of about 42nM or of about 29 nM.

[0085] Item 7: A nucleic acid construct (NAC), comprising a nucleic acid sequence encoding a p53 binding protein of any one of items 1 to 6.

[0086] Item 8: The NAC of item 7, wherein the nucleic acid is selected from DNA, RNA or a DNA / RNA hybrid.

[0087] Item 9: The NAC of item 7 or 8, which is an mRNA construct suitable for cellular delivery and translation of the encoded DARPin, or is a DNA construct comprising one or more sequence elements, such as a promoter, enhancer etc., for cellular expression of the DARPin.

[0088] Item 10: A (recombinant) cell, comprising a p53 binding protein of any one of items 1 to 6 or a NAC of any one of items 7 to 9.

[0089] Item 11: A pharmaceutical composition, comprising a p53 binding protein of any one of items 1 to 6, a NAC of any one of items 7 to 9, or a cell of item 10, optionally together with a pharmaceutically acceptable carrier or excipient.

[0090] Item 12: The pharmaceutical composition of item 11 , which is a lipid nanoparticle comprising a p53 binding protein of any one of items 1 to 6, a NAC of any one of items 7 to 9.

[0091] Item 13: A medicinal product for use in the treatment of a disease, comprising a p53 binding protein of any one of items 1 to 6, a NAC of any one of items 7 to 9, a cell of item 10, or a pharmaceutical composition of item 11 or 12.

[0092] Item 14: The medicinal product for use of item 13, wherein the disease is a proliferative disorder, preferably a cancer or tumor. Item 15: The medicinal product for use of any one of items 13 to 14, wherein the use comprises a stabilization of p53 protein in cells involved with the disease, preferably wherein the stabilization is a reduced E6-mediated protein degradation of p53.

[0093]

[0076]

[0094] BRIEF DESCRIPTION OF THE FIGURESAND SEQUENCES

[0095]

[0077] The figures show:

[0096]

[0078] Fig. 1 DARPin C10 inhibits the HPV E6 mediated degradation of p53. a, In vitro assay to investigate HPV E6-mediated degradation of p53. The HPV E6 protein is binding to the p53 DBD and recruits the E3-ubiquitin ligase E6AP, leading to ubiquitination and proteasomal degradation of p53. Myc-tagged p53 was in vitro translated in rabbit reticulocyte lysates (RRL) and co-incubated with a GST fusion of the HPV16-E6 protein (GST-E6), with or without the respective DARPin. Samples were analyzed using an a-Myc antibody (upper panel) or an a- GST antibody (lower panel). Co-incubation of p53 with GST did not show any degradation, whereas incubation with GST-E6 led to degradation of p53. Co-incubation of p53 with E6 and DARPin C10 prevented the HPV E6-mediated degradation of p53, while the control DARPin had no effect, b, Quantification of the degradation assay in (a). The relative protein level after 180 min, normalized to the protein level after 0 min, is shown on the y-axis. The bar diagram shows the mean values and the error bars the corresponding SD of three biological replicates. An ordinary one-way ANOVA was performed to assess the statistical significance, c, Pulldown of GFP-tagged E6 protein of HPV16 with immobilized p53 DBD, and competition with DARPin C10. Biotinylated p53 DBD was immobilized on beads and incubated with DARPin (DP) and GFP-E6. DARPin C10 efficiently blocked binding of E6 to the p53 DBD, while the control DARPin had no effect, d, Quantification of the pulldown experiment in (c). The relative pulldown efficiency normalized to the input samples is shown on the y-axis. The bar diagram shows the mean values and the error bars the corresponding SD of three biological replicates. An ordinary one-way ANOVA was performed to assess the statistical significance, e, Interaction study of the p53 DBD and DARPin C10 using ITC. The top diagram shows the raw measurement and the bottom diagram the integrated heat per titration step. The KD value for the interaction is given in the bottom right corner. The measurement was performed at 25 °C. f, Pulldown experiments of p53 family members with DARPin C10 and a non-binding control DARPin. The DARPins were biotinylated and immobilized on beads. H1299 cells were transiently transfected with Myc- tagged p53, ANp63a or TAp73a. Input signals are shown on the left, pulldown signals on the right. In the upper panel, the Myc-tag of the p53 family members is detected using an a-Myc antibody, in the lower panel the used biotinylated DARPins are detected using a streptavidin- HRP conjugate. The experiment was performed in biological triplicates, with one exemplary Western blot shown.

[0097]

[0079] Fig. 2 Investigation of the binding epitope of DARPin C10 on the p53 DBD. a, DNA pulldown experiments with p53 and an immobilized dsDNA oligomer containing the 20 bp binding site of the human p21 promotor. Pre-incubation of p53 with DARPin C10, or the control DARPin (DP), do not inhibit p53 interaction with DNA. b, Quantification of the pulldown experiment in (a). The relative pulldown efficiency normalized to the input samples is shown on the y-axis. The bar diagram shows the mean values and the error bars the corresponding SD of three biological replicates. An ordinary one-way ANOVA analysis was performed to assess the statistical significance, c, Crystal structure of DARPin C10 (blue) in complex with the p53 DBD (red) shown in two different orientations rotated by 90°. Upper panel, cartoon representation, lower panel, space filling model, d, Comparison of a crystal structure of HPVDE6 protein bound to the p53 DBD (upper part, PDB: 4XR8) and the crystal structure of DARPin C10 bound to the p53 DBD (this work, lower part). The DBD is shown in the same orientation. Comparison of both structures shows that DARPin C10 binds to the same region of the p53 DBD as HPV E6, thereby blocking the p53-E6 interaction, e, Superimposition of the crystal structure shown in (c) with a DNA complex indicates that DARPin C10 does not block the DNA-binding surface of the p53 DBD (PDB: 4KMD)22. f, Superimposition of the crystal structure shown in (c) with a crystal structure of the p53 DBD bound to a full consensus site as a self-assembled tetramer (PDB:4KMD), suggesting that none of the DBD-DBD interfaces are blocked by DARPin binding.

[0098]

[0080] Fig. 3 Reactivation of p53 by inhibition of its HPV E6 mediated degradation, a, Transactivation assay in HPV18 positive HeLa cells with a luciferase expression construct under the control of a pBDS-2 promotor. Increasing amounts of DARPin C10 or control DARPin (eDP) were transiently transfected in HeLa cells. An increase in the transcriptional activity regulated by p53 can be observed with increasing amounts of DARPin C10, whereas the control DARPin had no effect. The bar diagram shows the mean values and the error bars the corresponding SD of three biological replicates. An ordinary one-way ANOVA was performed to assess the statistical significance, b, c, Same assay as in (a) but with a luciferase expression construct under control of a PUMA promotor (b) or a p21 promotor (c). d, Exemplary Western blot of the experiments in (a-c) detecting the p53 protein level using the a-p53 antibody DO-I (Santa Cruz Biotechnology). The level of vinculin served as a loading control. Expression of C10 led to a significant stabilization of p53. e-h, Same experiments as in (a-d) but with HPV16 positive SiHa cells, i, j, Transactivation assay with endogenous p53 in HPV-negative U-2 OS cells on the pBDS-2 promotor (i) and the PUMA promotor (j). U-2 OS cells carry an amplification of the MDM2 gene, leading to degradation of p53. DARPin C10 does not interfere with the MDM2-mediated degradation of p53. The MDM2 inhibitor Nutlin-3a restores transcriptional activity of p53 by inhibiting its degradation, k, Exemplary Western blot of the experiments in (i, j) detecting the p53 protein level using the a-p53 antibody DO-I (Santa Cruz Biotechnology). The level of vinculin served as a loading control.

[0099]

[0081] Fig. 4 Reactivation of p53 in HPV-positive cells results in upregulation of pro- apoptotic p53 target genes, a, RT-qPCR analysis of the expression of the PUMA gene in HPV18-positive HeLa cells expressing either no DARPin, control DARPin (eDP), DARPin C10 or the inhibitory DARPin G4. Total RNA was isolated from the cells and reverse-transcribed into cDNA prior to analysis by quantitative PCR. Gene expression was referenced to the housekeeping gene HPRT-1. b-d, Same experiment as in (a) but with the p53 target genes NOXA (b), p21 (c) and MDM2 (d). e-h, RT-qPCR analysis of the expression of the p53 target genes PUMA (e), NOXA (f), p21 (g) and MDM2 (h) in HPV16-positive SiHa cells expressing either no DARPin, control DARPin, DARPin C10 or the inhibitory DARPin G4. i-l, Same experiment as in (a-d) but with HPV-negative U-2 OS cells carrying an amplification of the MDM2 gene, leading to degradation of p53. DARPin C10 does not interfere with the MDM2- mediated degradation of p53. In (a-l) the bar diagrams show the mean values and the error bars the corresponding SD of three biological replicates. An ordinary one-way ANOVA was performed to assess the statistical significance.

