Modified BCL9 mimetic peptide

Abstract

To provide modified BCL9 mimetic peptides.SOLUTION: Provided are BCL9 mimetic peptides having a modified α-helical homology domain-2 (HD2) region and, optionally, a cell-penetrating region, compositions comprising the BCL9 mimetic peptides, and methods of inhibiting proliferation of and / or promoting cytotoxicity in a neoplastic cell using the BCL9 mimetic peptides.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 870,938, filed on 5 July 2019.

[0002] Sequence List This application includes an electronically submitted sequence listing in ASCII format, which is incorporated herein by reference in its entirety. The above ASCII copy was created on July 6, 2020, is named Sapience_004_WO1_SL.txt, and has a size of 49,081 bytes. [Background technology]

[0003] B-cell CLL / lymphoma 9 (BCL9) is a protein that acts as a co-activator for β-catenin-mediated transcription. BCL9 is overexpressed in many tumors and enhances β-catenin signaling in cancer cells but not in normal cells that give rise to tumors (Zhan et al. 2017). BCL9 interacts with β-catenin via its α-helical homology domain-2 (HD2). Previous studies have shown that interfering with the BCL9 / β-catenin interaction using hydrocarbon stapled BCL9 peptides suppresses the transcription of Wnt target genes that regulate the proliferation, migration, invasion, and metastatic potential of tumor cells (Takada et al., 2012, WO2017 / 062518). [Overview of the project]

[0004] Some of the main aspects of the present invention are summarized below. Further aspects are described in the sections on embodiments, examples, drawings, and claims for carrying out the invention of this disclosure. The descriptions in each section of this disclosure are intended to be interpreted in conjunction with the other sections. Furthermore, various combinations of the various embodiments described in each section of this disclosure are intended to be included within the scope of the present invention.

[0005] The present invention provides a BCL9-mimicking peptide containing a modified BCL9α-helical homology domain-2 (HD2) region. In one embodiment, the present invention provides a BCL9 mimetic peptide comprising a modified BCL9α-helical homology domain-2 (HD2) region, wherein the modified BCL9 HD2 region comprises a variant of the amino acid sequence LSQEQLEHRERSLQTLRDIQRMLF (SEQ ID NO: 1), and the variant is modified at one or more positions of SEQ ID NO: 1 by: (i) substituting E7 with R; (ii) substituting R11 with E; (iii) substituting S12 with A; (iv) substituting Q14 with A or E; (v) substituting T15 with A; (vi) substituting D18 with A or R; (vii) substituting I19 with L; (viii) substituting R21 with E; (ix) substituting M22 with A or L; and (x) adding W, 1-Nal, or 2-Nal at position 25. The BCL9 mimetic peptide may further include modifications such as substituting F24 with W, 1-Nal, or 2-Nal, and / or cleaving 1 to 15 consecutive amino acids starting from L1 in SEQ ID NO: 1. In one embodiment, the modified BCL9 HD2 region includes an amino acid sequence selected from the group consisting of LSQEQLEHRERSLATLRAIQRMLF (SEQ ID NO: 3), LSQEQLRHREESLETLRRIQEMLF (SEQ ID NO: 4), LSQEQLEHRERALQALRAIQRALF (SEQ ID NO: 5), and ALQALRAIQRALF (SEQ ID NO: 6). Furthermore, retroinverso BCL9 containing a D-amino acid in an amino acid sequence that is inverted compared to the amino acid sequences disclosed herein may also be included. It also includes L9-mimicking peptides.

[0006] One embodiment of the present invention is a BCL9 mimetic peptide comprising a modified BCL9 α-helical homology domain-2 (HD2) region, wherein the modified BCL9 HD2 region is a D-amino acid sequence comprising a variant of the D-amino acid sequence FLMRQIDRLTQLS (SEQ ID NO: 7), and the variant is modified in such a way that at one or more positions of SEQ ID NO: 7, the following changes are made: (i) F1 is replaced with L or W; (ii) M3 is replaced with A, E, L or V; (iii) R4 is replaced with O (ornithine); (iv) I6 is replaced with L; (v) D7 is replaced with A or E; (vi) R8 is replaced with A; (vii) T10 is replaced with A, K, Q or R; (viii) Q11 is replaced with A, K or R; (ix) S13 is replaced with A. In certain embodiments, the BCL9-mimicking peptide further comprises W, F, R, 1-Nal, or 2-Nal at the N-terminus of the peptide, which is either the D-isomer or the L-isomer.

[0007] In one embodiment, the modified BCL9 HD2 region is FLMRQIDRLTQLA (sequence number 8), FLMRQLDRLTQLA (sequence number 9), FLARQLARLAQLA (sequence number 10), WLARQLARLAQLA (sequence number 11), WWLARQLARLAQLA (sequence number 12), FLMEQLRRLTELA (sequence number 13), FLAEQLRRLAELA (sequence number 14), WLAEQLRRLAELA (sequence number 15), WWLARQLERLAQLA (sequence number 16), 1-Nal-WLARQLARLRQLA (sequence number 17), FLLRQIDRLTQLA (sequence number 18), FLLRQLDRLTQLA (sequence number 19), FLLRQLERLTQLA (sequence number 20), WWLLRRQLARLAQLA (sequence number 102), 2-Nal-WLA It contains a D-amino acid sequence selected from the group consisting of RQLARLAQLA (SEQ ID NO: 115), FWLARQLARLAQLA (SEQ ID NO: 116), WWLARQLARLRQLA (SEQ ID NO: 117), WFLARQLARLAQLA (SEQ ID NO: 118), WLLARQLARLAQLA (SEQ ID NO: 119), WWLERQLARLAQLA (SEQ ID NO: 120), WWLARQLARLQQLA (SEQ ID NO: 122), WWLARQLERLARLA (SEQ ID NO: 123), WWLARQLERLRRLA (SEQ ID NO: 124), WWLARQLARLKQLA (SEQ ID NO: 125), WWLARQLERLAKLA (SEQ ID NO: 126), WWLVRQLARLAQLA (SEQ ID NO: 127), and WWLAOQLAOLAQLA (SEQ ID NO: 140).

[0008] In one embodiment, the BCL9 mimetic peptide of the present invention includes a modified BCL9 α-helical homology domain-2 (HD2) region with mixed chirality. In a particular embodiment, the modified BCL9 HD2 region is (i)F D R L [WLARQLARLAQLA] D (Sequence ID 103), (ii)F D R L [WLVRQLARLAQLA] D (Sequence ID 104), (iii)F D W L[WLVRQLARLAQLA] D (SEQ ID NO: 105), (iv) F D W L [WLARQLARLAALA] D (SEQ ID NO: 106), (v) F D W L [WLARQLAALAQLA] D (SEQ ID NO: 107), (vi) W L -[WLARQLARLAQLA] D (SEQ ID NO: 108), (vii) W L -[WLARQLARLRQLA] D (SEQ ID NO: 109), (viii) W L -[WLARQLERLRRLA] D (SEQ ID NO: 110), (ix) W L -[WLARQLERLARLA] D (SEQ ID NO: 111), (x) F L -[WLARQLARLAQLA] D (SEQ ID NO: 112), (xi) R L -[WLARQLARLAQLA] D (SEQ ID NO: 113), (xii) F D -W L -[WLARQLARLAQLA] D (SEQ ID NO: 114), and W<00​​​​​​​​​​

[0010] In some embodiments, the BCL9 mimetic peptide comprises an N-terminal group selected from the group consisting of acetyl, naphthyl, octanoyl, phenyl and isovaleryl, and / or the BCL9 mimetic peptide comprises a C-terminal amide group.

