Polypeptides comprising histone cryptographic reader domains and uses thereof

Abstract

The present invention relates to a polypeptide comprising a histone cipher reader domain and a use thereof, and more particularly, to a polypeptide and a use thereof in the prevention or treatment of cancer, the polypeptide comprising a PHD domain of PHF6 (PHD finger Protein 6), optionally comprising DPF3b (Double PHD finger 3b) or BPTF (Bromodomain PHD finger transcription factor), or a mutant thereof, and to a use thereof in the prevention or treatment of cancer, and more particularly, to a polypeptide comprising a PHD domain of PHF6 (PHD finger Protein 6), optionally comprising DPF3b (Double PHD finger 3b) or BPTF (Bromodomain PHD finger transcription factor), or a mutant thereof.

Description

Technical Field

[0001] The present invention relates to a polypeptide comprising a histone code reader domain and uses thereof, and in particular, to a polypeptide and uses thereof in the prevention or treatment of cancer, wherein the polypeptide comprises PHF6 (PHD finger protein 6), optionally comprising the PHD domain of DPF3b (Double PHD fingers 3b) or a mutant thereof. Background Art

[0002] The DNA of eukaryotic cells is surrounded by proteins called histones, which protect and regulate DNA. The structure of DNA surrounded by histones is called chromatin, which plays an important role in regulating gene expression. Although eukaryotic cells contain the same genetic components, the ability to maintain a distinct phenotype can be ensured by various chemical modifications that occur in chromatin-associated proteins, DNA, and histones.

[0003] Epigenetics refers to changes in gene expression patterns that occur as a result of inherited changes in the genome without affecting the core DNA sequence. Even though these changes do not affect the basic DNA sequence, they play an important role in the specific identity of the cell and are involved in regulating important cellular processes. Chromatin in the cell nucleus exists in two states: closed chromatin (heterochromatin) associated with transcriptional repression and open chromatin (euchromatin) that favors transcription. This level of chromatin regulation depends largely on post-translational modifications of core histone proteins and covalent modifications of DNA.

[0004] The nucleosome, a basic component of chromatin, is composed of an octamer of histone proteins (dimers of H3 and H4 and dimers of H2A and H2B) and forms a compact central structure around 147 base pairs of DNA. Histone proteins have N-terminal tails protruding from the center and undergo a variety of modifications after translation. This modification, together with direct modification of DNA, constitutes the epigenetic code (Epigenetic mediation of environmental influences in major psychotic disorders, Schizophr Bull. 2009 Nov; 35 (6): 1045-56).

[0005] The salient feature of this code is the ability to adapt and cope with environmental changes that occur during the life cycle (the growth of an organism from a fertilized egg to an adult). Four types of modifications of cytosine residues have been identified in DNA, including methylation, hydroxymethylation, formylation, and carboxylation. In addition, more than 10 types of histone modifications have been identified. According to epigenetics, the core elements associated with this modification include writers and readers, wherein the writers include various enzymes that can modify nucleotide bases and specific amino acid residues of histones, and the readers are proteins of various ranges with special domains that can recognize specific epigenetic marks of loci.

[0006] In terms of writer function, modification of DNA and histone proteins is achieved by adding a variety of chemical groups using a variety of enzymes. Although a variety of modifications can be performed, it is possible to focus on the two most widely studied epigenetic modifications, namely methylation and acetylation. Both DNA and histone proteins are susceptible to methylation, while acetylation is only associated with histones. These two modifications usually regulate gene expression patterns in cells through transcriptional activation or repression. Epigenetic writers include DNA methyltransferases, histone lysine methyltransferases, protein arginine methyltransferases, and histone acetyltransferases.

[0007] In terms of reader function, multiple modifications based on epigenetic writers need to be recognized by other cellular proteins so that they can recognize and mediate the effect. In mammalian cells, multiple protein domains have evolved as readers of the epigenome, binding to this modification in mammalian cells. Several chromatin variants can function as epigenome readers due to the presence of specialized domains that recognize and bind to multiple covalent modifications present in DNA and histones. Of interest are protein domains that recognize methylated DNA. Of interest are domains that recognize and bind to methylated DNA and two histone modifications, methylation and acetylation.

[0008] Most histone modifier proteins rarely perform two functions. Compared with the enhancement of writer function, enhancing reader function may have fewer side effects. Engineering of reader domains can not only enhance the effect of cancer treatment, but also suggest the possibility of controlling new therapeutic targets.

[0009] On the other hand, enhancing writer functions can only improve existing functions and does not suggest new therapeutic targets.In summary, enhancing reader functions can bring great benefits.

[0010] Against this technical background, the inventors of the present application identified a polypeptide comprising a histone code reader domain and its use in the prevention or treatment of cancer, thereby completing the present invention. Summary of the invention Technical Problems to be Solved by the Invention

[0011] The object of the present invention is to provide a polypeptide comprising a histone code reader domain.

[0012] The object of the present invention is to provide a nucleic acid encoding the polypeptide.

[0013] The object of the present invention is to provide an mRNA encoding the polypeptide.

[0014] The object of the present invention is to provide an isolated cell that produces the nucleic acid.

[0015] The object of the present invention is to provide a delivery vehicle comprising the mRNA.

[0016] The object of the present invention is to provide a composition for preventing or treating cancer, which comprises the polypeptide, the nucleic acid or the mRNA. Technical Solution

[0017] In order to achieve the above object, the present invention provides a polypeptide comprising the following structure:

[0018] The polypeptide is a polypeptide represented by a D1-D2-D3 structure.

[0019] (a) D1 is the PHD1 domain of PHD Finger Protein 6 (PHF6);

[0020] (b) the D2 is the PHD2 domain of PHF6 or a mutant thereof; and

[0021] (c) D3 comprises one or more selected from the group consisting of:

[0022] (i) D2;

[0023] (ii) a fragment of PHF6 containing D2;

[0024] (iii) PHD1 domain of DPF3b (Double PHD fingers 3b);

[0025] (iv) the PHD2 domain of DPF3b or a mutant thereof; and

[0026] (v) PHD1 domain and PHD2 domain of DPF3b or mutants thereof.

[0027] The present invention provides a nucleic acid encoding the polypeptide.

[0028] The present invention provides an mRNA encoding the polypeptide.

[0029] The invention provides an isolated cell producing the nucleic acid.

[0030] The present invention provides a delivery system comprising the mRNA.

[0031] The present invention provides a composition for preventing or treating cancer, comprising the polypeptide, nucleic acid, mRNA, cell or delivery body. The present invention provides a method for preventing or treating cancer, comprising: administering the polypeptide, nucleic acid, mRNA, cell or delivery body to an individual. The present invention provides a use of the polypeptide, nucleic acid, mRNA, cell or delivery body in preparing a preparation for preventing or treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a schematic diagram showing the PHF6-based engineering for preparing chromatin Epi-glue.

[0033] Figure 2 is a schematic diagram showing the details of yeast display used to improve the binding affinity of the PHF6 reader domain.

[0034] Figure 3 The graph shows the results of testing the binding affinity of H2B and H2BK12Ac peptides of ePHD2 using a yeast display system.

[0035] Figure 4 are results showing construction of a library and screening of the library for binding affinity maturation of the reader domain.

[0036] Figure 5 The graph shows the results of individual analyses of five ePHD2 mutants.

[0037] Figure 6 is an example showing the preparation of Epi-glue protein by replacing the reader domain of PHF6 with another epigenetic reader domain.

[0038] Figure 7 The graph shows the results of analyzing the peak shift according to the H2B peptide concentration titration.

[0039] Figure 8The graph shows the cell proliferation results of cells expressing ePHD2 (T208) after gene transfer using lentivirus.

[0040] Fig. 9 The figure shows western blot data of cells expressing ePHD2 (T208) after gene transfer using lentivirus.

[0041] Fig.10 The results confirm the effect of expressing in breast cancer cells using mRNA synthesized by in vitro transcription and suppressing cell proliferation.