[0100]

[0082] Fig. 5 RNAseq. a-c, Volcano plots showing differentially regulated genes in the indicated pair-wise comparisons using a fold change of 2 (abs(log2 FC)>1) and an adjusted p- value < 0.05 as significance thresholds. Significantly down-regulated genes are colored in light blue, upregulated genes in light red, and their numbers are stated in parentheses. Significantly upregulated genes included in the HALLMARK_P53_PATHWAY or FISCHER_DIRECT_P53_TARGETS_META_ANALYSIS gene sets (MSigDB, version 7.1) are shown in red. Gene name labels identify selected p53 target genes, d, e, Gene Set Enrichment Analysis (GSEA) results of HeLa cells expressing DARPin C10 versus control, d, Bar graph shows normalized enrichment scores (NES) for the top 15 enriched or depleted gene sets from the H, 02, C6, and 07 MSigDB collections, e, Enrichment plots of exemplary p53-related MSigDB gene sets with their normalized enrichment scores (NES), nominal p-value (NOM p) and False Discovery Rate q-value (FDR q).

[0101]

[0083] Fig. 6 Reactivation of p53 in HPV-positive cells results in a decreased cell viability, a, Immunofluorescence staining to analyze expression of p53 in HeLa cells stably expressing DARPin 010 (left column) or control DARPin (eDP) (right column). Cells were fixed with formaldehyde and incubated with anti-Myc (Abeam) antibody to detect DARPin expression and a-p53 antibody DO-I (Santa Cruz Biotechnology) to detect p53 expression, followed by incubation with the secondary antibodies Alexa Fluor 568 anti-rabbit (Life Technologies) and Alexa Fluor 647 anti-mouse (Life Technologies), b, c, Same experiment as in (a) but with SiHa cells stably expressing C10 or control DARPin (b) or U-2 OS cells stably expressing either C10 or control DARPin (c). d, Cell viability of HeLa cells stably expressing DARPin C10 (red) or control DARPin (blue). Viability was monitored over 80 h using the Real Time Gio MT cell viability assay (Promega). Cells expressing DARPin C10 showed a significantly reduced cell viability, e, Same assay as in (d) but with HPV16-positive SiHa cells. Again, cells expressing C10 showed a significantly decreased viability compared to cells expressing control DARPin. f, Same assay as in (d) but with HPV-negative U-2 OS cells that carry an amplification of the MDM2 gene, leading to degradation of p53. DARPin C10 had no significant impact on the viability of these cells. All data were normalized to the cell viability after 1 h. Standard deviations of biological triplicates are shown by dotted lines.

[0102]

[0084] Fig. 7 Reactivation of destabilized p53 mutants by DARPin C10 or DARPin C10H82R. The indicated p53 mutants were co-expressed with control DARPin, DARPin C10 or DARPin C10 H82R and the transcriptional activity on the pBDS-2 promoter was assessed using a luciferase reporter assay. The heatmap shows the fold induction on the promoter relative to wild-type p53. The assay was performed in biological triplicates with the mean value shown in the heatmap.

[0103]

[0085] Fig. 8 ITC measurements with the triple mutant DARPin (left) and the quadruple mutant DARPin (right). The measurement temperature was 30°C.

[0104]

[0086] Fig. 9 Thermodynamic parameters measured with ITC for the original variant (C10 WT) and the two mutants.

[0105]

[0087] Fig. 10 Detail of the crystal structure of the DARPin triple mutant - p53 DBD complex with focus on the side chain of Trp 19.

[0106]

[0088] Fig. 11 Reactivation of transcriptional activity of various p53 mutants. Activity is measured relative to the level of wild-type p53. The triple mutant DARPin can reactivate more mutants and to higher levels than other versions of the DARPin. Some mutants, such as R273H and R248W, have a mutated DNA binding interface and therefore cannot be reactivated. Others, such as R175H and H179Y, lose the Zn ion and therefore cannot be reactivated either.

[0107]

[0089] Fig. 12 Reactivation assays in transiently transfected cells with the mutations R158L (a), R158G(b), R158H (c), V157F (d). The reactivation of mutated p53 was measured in the specified cell lines using qPCR after expression of the DARPin. In addition, experiments were conducted with Nutlin-3a, which showed synergistic effects.

[0108]

[0090] The sequences show:

[0109]

[0091] SEQ ID NOs. 1 shows DARPin C10 (KD=250 nM) GSDLGKKLLEAAWHGQDDEVRILMANGADVNATDQSGMTPLHLAAWRGHLEIVEVLLKTGAD

[0110] VNAIDRWGKTPLHLAARIGHLEIVEVLLKAGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAA

[0111]

[0092] SEQ ID NOs. 2 shows DARPin C10_H82R (KD=72nM)

[0112] GSDLGKKLLEAAWHGQDDEVRILMANGADVNATDQSGMTPLHLAAWRGHLEIVEVLLKTGAD

[0113] VNAIDRWGKTPLHLAARIGRLEIVEVLLKAGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAA

[0114]

[0093] SEQ ID NOs. 3 shows DARPin C10_ Q16E,D18N,H82R (KD=42 nM)

[0115] GSDLGKKLLEAAWHGEDNEVRILMANGADVNATDQSGMTPLHLAAWRGHLEIVEVLLKTGADV

[0116] NAIDRWGKTPLHLAARIGRLEIVEVLLKAGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAA

[0117]

[0094] SEQ ID NOs. 4 shows DARPin C10_triple mutant (Q16E, E19R, H82R) (KD=29 nM)

[0118] GSDLGKKLLEAAWHGEDDRVRILMANGADVNATDQSGMTPLHLAAWRGHLEIVEVLLKTGADV

[0119] NAIDRWGKTPLHLAARIGRLEIVEVLLKAGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAA

[0120]

[0095] SEQ ID NOs. 5 shows DARPin C10_Q16E (KD=139 nM)

[0121] GSDLGKKLLEAAWHGEDDEVRILMANGADVNATDQSGMTPLHLAAWRGHLEIVEVLLKTGADV

[0122] NAIDRWGKTPLHLAARIGHLEIVEVLLKAGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAA

[0123]

[0096] SEQ ID NOs. 6 shows DARPin C10_double mutant (Q16E, D18N) (KD=118 nM)

[0124] GSDLGKKLLEAAWHGEDNEVRILMANGADVNATDQSGMTPLHLAAWRGHLEIVEVLLKTGADV

[0125] NAIDRWGKTPLHLAARIGHLEIVEVLLKAGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAA

[0126]

[0097] SEQ ID NOs. 7 shows DARPin C10_double mutant (Q16E, E19R) (KD=103 nM)

[0127] GSDLGKKLLEAAWHGEDDRVRILMANGADVNATDQSGMTPLHLAAWRGHLEIVEVLLKTGADV

[0128] NAIDRWGKTPLHLAARIGHLEIVEVLLKAGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAA

[0129]

[0098] SEQ ID NOs. 8 shows DARPin C10_triple mutant (Q16E, N18R, H82R) (KD=46 nM)

[0130] GSDLGKKLLEAAWHGEDREVRILMANGADVNATDQSGMTPLHLAAWRGHLEIVEVLLKTGADV

[0131] NAIDRWGKTPLHLAARIGRLEIVEVLLKAGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAA

[0132]

[0099] SEQ ID NOs. 9 shows DARPin C10_triple mutant (Q16E, R19W, H82R) (KD=3 nM)

[0133] GSDLGKKLLEAAWHGEDDWVRILMANGADVNATDQSGMTPLHLAAWRGHLEIVEVLLKTGAD

[0134] VNAIDRWGKTPLHLAARIGRLEIVEVLLKAGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAA

[0135]

[0100] SEQ ID NOs. 10 shows DARPin C10_quadruple mutant (Q16E, D18N, R19W, H82R)

[0136] (KD=3 nM)

[0137] GSDLGKKLLEAAWHGEDNWVRILMANGADVNATDQSGMTPLHLAAWRGHLEIVEVLLKTGAD

[0138] VNAIDRWGKTPLHLAARIGRLEIVEVLLKAGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAA EXAMPLES

[0139]

[0101] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.