[0011] In one aspect, the BCL9 mimetic peptide of the present invention is for use in inhibiting proliferation and / or promoting cytotoxicity in neoplastic cells.

[0012] A further aspect of the present invention provides a composition comprising the BCL9 mimetic peptide of the present invention, such as a pharmaceutical composition; a kit comprising the BCL9 mimetic peptide of the present invention; and a nucleic acid molecule encoding the BCL9 mimetic peptide of the present invention.

[0013] The present invention further provides a method of inhibiting proliferation and / or promoting cytotoxicity in neoplastic cells, the method comprising contacting the neoplastic cells with the BCL9 mimetic peptide of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] [Figure 1] It shows that the modified BCL9 mimetic peptide of the present invention antagonizes β-catenin and exhibits anti-proliferative activity in cultured MCF7 breast cancer cells. [Figure 2A] It shows that the retro-inverso BCL9 mimetic peptide of the present invention exhibits anti-proliferative activity in cultured MCF7 breast cancer cells. Cytotoxicity data of peptide BCL-26 (Figure 2A) and peptide BCL-27 (Figure 2B) are shown. [Figure 2B] It shows that the retro-inverso BCL9 mimetic peptide of the present invention exhibits anti-proliferative activity in cultured MCF7 breast cancer cells. Cytotoxicity data of peptide BCL-26 (Figure 2A) and peptide BCL-27 (Figure 2B) are shown. [Figure 3A]This study demonstrates that the retroinverso BCL9 mimetic peptide of the present invention exhibits antitumor activity in an MCF7 mammary cancer mouse model. Data points represent the mean ± standard error. BCL-26 (12.5 mg / kg) was administered to nude mice two days after tumor inoculation (Figure 3A, p<0.0001). [Figure 3B] This study demonstrates that the retroinverso BCL9 mimetic peptide of the present invention exhibits antitumor activity in an MCF7 mammary cancer mouse model. Data points represent the mean ± standard error. BCL-87 (5 mg / kg) was administered 21 days after tumor inoculation (Figure 3B, p<0.005). [Figure 3C] This study demonstrates that the retroinverso BCL9 mimetic peptide of the present invention exhibits antitumor activity in an MCF7 mammary cancer mouse model. Data points represent the mean ± standard error. Two concentrations (1 mg / kg and 5 mg / kg) of BCL-87 and BCL-27 were administered 14 days after tumor inoculation (Figure 3C, p ≤ 0.0004 compared to control for all tested peptides). [Modes for carrying out the invention]

[0015] Unless otherwise indicated, the implementation of this invention will involve the use of prior art in pharmaceuticals, formulation science, protein chemistry, cell biology, cell culture, molecular biology, microbiology, recombinant DNA, and immunology, which falls within the scope of the skill of those skilled in the art.

[0016] To make the present invention more easily understandable, some terms are first defined. Further definitions are given throughout this disclosure. Unless otherwise defined, all scientific and technical terms used herein are generally understood by those skilled in the art relating to the present invention. It has the same meaning as being done.

[0017] No heading provided herein is a limitation of any of the various aspects or embodiments of the invention that may be made by referring collectively to this specification. Therefore, the terms defined immediately below are more fully defined by referring to the entire specification.

[0018] All references cited herein are incorporated herein by reference in their entirety. In addition, any manufacturer's instructions or catalogs for any product cited or mentioned herein are incorporated herein by reference. Any documents incorporated herein by reference, or any teachings therein, may be used in the practice of the present invention. Documents incorporated herein by reference are not considered prior art.

[0019] I. Definition Any expressions or technical terms used in this disclosure are intended to be descriptive, not restrictive, so that a person skilled in the art would interpret them in light of the teachings and instructions provided herein.

[0020] As used herein and in the claims set forth herein, the singular forms “a,” “an,” and “the” imply a plural meaning, unless the context clearly indicates otherwise. The terms “a” (or “an”), as well as “one or more” and “at least one,” may be used interchangeably.

[0021] Furthermore, “and / or” should be considered to specifically disclose each of the two specified features or components, with or without the other. Thus, the term “and / or” as used in phrases such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). Similarly, the term “and / or” as used in phrases such as “A, B and / or C” is intended to include A, B and C; A, B or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0022] In any instance where an embodiment is described using the word “includes,” other similar embodiments described using the words “consist of” and / or “essentially consist of.”

[0023] Units, prefixes, and symbols are expressed in their International System of Units (SI) approved forms. Numerical ranges include the digits defining the range, and any individual values ​​provided herein may serve as endpoints to ranges that include other individual values ​​provided herein. For example, a set of values, e.g., 1, 2, 3, 8, 9, and 10, is also a disclosure of numerical ranges such as 1–10, 1–8, and 3–9. Similarly, a disclosed range is a disclosure of each individual value included within the range. For example, the explicitly stated range 5–10 is also a disclosure of 5, 6, 7, 8, 9, and 10.

[0024] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to mean polymers of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, and may have non-amino acid intersperses. Unless otherwise specified, amino acid residues are represented herein using three-letter and one-letter abbreviations, as used in the art, for example, as abbreviations for uncommon or unnatural amino acids as shown herein. Amino acids are L-amino acids unless preceded by a “D” or in lowercase. Peptides are represented using a single or linked amino acid abbreviation. Unless specifically indicated, peptides are shown with the N-terminus to the left. Furthermore, the array is written from the N-terminus to the C-terminus.

[0025] Polypeptides, peptides, and proteins may include natural or synthetic modifications, such as disulfide bonds, lactam crosslinking, glycosylation, lipidation, acetylation, acylation, amidation, phosphorylation, or other operations or modifications, such as conjugation with labeling components or addition of protecting groups. Furthermore, polypeptides containing, for example, one or more analogues of amino acids (including aminoisobutyric acid (Aib), unnatural amino acids such as naphthylalanine (Nal)), and polypeptides containing or consisting of D-amino acids and other modifications known in the art. Polypeptides may be in one or more salt forms. Preferred salt forms include acetates, chlorides, or trifluoroacetates. In some embodiments, polypeptides may exist as single-chain, covalent dimers, or non-covalently associated chains. Polypeptides may also be in cyclic form. Cyclic polypeptides can be prepared, for example, by crosslinking free amino acids with free carboxyl groups. The formation of cyclic compounds can be achieved by treatment with a dehydrating agent with suitable protection as needed. The reaction from open-chain (linear) to cyclic form may involve intramolecular cyclization. Cyclic polypeptides can also be prepared by other methods known in the art, for example, using one or more lactam bridges, hydrogen bond substitutes (Patgiri et al. 2008), hydrocarbon staples (Schafmeister et al. 2000), triazole staples (Le Chevalier Isaad et al. 2009), or disulfide bridges (Wang et al. 2006). The spacing between bridges or staples may be, for example, 3, 4, 7, or 8 amino acids.