[0042] Fig.11 The results confirmed that the expression of tumor suppressive miRNA in the chromatin Epi-glue T208 (Chromatin Epi-glue T208 (DR3)) sample was increased.

[0043] Fig.12 The results confirmed that the expression of chemokine among cytokines was increased in the Chromatin Epi-glue T208 (Chromatin Epi-glue T208 (DR3)) sample.

[0044] Fig.13 The results show that the in vivo efficacy of ePHD2(T208) was tested using an immunodeficient nude mouse model.

[0045] Fig.14 The results show that the efficacy of chromatin epi-glue was verified in an immunodeficient nude mouse xenograft model.

[0046] Fig.15 The results show that the expression of genes related to chemokines was confirmed in the T208 (DR3) sample.

[0047] Fig.16 The results show that the growth inhibitory effect of Epi-glue protein was confirmed using lymphoblast K562 derived from a chronic myeloid leukemia patient (CML patient) and promyeloblast HL-60 derived from an AML patient.

[0048] Fig.17The results show that the growth inhibitory effect of Epi-glue protein was confirmed using epithelial cancer D283 derived from a medulloblastoma patient and epithelial cancer HCT116 cell line derived from a colorectal carcinoma patient.

[0049] Fig.18 The results show that the growth inhibitory effect of Epi-glue protein was confirmed using RPMI6666 lymphoblasts derived from a Hodgkins Disease patient, U-87MG derived from a Likely Glioblastoma patient, and Daoy cell line derived from a Desmoplastic Cerebellar Medulloblastoma patient.

[0050] Fig.19 is a schematic diagram showing the design according to the linker length in order to verify the difference in efficacy caused by the length difference (of the linker) between the two ePHD2 domains.

[0051] Fig. 20 The graph shows the results of a cell proliferation assay and cell migration inhibition according to the length of the linker between ePHD2 domains. DETAILED DESCRIPTION

[0052] Unless otherwise defined, all technical terms and scientific terms used in this specification have the same meanings as those commonly understood by those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature used in this specification is a well-known and commonly used method in the art.

[0053] The inventors of the present invention discovered a polypeptide comprising the PHD1 domain and the PHD2 domain of PHF6 (PHD Finger Protein 6) or a mutant thereof during the process of studying epigenetic therapeutic drugs for treating cancer as a candidate group. The polypeptide is not an irreversible reaction (such as genome editing), but can be reversibly regulated, and the therapeutic intensity can be adjusted by introducing mutations, etc.

[0054] A histone modifier protein performs two functions, a reader and a writer, and the intensity of its activity can be adjusted by modifying them separately. In particular, the safety of drugs can be improved by strengthening the reader function, and side effects can be reduced by optimizing the effect, which is optimized by mutations in the reader domain.

[0055] Engineering of reader domains may open up new possibilities for therapeutic targeting and allow the introduction of mutations for reversible regulation and modulation of therapeutic intensity.

[0056] Based on this, the present invention relates to a histone code reader domain, specifically, to a polypeptide that repeatedly comprises a domain of PHD finger protein 6 (PHD Finger Protein 6, PHF6) or a mutant thereof and / or a domain of double PHD fingers 3b (DoublePHD fingers 3b, DPF3b) or a mutant thereof, or prepared by swapping between domains.

[0057] Specifically, the present invention relates to a polypeptide represented by a D1-D2-D3 structure.

[0058] (a) D1 is the PHD1 domain of PHD Finger Protein 6 (PHF6);

[0059] (b) the D2 is the PHD2 domain of PHF6 or a mutant thereof; and

[0060] (c) D3 comprises one or more selected from the group consisting of:

[0061] (i) D2;

[0062] (ii) a fragment of PHF6 containing D2;

[0063] (iii) PHD1 domain of Double PHD fingers 3b (DPF3b);

[0064] (iv) the PHD2 domain of DPF3b or a mutant thereof; and

[0065] (v) PHD1 domain and PHD2 domain of DPF3b or mutants thereof.

[0066] "Domain" may refer to an identifiable unit or module that constitutes a polypeptide. Some small polypeptides may consist of a single domain, but may contain multiple domains. Domains that may be modular building blocks may have structural or functional autonomy and / or independence.

[0067] "Swapping" may refer to the mutual exchange of segments between two or more domains. The polypeptides based thereon may be rearranged by binding domains or gene fragments encoding the domains. When swapping occurs between multiple domains, in terms of structure, the remaining domains may be included in the same manner except for the exchange and modification points based on the binding and cleavage between the domains. In this way, polypeptides with altered structures or lengths may be generated.

[0068] "Polypeptide" is used interchangeably with "protein" to refer to a polymer of amino acid residues, and more than one amino acid residue applies not only to polymers that are artificial chemical mimetics of naturally occurring amino acids, but also to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0069] "Amino acid" may refer to naturally occurring amino acids, synthetic amino acids, amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids include amino acids encoded by the genetic code and amino acids that are later modified (e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine). Amino acid analogs include compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbons attached to hydrogen, carboxyl, amino groups, and R groups, for example, including compounds such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but may retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics have a structure that is different from the general chemical structure of amino acids, but include compounds that function in a manner similar to naturally occurring amino acids. The terms "non-naturally occurring amino acids" and "non-natural amino acids" include amino acid analogs, synthetic amino acids, and amino acid mimetics that are not found in nature.

[0070] As an epigenome reader, the "histone code reader" can recognize a variety of post-translational modifications (PTMs) and play a platform role in loading a variety of epigenome regulatory factors, thereby playing a core role in histone modification crosstalk. It has multiple domains for reading specific PTMs, including bromodomains for acetylation and chromodomains for methylation.

[0071] The domain of the histone code reader is a PHD finger (PHD Finger), which can recognize the methylated lysine of histones (such as H3K4me2 / 3, which is mainly associated with the transcription start site of active genes). PHD fingers can read the bound histone PTM and play an important role in histone modification crosstalk. PHD fingers belong to the zinc binding superfamily, and the tandem PHD domain can detect two zinc ions through conserved cysteine ​​and histidine residues.

[0072] PHD finger-containing proteins (PHFs) have multiple functions and structures, and can interact with histone modifications through plant homeodomains (PHDs) to play the role of epigenetic factors. PHF6, which has two expanded PHDs in PHF, can interact with histones to play the role of transcription factors, and plays the role of a reader of histone modifications, and can specifically recognize histone H2BK12 acetylation (H2BK12Ac).

[0073] For example, the PHF6 may include the sequence of SEQ ID NO:1.

[0074] [SEQ ID NO: 1]

[0075] MSSSIEQKKGSTRQRKCGFCKSSNRDKECGQLLISENQKVAAH HKCMLFSSALVSSHSDNESLGGFSIEDVQKEIKRGTKLMCSLCHCPGATIGCDVKTCHRTYHYHCALHDKAQIREKPSQGIYMVYCRKHKKTAHNSEADLEESFNEHELEPSSPKTKKKSRKGRPRKTNLKGLPEDSRSTSSHGTDEMESSSYRDRSPHRS SPNDTRPKCGFCHVGEEENEARGKLHIFNAKKAAAHYKCMLFSSGTVQLTTTSRAEFGDFDIKTVLQEIKRGKRMKCTLCSQPGATIGCEIKACVKTYHYHCGVQDKAKYIENMSRGIYKLYCKNHSGNDERDEEDEERESKSRGRVAIDQQLTQQQLNGN.

[0076] The PHD domain of PHD Finger Protein 6 (PHF6) is a protein domain composed of two β-chains and multiple short α-helical segments, and a stable structure is achieved through two zinc finger motifs. The PHD domain can be composed of two or more connected and recognize and bind to specific histone modifications, and interact with another domain that binds to histones to play the role of epigenetic factors.

[0077] The present invention is a polypeptide represented by a D1-D2-D3 structure.

[0078] (a) D1 is the PHD1 domain of PHD Finger Protein 6 (PHF6);

[0079] (b) the D2 is the PHD2 domain of PHF6 or a mutant thereof; and

[0080] (c) D3 is a fragment of PHF6 including D2 or containing D2.