[0140]

[0102] The examples show:

[0141]

[0103] Example 1: DARPin C10 inhibits HPV E6 induced degradation of p53

[0142]

[0104] To identify highly specific binders for the p53 DBD the inventors used a strategy similar to the one described previously for the selection of DARPins targeting the p63 domains20and the hetero-tetramer of p63 and p73. Ribosome display of the DARPin library was used for the selection of binders to p53 DBD (amino acids 94-294), followed by screening of individual clones (see Methods). Initial screening using homogenous time-resolved fluorescence (HTRF) yielded several DARPins binding to the p53 DBD, which were further characterized for their ability to inhibit the degradation by the HPV E16 protein. To address this question, the inventors used an in vitro degradation assay by expressing p53 in rabbit reticulocyte lysates (RRL) followed by incubation with GST-fused HPV16 E6 protein alone or GST-fused E6 protein and DARPin. Fusion to GST was necessary to render the E6 protein soluble when expressed by this method. Incubation with GST itself served as negative control, and protein levels were analyzed by Western blot. As expected, incubation of p53 with HPV16 E6 led to complete degradation of p53 within 180 min. The non-binding control DARPin E3_5 had no inhibitory effect on this degradation (Fig. 1a, b). In contrast, DARPin C10 efficiently inhibited degradation of p53, while other DARPins showed less or no protection (Supplementary Fig. 1a, b). Quantification of the protein level after 180 min indicated that incubation with DARPin C10 resulted in the same p53 level as in the GST-control sample in which no degradation takes place (Fig. 1b). To investigate if the inhibitory effect is dependent on the HPV strain, the inventors repeated the assay with HPV18-E6 and HPV35-E6, which returned the same results as for HPV16 E6 (Supplementary Fig. 1c, d). Based on these results, the inventors decided to further concentrate on characterizing the DARPin C10.

[0143]

[0105] The inventors hypothesized that inhibition of degradation of p53 was most likely due to the displacement of the E6 protein from p53 by the DARPin. To verify this assumption, the inventors performed a competitive pulldown assay in which biotinylated p53 DBD was immobilized on magnetic streptavidin beads and incubated with GFP-fused HPV16 E6 alone or in combination with DARPin. Pulldown of GFP-fused HPV16 E6 protein was detected for the samples containing no DARPin or the non-binding control DARPin, while co-incubation with DARPin C10 resulted in a significant reduction of the interaction of the p53 DBD with HPV16 E6 (Fig. 1c, d).

[0144]

[0106] Example 2: Affinity and selectivity of binding

[0145]

[0107] The inventors characterized the binding of DARPin C10 to the p53 DBD using isothermal titration calorimetry (ITC) with the isolated p53 DBD expressed in E. coli. Titration of the p53 DBD with DARPin C10 yielded a dissociation constant of KD = 243 nM (Fig. 1e) at 25°C or 180 nM at 15°C. Due to the high degree of sequence conservation in the DBDs of all p53 family members the inventors wanted to determine the specificity of this DARPin and measured binding to the DBDs of p63 and p73 as well, but could not detect any interaction, demonstrating the high specificity of DARPin C10. Titration of the DBDs of all p53 family members with a nonbinding control DARPin did not show any interaction either. Next, the inventors investigated if the specific interaction of DARPin C10 with the p53 DBD is preserved in the context of the full- length protein in cell culture lysate. Hence, the inventors overexpressed full-length p53, ANp63a and TAp73a in H1299 cells, and cell lysates were incubated with DARPin C10 or control DARPin immobilized on streptavidin magnetic beads. For DARPin C10, a strong pulldown of p53 was detected, while no interaction with ANp63a or TAp73a was observed. The control DARPin did not interact with any of the p53 family members (Fig. 1f). In combination, these assays demonstrated that DARPin C10 was a highly specific binder of p53.

[0146]

[0108] Example 3: Identification of the interaction interface of DARPin C10 on the p53 DBD

[0147]

[0109] Protection from degradation is a necessary but not sufficient condition for reactivation of p53 in HPV-infected cells. Binding of the DARPin must also allow high-affinity binding of p53 to the DNA, which requires not only direct interaction with the DNA but also contacts between the DBDs that enable cooperative binding, the inventors investigated if DARPin C10 interfered with these interactions by performing pulldown assays in which the inventors incubated dsDNA comprising the human p21 promotor immobilized on streptavidin magnetic beads with p53 expressed in H1299 cells. Pre-incubation of the lysate with DARPin C10 or control DARPin did not result in a significant reduction of the pulldown efficiency compared to the pulldown without any DARPin (Fig. 2a, b). This result indicated that DARPin C10 recognizes an epitope on the DBD different from the epitope of the previously characterized DARPin G4 that blocks interaction with the DNA.

[0148]

[0110] To directly identify the interaction site of DARPin C10 on the p53 DBD, the inventors determined a high-resolution (1.5 A) crystal structure of the complex (Fig. 2c,). This structure revealed that the interaction interface of DARPin C10 is located on the edge of the central - sandwich, below the loop-sheet-helix motif, largely overlapping with the E6 binding site, proving that the DARPin, like the E6 protein, binds to a site different from the DNA-binding surface (Fig. 2d). A superimposition of the structure of the complex with a crystal structure of the p53 DBD bound to DNA (PDB: 3KMD) further illustrated this finding (Fig. 2e, f) and also indicated that the important DBD-DBD contacts, both within and between DNA half-sites, are not affected by binding of the DARPin. The p53-DARPin binding interface (with an interface area of 780 A2) is characterized by a central hydrophobic patch with three interacting tryptophan side chains, one from p53 and two from the DARPin (p53-Trp146, C10-Trp13, and C10-Trp46) flanked by intermolecular salt-bridge networks involving p53 residues Arg110 and Asp148. These key interacting residues are not conserved between p53 family members. Trp146 at the center of the interface, for example, is replaced by a lysine in p63 and p73, and the salt-bridge forming Arg110 is replaced by a negatively charged amino acid in the other two family members, explaining the high specificity of DARPin C10 for p53.

[0149]

[0111] Example 3: DARPin C10 restores p53 activity in HPV-positive cell lines

[0150]

[0112] Having shown that DARPin C10 protects p53 from degradation by the E6 protein and does not inhibit DNA binding suggested that the transcriptional activity of p53 can be restored by the DARPin in HPV-infected cells. Therefore, the inventors investigated the effect of DARPin C10 on the transcriptional activity of p53 in HeLa cells, which are HPV18 positive, being derived from a cervical cancer patient. For this purpose, a luciferase-based transactivation assay was performed by transient co-transfection of empty pcDNA3.1(+) plasmid or plasmids coding for DARPin C10 or the control DARPin together with the respective reporter plasmids into HeLa cells. No transactivation on the pBDS-2, PUMA or p21 promotor was observed for the samples containing empty vector or control DARPin, while increasing transcriptional activity was observed with increasing amounts of DARPin C10 on all promotors (Fig. 3a-c). Additionally, the p53 protein level was monitored by Western blot. Nearly no p53 could be detected in samples containing no DARPin or control DARPin. However, a significant stabilization of p53 was observed already with the lowest transfected amount of DARPin C10 (100 ng plasmid DNA), which further increased with increasing amounts of DARPin C10 (Fig. 3d). To exclude that the observed effect is cell-type dependent, the assay was repeated using SiHa cells, which are HPV16 positive. Similar to the HeLa cells, increasing transactivation was observed with increasing amounts of DARPin C10, but the overall activity was lower compared with HeLa cells (Fig. 3e-g). This also correlated with a lower p53 protein level observed by Western blot (Fig. 3h). In addition, the transactivation assay was repeated with HPV-negative U-2 OS cells to eliminate the possibility that DARPin C10 interfered with the MDM2 feedback loop. In this assay, treatment of the cells with the MDM2 inhibitor Nutlin-3a for 6 h was included as a positive control. Transient expression of DARPin C10 in those cells did not lead to significantly higher transactivation compared to samples containing no DARPin or control DARPin, while treatment with Nutlin-3a resulted in drastically increased transactivation (Fig. 3i, j). Furthermore, no stabilization of p53 on the protein level was observed for control DARPin or DARPin C10, while a significantly higher protein level was observed after treatment with Nutlin-3a (Fig. 3k). These results indicate that DARPin C10 is a potent reactivator of p53 in HPV-positive cells, without interfering with the MDM2-feedback loop, which is important for p53 regulation in healthy cells.

[0151]

[0113] To further characterize the reactivation of p53 regarding other pro-apoptotic target genes, the inventors performed real-time quantitative PCR. For this purpose, control DARPin, DARPin C10 or DARPin G4, which inhibits p53’s transcriptional activity, were transiently transfected in HeLa (HPV18 positive), SiHA (HPV16 positive) or U-2 OS (HPV negative) cells, and total mRNA was extracted 24 h after transfection and reverse transcribed into cDNA. Cells transfected with empty pcDNA3.1(+) vector served as a reference. In case of the HeLa cells, expression of DARPin C10 led to a significant increase of the expression of the p53 target genes PUMA, NOXA, p21 , and MDM2, while the non-binding control DARPin and the inhibitory DARPin G4 had no effect (Fig. 4a-d). In SiHa cells, a significant increase of p53 target gene expression was observable for PUMA, p21, and MDM2, but not for NOXA (Fig. 4e-h). However, the fold change was much lower than in the experiments performed with the HeLa cells. The non-binding control DARPin had no effect on the expression of the investigated p53 target genes, while expression of the inhibitory DARPin G4 led to a slightly reduced gene expression of NOXA (Fig. 4f). To exclude the possibility that p53 target gene expression is changed by interference with the MDM2 feedback loop, the experiment was repeated with HPV-negative U- 2 OS cells. The MDM2 inhibitor Nutlin-3a was again included as a positive control. No change of the fold expression of the p53 target genes was observed for the non-binding control DARPin and DARPin C10, while expression of DARPin G4 led to a reduced expression of NOXA (Fig. 4i-l). Overall, these experiments convincingly showed that DARPin C10 is a potent reactivator of p53 activity in HPV-positive cells that does not interfere with the important regulatory MDM2 negative-feedback loop.