[0026] The term "mutant" refers to a polypeptide having one or more amino acid substitutions, deletions, and / or insertions compared to a reference sequence. Deletions and insertions may be internal and / or at one or more terminals. Substitutions may include the substitution of one or more amino acids by similar or homologous amino acids or dissimilar amino acids. For example, some mutants contain alanine substitutions at one or more amino acid positions. Other substitutions include conservative substitutions that have little or no effect on the overall net charge, polarity, or hydrophobicity of the protein. Some mutants contain non-conservative substitutions that change the charge or polarity of amino acids. Substitutions may be by either L-type or D-type amino acids.

[0027] "Retroinverso" polypeptides have an inverted amino acid sequence compared to the natural L-amino acid sequence and are composed of D-amino acids (with reversed α-center chirality of the amino acid subunits), which helps maintain a side-chain topology similar to that of the original L-amino acid peptide.

[0028] As used herein, the term “conservative substitution” refers to the replacement of one or more amino acids with other biologically similar residues. Examples include substitutions of amino acid residues with similar properties, such as small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids, and aromatic amino acids. For further information on phenotypic silent substitutions in peptides and proteins, see, for example, Bowie et al., Science 247:1306-1310 (1990). The following table classifies conservative amino acid substitutions by their physicochemical properties: I for neutral and / or hydrophilic, II for acidic and amide, III for basic, IV for hydrophobic, and V for aromatic bulky amino acids. [Table 1]

[0029] The table below classifies conserved amino acid substitutions by their physicochemical properties: VI is neutral or hydrophobic, VII is acidic, VIII is basic, IX is polar, and X is aromatic. [Table 2]

[0030] Methods for identifying conserved nucleotide and amino acid substitutions that do not affect protein function are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993), Kobayashi et al., Protein Eng. 12(10):879-884 (1999), and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0031] The term “identical” or “identity” percentage for two or more nucleic acids or polypeptides means that two or more sequences or subsequences are the same, or have the same percentage of identical nucleotide or amino acid residue designations when compared and aligned (with gaps introduced as necessary) to obtain the greatest possible match without considering any conserved amino acid substitutions as part of sequence identity. Identity percentages can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software that can be used to obtain amino acid or nucleotide sequence alignments are known in the art.

[0032] One such non-restrictive example of a sequence alignment algorithm is described in Karlin et al., Proc. Natl. Acad. Sci., 87:2264-2268 (1990), which is Karlin et al., Proc. Nat It has been modified in l.Acad.Sci.,90:5873-5877(1993) and incorporated into the NBLAST and XBLAST programs (Altschul et al., Nucleic Acids Res.,25:3389-3402(1991)). In some embodiments, gapped BLAST, as described in Altschul et al., Nucleic Acids Res. 25:3389-3402(1997), may be used. BLAST-2, WU-BLAST-2 (Altschul et al., Methods in Enzymology,266:460-480(1996)), ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or Megalign (DNASTAR) are other publicly available software programs that can be used to align sequences. In one embodiment, the percentage of identity of two nucleotide sequences is determined using the GAP program in the GCG software package (e.g., using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 90, and length weights of 1, 2, 3, 4, 5, or 6). In an alternative embodiment, the percentage of identity of two amino acid sequences may be determined using the GAP program in the GCG software package incorporating the algorithm of Needleman and Wunsch (J.Mol.Biol.(48):444-453(1970)) (e.g., using either the BLOSUM 62 matrix or the PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, or 5). Alternatively, in one embodiment, the percentage of identity of a nucleotide or amino acid sequence is determined using the algorithm of Myers and Miller (CABIOS 4:11-17(1989)). For example, identity percentage can be determined using the ALIGN program (version 2.0) and PAM120 along with a residue table, a 12-gap length penalty, and a 4-gap penalty.Those skilled in the art can determine appropriate parameters for maximum alignment using specific alignment software. In some embodiments, the default parameters of the alignment software are used. Other means for calculating identity include the methods described in Computational Molecular Biology (Lesk ed., 1988), Biocomputing: Informatics and Genome Projects (Smith ed., 1993), Computer Analysis of Sequence Data, Part 1 (Griffin and Griffin eds., 1994), Sequence Analysis in Molecular Biology (G. von Heinje, 1987), Sequence Analysis Primer (Gribskov et al. eds., 1991), and Carillo et al., SIAM J. Applied Math., 48:1073 (1988).

[0033] As used herein, “polynucleotide” may include one or more “nucleic acids,” “nucleic acid molecules,” or “nucleic acid sequences,” and refers to a polymer of nucleotides of any length, including DNA and RNA. A polynucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and / or their analogues, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase. A polynucleotide may include modified nucleotides, such as methylated nucleotides and their analogues. The foregoing applies to all polynucleotides referred to herein, including RNA and DNA.

[0034] "Isolated" molecules are those in forms not found in nature, and include purified molecules.

[0035] "Labeling" refers to the process of directly or indirectly conjugating a molecule to create a "labeled" molecule. The label is a detectable compound that can be labeled. The label may be detectable on its own (e.g., radioisotope labeling or fluorescent labeling), or indirectly, for example, by catalyzing a detectable chemical change in the substrate compound or composition (e.g., enzymatic labeling), or by other indirect detection means (e.g., biotinylation).

[0036] "Binding affinity" generally refers to the sum of the strength of non-covalent interactions between a single binding site of a molecule and its binding partner (e.g., a receptor and its ligand, an antibody and its antigen, or two monomers forming a dimer). Unless otherwise specified, "binding affinity," as used herein, refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair. The affinity of molecule X to its partner Y is usually expressed by the dissociation constant (K D ) can be expressed by. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity partners tend to bind slowly and dissociate quickly, while high-affinity partners tend to bind more quickly and remain bound for a longer period.

[0037] The affinity or avidity of a molecule to its binding partner can be experimentally determined using any suitable method known in the art, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or kinetic analysis (e.g., KINEXA®, BIACORE®, or OCTET® analysis). Direct and competitive binding assays can be readily employed. (e.g., Berzofsky et al., “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, WE, ed., Raven Press: New York, NY (1984), Kuby, Immunology, WH Freeman and See Company: New York, NY (1992). The measured affinity of a particular bond pair interaction may vary when measured under different conditions (e.g., salt concentration, pH, temperature). Therefore, affinity and other binding parameters (e.g., K D Or Kd, K on , K off The measurement of ) is performed using a standard solution and standard buffer of the binding partner, as is known in the art.