[0081] According to the present invention, novel polypeptides were designed based on a strategy of duplicating the PHD2 (reader) domain of PHF6.

[0082] At this time, from the N-terminus to the C-terminus, D1-D2-D3 may have a structure including D2 at the C-terminus of D1 and D3 at the C-terminus of D2. Specifically, from the N-terminus to the C-terminus, D1-D2-D3 may include the PHD1 domain of PHF6-the PHD2 domain of PHF6 or a mutant thereof-the PHD2 domain of PHF6 or a mutant thereof, or a fragment of PHF6 containing the same.

[0083] The D1 is the PHD1 domain of PHF6, for example, and may include the sequence from position 17 to position 131 in SEQ ID NO: 1. Specifically, the D1 may include the sequence of SEQ ID NO: 2.

[0084] [SEQ ID NO: 2]

[0085] CGFCKSNRDKECGQLLISENQKVAAHHKCMLFSSALVSSHSD NESLGGFSIEDVQKEIKRGTKLMCSLCHCPGATIGCDVKTCHRTY HYHCALHDKAQIREKPSQGIYMVYCRKH.

[0086] The D2 is the PHD2 domain of PHF6 or a mutant thereof, for example, the PHD2 domain of D2 may include the sequence from position 208 to position 329 in SEQ ID NO: 1. Specifically, the D2 may include the sequence of SEQ ID NO: 3.

[0087] [SEQ ID NO: 3]

[0088] TRPKCGFCHVGEEENEARGKLHIFNAKKAAAHYKCMLFSSG TVQLTTTSRAEFGDFDIKTVLQEIKRGKRMKCTLCSQPGATIGCEI KACVKTYHYHCGVQDKAKYIENMSRGIYKLYCKNH.

[0089] The PHF6 PHD2 domain mutant refers to one or more amino acids being substituted and / or modified, and thus different from the amino acid sequence of the PHF6 PHD2 domain that is not mutated, but in a state of retaining functions or properties. In the amino acid sequence, one or more amino acids may be modified, and compared with the properties of the domain before mutation, the properties may be increased, or the properties may be unchanged or reduced.

[0090] The substitution and / or modification include deletion, insertion and / or substitution of amino acid sequence residues. This amino acid mutation is achieved based on the relative similarity of the amino acid side chain substitution products, for example, hydrophobicity, hydrophilicity, charge and size. According to the analysis of the size, shape and type of the amino acid side chain substitution products, it can be seen that arginine, lysine and histidine are all residues with positive charges, alanine, glycine and serine have similar sizes, and phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine can be respectively referred to as biological functional equivalents.

[0091] It should be interpreted as comprising a sequence that exhibits substantial identity with the sequence of the present invention. The so-called substantial identity refers to: aligning the sequence of the present invention described above with any other sequence in a manner as corresponding as possible, and when using an algorithm commonly used in the art to analyze the aligned sequence, a sequence that exhibits at least 90% homology, most preferably, exhibits at least 95%, 96% or more, 97% or more, 98% or more, 99% or more homology. The alignment method for sequence comparison is a method well known in the art. Compared with the explicit sequence or the entire sequence recorded in this specification, it may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% homology.

[0092] At this time, from the N-terminus to the C-terminus, D1-D2-D3 may have a structure in which the C-terminus of D1 includes D2, and the C-terminus of D2 includes D3. Specifically, from the N-terminus to the C-terminus, D1-D2-D3 may include the PHD1 domain of PHF6-the PHD2 domain of PHF6-the PHD2 domain of PHF6.

[0093] For example, the PHF6 PHD2 domain mutant may comprise one or more substitutions selected from the following in SEQ ID NO: 1:

[0094] C212Y;

[0095] G218R;

[0096] I230T;

[0097] F231S;

[0098] N232K;

[0099] T253I;

[0100] Q270K or Q270R;

[0101] C280S;

[0102] I290V;

[0103] E293G or E293A;

[0104] Y301H;

[0105] C326R or C326S; and

[0106] S346R.

[0107] The PHF6 PHD2 domain mutant may contain any combination of the above-mentioned specific substitutions in SEQ ID NO: 1. It may contain 1, 2, 3, 4, 5 or 6 amino acid substitution combinations among the above-mentioned specific substitutions, but is not limited thereto.

[0108] Specifically, the PHD2 domain mutant of PHF6 may comprise the following amino acid substitutions in SEQ ID NO:1.

[0109] V1: T253I, Q270K, E293G, S346R

[0110] V2: Q270R, E293A

[0111] V3: Q270K, S284R, Y322H, Q359R

[0112] V4: C212Y, G218R, I230T, C280S, I290V, C326R; or

[0113] V5: F231S, N232K, Y301H, Q270R, C326S.

[0114] Specifically, the PHD2 domain mutant of PHF6 may include V1: T253I, Q270K, E293G and S346R.

[0115] At this time, from the N-terminus to the C-terminus, D1-D2-D3 may have a structure including D2 at the C-terminus of D1 and D3 at the C-terminus of D2. Specifically, from the N-terminus to the C-terminus, D1-D2-D3 may include a PHD1 domain of PHF6, a PHD2 domain mutant of PHF6 including a V1 mutation, a PHD2 domain mutant of PHF6 including a V1 mutation, or a fragment of PHF6 including the same.

[0116] Specifically, the PHF6 PHD2 domain mutant may comprise V2 mutations (Q270R and E293A).

[0117] At this time, from the N-terminus to the C-terminus, D1-D2-D3 may have a structure including D2 at the C-terminus of D1 and D3 at the C-terminus of D2. Specifically, from the N-terminus to the C-terminus, D1-D2-D3 may include a PHD1 domain of PHF6, a PHD2 domain mutant of PHF6 including a V2 mutation, a PHD2 domain mutant of PHF6 including a V2 mutation, or a fragment of PHF6 including the same.

[0118] The fragment of PHF6 containing D2 in D3 may be cleaved before one or more amino acid residues selected from the group consisting of positions 132 to 210 in the sequence of SEQ ID NO: 1. The fragment of PHF6 containing D2 in D3 may be cleaved at the N-terminus of one or more amino acid residues selected from the group consisting of positions 132 to 210 in the sequence of SEQ ID NO: 1.

[0119] For example, the cleavage before one or more amino acid residues selected from the group consisting of positions 132 to 210 in the sequence of SEQ ID NO: 1, for example, means: based on the N-terminus in the sequence of SEQ ID NO: 1, cleavage and removal of amino acid residues from position 1 to amino acid residues 131 (amino acid residues before amino acid residue 132). Another example means: based on the N-terminus in the sequence of SEQ ID NO: 1, cleavage and removal of amino acid residues from position 1 to amino acid residues 209 (amino acid residues before amino acid residue 210).

[0120] The fragment of PHF6 comprising D2 in D3 may be cleaved before the amino acid residue at position 134T or position 177E in the sequence of SEQ ID NO:1.

[0121] For example, cleavage before amino acid residue 134T in the sequence of SEQ ID NO: 1 means: based on the N-terminus of the sequence of SEQ ID NO: 1, cleavage and removal of amino acid residues from position 1 to position 133 (amino acid residues before amino acid residue 134T). In addition, for example, based on the N-terminus of the sequence of SEQ ID NO: 1, cleavage and removal of amino acid residues from position 1 to position 176 (amino acid residues before amino acid residue 177E).

[0122] The polypeptide may have a structure in which the reader domain is repeated at the C-terminus of PHF6. As for the function of the reader domain, the role of the linker is unknown, so the following three candidate groups were prepared. The mutation positions are marked based on SEQ ID NO: 1, which is the sequence of PHF6.

[0123] Candidate 1: Repeated sequence from 134T to ePHD2 including the entire linker connecting ePHD1 and ePHD2.

[0124] Candidate 2: Repeats of the sequence from 177E to ePHD2, which contains sequences in the linker sequence that have features conserved among vertebrate species.

[0125] Candidate 3: Only the reader domain excluding the linker sequence is repeated (starting from 208T).

[0126] Mutants found by binding affinity maturation techniques can be used.