[0152]

[0114] Example 4: Potential cellular delivery of DARPins via an mRNA / lipid nanoparticle approach

[0153]

[0115] The use of biotherapeutics like DARPins in the clinic has so far been limited to extracellular applications (see below) as their intracellular application is hampered by their inability to cross biological membranes, requiring delivery systems for the DARPin or its genetic information. A variety of viral and non-viral gene delivery systems have been developed, both for DNA and for RNA. One of the non-viral methods is the delivery of mRNA packaged in lipid nanoparticles that has been used for vaccine delivery. Its use for the delivery of therapeutics, requiring much higher concentrations than vaccines, is still in early stages of development. As a proof of principle that such an approach can work, the inventors have generated mRNA from the expression plasmid of the DARPin C10 as well as from the control DARPin and a GFP expression plasmid. For higher stability the mRNA contained pseudouridine and 5-methyl cytosine. Transient transfection of HeLa, SiHa and U-2 OS cells with mRNA packaged in Lipofectamine MessengerMax (Invitrogen) showed a very high transfection efficiency. Immunofluorescence staining revealed stabilization of p53 in HeLa and SiHa cells transfected with DARPin C10 but not with the control DARPin. In U-2 OS cells the stabilizing effect was minimal. A qPCR analysis of selected p53 target genes in the transfected HeLa cells demonstrated an upregulation of the genes coding for PUMA, NOXA, p21, and Mdm2. These results demonstrate that mRNA can be used to express DARPin C10, at least in cell culture.

[0154]

[0116] Example 5: DARPin C10 reactivated p53-dependent transcription

[0155]

[0117] p53 regulates several hundred target genes beyond p21 , MDM2, PUMA, and NOXA that synergistically contribute to its tumor suppressive activity, the inventors therefore profiled the transcriptional changes induced by DARPin C10 in HPV-positive HeLa and HPV-negative U-2- OS cells at a genome-wide scale by RNAseq using the non-binding DARPin as a negative and Nutlin-3a as a positive control. For this purpose, the inventors generated stable cell lines expressing either DARPin C10 or the non-binding control DARPin. DARPin C10 induced profound transcriptional alterations in HeLa cells, with a total of 561 genes significantly up- and 109 genes downregulated (Fig. 5a). In HPV-negative U-2 OS cells, a similar reprogramming of the transcriptome was only observed in response to Nutlin-3a treatment, but not by expression of DARPin C10 (Fig. 5b, c). Many of the genes activated by DARPin C10 in HeLa cells are canonical p53 target genes, including MDM2, BTG2, FAS, and FDXR (Fig. 5a, Supplementary Table 3). Moreover, gene set enrichment analysis identified various p53-target gene sets and p53-related DNA damage signatures as the most significantly upregulated pathways (Fig. 5d, e). Consistent with p53-mediated repression of cell-cycle genes via the DREAM complex, various Myc-regulated cell-cycle gene sets were among the most strongly depleted signatures. Together, this transcriptome-wide profiling confirmed a broad and highly efficient activation of the p53 tumor suppressor pathway specifically in HPV-positive HeLa cells by DARPin C10.

[0156]

[0118] Further, Fig? shows reactivation a selection of destabilized p53 mutants using DARPin of the invention. In particular a DARPin of the invention comprising a H82R mutation further enhances the stabilization of p53.

[0157]

[0119] Example 6: Expression of DARPin C10 in HPV-positive cells resulted in reduced cell viability

[0158]

[0120] As expression of DARPin C10 in HPV-positive cells resulted in an increased expression of pro-apoptotic p53 target genes, the question arose whether expression of this DARPin also results in reduced cell viability. Immunofluorescence (IF) was measured to assess expression and nuclear localization of the DARPins. IF-staining of HeLa cells showed that the DARPins were mainly located in the nucleus (Fig. 6a). Furthermore, p53 expression was detected in cells that expressed DARPin C10. The same experiment was performed with SiHa cells, leading to the same result (Fig. 6b). However, the p53 protein level in the nucleus was lower compared to that in HeLa cells. As a control, HPV-negative U-2 OS cells expressing DARPin C10 or control DARPin were generated. IF-staining indicated that both DARPins were present in the nucleus of these cells as well, but no increase of p53 protein level was detected in cells expressing DARPin C10 compared to cells expressing control DARPin (Fig. 6c). Cell viability was measured for 80 h, revealing that HeLa cells expressing DARPin C10 proliferated at similar level compared to cells expressing control DARPin in the first few hours, but then cell growth slowed down, and cell viability began to decrease. In contrast, no reduction of cell viability was detectable for cells expressing control DARPin over the entire experimental period (Fig. 6d). In comparison to HeLa cells, SiHa cells expressing control DARPin or DARPin C10 grew more slowly. For cells expressing DARPin C10, a reduction of cell viability was detectable after approximately 50 h, while the cells expressing control DARPin continued to grow (Fig. 6e). As in the transactivation assays and qPCR studies, HPV-negative U-2 OS cells served as negative control. The viability assays performed with these cells showed a slight reduction of the proliferation rate but no reduction of cell viability for cells expressing either control DARPin or DARPin C10, suggesting that none of the DARPins caused toxicity in these cells (Fig. 6f).

[0159]

[0121] Materials and Methods

[0160]

[0122] Selection and screening of DARPin binders specific for the p53 DNA-binding domain

[0161]

[0123] To generate DARPin binders, Escherichia coli expression plasmids of E. coli biotin ligase BirA and p53 DBD-OD (aa 94-363 of full length p53; aa 94-294 correspond to the sequence specific DBD, amino acids 325-355 correspond to the oligomerization domain (OD)) containing an Avi-tag were co-transformed in BL21 (DE3) Rosetta cells (SGC Frankfurt) for protein production and in vivo biotinylation. Cells were grown in 2xYT media supplemented with 100 pM ZnCh and 10 pM Biotin to an OD of 0.8. Protein expression was induced with 0.6 mM IPTG for 16 h at 16 °C. Cells were harvested by centrifugation, resuspended in IMAC A buffer (50 mM HEPES, pH 7.4, 400 mM NaCI, 20 mM p-mercaptoethanol, 10 pM ZnCh) supplemented with DNAse (Sigma), RNAse (Sigma) and self-made protease inhibitors (protease inhibitor cocktail (PIC) (100x): 250 mM AEBSF, 25 mM leupeptin, 25 mM bestatin, 0.75 mM aprotinin, 12.5 mM E-64 and 2.5 mM pepstatin A dissolved in 50% methanol at 4 °C; solvent was evaporated under vacuum; stored at -20 °C; before use the PIC pellet was resuspended in 1 ml Milli-Q H2O or buffer; all chemicals were bought from Carl-Roth GmbH & Co. KG, Germany) and lysed by sonication. The lysate was cleared by centrifugation at 4 °C, the supernatant was supplemented with 30 mM imidazole and applied onto a pre-equilibrated immobilized metal ion affinity chromatography (IMAC) column (HiTrap IMAC Sepharose FF, Cytiva). Bound protein was washed with IMAC A buffer supplemented with 50 mM imidazole and eluted by a step gradient with IMAC A buffer supplemented with 300 mM imidazole. The eluted protein was then simultaneously dialyzed to IMAC A buffer and digested with TEV protease. After the dialysis step, heparin affinity chromatography (HAC) was performed to separate the p53 DBD-OD from any impurities. Prior to loading the protein solution on a HiTrap heparin HP column (Cytiva), the protein solution was diluted 1 :8 in HAC buffer A (25 mM HEPES pH 7.4, 0.5 mM TCEP). Bound protein was eluted by applying an increasing gradient of HAC buffer B (25 mM HEPES pH 7.4, 1000 mM NaCI, 0.5 mM TCEP) using an AKTA purifier system at 4 °C. Central peak fractions were pooled, concentrated and loaded onto a HiLoad Superdex 75 16 / 600 column (Cytiva) using an AKTA purifier system at 4 °C. Purity and molecular weight of the purified proteins were monitored by SDS-PAGE and LC-ESI-TOF-mass spectrometry.

[0162]

[0124] The biotinylated p53 (aa 94-363) containing the DBD and OD domains was immobilized on either MyOne T1 streptavidin-coated beads (SA; Pierce) or Sera-Mag neutravidin-coated beads (NA, GE Healthcare), depending on the particular selection round, and these beads were alternated. Ribosome display selections were performed essentially as described19, but using a semi-automatic KingFisher Flex MTP 96-well platform.