[0038] An "active agent" is a component intended to produce biological activity. Active agents can exist in association with one or more other components. An active agent that is a peptide is sometimes called an "active peptide."

[0039] The "effective dose" of an active agent is the amount sufficient to accomplish the specifically stated purpose.

[0040] The term "pharmaceutical composition" refers to a formulation that is in a form that enables the biological activity of an active ingredient to be effective and does not contain any additional ingredients that are unacceptably toxic to the subject to which the composition is to be administered. Such a composition may be sterile and may contain a pharmaceutically acceptable carrier, such as physiological saline. A suitable pharmaceutical composition may contain one or more buffers (e.g., acetic acid, phosphoric acid, or citrate buffers), surfactants (e.g., polysorbates), stabilizers (e.g., polyols or amino acids), preservatives (e.g., sodium benzoate), and / or other commonly used solubilizers or dispersants.

[0041] The terms “inhibit,” “block,” and “suppress” are used interchangeably and mean any statistically significant reduction in occurrence or activity, including complete blockade of occurrence or activity. For example, “inhibition” may mean a reduction of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of activity or occurrence. An “inhibitor” is a molecule, factor, or substance that results in a statistically significant reduction in the occurrence or activity of a process, pathway, or molecule.

[0042] A "neoplastic cell" or "neoplasm" is typically a cell or tissue that has grown abnormally compared to normal cells or tissues of the same type, resulting from some form of mutation / transformation. Neoplasms include morphological abnormalities and pathological growth. Neoplastic cells can be benign or malignant. Malignant neoplasms, or cancers, are distinguished from benign neoplasms in that they exhibit loss of cell differentiation and orientation and have invasive and metastatic characteristics.

[0043] II. BCL9-mimicking peptides and compositions BCL9-mimicking peptide BCL9 is a 149 kDa eukaryotic protein involved in signal transduction via the Wnt pathway. BCL9 binds to β-catenin and promotes its transcriptional activity. The β-catenin-binding region of BCL9, or "HD2 domain," is a 24-residue α-helix (SEQ ID NO: 1) located at amino acids 351-374 of BCL9. The complete amino acid sequence of wild-type human BCL9 is shown in NCBI accession number NP_004317.2.

[0044] Peptide ST-BC1 (SEQ ID NO: 2) is a cyclic mutant of the natural BCL9 HD2 domain, having a lactam crosslink between residues 14 and 18. Previous studies have shown that analogues of ST-BC1 having a hydrocarbon crosslink between residues 14 and 18 inhibit Wnt transcriptional activity in human colon cancer cells and exhibit antitumor activity in mouse models (Takada et al. 2012). The inventors have discovered that non-conservative linear mutants of ST-BC1 induce cell death in neoplastic cells and reduce tumor volume in animal models. The discovery that the BCL9-derived peptide of the present invention retains the ability to specifically target and kill neoplastic cells, despite having multiple non-conservative amino acid substitutions to the wild-type BCL9 HD2 region, was unpredictable before the present invention. Furthermore, the retroinverso mutant not only exhibited activity but also possessed comparable activity to ST-BC1 and the linear "L" mutant, which was also unpredictable.

[0045] The present invention provides a BCL9 mimetic peptide having a modified BCL9 HD2 region and optionally a cell membrane permeable region. The BCL9 peptide of the present invention is a “mimicking” in the sense that it can interfere with or inhibit wild-type BCL9 activity in cells into which it is introduced. More specifically, the BCL9 mimetic peptide of the present invention can bind to β-catenin and compete with the binding of native BCL9 to β-catenin. In some embodiments, the BCL9 mimetic peptide can downregulate the expression of one or more members of the Wnt signaling pathway, e.g., axyn, CD44, c-Myc, cyclin D1, LEF1, LGR5, survivin, and VEGF-A. In some embodiments, the BCL9 mimetic peptide can inhibit cell proliferation, angiogenesis, and / or cell migration. BCL9 activity can be evaluated by any of several assays known in the art, including the cell killing assay described herein (Kawamoto et al. 2009, WO2017 / 062518).

[0046] The "modified BCL9 HD2 region" is a sequence derived from the wild-type BCL9 HD2 region, having at least one addition, deletion, or substitution compared to the wild-type BCL9 HD2 sequence. The modified BCL9 HD2 region preferably includes a peptide corresponding to at least positions 16-23 of SEQ ID NO: 1 and containing at least one addition, deletion, or substitution compared to SEQ ID NO: 1. The modified BCL9 HD2 region may include, for example, the amino acid sequences shown in Table 1. The natural BCL9 HD2 sequence (SEQ ID NO: 1) is shown as a reference point. Substitutions in SEQ ID NO: 1 are shown in underlined bold. [Table 3]

[0047] The modified BCL9 HD2 region may be in a retroinverso form and may have, for example, the D-amino acid sequence X1LX2X3QLX4X5LX6X7LA (SEQ ID NO: 142), where each amino acid at positions 1-13 is independently selected from those shown in Table 2. [Table 4]

[0048] In the D-amino acid HD2 domain sequences shown in Table 2, only one of position 4 or position 8 can be alanine; that is, if position 4 is A, then position 8 is not A, and vice versa. The BCL9 HD2 region may optionally contain a D-amino acid or L-amino acid selected from the group consisting of F, 1-Nal, 2-Nal, R, and W at position-1. Furthermore, the BCL9 HD2 region may optionally contain a D-amino acid or L-amino acid selected from the group consisting of F, 1-Nal, 2-Nal, and W at position-2. In one embodiment, if position-1 is R, then position 1 is F or W, and / or position-2 is F, 1-Nal, 2-Nal, or W.

[0049] Table 3 shows specific examples of the retroinverso BCL9 HD2 region. The retroinverso sequence (SEQ ID NO: 7) of the wild-type BCL9 HD2 region is shown as a reference point. Substitutions in SEQ ID NO: 7 are indicated in underlined bold. [Table 5]

[0050] The modified BCL9 HD2 region may contain additional D-amino acids corresponding to the complete retroinverso sequence of SEQ ID NO: 1. For example, the modified BCL9 HD2 region may contain a D-amino acid sequence [ka] This may include, where substitutions and additions to the retroinverso wild-type BCL9 HD2 sequence are indicated in underlined bold.

[0051] The modified BCL9 HD2 region may contain mixed-chirality amino acids, such that one or more amino acids in the peptide are L-isomers and one or more amino acids are D-isomers. For example, an L-peptide may contain one or more D-amino acids. Similarly, a retroinverso-D-peptide may contain one or more L-amino acids. In one embodiment, the BCL9 HD2 region is: [ka] The sequence is selected from the group consisting of the following, where the subscripts D and L represent the chirality of the amino acids, and substitutions and additions to the retroinverso wild-type BCL9 HD2 sequence (SEQ ID NO: 7) are shown in underlined bold.