[0127] Candidate 4: The wild type reader domain was replaced with mutant 1 (T253I, Q270K, E293G and S346R).

[0128] Candidate 5: Replace the biotype reader domain with mutant 2 (Q270R and E293A).

[0129] Candidate 6: In construct #7, the reader domain was replaced with mutant 1, which resulted in increased ubiquitination efficiency.

[0130] Specifically, the polypeptide may include the sequence of SEQ ID NO:6.

[0131] MSSSIEQKKGSTRQRKCGFCKSSNRDKECGQLLISENQKVAAH HKCMLFSSALVSSHSDNESLGGFSIEDVQKEIKRGTKLMCSLCHCPGATIGCDVKTCHRTYHYHCALHDKAQIREKPSQGIYMVYCRKHKKTAHNSEADLEESFNEHELEPSSPKTKKKSRK GRPRKTNLKGLPEDSRSTSSHGTDEMESSSYRDRSPHRSSPNDTRPKCGFCHVGEEENEARGKLHIFNAKKAAAHYKCMLFSSGTVQLTTTSRAEFGDFDIKTVLQEIKRGKRMKCTLCSQPG ATIGCEIKACVKTYHYHCGVQDKAKYIENMSRGIYKLYCKNHSGNDERDEEDEERESKSRGRVAIDQQLTQQQLNGNHMGSGGGGPGTRPKCGFCHVGEEENEARGKLHIFNAKKAAAHYKC MLFSSGTVQLTTTSRAEFGDFDIKTVLQEIKRGKRMKCTLCSQPGATIGCEIKACVKTYHYHCGVQDKAKYIENMSRGIYKLYCKNHSGNDERDEEDEERESKSRGRVAIDQQLTQQQLNGN.

[0132] In addition, the present invention screened out a new domain with existing H2BK12Ac reading ability and swapping. Based on this, the present invention is a polypeptide represented by a D1-D2-D3 structure.

[0133] Wherein, (a) the D1 is the PHD1 domain of PHD Finger Protein 6 (PHF6);

[0134] (b) the D2 is the PHD2 domain of PHF6 or a mutant thereof; and

[0135] (c) D3 comprises one or more selected from the group consisting of:

[0136] (iii) PHD1 domain of Double PHD fingers 3b (DPF3b);

[0137] (iv) the PHD2 domain of DPF3b or a mutant thereof; and

[0138] (v) PHD1 domain and PHD2 domain of DPF3b or mutants thereof.

[0139] The description of D1 and D2 can be applied in the same manner as above. D3 includes a DPF3b fragment comprising the following structure: PHD1 domain, PHD2 domain or a mutant thereof, or PHD1 domain and PHD2 domain or a mutant thereof of double PHD fingers 3b (DPF3b).

[0140] The plant homeodomain (PHD) of DPF3b refers to (finger) can act as a reader of acetylated histones. The DPF3b is a member of the d4 protein family and consists of an N-terminal 2 / 3 domain, a zinc finger such as a C2H2-Kruppel-like factor, and a C-terminal tandem PHD zinc finger. The DPF3b interacts with histones H3 and H4 through lysine and in an acetylation-dependent manner. The tandem PHD fingers of DPF3b bind to the N-terminal tail of histone H3, and acetylation of H3 lysine 14 (H3K14ac) strengthens the interaction, while methylation of lysine 4 (H3K4me) inhibits binding.

[0141] The first PHD finger (PHD1) domain of DPF3b binds to H3K14ac, while the second PHD finger (PHD2) domain binds to the H3R2-K4 region. DPF3b-PHD2 is similar to H3K4me0 binding PHD fingers (BHC80, AIRE and DNMT3L) and shares a combination of acidic and hydrophobic residues in the H3K4me0 binding pocket. However, the H3K14ac binding pocket of DPF3b-PHD1 is located on the other side of the central β-sheet and uses a set of residues that do not overlap with the positions used to form H3K4 or H3R2 binding sites. Nevertheless, the H3K14ac binding site follows the same principle of using a Zn-coordinated Cys residue or a structurally rigid site near the β2 strand. (Trends Biochem Sci. 2011 Jul; 36 (7): 364–372. The PHD Finger: A Versatile Epigenome Reader).

[0142] For example, the PHD1 domain of DPF3b may include the sequence of SEQ ID NO: 4. For example, the PHD2 domain of DPF3b may include the sequence of SEQ ID NO: 5.

[0143] [SEQ ID NO: 4]

[0144] YCDFCLGGSNMNKKSGRPEELVSCADCGRSGHPTCLQFTLN MTEAVKTYKWQCIEC.

[0145] [SEQ ID NO: 5]

[0146] SCILCGTSENDDQLLFCDDCDRGYHMYCLNPPVAEPPEGSWS CHLCWELLKEKASAFGCQA.

[0147] The DPF3b PHD2 domain mutant refers to one or more amino acids being substituted and / or modified, and thus different from the amino acid sequence of the non-mutated DPF3b PHD2 domain, but in a state of retaining functions or properties. In the amino acid sequence, one or more amino acids may be modified, and compared with the properties of the domain before mutation, the properties may be increased, unchanged or reduced.

[0148] The substitution and / or modification include deletion, insertion and / or substitution of amino acid sequence residues. This amino acid mutation is achieved based on the relative similarity of the amino acid side chain substitution products, for example, hydrophobicity, hydrophilicity, charge and size. According to the analysis of the size, shape and type of the amino acid side chain substitution products, it can be seen that arginine, lysine and histidine are all residues with positive charges, alanine, glycine and serine have similar sizes, and phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine can be respectively referred to as biological functional equivalents.

[0149] It should be interpreted as comprising a sequence showing substantial identity with the sequence of the present invention. The so-called substantial identity refers to: aligning the sequence of the present invention described above with any other sequence in a manner as corresponding as possible, and when using an algorithm commonly used in the art to analyze the aligned sequence, a sequence showing at least 90% homology, most preferably, showing at least 95%, more than 96%, more than 97%, more than 98%, more than 99% homology. The alignment method for sequence comparison is a method well known in the art. Compared with the explicit sequence or the entire sequence recorded in this specification, it may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% homology.