[0163]

[0125] The library included N3C-DARPins with the original randomization strategy as reported but used a stabilized C-cap. Additionally, the library was a mixture of DARPins with randomized and non-randomized N- and C- terminal caps, respectively. Successively enriched pools were cloned as intermediates in a ribosome display-specific vector. Selections were performed over four rounds with decreasing target concentration and increasing washing steps to enrich for binders with high affinities. In addition, a prepanning with BSA blocked SA or NA beads was performed to eliminate unspecific DARPins in rounds two to four.

[0164]

[0126] DARPin screening

[0165]

[0127] The final enriched pool was cloned as fusion construct into a bacterial pQE30 derivative vector with an N-terminal MRGS(H)8 tag and C-terminal FLAG tag via unique BamHI x Hindlll sites containing a T5lac promoter and laclq for expression control.

[0166]

[0128] After transformation of E. coli XL1-blue, 380 single DARPin clones for p53 (aa 94-363) were expressed in 1-mL scale in deep-well plates by addition of IPTG (isopropyl p-D-1- thiogalactopyranoside), cells were harvested by centrifugation, and lysed by addition of B-Per Direct detergent plus lysozyme and nuclease (Pierce). The lysates were cleared by centrifugation. These bacterial crude extracts of single DARPin clones were subsequently used in a homogeneous time resolved fluorescence (HTRF)-based screen to identify potential binders. Binding of the FLAG-tagged DARPins to streptavidin-immobilized biotinylated p53 (aa 94-363) was measured using FRET (donor: streptavidin-Tb cryptate (610SATLB, Cisbio), acceptor: mAb anti-FLAG M2-d2 (61FG2DLB, Cisbio). Further HTRF measurement against ‘No Target’ allowed for discrimination of p53 (aa94-363)-specific hits. Additionally, p53 DBD-specific hits were confirmed by using biotinylated p53 DBD (aa 94-294) lacking the OD. Experiments were performed at room temperature in white 384-well Optiplate plates (PerkinElmer) using the Taglite assay buffer (Cisbio) at a final volume of 20 pl per well. FRET signals were recorded after an incubation time of 30 min using a Varioskan LUX Multimode Microplate (Thermo Scientific). HTRF ratios were obtained by dividing the acceptor signal (665 nm) by the donor signal (620 nm) and multiplying this value by 10,000 to derive the 665 / 620 ratio. The background signal was determined by using reagents in the absence of DARPins.

[0167]

[0129] Characterization of purified DARPins

[0168]

[0130] From the identified binders, 32 clones binding to both p53 DBD-containing targets were sequenced, and single clones were identified. For the selection of p53 DBD-specific DARPins, 23 DARPins were unique and single clones were expressed on a 1-ml scale. Cells were lysed using Cell lytic B reagent (Sigma), lysozyme and Pierce nuclease, and purified using a 96-well IMAC column (HisPur™ Cobalt plates, Thermo Scientific). DARPins after IMAC purification were analyzed at a concentration of 10 pM on a Superdex 75 5 / 150 GL column (GE Healthcare) using an Akta Micro system (GE Healthcare) with PBS containing 400 mM NaCI as the running buffer. Out of the panel of 23 p53 DBD-binding DARPins, 3 were chosen for further analysis: DARPin C10 (006-627-1801-C10), DARPin B12 (006-627-1803-B12), and DARPin F12 (006- 627-1804-F12).

[0169]

[0131] Cell culture

[0170]

[0132] HeLa, SiHa and U-2 OS cell lines were cultured in DMEM (Gibco), supplemented with 10% FBS (Capricorn Scientific), 1 mM pyruvate (Gibco), 100 U / ml penicillin (Gibco) and 100 pg / ml streptomycin (Gibco) at 37°C and 5% CO2. HeLa cells were obtained from CLS Cell Lines Service GmbH. U-2 OS cells were a gift from Prof. Dr. Ivan Dikic (IBCII, Goethe University, Frankfurt am Main, Germany). SiHa cells were obtained from Biozol. T-REx HeLa and T-REx U- 2 OS cell lines were obtained from Christian Behrends (Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-University (LMU), Munich, Germany) and were cultured in DMEM medium (Gibco), containing 10% FBS (Capricorn Scientific), 4 pg / ml blasticidin (Gibco), 333 pg / ml Zeocin (Gibco), 100 U / ml penicillin (Gibco), 100 pg / ml streptomycin (Gibco) and 1 mM pyruvate (Gibco) at 37°C and 5% CO2. The cell lines used in this study were frequently tested for mycoplasma contaminations.

[0171]

[0133] For recombinant protein expression, cells in DMEM without antibiotics were transfected using Lipofectamine 2000 as transfection reagent according to the manufacturer’s recommendations. 6 h after transfection, the medium was exchanged to standard culturing medium with antibiotics.

[0172]

[0134] Generation of stable cell lines expressing DARPin C10 or control DARPin

[0173]

[0135] HeLa and U-2 OS cell lines stable expressing DARPin C10 or the control DARPin E3_5 were generated using the Flp-ln T-Rex system (Thermo Fisher Scientific) for homologous recombination. After two weeks of culturing T-REx HeLa or T-Rex U-2 OS cells, they were transfected with pcDNA5 / FRT / TO (Thermo Fisher Scientific) containing the respective DARPin and with pOG44 (Thermo Fisher Scientific) containing the Flp recombinase according to the manufacturer’s recommendations. After transfection, the medium was exchanged to DMEM supplemented with 10% tetracycline-free FBS (BioCell). The next day after transfection, cells were reseeded in 15-cm dishes, and 24 h after cell transfer, the medium was exchanged to selection medium (DMEM supplemented with 10% tetracycline-free FBS, 4 pg / ml blasticidin, 200 pg / ml hygromycin (Thermo Fisher Scientific), 100 U / ml penicillin, 100 pg / ml streptomycin and 1 mM pyruvate. Cells were cultured for 10-14 d until a non-transfected control showed no viable cells. Six single colonies of each cell line were isolated, cultured, and inducible expression of the desired protein was tested by fluorescence staining. Protein expression was induced by adding 1 pg / mL tetracycline (Thermo Fischer Scientific) to the selection medium for 24 h.

[0174]

[0136] SiHa cell lines stably expressing DARPin C10 or control DARPins were generated using the PiggyBac Transposon system (System Biosciences) according to the manufacturer’s instructions. In brief, cells were transfected with 200 ng PiggyBac Transposase vector (PB210PA-1, System Biosciences) and 500 ng PB Cumate switch Transposon vector (PBQM812A-1) containing the respective DARPins. 6 h after transfection, cells were split into multiple wells of a 6-well plate and grown to confluency. Puromycin selection (2.5 pg / ml) was applied for 3-5 d to establish positively transposed cells. Inducible expression of the desired protein was tested by fluorescence staining using an anti-Myc antibody to detect the tag of the DARPin. Expression was induced by addition of 30 pg / ml Cumate (PBQM100-A, System Biosciences) to the medium for 24 h.

[0175]

[0137] Molecular Cloning

[0176]

[0138] For recombinant protein expression of DARPins, HPV E6 and all DBD constructs, a pET15b vector (Novagen, Merck KGaA) was used. Inserts generated by PCR were introduced into pET-15b-His10-TEV (N-terminal His10-tag followed by a tobacco etch virus (TEV) protease cleavage side), pET-15b-His10-TEV-Avi (N-terminal His10-tag followed by a TEV protease cleavage side and Avi-tag), pET-15b-GFP-His8-TEV (N-terminal GFP followed by a Hiss-tag and a TEV protease cleavage side) or pGEX-6P-2-His8-TEV (N-terminal GST-tag followed by Hiss-tag and TEV protease cleavage side) by subcloning using BamHI and Xhol restriction sites. For transient expression in mammalian cells, PCR-generated inserts were introduced into pcDNA3.1 (+) Myc (Invitrogen, Thermo Fisher Scientific) by subcloning using BamHI and Xhol restriction sites.