[0052] Variants of these sequences are also included in the scope of the present invention. The BCL9 mimetic peptides of the present invention may have an HD2 region having at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequences disclosed herein.

[0053] In embodiments where the BCL9 mimetic peptide includes another active peptide, such as a cell membrane permeable region or an RGD-like sequence, the active peptide is functionally linked to the modified BCL9 HD2 region. In some embodiments, the active peptide is covalently linked to the modified BCL9 HD2 region by, for example, a peptide bond, a disulfide bond, a thioether bond, or a linker known in the art. Exemplary linkers include, but are not limited to, substituted alkyls, substituted cycloalkyls, polyethylene glycols, and their derivatives. The linker is cleaved after the peptide has been delivered into the cell. This may be possible. A "fusion" is sometimes referred to as a compound in which the active peptide and the modified BCL9 HD2 region are directly linked by an amide bond. The fusion may contain the amino acid linker sequence described above for the active peptide between the active peptide and the modified BCL9 HD2 region. The active peptide may be linked to the N-terminus or C-terminus of the modified BCL9 HD2 region, or via residue side chains. The active peptide and the modified BCL9 HD2 region may have the same or opposite chirality.

[0054] The cell membrane-permeable BCL9 mimetic peptides of the present invention may comprise any combination of the cell membrane-permeable and modified BCL9 HD2 regions disclosed herein. Non-limiting examples of such peptides are shown in Table 4. The cell membrane-permeable regions are italicized. Peptide BCL-21 contains the native BCL9 HD2 sequence and is inefficient in inhibiting cell proliferation. Substitutions for the HD2 sequence are shown in underlined bold text. [Table 6]

[0055] This also includes retroinverso forms of BCL9-mimicking peptides. Exemplary embodiments of cell membrane-permeable retroinverso BCL9-mimicking peptides are shown in Table 5. [Table 7]

[0056] Cell membrane permeability and RGD-like regions are italicized. Substitutions and additions to the retroinverso wild-type BCL9 HD2 sequence (SEQ ID NO: 7) are shown in underlined bold. The present invention also includes peptides containing the BCL9 HD2 region shown in Table 5 and a different active peptide, such as a different cell membrane permeability region, and peptides containing the BCL9-HD2 region shown in Table 5 but without an active peptide.

[0057] The BCL9-mimicking peptides of the present invention may contain amino acids with mixed chirality, such that one or more amino acids in the peptide are L-isomers and one or more amino acids are D-isomers. Table 6 shows non-limiting examples of BCL9-mimicking peptides having mixed chirality. [Table 8]

[0058] The subscripts D and L represent the chirality of the amino acids. The cell membrane permeable region is italicized. Substitutions and additions to the retroinverso wild-type BCL9 HD2 sequence (SEQ ID NO: 7) are shown in underlined bold. The present invention also includes peptides containing the BCL9 HD2 region shown in Table 6 and a different active peptide, such as a different cell membrane permeable region, and peptides containing the BCL9-HD2 region shown in Table 6 but without an active peptide.

[0059] The BCL9 mimetic peptides of the present invention include peptides having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with sequences disclosed herein.

[0060] The length of the BCL9-mimicking peptide of the present invention is preferably 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids, and includes a range having any of these lengths as endpoints, for example, 13 to 35 amino acids.

[0061] BCL9-mimicking peptides may have modified N-terminus and / or modified C-terminus. For example, BCL9-mimicking peptides may optionally contain an N-terminal acetyl group and / or a C-terminal amide group. Other examples of optional N-terminal and / or C-terminal groups include hydrophobic groups, e.g., linear or cyclic C2-C 18 Examples include aliphatic or aromatic hydrocarbons, naphthyl groups, phenyl groups, octanoyl groups, and valeryl groups, including isovaleryl groups. In some embodiments, the BCL9 mimetic peptide includes a linker or spacer between the peptide and the hydrophobic group. Examples of such linkers or spacers include aminohexanoic acid, beta-alanine, substituted alkyl groups, substituted cycloalkyl groups, and polyethylene glycol.

[0062] The BCL9-mimicking peptide of the present invention may optionally be cyclic. For example, the BC of the present invention L9-mimicking peptides may contain one or more lactam crosslinks. These lactam crosslinks are preferably, but not necessarily, formed between side chains spaced 3, 4, 7, or 8 amino acid residues apart (i.e., BxxB, BxxxB, BxxxxxxB, BxxxxxxxB). For example, lactam crosslinks may be formed between the side chains of Asp or Glu and Lys or Orn. ​​Amino acid substitutions may be made at the lactam crosslink site to facilitate linking.

[0063] The BCL9 mimetic peptide of the present invention may optionally include one or more epitopes and / or affinity tags for purification or detection, etc. Non-limiting examples of such tags include FLAG, HA, His, Myc, GST, etc. The BCL9 mimetic peptide of the present invention may optionally include one or more labels.

[0064] In one embodiment, the present invention provides a composition, such as a pharmaceutical composition, comprising the BCL9 mimetic peptide of the present invention and optionally further comprising one or more carriers, diluents, excipients, or other additives.

[0065] The BCL9 mimetic peptides and compositions provided herein, as well as kits optionally including instructions for use, are also within the scope of the present invention. A kit may further contain at least one additional reagent and / or one or more additional active agents. A kit typically includes a label indicating the intended use of the kit's contents. In this regard, the term “label” includes any written or recorded information on or provided with the kit, or otherwise accompanying the kit.

[0066] The BCL9-mimicking peptide of the present invention may be used to inhibit proliferation and / or promote cytotoxicity in neoplastic cells. Proliferation and cytotoxicity may be measured by known assays, including the cell-killing assay described herein.

[0067] Cell targeting The BCL9-mimicking peptide of the present invention can be introduced into target cells by methods known in the art. The selected introduction method may be determined, for example, depending on the intended application.

[0068] In some cases, DNA or RNA encoding the BCL9 mimetic peptide is delivered to target cells and expressed there. Depending on the application, delivery can be achieved via any suitable vector. Examples of vectors include plasmids, cosmids, phages, bacteria, yeast, and constructed viral vectors, such as retroviral vectors including lentiviruses, adenoviruses, adeno-associated viruses, and enveloped pseudotyped viruses. Vectors can be introduced into cells, for example, using nanoparticles, hydrodynamic delivery, electroporation, sonication, calcium phosphate precipitation, or cationic polymers, such as DEAE-dextran. Vectors may also be complexed with lipids, for example, encapsulated in liposomes, or associated with cationic condensing agents.