[0150] According to a specific embodiment of the present invention, the PHD1 domain of PHF containing the PHD2 (reader) domain of PHF6 is confirmed - ePHD2 (T208) - ePHD2 The H2BK120Ubrk function as a writer was enhanced in the candidate group of polypeptides (T208) and polypeptides comprising the PHD1 domain of PHF6-PHF6ePHD2-DPF3b PHD1 and / or PHD2 domains swapping the reader domain (hereinafter referred to as Epi-glue), indicating that the enhanced function of the reader domain can lead to the enhanced function of the writer. The present invention relates to a nucleic acid encoding the polypeptide. The so-called "nucleic acid" refers to a nucleic acid molecule that is separated from the source cell to about 50% or more of the protein, lipid, carbohydrate or other substance found in nature with the nucleic acid, or a nucleic acid molecule that is operably linked to a polynucleotide that is not linked in nature, or a nucleic acid molecule that is part of a larger polynucleotide sequence that does not occur in nature. Specifically, in the isolated nucleic acid molecule, there is substantially no other contaminating nucleic acid molecule, or other contaminants found in the natural environment that inhibit its use in the production of the polypeptide or its therapeutic, diagnostic, preventive or research purposes. "Polynucleotide", "nucleic acid", "nucleic acid molecule", "nucleic acid oligomer", "oligonucleotide", "nucleic acid sequence" and "nucleic acid fragment" may include, but are not limited to, nucleotides, deoxyribonucleotides or ribonucleotides in the form of polymers that are used interchangeably and may have covalent bonds of various lengths to each other, or their analogs, derivatives or modifications. Different polynucleotides may have different three-dimensional structures and may perform various functions, known or unknown. For example, polynucleotides may include genes, gene fragments, exons, introns, DNA between genes (including but not limited to heterogeneous DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, sequence-separated DNA, sequence-separated RNA, nucleic acid probes and primers, but are not limited thereto. The polynucleotide may generally include four nucleotide bases, namely, adenine (A), cytosine (C), guanine (G), and thymine (T) (when the polynucleotide is RNA, thymine (T) is represented by uracil (U)). A "sequence" is an alphabetical representation of a molecule, and such representation can be entered into a database of a computer having a central processing unit and can be used in bioinformatics applications such as functional genomics and homology searches. A polynucleotide may optionally contain one or more non-standard nucleotides, nucleotide analogs and / or modified nucleotides. "Nucleic acid" may comprise deoxyribonucleotides or ribonucleotides and polymers thereof or their complements in single-stranded, double-stranded or multi-stranded form. "Polynucleotide" may comprise a linear sequence of nucleotides. "Nucleotide" generally refers to a single unit of a polynucleotide. A nucleotide may be a ribonucleotide, a deoxyribonucleotide, or a modified form thereof. Examples of polynucleotides may include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA (including siRNA), and hybrid molecules having a mixture of single-stranded and double-stranded DNA and RNA. The nucleic acid may have a broad meaning including DNA (gDNA and cDNA) and RNA molecules, and may be mRNA. Nucleotides, which are the basic building blocks of nucleic acids, include not only natural nucleotides, but also analogs with modified sugar or base sites. The sequences of the nucleic acids encoding the heavy and light chain variable regions of the present invention may be modified. The modifications include addition, deletion, or non-conservative or conservative substitutions of nucleotides. The nucleic acid can be injected in the form of ribonucleic acid (such as messenger ribonucleic acid mRNA). The nucleic acid of the present invention can be in the form of mRNA. Thus, transient protein expression can be achieved. The expression level of a DNA molecule within a cell can be determined based on the amount of the corresponding mRNA present in the cell or the amount of protein encoded by the corresponding DNA produced by the cell. The composition may include a delivery vehicle for delivering the expressed mRNA. For example, the mRNA can be delivered by a vector. The vector can be a viral vector. The viral vector can be a retrovirus, an adenovirus, a parvovirus (e.g., an adeno-associated virus (AAV)), a coronavirus, a negative-strand RNA virus such as an orthomyxovirus (e.g., influenza virus, a rhabdovirus (e.g., rabies and vesicular stomatitis virus)), a positive-strand RNA virus such as a paramyxovirus (e.g., measles and Sendai, an alphavirus and a picornavirus), and a herpesvirus (e.g., Herpes Simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus), a double-stranded DNA virus including an adenovirus, a poxvirus (e.g., vaccinia, fowlpox or canarypox), etc. The vector can be delivered into the body or into cells not only through electroporation, lipofection, viral vectors and nanoparticles, but also through protein translocation domain (PTD) fusion protein method and the like. For example, the expressed mRNA can be delivered via nanoparticles. The mRNA of the expression can be delivered by liposomes (liposome), lipid nanoparticles (lipid Nano-Particle, LNP) or a variety of nanoparticles. Liposomes or LNPs include cationic lipids, non-cationic lipids or neutral lipids, etc., and also include polyethylene glycol (polyethylene glycol, PEG) or cholesterol and other lipids. This mRNA delivery body is specifically recorded in U.S. Patent Publication No. 2018 / 0311176, No. 2019 / 0032051, No. 2021 / 0046192, International Patent Publication No. WO2018 / 081480, No. WO2020 / 097540, No. WO2020 / 097548, No. WO2021 / 007278, etc., and these contents are incorporated by reference into the present invention. In order to prevent the aggregation of particles occurring during the mRNA delivery process, PEG fat may be included. PEG fat is specifically shown by U.S. Patent Publication Nos. 2018 / 0311176 and 2019 / 0032051, for example, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer) or a mixture thereof.As a non-limiting example, PLGA can be conjugated with a lipid-terminated PEG to form PLGA-DSPE-PEG, wherein the PEG lipid is selected from PEG-c-DOMG and 1,2-Dimyristoyl-sn-glycerol, methoxypolyethylene Glycol (PEG-DMG), 1,2-Distearoyl-sn-glycerol, methoxypolyethylene Glycol (PEG-DSG), PEG-c-DOMG, 1,2-Distearoyl-sn-glycerol, methoxypolyethylene Glycol (PEG-DSG), 1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene Glycol (PEG-DSG), 1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene Glycol (PEG-DPG)), PEG-lipid conjugates (e.g., PEG coupled to dialkyloxypropyls) (e.g., PEG-DAA conjugates), PEG coupled to diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines and PEG conjugated to ceramides), cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates, polyamide oligomers (e.g., ATTA-lipid conjugates) and mixtures thereof.In some embodiments, the PEG can be PEG-dilauryloxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG-dipalmityloxypropyl (C16), PEG-distearyloxypropyl (C18), PEG-c-DOMG, PEG-DMG or a mixture thereof, but is not limited thereto. Peptides can be used for the mRNA delivery. The peptide should have a cation for electrostatic interaction with the anionic phosphate group of the nucleic acid, and may contain positively charged amino acids in order to generate electrostatic interaction with the phosphate group. Cell penetrating peptides (CPPs) can also be cationic molecules that hold promise for delivering mRNA. Depending on the situation, in addition to the liposome or lipid nanoparticle (LNP), a peptide may be included in the mRNA delivery process. The peptide can provide a nucleic acid packaging function and can prevent DNA or RNA from being degraded inside or outside the cell. The composition may also include more than one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier needs to be compatible with the active ingredient of the present invention, and can be mixed with one or more components in saline, sterile water, Ringer's solution, buffered saline, dextrose (dextrose) solution, maltodextrin solution, glycerol, ethanol and these components, and other conventional additives (antioxidants, buffers and antibacterial agents, etc.) can be added as needed. In addition, diluents, dispersants, surfactants, adhesives and lubricants can be added to be formulated into injection forms (such as aqueous solutions, suspensions and emulsions, etc.). In particular, preferably, the formulation of freeze-dried (lyophilized) form is provided. In order to prepare freeze-dried formulations, methods known in the art of the present invention can be used, and stabilizers for freeze drying can also be added. Further, the method suitable in the art or the method disclosed in Remington's Pharmaceutical Science (Remington'spharmaceutical Science, Mack Publishing Company, Easton PA, Pennsylvania) can be used, and formulation can be carried out in a preferred manner according to each disease or according to the composition. The content of the active ingredient etc. contained in the composition of the present invention and the method of administration can be determined by a person of ordinary skill in the art based on the symptoms of the patient and the severity of the disease. In addition, it can be formulated in various forms (powders, tablets, capsules, liquids, injections, ointments and syrups, etc.), and can also be provided as a unit-dosage or multi-dosage container (e.g., sealed ampoules and bottles, etc.). Composition of the present invention can be administered orally or parenterally. The route of administration of composition of the present invention can be, for example, bronchial, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intracardiac, percutaneous, subcutaneous, intraperitoneal, enteral, sublingual or topical administration, but is not limited thereto. The dosage of composition of the present invention can have different ranges according to the severity of patient's body weight, age, sex, health condition, diet, administration time, method, excretion rate or disease etc., and this can be easily determined by those of ordinary skill in the art. In addition, for clinical use, known technology can be used, composition of the present invention is mixed with suitable dosage form. DNA can be easily isolated or synthesized using conventional procedures. A variety of vectors are available. Typically, vector components include one or more of the following structures: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, but are not limited thereto. The term "vector" used in this specification refers to a means for expressing a target gene in a host cell, including plasmid vectors, cosmid vectors, viral vectors (such as phage vectors, adenovirus vectors, retrovirus vectors and adeno-associated virus vectors, etc.). "Operably linked" refers to the functional association between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the control sequence regulates the transcription and / or decoding of the other nucleic acid sequence. When prokaryotic cells are used as hosts, they usually contain a strong promoter capable of transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiating decoding, and a transcription / decoding termination sequence. In addition, for example, when eukaryotic cells are used as hosts, promoters derived from mammalian cell genomes (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, promoter of Epstein-Barr virus (EBV), and promoter of respiratory syncytial virus (RSV)) can be used, and generally have a polyadenylation sequence as a transcription termination sequence. As isolated cells containing the nucleic acid, prokaryotic host cells such as Bacillus strains (such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis), Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis and Staphylococcus (e.g., Staphylococcus carnosus) can be used. Examples of useful host cell strains may include COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S or HT1080, but are not limited thereto. For example, the cell may comprise an immune cell for