[0177]

[0139] Protein expression and purification

[0178]

[0140] DARPins. DARPins were expressed in E. coli and purified as described before. In brief, the respective pET-15b expression plasmid was transformed into E. coli BL21(DE3) Rosetta cells. Cells were grown in 2xYT medium until an optical density of 0.8 was reached. Protein expression was induced with 0.6 mM IPTG for 16 h at 16 °C. Cells were harvested by centrifugation, resuspended in IMAC A buffer (50 mM HEPES, pH 7.2, 400 mM NaCI) supplemented with RNAse (Sigma), DNAse (Sigma), lysozyme (Sigma), and self-made protease inhibitors, and were lysed by sonification. After clearing the lysate by centrifugation, the supernatant was supplemented with 30 mM imidazole and loaded on a pre-equilibrated immobilized metal affinity chromatography (IMAC) column (HiTrap IMAC Sepharose FF, Cytiva) following an IMAC purification protocol. Bound protein was washed with IMAC A buffer supplemented with 50 mM imidazole and eluted by a step gradient with IMAC A buffer supplemented with 300 mM imidazole. The eluted protein was simultaneously dialyzed to IMAC A buffer and digested with TEV protease (self-made). TEV protease and undigested protein were separated by a reverse IMAC step. The purified proteins were further purified and buffer-exchanged by size-exclusion chromatography (SEC) with SEC buffer (50 mM Tris, pH 8, 150 mM NaCI, 0.5 mM TCEP) using a Superdex 75 10 / 300 column (Cytiva) using an AKTA purifier system at 4°C. Central peak fractions were collected, concentrated to a concentration of 300 to 500 pM (Amicon Ultra Centrifugal Filters, Millipore) and flash-frozen in liquid nitrogen prior to storage at -80°C until use. Purity and molecular weight of purified proteins was monitored by SDS-PAGE and LC-ESI-TOF-mass spectrometry.

[0179]

[0141] p53 DBD and HPV E6 protein. The pET-15b-derived expression plasmids were transformed and expressed in E. coli BL21 (DE3) Rosetta cells as described before. HPV E6 proteins were expressed either as GFP or as GST-fusion proteins and purified as described for the DARPins. For DBD and E6 proteins, the expression medium was supplemented with 100 pM ZnCh, and IMAC buffers were supplemented with 20 mM p-mercaptoethanol and 10 pM ZnCh. Cell lysis, IMAC, dialysis and TEV-digestion were performed as described for the DARPins. For the p53 DBD, heparin affinity chromatography (HAC) was performed after the dialysis step to separate the p53 DBD from any impurities. Prior to loading the protein solution on a HiTrap heparin HP column (Cytiva), the protein solution was diluted 1 :8 in HAC buffer A (25 mM HEPES pH 7.4, 0.5 mM TCEP). Bound protein was eluted by applying an increasing gradient of HAC buffer B (25 mM HEPES pH 7.4, 1000 mM NaCI 0.5 mM TCEP) using an AKTA purifier system at 4°C. Central peak fractions were pooled, concentrated and loaded onto a HiLoad Superdex 75 16 / 600 column (Cytiva) using an AKTA purifier system at 4°C. Purity and molecular weight of the purified proteins was monitored by SDS-PAGE and LC-ESI-TOF-mass spectrometry.

[0180]

[0142] DARPin biotinylation

[0181]

[0143] In vitro biotinylation of Avi-tagged DARPins was performed as described previously. In brief, the E. coli biotin ligase BirA was subcloned into a pET15b-GFP-His8-TEV E. coli expression vector. GFP-BirA was expressed and purified as described before, except for a TEV cleavage and reverse IMAC step.

[0182]

[0144] DARPins containing an Avi-Tag were enzymatically biotinylated in vitro by mixing them with GFP-BirA in a 1 :50 molar ratio in SEC buffer supplemented with 10 mM ATP, 10 mM MgCh, 0.5 mM biotin, followed by 16 h incubation at 16°C. For separation, the reaction mix was applied onto a Superdex 75 10 / 300 column (Cytiva). DARPin fractions were pooled and analyzed by LC-ESI-TOF-mass spectrometry. Only DARPins showing 100% labelling efficacy were used for experiments.

[0183]

[0145] Pulldown assays

[0184]

[0146] DARPin pulldown assays. Recombinant target proteins were expressed in H1299 cells, and respective cell lysates were generated as described before.20Biotinylated DARPins were immobilized on pre-equilibrated magnetic Dynabeads MyOne Streptavidin T1 (Thermo Fisher Scientific) in pulldown (PD) wash buffer (50 mM Tris pH 8, 150 mM NaCI, 0.1 % (v / v) Tween- 20), rotating for 2 h at 4°C. Unbound DARPin was removed by washing three times with PD wash buffer, and beads were resuspended in the same volume of PD wash buffer as before to maintain the bead concentration. Per sample, 10 pl DARPin-loaded beads were mixed with cell lysate, 1x complete protease inhibitor (Roche), and the total volume was adjusted to 1000 pl with PD wash buffer. Samples were incubated by rotating overnight at 4°C. The next day, the beads were washed five times with 1000 pl PD wash buffer, and bound proteins were eluted by incubating with LDS buffer at 70°C for 10 min. Samples were analyzed by Western blot as described before. All pulldown experiments in this study were performed as biological triplicates unless stated otherwise.

[0185]

[0147] Competitive pulldown of HPV E6. Avi-tagged p53 DBD (amino acids 94-294) was expressed in E. coli and purified as described above followed by in vitro biotinylation. An excess of biotinylated p53 DBD was incubated by rotating with Dynabeads MyOne Streptavidin T1 (Thermo Fisher Scientific) for 1 h at 4°C, and unbound protein was removed by washing three times with PD wash buffer. Per sample, 10 pl pre-loaded beads, 10 pM GFP-fused HPV E6 protein, 15 pM DARPin and 1x complete protease inhibitor (Roche) were mixed and the total volume was adjusted to 1000 pl with PD wash buffer. Samples were incubated rotating overnight at 4°C. The next day, the beads were washed five times with 1000 l PD wash buffer and bound proteins were eluted with LDS buffer boiling at 70°C for 10 min. Samples were analyzed by Western blot as described before. GFP-fused E6 protein was detected using an anti-GFP antibody. The pulldown was performed in triplicates and signals were quantified using ImageLab (version 6.1, Bio-Rad).

[0186]

[0148] Isothermal titration calorimetry

[0187]

[0149] All titration experiments were performed using a MicroCai VP-ITC microcalorimeter (Malvern Instruments Ltd, UK). DARPins and p53 family DBDs were dialyzed against ITC buffer (50 mM HEPES pH 7.4, 150 mM NaCI, 0.5 mM TCEP). DBDs were titrated to constant concentrations of DARPin in 25 injections of 10 pl each, with a spacing time of 250 s and a stirring speed of 307 rpm. Measurements were performed at 15°C or 25°C, with the reference power set to 25 pCal / s. NITPIC was used for unbiased baseline calculation and curve integration. Thermodynamic parameters and final binding affinities were calculated using SEDPHAT, assuming an AB hetero-association model. The first data point was excluded from the analysis. Final figures were generated using GUSSI.

[0188]

[0150] Protein crystallization and structure determination

[0189]

[0151] The protein complex for crystallization was prepared by mixing DARPin C10 and the p53 DBD at a 1:1 molar ratio in SEC buffer, each at at 60 pM protein concentration. The protein mix was incubated overnight at 4°C, and the formed complex was separated from unbound proteins by SEC using a Superdex 75 10 / 300 column. Central peak fractions corresponding to the protein complex were pooled, concentrated to 2.5 mg / ml and analyzed by SDS-PAGE as well as LC-ESI-TOF-mass spectrometry. Crystals of the DARPin C10-p53 DBD complex were grown at 293 K using the sitting drop vapor diffusion technique with a Mosquito crystallization robot (TTP Labtech, Royston, UK). The protein solution was mixed with reservoir solution (25 % w / v PEG 3350) at a 2:1 ratio (final drop volume 200 nL). Crystals were cryo- protected with mother liquor supplemented with 23% ethylene glycol and flash-frozen in liquid nitrogen. An X-ray diffraction data set was collected at 100 K at beamline X06SA of the Swiss Light Source, Villigen, Switzerland. The diffraction data were integrated with the program XDS and scaled with AIMLESS, which is part of the CCP4 package. The structure of the p53-DARPin complex was then solved by molecular replacement with PHASER using structures of the p53 DBD (PDB entry 2XWR) and DARPin 8F1 (PDB entry 7Z73) as search models. Model building and refinement was then performed using iterative cycles of manual model building in COOT and refinement in PHENIX. Validation of the final model was performed using MolProbity. X-ray data collection and refinement statistics are listed in Supplementary Table S2. Interface areas were calculated using the PISA server (http: / / www.ebi.ac.uk / pdbe / prot_int / pistart.html) and are defined as the difference in total accessible surface areas of isolated and interfacing structures divided by two. Structural figures in this paper were prepared with PyMOL (www.pymol.org).

[0190]

[0152] Gel electrophoresis and Western blotting

[0191]

[0153] Purified proteins were mixed with SDS loading buffer (250 mM Tris, pH 8.0, 7.5% (w / v) SDS, 25% (w / v) glycerol, 12.5% (v / v) p-mercaptoethanol, 0.025% (w / v) bromophenol blue), denatured at 95 °C and separated on manually prepared discontinuous 4-16% Tris-glycine gels. The gels were subsequently stained using Quick Coomassie Stain (NeoBiotech) according to the manufacturer’s recommendations.