[0069] The BCL9-mimicking peptide of the present invention can be delivered to cells via mechanisms utilizing cell receptors. Examples of such mechanisms include antibody-drug conjugates, chimeric antigen receptors, multiple antigen presentation (MAP) systems, and integrin targeting, as well as RGD-like sequences. Examples of RGD-like sequences include GRGDS (SEQ ID NO: 28) and GRGDNP (SEQ ID NO: 29). The BCL9-mimicking peptide of the present invention may comprise one or more RGD-like sequences, e.g., two, three, four, or five, linked together as described herein or by any method known in the art. One or more RGD-like sequences may be incorporated into the N-terminal or C-terminal side of the BCL9 HD2 region. Such RGD-like sequences may also be retroinvaded independently of each other and the BCL9 HD2 region. The sequence may also be in luso form. One detailed example of a retroinverso RGD-like sequence is PSDGRG (SEQ ID NO: 75). Alternatively, the BCL9-mimicking peptide may be delivered to cells by encapsulation in vesicles, such as exosomes or liposomes, or micelles. Another method for introducing the BCL9-mimicking peptide into cells is by cyclization, for example, using hydrocarbon staples (Bernal et al. 2007, Bird et al. 2016), or other cyclization methods known in the art.

[0070] A certain BCL9-mimicking peptide of the present invention comprises a cell membrane permeable domain or a cell membrane permeable peptide (CPP). The terms “cell membrane permeable domain,” “cell membrane permeable region,” and “cell membrane permeable peptide” are used interchangeably herein.

[0071] CPPs are short peptides (typically about 6–40 amino acids) that can cross the cell membrane. Many CPPs can cross the blood-brain barrier (BBB). In some embodiments, CPPs are 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 amino acids in length, and include a range having any of those lengths as endpoints, e.g., 10–30 amino acids. CPPs have the ability to transport covalently or non-covalently linked molecular cargoes, such as polypeptides, polynucleotides, and nanoparticles, across the cell membrane and the BBB. The transfer may be endocytosis via the transfer or energy-independent (e.g., non-endocytosis). Numerous CPPs have been described and characterized in the literature (for example, Handbook of Cell-Penetrating Peptides (2d ed.Ulo Langel ed.,2007), Herve et al.2008, Heitz et al.2009, Munyendo See et al. 2012, Zou et al. 2013, and Krautwald et al. 2016. The CPP database, which was supervised by Gautam et al., is managed at crdd.osdd.net / raghava / cppsite (Gautam et al. (al. 2012).

[0072] Peptides referred to as nuclear localization sequences (NLS) are a subset of CPPs. Classical NLSs contain one (uniform) or two (binary) basic amino acid regions. The consensus sequences for classical uniform and binary NLS are K(K / R)X(K / R)(SEQ ID NO: 30) and (K / R)(K / R)X, respectively. 10-12 (K / R) 3 / 5(SEQ ID NO: 31), where 3 / 5 indicates that at least three of the five consecutive amino acids are lysine or arginine (Kosugi et al. 2009). The NLS sequence PKKKRKV from the SV40 large T antigen (SEQ ID NO: 57) is an example of a classic one-dimensional NLS, while the NLS sequence KRPAATKKAGQAKKK from nucleoplasmin (SEQ ID NO: 44) is an example of a classic two-dimensional NLS (Lange et al. 2007, Kosugi). (et al. 2009). Many non-classical NLSs exist, such as those derived from ribonucleoproteins (RNPs) hnRNP A1, hnRNP K, and U snRNP (Mattaj et al. 1998).

[0073] Non-limiting examples of CPPs suitable for use in the present invention include protein-derived peptides, such as those derived from Drosophila antennapedia transcription factors (penetratin and its derivatives RL-16 and EB1) (Derossi et al. 1998, Thoren et al. 2000, Lundberg et al. 2007, Alves et al. 2008), those derived from HIV-1 trans-transcriptional activator (Tat) (Vives et al. 1997, Hallbrink et al. 2001), those derived from rabies virus glycoprotein (RVG) (Kumar et al. 2007), and those derived from herpes simplex virus VP22 (Elliott et al. (1997), derived from antimicrobial proteoprotein 1 (SynB) (Rousselle et al. 2001), derived from rat insulin 1 gene enhancer protein (pIS1) (Kilk et al. 2001, Magzoub et al. 2001), derived from mouse vascular endothelial cadherin (pVEC) (Elmquist et al. Examples include those derived from human calcitonin (hCT) (Schmidt et al. 1998) and those derived from fibroblast growth factor 4 (FGF4) (Jo et al. 2005). Suitable CPPs for use in the present invention also include synthetic and chimeric peptides, such as transportan (TP) and its derivatives (Pooga et al. 1998, Soomets et al. 2000), membrane transition sequences (MTS) (Brodsky et al. 1998, Lindgren et al. 2000, Zhao et al. 2001), such as MPS peptides (also known as fusion sequence peptides or FBP) (Chaloin et al. 1998), sequence signaling peptides (SBP) (Chaloin et al. 1997), model amphiphilic peptides (MAP) (Oehlke et al. 1998, Scheller et al. 1999, Hallbrink et al. 2001), transition peptide 2 (TP2) (Cruz et al. 2013), MPG (Morris et al. 1997, Kwon et al. 2009), Pep-1 (Morris et al. al.2001, Munoz-Morris et al.2007), as well as polyarginines (e.g., R7-R 12 (Sequence ID 144) (Mitchell et al. 2000, Wender et al. 2000, Futaki et al. 2001, Suzuki) (et al. 2002) is another example. While not limited to specific sequences, representative sequences are shown in Table 7. [Table 9-1] [Table 9-2]

[0074] Since the function of CPP depends on its physical properties rather than sequence-specific interactions, it can have reverse sequence and / or reverse chirality, as shown in Table 7 and / or known in the art. For example, retroinverso forms of CPP (reverse sequence and reverse chirality) are suitable for use in the present invention. An example of retroinverso CPP is the D-amino acid sequence KKWKMRRNQFWIKIQR ( Examples include sequences with sequence number 71), KKWKMRRNQFWLKLQR (sequence number 72), RRRQRRKKRGY (sequence number 73), KLTPV (sequence number 74), or OLTPV (sequence number 143). Variants of these sequences having one or more amino acid additions, deletions, and / or substitutions that retain the ability to cross the cell membrane and / or BBB are also suitable for use in the present invention. The BCL9-mimicking peptides of the present invention may contain a cell membrane permeable domain having at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the exemplary sequences shown in Table 7. The effects of amino acid additions, deletions, and / or substitutions on the cell membrane permeability of the CPP can be tested using methods known in the art.

[0075] III.Preparation method The BCL9-mimicking peptide of the present invention can be chemically synthesized, for example, using solid-phase peptide synthesis or solution-based peptide synthesis, or expressed using recombinant methods. Synthesis or expression may be carried out as peptide fragments, which may then be chemically or enzymatically linked.

[0076] Accordingly, nucleic acid molecules encoding the BCL9-mimicking peptide of the present invention are also provided. Such nucleic acids can be constructed by chemical synthesis using oligonucleotide synthesizers. The nucleic acid molecules of the present invention can be designed based on the amino acid sequence of the desired BCL9-mimicking peptide and the selection of codons that are advantageous in the host cell that will produce the recombinant BCL9-mimicking peptide. Standard methods can be applied to synthesize nucleic acid molecules encoding the BCL9-mimicking peptide of interest.