treatment. The nucleic acid may be introduced into an immune cell. For example, the cell may be selected from the group consisting of a T cell, a NK cell, a cytokine induced killer cell (CIK), an activated cytotoxic T lymphocyte (CTL), a macrophage, a tumor-infiltrating lymphocyte (TIL) and a dendritic cell, but is not limited thereto. The present invention is a composition for preventing or treating cancer, the composition comprising the polypeptide, nucleic acid, mRNA, cell or delivery body. The present invention is a method for preventing or treating cancer, the method comprising: administering the polypeptide, nucleic acid, mRNA, cell or delivery body to an individual. The present invention provides a use of the polypeptide, nucleic acid, mRNA, cell or delivery body in preparing a drug for preventing or treating cancer. For example, the cancer may be a solid cancer including breast cancer. According to the present invention, expression was performed in breast cancer cells using mRNA synthesized by in vitro transcription. Under the condition of expressing a polypeptide (Chromatin epi-glue T208) comprising the PHD1 domain of PHF6-ePHD2(T208)-ePHD2(T208), the effect of inhibiting cell proliferation was confirmed. Compared with other molecules that promote proliferation or have no effect, a particularly significant reduction was shown, so a method of delivering it to cells was designed. Thus, evidence that Chromatin Epi-glue T208 can be used as a drug for breast cancer was confirmed. In addition, for example, the cancer may be a blood cancer, medulloblastoma, colon cancer or head and neck cancer. According to the present invention, expression is performed in breast cancer cells using mRNA synthesized by in vitro transcription. It is confirmed that various candidate substances of the present invention have the effect of inhibiting cell proliferation. The pharmaceutically acceptable carriers included in the composition of the present invention are commonly used carriers in the formulation process, including lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, but are not limited thereto. In addition to the above ingredients, the composition of the present invention may also include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. The pharmaceutical composition of the present invention can be administered orally or parenterally. When administered parenterally, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and rectal administration. When administered orally, since proteins or peptides are digestible, oral compositions should be formulated to coat the active agent or protect it from decomposition in the stomach. In addition, the pharmaceutical composition can be administered by any device that can move the active substance to the target cells. The appropriate dosage of the composition of the present invention may vary according to factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity. Usually, a skilled doctor can easily determine and prescribe the effective dosage for the desired treatment or prevention. For example, the dosage of the pharmaceutical composition of the present invention is 0.0001 mg / kg to 100 mg / kg per day. In this specification, the term "pharmaceutical effective dose" refers to a sufficient amount for preventing or treating cancer or autoimmune diseases. The pharmaceutical composition of the present invention can be formulated using pharmaceutically acceptable carriers and / or excipients according to methods that can be easily implemented by those skilled in the art to which the present invention belongs, and can be prepared in a unit dosage form or loaded into a multi-capacity container for preparation. In this case, the dosage form can be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or can be in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and can also contain a dispersant or stabilizer. The pharmaceutical composition of the present invention can be administered orally or parenterally. When administered parenterally (e.g., intravenously, subcutaneously, ophthalmically, intraperitoneally, intramuscularly, orally, rectally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracistenally, intracapsularly, intranasally or by aerosol administration), the pharmaceutical composition may include, for example, a portion of an aqueous or physiologically applicable body fluid suspension or solution. Since the carrier or vehicle is physiologically acceptable, it can be added to the composition and delivered to the patient. Therefore, as a carrier for the formulation that is the same as the body fluid, physiological saline can generally be included. When administered orally, since proteins or peptides are digestible, oral compositions should be formulated to coat the active agent or protect it from decomposition in the stomach. In addition, the pharmaceutical composition can be administered by any device that can move the active substance to the target cells. The frequency of administration varies according to pharmacokinetic parameters. Typically, a clinician will use the pharmaceutical composition until the dosage of the target effect is reached. Therefore, the pharmaceutical composition can be used in a single dose, with a dosage of more than twice at a time interval, or in a continuous injection mode by an implantation device or a catheter. The further accuracy of the suitable dosage is easily achieved by those skilled in the art, and it is included in the scope of their daily execution. The appropriate dosage of the composition of the present invention may vary according to factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity. Usually, a skilled doctor can easily determine and prescribe the effective dosage for the desired treatment or prevention. For example, the dosage of the pharmaceutical composition of the present invention is 0.0001 mg / kg to 100 mg / kg per day. In this specification, the term "pharmaceutical effective dose" refers to a sufficient amount for preventing or treating cancer or autoimmune diseases. Hereinafter, the present invention will be described in more detail by way of examples. These examples are only used to illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention should not be interpreted as being limited by these examples. Example 1: PHF6-based engineering for preparing chromatin Epi-glue The PHF6-based engineering for preparing chromatin Epi-glue is as follows ( Figure 1 ): 1. Engineering (1) Binding affinity engineering The binding affinity of the PHF6 reader domain was improved. Yeast display technology was used. The specific content of yeast display, such as Figure 2 As shown. The binding affinity comparison effectiveness of the yeast display system was evaluated. The binding affinity of ePHD2 comprising the sequence of SEQ ID NO: 3 to H2B and H2BK12Ac peptides was tested using the yeast display system. Figure 3 The left panel of FIG. 1 confirms that a peak of significantly increased binding signal appears in yeast treated with H2B peptide. Yeast expressing ePHD2 and PHF6 including the sequence of SEQ ID NO: 1 show the same trend. In the case of the empty vector (empty_pCTcon2, an empty plasmid without a protein sequence), there is almost no binding signal. Microscale thermophoresis (MST) utilizes the difference in particle mobility according to the temperature gradient between the ligand bound complex and unbound protein states of a protein. The protein was labeled with fluorescent molecules, and the difference in fluorescence signals of samples with different ligand concentrations was used to distinguish whether the protein and the ligand were bound. Thus, the binding affinity (Kd) was analyzed. Figure 3 The middle figure is a dot plot. When treated with H2B peptide, the peptide binding signal of the expression population in yeast expressing ePHD2 and PHF6 increased significantly (see orange box). Therefore, it was determined that the reader domain of PHF6 specifically recognized the H2B peptide. according to Figure 3 The right panel shows the results of comparing the binding affinity of H2B and H2BK12Ac peptides using MST after the reader domain (ePHD2) was expressed and purified in E. coli. Similar to the yeast display results, the H2B peptide showed a higher binding affinity than the H2BK12Ac peptide. (2) Library construction and screening For reader domain binding affinity maturation techniques, e.g. Figure 4As shown, a library was prepared, using the strategy of TSA (Tyramide signal amplification) added as described in (1) project, after marking the yeast library (yeast library), the fluorescence activated cell sorting technique (Fluorescence activated cell sorting, FACS) (which is a flow cytometry method for only classifying cells with target fluorescence intensity) used in a manner that allows fluorescently labeled cells to flow through one by one and detect fluorescent signals was used for screening. Together with the recovery process, the above process was repeated. The library did not know the residues involved in binding to the H2B peptide, so random mutagenesis was given by error-prone PCR. The FACS process was repeated until the peptide binding signal increased and saturated. according to Figure 4 According to the flowchart described above, FACS was performed a total of 7 times in such a manner that the peptide concentration was decreased from 50 μM to 10 μM. It can be found that compared with the initial library ( Figure 4 The lower middle dot plot of ( ) ) was repeated in the 7th round of FACS ( Figure 4 The population with high peptide binding signal appeared after the 7th round of repetitions, as the peptide binding signal did not increase any more, so the FACS was not repeated. The results of the seventh round of sequencing revealed five mutants (enriched variants). (3) Individual analysis of the five ePHD2 mutants It is an ePHD2 mutant including the sequence of SEQ ID NO: 3. The mutation positions and amino acids of the five mutants found after the seventh round of repetition are as follows ( Figure 5 Upper right). The mutation positions are marked based on SEQ ID NO: 1 of the sequence of PHF6. V1: T253I, Q270K, E293G, S346R; V2: Q270R, E293A; V3: Q270K, S284R, Y322H, Q359R; V4: C212Y, G218R, I230T, C280S, I290V, C326R; or V5: F231S, N232K, Y301H, Q270R, C326S. according to Figure 5 In the middle upper part of the figure, each mutant was expressed in yeast to confirm whether the peptide binding signal was expressed in the same system. All five mutants showed higher peptide binding signals than the wild type. according to Figure 5 In the upper left part of the figure, mutants 1, 2, and 3 were expressed and purified in E. coli and their binding affinities were measured using MST. Mutant 2 showed a lower Kd value for H2B peptide than the wild type. It was determined that mutant 2 had improved binding affinity. Mutants 4 and 5 had mutations in cysteine ​​involved in zinc chelation, so they were excluded as they could not maintain the original structure of ePHD2. ePHD2 is a zinc finger domain that folds by chelating three zinc ions. Mutation preference analysis results, such as Figure 5 As shown in the lower left part of st The 1st library (1) was sorted and sequenced by sorting 4, 5, and 7, and the mutation preference was analyzed. Common and frequently occurring mutation sites were found. st The library is a library prepared by the error-prone PCR mentioned in item (1) and has a size of about 1 × 10 7 The library size (library size). It was confirmed that the mutation frequencies at sites 270 and 293 increased with repeated FACS. like Figure 5 As shown in the lower right part of the figure, based on the first library (1 st The mutation frequency information obtained from the second library (2 nd Its purpose is to understand the mutation preference of a specific site. The 2nd Library (2 nd library) was screened in the 1st library sorting 4 (1 stIn library sort 4, positions 270, 293, 326, 334 and 359 showed high mutation frequencies. Sort 4 is a library where the peptide binding signal begins to be higher than the wild-type signal. The NNK codon was inserted as a primer at 5 sites to construct a site saturation mutagenesis library. After one MACS and three FACS, the sequencing results were analyzed. Amino acid preferences of specific properties were observed in Q270 and E293. In Q270, the