[0192]

[0154] Samples for immunoblotting were either mixed with SDS loading buffer or NuPAGE LDS buffer (Thermo Fisher Scientific) supplemented with DTT, denatured at 95 °C and applied on 4- 15 % Mini-PROTEAN TGX Stain-Free Precast Protein gels (Bio-Rad). The gels were transferred using the TransBlot Turbo Transfer System (Bio-Rad) according to the manufacturer’s recommendations. Membranes were blocked for 1 h in blocking buffer (TBS, 0.05 % (v / v) Tween-20, 5% skim milk powder, Sigma-Aldrich) and incubated with primary antibody in blocking buffer overnight shaking at 4°C. Membranes were washed three times with TBS-T, followed by incubation with secondary antibody in blocking buffer under shaking for 1 h at room temperature. Afterwards, membranes were washed three times with TBS-T and analyzed by adding Amersham ECL Prime WB Detection Reagent (Cytiva). Quantification of Western blot signals was performed using ImageLab (version 6.1 , Bio-Rad).

[0193]

[0155] The following antibodies and dilutions were used: anti-myc (1:2000, clone 4A6, Millipore), anti-p53 (1:500, DO-I, Santa Cruz Biotechnology), anti-vinculin (1:2000, clone 7F9, Santa Cruz Biotechnology), goat anti-mouse HRP (1:5000, A9917, Sigma Aldrich).

[0194]

[0156] HPV p53 degradation assay

[0195]

[0157] Full-length TAp53a was in vitro translated using the TNT T7 Quick coupled Transcription / Translation system (Promega). For in vitro translation, pcDNA3.1(+) containing the TAp53a gene was diluted to 100 ng / pl, mixed with rabbit reticulocyte lysate (RRL) in a 1:4 ratio and incubated at 30 °C for 90 min. The reaction was stopped by addition of benzonase (Millipore) for 30 min. Afterwards, 22.5 pl RRL were mixed with 2 pM GST or GST-E6 and 10 pM DARPin. The sample volume was adjusted to 60 pl with 2x reaction buffer (50 mM Tris pH 7.5, 200 mM NaCI, 4 mM DTT). The reaction mix was then incubated at 25 °C, and samples were taken at different time points by mixing 5 pl reaction mix with 25 pl 5x SDS sample buffer and boiling at 95 °C for 1 min. Degradation of p53 was analyzed by Western blot using an a- Myc antibody. The degradation assay was performed in biological triplicates.

[0196]

[0158] Transactivation assay

[0159] For transactivation assays, HeLa, U-2 OS or SiHa cells were seeded in 12-well plates and transfected using Lipofectamine 2000 as transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. All transfection mixes included 200 ng of pRL-CMV (Promega) for constitutive expression of Renilla luciferase, 200 ng of a reporter plasmid with firefly luciferase under the control of a specific promotor and varying amounts of pcDNA3.1(+) with the respective DARPin. For each assay, an empty vector control comprising only empty pcDNA3.1 (+) was transfected to determine the fold induction without presence of any DARPin. 24 h after transfection, cells were washed with PBS (Gibco), detached with Accutase and reseeded into white Nunc 96-well microplates (Thermo Fisher scientific) in quadruplicates. The assay was performed using the Dual-Glo Luciferase reporter assay kit (Promega) according to the manufacturer’s instructions, and firefly as well as Renilla luciferase fluorescence was measured using a Spark plate reader (Tecan). The remaining sample was centrifuged for 5 min at 500 g, pelleted cells were mixed with 1x SDS-loading buffer, and p53 protein levels were analyzed by Western blot using the anti-p53 (DO-I) antibody (Santa Cruz Biotechnology). The experiment was repeated in three biological replicates, and the ratio of firefly to Renilla luciferase signal was normalized to empty vector control for each biological replicate. Statistical significance was assessed by ordinary one-way ANOVA (n.s.: P > 0.05, *P < 0.05, **P < 0.01 , ***P < 0.001 , ****p < 0.0001) using Prism (Version 8.2.1 , GraphPad).

[0197]

[0160] Quantitative PCR (qPCR)

[0198]

[0161] For qPCR analysis of the expression of p53 target genes, HeLa, U-2 OS or SiHa cells were transfected with pcDNA3.1(+) plasmids containing the respective DARPin using Lipofectamine 2000 as transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. 24 h after transfection, cells were washed with PBS, detached and mRNA was isolated using the RNease mini kit (Qiagen). Reverse transcription of mRNA was performed using the SuperScript™ IV VILO™ mastermix with the ezDNase™-enzyme kit (Thermo Fisher Scientific). Both kits were used according to the manufacturer’s instructions. Quantitative PCR was performed in technical triplicates using the TaqMan fast advanced Master Mix and the respective TaqMan Assay (Thermo Fisher Scientific) using a QuantStudio 5 Real Time PCR system (Thermo Fisher Scientific). Target gene expression was referenced to the house-keeping gene HPRT-1. The experiment was repeated in three biological replicates, and statistical significance was assessed by ordinary one-way ANOVA (n.s.: P > 0.05, *P < 0.05, **P < 0.01 , ***P < 0.001 , ****P < 0.0001) using Prism (Version 8.2.1 , GraphPad).

[0199]

[0162] Preparation of mRNA by in vitro transcription

[0200]

[0163] For IVT PCR-generated inserts of DARPin C10, control DARPin and GFP were introduced into a modified pcDNA3.1 plasmid in which the 5’ UTR of human p-globin was introduced between the T7 promotor and the DARPin insert using Nhel and Hindi 11. Additionally, the 3’UTR of human p-globin followed by a 120-bp poly(A) tail was cloned behind the DARPin insert using Kpnl and Spel. Plasmids were linearized using Spel and in vitro transcribed using the HiScribe T7 ARCA mRNA Kit (New England Biolabs) with the modified nucleotides pseudo- UTP and 5’methyl-CTP according to the manufacturer’s instructions. RNA was purified using the RNeasy mini kit (Qiagen). RNA concentration was determined by measuring the absorption at 260 nm and RNA quality was confirmed by running a 1% agarose gel.

[0201]

[0164] mRNA transfection

[0202]

[0165] Cells were transfected with the respective mRNA using Lipofectamine MessengerMax (Invitrogen) according to the manufacturer’s instructions. RNA isolation, reverse transcription and qPCR were performed as described above.

[0203]

[0166] Immunofluorescence Staining

[0204]

[0167] 24 hours after transfection or induction of protein expression, cells were washed twice with PBS and fixed with PFA for 10 minutes at room temperature. Fixed cells were washed twice with PBS and permeabilized with PBS-T (PBS supplemented with 0.1 % Triton X-100) for 5 minutes two times. Permeabilized cells were blocked with blocking buffer (PBS-T supplemented with 1 % BSA) for 20 minutes at room temperature. Blocked cells were incubated with rabbit anti-myc (1:500, Abeam ab9106) and mouse anti-p53 (1:250, DO-I, Santa Cruz) antibodies in blocking buffer overnight at 4°C. Cells were washed three times with PBS-T and incubated with Alexa Fluor 568 anti-rabbit (1 :200, A10042, Life technologies) and Alexa Fluor 647 anti-mouse antibody (1:200, A31571, Life Technologies) in blocking buffer for 2 h at room temperature. Slides were washed three times with PBS-T and mounted using Mowiol (Carl Roth) mounting medium which was supplemented with DAPI (Thermo Fisher Scientific). Detailed recipes of the mounting medium can be found at CSH protocols (htp: / / cshprotocols.cshlp.Org / content / 2006 / 1 / pdb. red 0255). The slides were dried for at least one day before imaging with a LSM 780 confocal laser scanning microscope (Zeiss).

[0205]

[0168] RNAseq

[0206]

[0169] RNA was isolated using the RNeasy Mini Kit (QIAGEN, 74106) according to the manufacturer’s protocol. RNA quality assessment was performed using the Experion RNA StdSens Analysis Kit (Bio-Rad, 700-7103). RNAseq libraries were prepared from total RNA with the Lexogen QuantSeq 3'-mRNA Library Prep Kit FWD for Illumina (Lexogen, 015.24) in combination with the UMI Second Strand Synthesis Module for QuantSeq FWD (Illumina, Read 1) (Lexogen, 081.96) following the manufacturer's protocol. Sequencing library quality was checked on a Bioanalyzer 2100 using the Agilent High Sensitivity DNA Kit. Pooled sequencing libraries were quantified and sequenced on the NextSeq 550 platform (Illumina) with 75-base single reads.