[0077] Nucleic acids encoding specific BCL9-mimicking peptides can be inserted into expression vectors as soon as they are prepared and functionally ligated to expression regulatory sequences suitable for peptide expression in a desired host. To obtain high expression levels of BCL9-mimicking peptides, the nucleic acids can be functionally ligated or associated to transcriptional and translational expression regulatory sequences that function in a selected expression host.

[0078] A variety of expression host / vector combinations can be employed for any of those known in the art. Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression regulatory sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, e.g., E. coli plasmids including pCR1, pBR322, pMB9 and their derivatives, plasmids with a broader host range, e.g., M13, and filamentous single-stranded DNA phages.

[0079] Suitable host cells include prokaryotes, yeasts, insects, or higher eukaryotic cells under the control of an appropriate promoter. Prokaryotes include Gram-negative or Gram-positive organisms, such as E. coli or bacilli. Higher eukaryotic cells can be established or derived from mammalian cell lines, including Pichia pastoris, 293 cells, COS-7 cells, L cells, C127 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, HeLa cells, and BHK cells. A cell-free transition system can also be employed. [Examples]

[0080] Embodiments of this disclosure may be further defined by reference to the following non-limiting examples. It will be apparent to those skilled in the art that many modifications to both the materials and methods can be carried out without departing from the scope of this disclosure.

[0081] Example 1. BCL9 peptide exhibits antiproliferative activity in cancer cells in vitro. The inventors generated a panel of peptides containing a cell membrane permeable region and a BCL9 HD2 domain, and compared their activity with that of a previously described cyclic peptide (Takada et al. 2012) and ST-BC1. The BCL9 peptides described in this example are summarized in Table 8. [Table 10]

[0082] ST-BC1 contains lactam crosslinks between the underlined residues in bold. The other five peptides are linear in nature, possessing a TAT cell membrane permeable region shown in italics. Peptide BCL-21 contains the native HD2 domain, while each of peptides BCL-22 to 25 has amino acid substitutions in the native BCL9 HD2 domain. In addition to the substitutions, peptide BCL-25 also has a shortened HD2 domain compared to the native sequence.

[0083] 2.5 × 10 4 A 96-well dish contained 150 μL of MEM medium and 10% fetal bovine serum (FBS) with MCF-7 breast cancer cells at a cell / well density. Lyophilized BCL9 peptide was reconstituted to a concentration of 10 mg / mL in 270 mM trehalose buffer, and 50 μL of this was added to each well to achieve a final concentration range of 2.5–40 μM. Cells were incubated with the BCL9 peptide at 37°C for 96 hours.

[0084] Cell viability was quantified by spectrophotometric analysis using the Roche MTT cell proliferation assay kit according to the manufacturer's instructions. Briefly, cells were washed with PBS and incubated for 4 hours at 37°C in fresh MEM medium containing 10% FBS, 1% penicillin / streptomycin, and 1% non-essential amino acids, with 10 μL of MTT reagent added. After 4 hours, 100 μL of solubilization solution was added, and the cells were incubated overnight at 37°C with 5% carbon dioxide. Absorbance was measured at OD570 nm relative to OD650 nm. Absorbance is proportional to the number of viable cells. The percentage of absorbance relative to the untreated control was quantified and expressed as cell viability %.

[0085] The modified BCL9 peptide of the present invention, rather than BCL-21 which has the natural BCL9 HD2 domain sequence, demonstrated equal or greater antiproliferative activity compared to peptide ST-BC1 in MCF7 breast cancer cells (EC 50 Value <10 μM) (Figure 1). In functional assays, EC 50 This is the concentration that reduces the biological response by 50% of its maximum value. In the case of BLC9 peptide, EC 50 It is measured as the concentration that reduces cell viability by 50% of its maximum value. EC 50 This can be calculated by any number of means known in the art.

[0086] Example 2. Retroinverso BCL9 peptide exhibits antiproliferative activity in cancer cells in vitro. The inventors prepared a retroinversopeptide: D-amino acid sequence [ka] BCL-26 having a D-amino acid sequence [ka] BCL-27 possesses the following characteristics. The cell membrane permeable region derived from the Bax inhibitor peptide is shown in italics. In BCL-26, the HD2 domain sequence is shortened by only 11 amino acids and has substitutions at two positions compared to the native sequence. In BCL-27, the HD2 domain sequence is shortened by only 11 amino acids and has substitutions at six positions compared to the native sequence, and contains an additional N-terminal tryptophan.

[0087] The inventors investigated the cytotoxicity of peptides BCL-26 and BCL-27 using an assay in HL60 promyelocytic leukemia cells. The density of HL60 PML suspended cells in 150 μL of RPMI + 1.5% fetal bovine serum (FBS) placed in a 96-well dish was 3.5 × 10⁻⁶. 3Cells / well were defined. 50 μL of BCL-26 or BCL-27, reconstituted to a concentration of 10 mg / mL at 20 mM His, pH 7.5, was added to each well to achieve a final concentration range of 0–80 μM. Cells were incubated with the peptide at 37°C for 48 hours. Cell viability was quantified by flow cytometry using the abcam Annexin V FITC Apoptosis Detection Kit. Briefly, cells were washed with PBS and resuspended in 1x assay buffer containing Annexin V FITC and propidium iodide (PI). Annexin V detects apoptotic cells, and PI stains dead cells. After staining, apoptotic cells exhibit green fluorescence, dead cells exhibit red and green fluorescence, and viable cells exhibit little to no fluorescence. Cells were selected for analysis based on forward scattering (FSC) relative to side scattering (SSC), and annexin V-FITC binding was detected using a BD Accuri C6 Plus flow cytometer (Ex=488nm, Em=530nm) with a FITC signal detector. PI staining was analyzed using a phycoerythrin emission signal detector. 低 and PI 低 The percentage was quantified and expressed as viability %. The retroinversopeptide had activity comparable to that of the standard peptide tested in Example 1 (Figures 2A-2B).

[0088] The inventors investigated the cytotoxicity of further retroinverso and mixed chirality BCL9 mimetic peptides in HL60 cells using the above assay. The results are shown in Table 9. [Table 11-1] [Table 11-2]

[0089] The subscripts D and L represent amino acid chirality. Substitutions and additions compared to the retroinverso wild-type BCL9 HD2 sequence (SEQ ID NO: 7) are shown in underlined bold. Cell membrane permeability and RGD-like regions are italicized. As expected, BCL-12 exhibited no cytotoxic activity due to the lack of a sequence for cell membrane permeability.

[0090] The lack of cytotoxic activity by BCL-134 indicates that a positively charged amino acid is required at least one of position 4 or position 8 of the HD2 domain compared to SEQ ID NO: 7. (Compare the activity of BCL-133 with that of BCL-134.)