proportion of R and K with positive charge was high. In E293, the proportion of R with positive charge and A and G with hydrophobic side chain was high. 2. Swapping The reader domain of PHF6 was exchanged for an engineered reader domain, and an Epi-glue protein was prepared. Other epigenetic reader domains reported to recognize epigenetic histone marks were explored. The reader domain of PHF6 was replaced with other epigenetic reader domains, and an Epi-glue protein was prepared. Specific examples are shown in Figure 6 middle. (1) Domain exchange #1: The PHD1-PHD2 domain of DPF3b, which is known to recognize H3K14Ac, was ligated to the C terminus (Cterm) of PHF6. #2: The results show that PHD1 and PHD2 of DPF3b play a role in H3K14Ac binding and H3N-terminal binding, respectively (ref. Figure 6 Therefore, only PHD1 to PHF6, which are more directly involved in binding to H3K14Ac, can be linked. (2) Dual reader domain The avidity effect was achieved by repeating the reader domain at the C-terminus of PHF6. As for the function of the reader domain, the following three candidate groups were created because the role of the linker was unknown. The mutation sites are marked with reference to SEQ ID NO: 1 of the sequence of PHF6. #5: Repeat the sequence from 134T to ePHD2 including the entire linker connecting ePHD1 and ePHD2. #6: Repeating the sequence from 177E to ePHD2, which contains a sequence in the linker sequence that has features conserved among vertebrate species. #7: Repeat only the repeat reader domain excluding the linker sequence (starting from 208T). Mutants discovered by binding affinity maturation techniques may be used. #8: Replacement of the wildtype reader domain with mutant 1 (T253I, Q270K, E293G and S346R). #9: Replacement of the wild-type reader domain with mutant 2 (Q270R and E293A). #10: In construct #7, the reader domain was replaced with mutant 1, resulting in increased ubiquitination efficiency. #11: In construct #7, which resulted in increased ubiquitination efficiency, the reader domain was replaced with mutant 2. Example 2. Structural analysis of the binding of the reader domain to the H2B peptide (NMR) pass 1 H- 15 N HSQC, analyzed the binding structure of the reader domain and H2B peptide. The amide bonds in the protein were labeled with isotopes and measured. One peak represents the amide group of one residue. Therefore, information about the protein structure can be obtained by the peak position. In addition, the residues involved in the interaction with the ligand can be known by the peak shift according to the ligand titration. The peak shift according to the titration of H2B peptide concentration was analyzed, and the residues involved in H2B peptide binding were identified. Figure 7 The left side shows 1 H- 15 N HSQC. Residues marked in red = assigned residues among those showing peak shift (assigned residues were used in doi: 10.1074 / jbc.M113.535351), Figure 7The residues producing the peak shift in the ePHD2 (208-333) structure (doi: 10.1074 / jbc.M113.535351) are marked in red on the right side, and the mutation site of mutant 2 is marked in light green. Example 3. Affinity-enhancing Epi-glue screening New Epi-glue was designed by reader domain duplication. Reader domain duplication Epi-glue was designed to diversify the linker length between PHD1 and PHD2. 134T-364N: Includes the entire connector for connecting ePHD1 and ePHD2. 177E-364N: Contains sequences in the linker that are conserved among vertebrate species. 208T-364N: Connectors not included. The reader domain of DPF3b that recognizes H3K14Ac was used. According to the PHD1 of DPF3b: H3K14Ac binding (binding), PHD2: H3 N term binding (N term binding), as follows. Candidate 1. PHF6-DPF3b (246-378): The reader domains PHD1-PHD2 of DPF3b are all used. Candidate 2. PHF6-DPF3b(246-316): Only PHD1 directly involved in H3K14Ac binding was used. Example 4. Searching for a candidate group of chromatin epi-glue for treating breast cancer After gene delivery using lentivirus, cell proliferation was confirmed. In breast cancer expressing ePHD2(T208), significant inhibition of cell proliferation was confirmed ( Figure 8 ). Compared to other molecules that promoted proliferation or had no effect, a method for delivering it to cells was designed. The western blot data are shown in Fig. 9 middle. Fig. 9 The first row in the figure represents H2BK120ub, the second row represents H2B, and the third row represents housekeeping genes. Using markers, it was confirmed that the function was improved by extension and swapping of the reader domain after lentiviral infection. H2BK120Ub can be called the writer function of PHF6. This means that the activity is increased, so the probability of strengthening the specific ability is increased. In particular, the increase of the corresponding markers was confirmed in T208 and DPF3B PHD1 / 2, and the possibility of being a candidate for improvement was confirmed. In addition, expression was performed in breast cancer cells using mRNA synthesized by in vitro transcription. Fig.10 As shown, under the condition of expressing T208, the effect of inhibiting cell proliferation was confirmed. The mutants with induced point mutations in T208 did not show excellent efficacy. Therefore, evidence was confirmed that Chromatin Epi-glue T208 can be used as a drug for breast cancer. Example 5. Effect of ePHD2(T208) in breast cancer (in vitro) The tumor regulatory mechanism of T208 (DR3) was confirmed by NGS miRNA-seq and ATAC-seq. As a miRNA-seq sample, the plasmid vector transformed with T208 (DR3) was transiently expressed in the breast cancer cell line MDA-MB-231, and RNA was extracted with TRIzol after 48 hours. Next, NGS analysis was performed using the HiSeq250 platform. ATAC-seq samples were also prepared in the same way as miRNA-seq samples, and the library was prepared using the Illumina Tagment DNA Enzyme and Buffer Small Kit, and then NGS analysis was performed using the NovaSeq platform. The differentially expressed miRNAs that changed in the miRNA-seq analysis were compared with the peaks of the miRNA-seq that changed in the ATAC-seq. according to Fig.11 In the chromatin Epi-glue T208 (DR3) sample, the expression of tumor suppressive miRNA was confirmed to be increased. It was confirmed that the inhibitory mechanism of chromatin Epi-glue T208 on breast cancer can be achieved through miRNA. The tumor regulatory mechanism of T208(DR3) was confirmed by NGS RNA-seq and ATAC-seq. Bulk RNA-seq samples were prepared under the same conditions as miRNA-seq samples, i.e., RNA was extracted with TRIzol after transient expression of plasmid vector transformed with T208(DR3) in breast cancer cell line MDA-MB-231 for 48 hours. Next, NGS analysis was performed using the NovaSeq platform. Significant genes were screened by analyzing differentially expressed genes, and peak calling of promoter and enhancer regions was performed by ATAC-seq analysis and compared with RNA-seq data. like Fig.12 As shown, in the sample of Chromatin Epi-glue T208 (DR3), the expression of chemokine among cytokines was confirmed to be increased. The results show that Chromatin Epi-glue T208 has an additional function such as promoting immunity in addition to its inhibitory mechanism against breast cancer. Example 6. Effect of ePHD2 (T208) in an in vivo xenograft model To analyze the in vivo efficacy, experiments were performed using an immunodeficient nude mouse model. Fig.13 As shown, MCF7 cells were seeded by subcutaneous injection. Next, 10ug of LNP or the same volume of PBS was injected every 2 days in 4 mice per condition. After two weeks of monitoring, the mice were sacrificed and the tumor size was compared. The results of the efficacy of Chromatine Epi-glue were verified by using an immunodeficient nude mouse xenograft model. Fig.14 As shown, it was confirmed that the growth of tumors was reduced compared with the control group (PBS group and GFP LNP group). It was confirmed that T208 (DR3) can also inhibit tumor formation in vivo. In addition, the target gene and protein were confirmed using a tumor sample grown in a xenograft model. After the tumor removed from the xenograft model was cut into a size of about 3 mm, 200 μl of TRIzol was added and homogenized using a homogenizer to confirm RNA, and then RNA was extracted. Next, cDNA was synthesized by reverse transcription reaction, and the expression of the expected chemokine was compared by quantitative PCR. For protein samples, 200 μl of EBC200 buffer was added and crushed using a homogenizer, and then fully dissolved by vortexing. As a result, whether β-catenin, a carcinogenic protein, was expressed was confirmed by SDS-PAGE and Western blot. like Fig.15 As shown, in the T208 (DR3) sample, the expression of genes related to chemokines increased, and it was confirmed that the expression of β-catenin, which had been confirmed previously, also decreased. Example 6. Efficacy in cancers other than breast cancer CCK8 assay was performed on other cells. Dojindo Cell counting kit-8 was used. For suspension cells, transfection was performed in 6 wells and then divided into 96 wells. For adherent cells, the media and mRNA reagent were replaced in 96 well plates and the assay was performed. Cell viability of mitochondria was measured by date. (1) Blood cancer As cell lines used in the experiment, lymphoblast K562 derived from CML patients and promyeloblast HL-60 derived from AML patients were used for confirmation. In these cells, T208 showed the best cell growth inhibitory effect. PHF6 also showed the same inhibitory pattern, which may indicate that mutations in PHF6 may show a poor prognosis in leukemia. according to Fig.16Compared with the control group, the cell survival rate was significantly reduced. From the perspective of RNA expression, if the expression of chemokines that attract immune cells is increased, it is considered to be significantly significant in vivo. (2) Medulloblastoma and colorectal cancer As cell lines used in the experiment, epithelial cancer D283 derived from a medulloblastoma patient and epithelial cancer HCT116 cell line derived from a colorectal carcinoma patient were used for confirmation. like Fig.17 As shown, T208 also exhibited excellent cell growth inhibition in the cells. Other candidates were also identified in the cell lines. (3) Efficacy of the T208 mutant As cell lines used in the experiment, confirmation was performed using lymphoblast RPMI6666 derived from a Hodgkins disease patient, U-87MG derived from a similar glioblastoma patient, and Daoy cell line derived from a desmoplastic cerebellar medulloblastoma patient. like Fig.18 As shown, in the cells, T208 showed some efficacy, but it was weaker than that of the previous cells. In addition, two V1V1 ( Figure 6 #10) and V2V2( Figure 6 The candidate group #11) is expected to have a maturation affinity and is expected to show better efficacy. In the cell line, the availability of other candidates V1V1 and V2V2 can be known. Example 7. Efficacy of ePHD2 (T208) according to linker length The purpose of this example is to verify the difference in efficacy depending on the length of the (linker) between the two ePHD2 domains. Fig.19As shown, the design was carried out according to the length of the joint, and cell proliferation and migration experiments for optimizing the length of the joint were performed. T208 (DR3) with various joint lengths was transiently transfected in breast cancer cells seeded in the same manner. Next, after inducing sufficient expression, the same number of cells were seeded in a 96-well plate, and the change of the fusion state (confluency) and the speed at which the wound scratch (wound scratch) was filled were compared using a live cell analysis system (Incucyte) device. like Fig. 20 As shown, the results of verifying the efficacy of the length of the linker between the ePHD2 domains showed that the cell proliferation assay (Cell proliferation assay: Fig. 20 The upper part) and cell migration experiment: Fig. 20 ) showed excellent efficacy in inhibiting the progression of breast cancer in the T208 (DR3) sample. Above, specific parts of the present invention are described in detail. It is obvious to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, the substantive scope of the present invention will be defined by the appended claims and their equivalents.