[0170] After sequencing, unique molecular identifiers (UM Is) were extracted from the obtained FASTQ files for sequenced reads, and the QuantSeq FWD-UMI 3' spacer corresponding to the first four nucleotides was removed. Trimmed reads were then aligned to the Homo sapiens Ensembl reference genome (revision 109, GRCh38), using the STAR RNAseq aligner (version 2.7.10a). Subsequently, UMI deduplication was done via UMI-tools (version 1.1.2). UMIs per gene were quantified, normalized to counts per million (CPM), and threshold filtering was applied to exclude genes with a CPM < 1 in all samples. Pairwise comparisons were performed, and differential expression was determined using DEseq2 (version 1.36.0). False discovery rate was controlled via Benjamini-Hochberg corrected p-values (alpha=0.05). Genes showing a log2FC > 1 and corrected p-values < 0.05 were considered differentially expressed. Volcano plots and bar charts were generated using the matplotlib library (version 3.6.2). Gene set enrichment analysis (GSEA) was performed using the GSEA software (version 4.3.2) and Molecular Signatures Database (MSigDB, version 7.1).

[0207]

[0171] Cell survival assay (cell viability assay)

[0208]

[0172] T -Rex HeLa cells, T-Rex U-2 OS cells or SiHa cells stably expressing DARPin C10 or control DARPin were seeded into white Nunc 96-well microplates (Thermo Fisher Scientific), and protein expression was induced as described before. 24 h after induction, the medium was exchanged to medium containing substrate and NanoLuc enzyme according to the manufacturer’s instructions using the RealTime- Gio MT assay kit (Promega). Luminescence was monitored continuously using a Spark plate reader (Tecan).

[0209]

[0173] Statistics and reproducibility

[0210]

[0174] Pulldown experiments, p53 degradation assays, transactivation assays, and qPCR experiments were performed in biological triplicates, and all individual data points are shown in the corresponding figures. The bar diagrams present the mean value and the error bar the SD. ITC measurements were performed twice, however, the determination of the KD values was based on a single measurement. The KD values and the 95% confidence interval were determined by SEDPHAT. The cell survival assays were performed in triplicates. Each data point presents the mean value and the error bar the SD.

[0211]

[0175] Example 7: Structural and biophysical characterization of triple mutant Q16E / R19W / H82R and the quadruple mutant Q16E / D18N / R19W / H82R

[0212]

[0176] Based on the crystal structure of DARPin C10 with the DNA binding domain of p53, the inventors were able to further increase the affinity between the two proteins. It was found that the triple mutant Q16E / R19W / H82R and the quadruple mutant Q16E / D18N / R19W / H82R of the DARPin show a very high affinity with a binding constant of approximately 3 nM at a measurement temperature of 30°C (figure 8). This means that the newly developed DARPins bind with a significantly higher affinity compared to the original DARPin (binding constant of 255 nM at 25°C).

[0213]

[0177] The difference becomes even more significant when comparing the affinities at 37°C. The dissociation constant of the original DARPin at this temperature is 896 nM. The affinity of the triple and quadruple mutants, on the other hand, remains almost unchanged with a dissociation constant of approximately 6 nM. This corresponds to an approximately 150-fold increase in affinity.

[0214]

[0178] This increase in affinity was achieved primarily by replacing the amino acid glutamic acid 19 with tryptophan. This leads to less negative binding entropy and thus to higher affinity combined with lower temperature dependence of the dissociation constants.

[0215]

[0179] Figure 9 shows the comparison of thermodynamic parameters measured with ITC for the original variant (C10 WT) and the two mutants.

[0216]

[0180] The crystal structure of the triple mutant in complex with the p53 DNA binding domain shows that the side chain of tryptophan 19 contributes to hydrophobic interactions, thus explaining the less negative binding entropy (figure 10).

[0217] Reactivation of p53 mutants in transiently transfected cells

[0218]

[0181] The triple mutant Q16E / R19W / H82R was tested in functional assays. To do this, the inventors overexpressed various p53 mutants in H1299 cells and measured their transcriptional activity after co-expression with the DARPin in a luciferase-based assay. These data were compared with the C10 wild-type DARPin and a first improved variant (H82R). The data show that the triple mutant leads to significantly higher activation of structural p53 mutants (figure 11). P53 mutations in the DNA binding interface, on the other hand, cannot be reactivated, as expected. The same applies to p53 mutants that lose the essential Zn ion.

[0219] Reactivation of p53 in cancer cell lines at the endogenous level

[0220]

[0182] The inventors measured the reactivation of p53 mutants directly in cancer cell lines. Cell lines with the mutations R158L, R158G, R158H, and V157F were used. In the reactivation assays in transiently transfected cells, the inventors observed that mutations at positions R158 and V157 can be reactivated particularly well by the DARPin (figure 12). This was confirmed in experiments with cancer cell lines at endogenous levels. These experiments have been carried out with the H82R variant of DARPin. The reactivation of mutated p53 was measured in the specified cell lines using qPCR after expression of the DARPin. In addition, experiments were conducted with Nutlin-3a, which showed synergistic effects.

Claims

CLAIMS1. A p53 binding protein, comprising a designed ankyrin repeat protein (DARPin), wherein the DARPin is capable of forming a binding interface with a DNA binding domain (DBD) of (human) p53, wherein said binding interface comprises amino acid position Trp146 of hp53.

2. The p53 binding protein of claim 1, wherein the DARPin comprises an amino acid sequence having not more than 90% sequence identity to the amino acid sequence shown in any one of SEQ ID NO: 1 to 4.

3. The p53 binding protein of claim 1, wherein the DARPin comprises an amino acid sequence having not more than 90% sequence identity to the amino acid sequence shown in any one of SEQ ID NO: 5 to 10.

4. The p53 binding protein of claim 1 to 3, wherein the DARPin has a sequence that when aligned with SEQ ID NO: 1 comprises an amino acid substitution at a corresponding position of SEQ ID NO: 1 selected from the group of Q16, D18, E19 and H82; and I or wherein the DARPin has a sequence that when aligned with SEQ ID NO: 1 comprises variant amino acids at two or three corresponding positions of SEQ ID NO: 1 selected from the group of Q16, D18, E19 and H82; and / or wherein the DARPin has a sequence that when aligned with SEQ ID NO: 1 comprises variant amino acids at corresponding positions Q16, D18 and H82 in SEQ ID NO: 1, or (preferably) comprises variant amino acids at corresponding positions Q16, E19 and H82 in SEQ ID NO: 1.

5. The p53 binding protein of claim 1 to 4, wherein the DARPin has a sequence that when aligned with SEQ ID NO: 1 does not comprise variant amino acids at corresponding positions Trp13 and Trp46 of SEQ ID NO:

16. The p53 binding protein of any one of claims 1 to 5, wherein the DARPin has an amino acid sequence selected from SEQ ID: 2 (H82R), 3 (Q16E,D18N,H82R) or 4 (Q16E, E19R, H82R), optionally with not more than 9 amino acid substitutions, additions and / or deletions.

7. The p53 binding protein of any one of claims 1 to 5, wherein the DARPin has an amino acid sequence selected from SEQ ID: 5 (Q16E), 6 (Q16E,D18N), 7 (Q16E, E19R), 8 (Q16E, N18R, H82R), 9 (Q16E, R19W, H82R) or 10 (Q16E, D18N, R19W, H82R) optionally with not more than 9 amino acid substitutions, additions and / or deletions.

8. The p53 binding protein of any one of claims 1 to 7, wherein the binding protein binds to p53 protein with a KD of less than 250nM, more preferably less than 100nM, more preferably of less than 80nM, most preferably of less than 50nM, and preferably of about 72nM, of about 42nM or of about 29nM.

9. The p53 binding protein of any one of claims 1 to 8, wherein the binding protein binds to p53 protein with a KD of about 3 nM.

10. A nucleic acid construct (NAC), comprising a nucleic acid sequence encoding a p53 binding protein of any one of claims 1 to 9.

11. The NAC of claim 10, wherein the nucleic acid is selected from DNA, RNA or a DNA / RNA hybrid.

12. The NAC of claim 10 or 11 , which is an mRNA construct suitable for cellular delivery and translation of the encoded DARPin, or is a DNA construct comprising one or more sequence elements, such as a promoter, enhancer etc., for cellular expression of the DARPin.

13. A (recombinant) cell, comprising a p53 binding protein of any one of claims 1 to 9 or a NAC of any one of claims 10 to 12.

14. A pharmaceutical composition, comprising a p53 binding protein of any one of claims 1 to 9, a NAC of any one of claims 10 to 12, or a cell of claim 13, optionally together with a pharmaceutically acceptable carrier or excipient.

15. The pharmaceutical composition of claim 14, which is a lipid nanoparticle comprising a p53 binding protein of any one of claims 1 to 9, a NAC of any one of claims 10 to 12.

16. A medicinal product for use in the treatment of a disease, comprising a p53 binding protein of any one of claims 1 to 9, a NAC of any one of claims 10 to 12, a cell of claim 13, or a pharmaceutical composition of claim 14 or 15.

17. The medicinal product for use of claim 16, wherein the disease is a proliferative disorder, preferably a cancer or tumor.

18. The medicinal product for use of any one of claims 16 to 17, wherein the use comprises a stabilization of p53 protein in cells involved with the disease, preferably wherein the stabilization is a reduced E6-mediated protein degradation of p53.

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