[0091] The lack of cytotoxic activity by BCL-138 suggests the necessity of at least one of the two N-terminal amino acids in the HD2 domain being hydrophobic. (Compare the activity of BCL-91b with that of BCL-138.)

[0092] Example 3. Retroinverso BCL9 peptide exhibits antitumor activity in vivo. In this experiment, the inventors investigated the effect of peptide BCL-26 on tumor volume in an MCF7 subcutaneous tumor model. Briefly, 2 × 10⁶ peptides were suspended in Matrigel in a 1:1 ratio. 6 Individual MCF7 breast cancer cells were transplanted into the axilla of NU / J mice by subcutaneous injection. Peptide BCL-26 was administered by subcutaneous injection at a dose of 12.5 mg / kg three times a week for three weeks. The drug was administered approximately 25 mm on the second day after tumor inoculation. 3 The average starting tumor volume was used. Peptide BCL-26 significantly reduced tumor volume compared to the vehicle (Figure 3). A).

[0093] The inventors also investigated the effects of the BCL9 mimetic peptide on established tumors. MCF7 cells were transplanted into mice as described above. Peptide BCL-87 was administered by subcutaneous injection at a dose of 5 mg / kg three times a week for three weeks. The drug was administered approximately 470 mm on the 21st day after tumor inoculation. 3The average starting tumor volume was used. Peptide BCL-87 significantly reduced tumor volume compared to the vehicle (Figure 3B).

[0094] The inventors further investigated the effects of different concentrations of mimetic peptides in established tumors. MCF7 cells were transplanted into mice as described above. Peptides BCL-27 and BCL-87 were administered by subcutaneous injection at doses of 1 mg / kg or 5 mg / kg three times a week for three weeks. The drug was administered approximately 340 mm on day 14 after tumor inoculation. 3 The average starting tumor volume was used. Both peptides significantly reduced tumor volume compared to vehicle and peptide controls in BCL-134 (Figure 3C). References Alves ID,et al.Membrane interaction and perturbation mechanisms induced by two cationic cell penetrating peptides with distinct charge distribution.Biochim.Biophys.Acta 1780:948-959(2008). Bernal F, et al. Reactivation of the p53 Tumor Suppressor Pathway by a Stapled p53 Peptide. J. Am. Chem. Soc. 129:2456-2457 (2007). 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Wender PA, et al.The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters.Proc.Natl.Acad.Sci.USA97:13003-13008(2000). Zhan T, et al.Wnt signaling in cancer.Oncogene 36:1461-1473(2017). Zhao Y, et al.Chemical engineering of cell penetrating antibodies.J.Immunol.Methods 254:137-45(2001). Zou LL, et al.Cell-penetrating peptide-mediated therapeutic molecule delivery into the central nervous system.Curr.Neuropharmacol.11:197-208(2013). *** The present invention is further described in the following claims.

Claims

1. A BCL9-mimicking peptide comprising a modified BCL9 α-helical homology domain-2 (HD2) region, wherein the modified BCL9 HD2 region is a D-amino acid sequence consisting of a variant of the D-amino acid sequence FLMRQIDRLTQLS (SEQ ID NO: 7), and the variant is a modified BCL9 HD2 region consisting of a sequence selected from the following: i. WWLARQLARLAQLA (Sequence ID 12), ii. FLMRQLDRLTQLA (Sequence ID 9), iii. WWLARQLERLAQLA (Sequence ID 16), iv. 1-Nal-WLARQLARLRQLA (Sequence ID 17), v. 2-Nal-WLARQLARLAQLA (Sequence ID 115), vi. FWLARQLARLAQLA (Sequence ID 116), vii. WWLARQLARLRQLA (Sequence ID 117), viiii. WFLARQLARLAQLA (Sequence ID 118), ix. WLLARQLARLAQLA (Sequence ID 119), x. WWLERQLARLAQLA (Sequence ID 120), xi. WWLARQLARLQQLA (Sequence ID 122), xii. WWLARQLERLARLA (Sequence ID 123), xiiii. WWLARQLERLRRRLA (Sequence ID 124), xiv. WWLARQLARLKQLA (Sequence ID 125), xv. WWLARQLERLAKLA (Sequence ID 126), xvi. WWLVRQLARLAQLA (Sequence ID 127), and xvii.WWLAOQLAOLAQLA (Sequence ID 140) Here, the BCL9-mimicking peptide has the effect of promoting cytotoxicity in neoplastic cells. The aforementioned BCL9-mimicking peptide.

2. The BCL9-mimicking peptide according to claim 1, further comprising a cell membrane permeable region, wherein the BCL9-mimicking peptide is a cell membrane permeable peptide.

3. The cell membrane permeable region has an amino acid sequence selected from the group consisting of YGRKKRRQRRR (SEQ ID NO: 61) and VPTLK (SEQ ID NO: 32), or The cell membrane permeable region has a D-amino acid sequence selected from the group consisting of RRRQRRKKRGY (SEQ ID NO: 73), KLTPV (SEQ ID NO: 74), PSDGRG (SEQ ID NO: 75), and OLTPV (SEQ ID NO: 143). The BCL9-mimicking peptide according to claim 2.

4. The BCL9 mimetic peptide according to any one of claims 1 to 3, wherein the peptide comprises an N-terminal group selected from the group consisting of acetyl, naphthyl, octanoyl, phenyl, and isovaleryl.

5. The BCL9-mimicking peptide according to any one of claims 1 to 4, wherein the peptide comprises a C-terminal amide group.

6. A composition comprising the BCL9 mimetic peptide described in any one of claims 1 to 4.

7. The composition according to claim 6, which is a pharmaceutical composition.

8. A kit comprising a BCL9-mimicking peptide according to any one of claims 1 to 5 or a composition according to claim 6 or 7.

9. An in vitro method for promoting cytotoxicity in neoplastic cells, The neoplastic cells are brought into contact with the BCL9 mimetic peptide described in any one of claims 1 to 5 or the composition described in claim 6 or 7. The method, including the method described above.

10. An in vitro method for inhibiting the proliferation of neoplastic cells, The neoplastic cells are brought into contact with the BCL9 mimetic peptide described in any one of claims 1 to 5 or the composition described in claim 6 or claim 7. The method, including the method described above.

11. A BCL9-mimicking peptide according to any one of claims 1 to 5 or a composition according to claim 6 or 7, for use in promoting cytotoxicity in neoplastic cells.

12. A BCL9-mimicking peptide according to any one of claims 1 to 5 or a composition according to claim 6 or 7, for use in inhibiting the proliferation of neoplastic cells.

13. Use of the BCL9 mimetic peptide according to any one of claims 1 to 5 or the composition according to claim 6 or claim 7 for the manufacture of a pharmaceutical product for promoting cytotoxicity in neoplastic cells.

14. Use of the BCL9 mimetic peptide according to any one of claims 1 to 5 or the composition according to claim 6 or claim 7 for the manufacture of a pharmaceutical for inhibiting the proliferation of neoplastic cells.

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