Claims

1. A polypeptide represented by a D1-D2-D3 structure, characterized in that: (a) D1 is the PHD1 domain of PHF6; (b) the D2 is the PHD2 domain of PHF6 or a mutant thereof; as well as (c) D3 comprises one or more selected from the group consisting of: (i) D2; (ii) a fragment of PHF6 containing D2; (iii) PHD1 domain of DPF3b; (iv) the PHD2 domain of DPF3b or a mutant thereof; as well as (v) PHD1 domain and PHD2 domain of DPF3b or mutants thereof.

2. The polypeptide according to claim 1, characterized in that The C-terminus of D1 includes D2, and the C-terminus of D2 includes D3.

3. The polypeptide according to claim 1, characterized in that The D1 comprises the sequence of SEQ ID NO:

2.

4. The polypeptide according to claim 1, characterized in that The PHD2 domain of D2 includes the sequence of SEQ ID NO:

3.

5. The polypeptide according to claim 1, characterized in that The fragment of PHF6 in D3 comprising D2 is cleaved before one or more amino acid residues selected from the group consisting of positions 132 to 210 in the sequence of SEQ ID NO:

1.

6. The polypeptide according to claim 5, characterized in that The fragment of PHF6 containing D2 is cleaved before the amino acid residue at position 134T or position 177E in the sequence of SEQ ID NO:

1.

7. The polypeptide according to claim 1, characterized in that The PHD2 domain mutant of PHF6 comprises one or more substitutions selected from the following in SEQ ID NO: 1: C212Y; G218R; I230T; F231S; N232K; T253I; Q270K or Q270R; C280S; I290V; E293G or E293A; Y301H; C326R or C326S; and S346R.

8. The polypeptide according to claim 1, characterized in that The sequence comprising SEQ ID NO:

6.

9. The polypeptide according to claim 1, characterized in that The PHD1 domain of DPF3b includes the sequence of SEQ ID NO:

4.

10. The polypeptide according to claim 1, characterized in that The PHD2 domain of DPF3b includes the sequence of SEQ ID NO:

5.

11. A nucleic acid, characterized in that Encodes the polypeptide according to claim 1.

12. An mRNA, characterized in that Encodes the polypeptide according to claim 1.

13. A cell, characterized in that The nucleic acid according to claim 11 is introduced.

14. A delivery vehicle, characterized in that Comprising the mRNA of claim 12.

15. The delivery vehicle according to claim 14, characterized in that The delivery vehicle includes nanoparticles, liposomes, lipid nanoparticles or carriers.

16. A composition for preventing or treating cancer, characterized in that ， Comprising the polypeptide of claim 1, the nucleic acid of claim 11, the mRNA of claim 12, the cell of claim 13 or the delivery body of claim 14.

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