Modified BCL9 mimetic peptides

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 to related applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 870,938, filed on July 5, 2019.

[0002] Sequence Listing This application contains a Sequence Listing submitted in ASCII format and hereby incorporated by reference in its entirety. The ASCII copy was created on July 6, 2020, named Sapience_004_WO1_SL.txt, and is 49,081 bytes in size.

Background Art

[0003] B - cell CLL / lymphoma 9 (BCL9) is a protein that functions 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 the 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 by using hydrocarbon - stapled BCL9 peptides to interfere with the BCL9 / β - catenin interaction, the transcription of Wnt target genes that regulate the proliferation, migration, invasion, and metastatic potential of tumor cells is suppressed (Takada et al., 2012, WO2017 / 062518).

Summary of the Invention

[0004] Some of the main aspects of the present invention are summarized below. Further aspects are described in the embodiments, examples, drawings, and claims sections of this disclosure. The descriptions in each section of this disclosure are intended to be interpreted in association with other sections. Furthermore, although the various embodiments described in each section of this disclosure can be combined in various ways, all such combinations are intended to be within the scope of the present invention.

[0005] The present invention provides a BCL9 mimetic peptide comprising a modified BCL9 α-helical homology domain-2 (HD2) region. In one embodiment, the present invention provides a BCL9 mimetic peptide comprising a modified BCL9 α-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 has the following modifications at one or more positions of SEQ ID NO: 1: (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; (x) adding W, 1-Nal or 2-Nal at position 25. The BCL9 mimetic peptide may further comprise the modification of substituting F24 with W, 1-Nal or 2-Nal and / or cleaving 1 to 15 consecutive amino acids starting from L1 of SEQ ID NO: 1. In one embodiment, the modified BCL9 HD2 region comprises 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, a retro-inverso BCL9 mimetic peptide containing D-amino acids with an amino acid sequence reversed as compared to the amino acid sequences disclosed herein is also included. A BCL9 mimetic peptide is also included.

[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 at one or more positions of SEQ ID NO: 7 as follows: (i) substituting F1 with L or W; (ii) substituting M3 with A, E, L or V; (iii) substituting R4 with O (ornithine); (iv) substituting I6 with L; (v) substituting D7 with A or E; (vi) substituting R8 with A; (vii) substituting T10 with A, K, Q or R; (viii) substituting Q11 with A, K or R; (ix) substituting S13 with A. In a particular embodiment, the BCL9 mimetic peptide further comprises W, F, R, 1-Nal or 2-Nal, either in the D-form or the L-form, at the N-terminus of the peptide.

[0007] In certain embodiments, the modified BCL9 HD2 region comprises a D - amino acid sequence selected from the group consisting of FLMRQIDRLTQLA (SEQ ID NO: 8), FLMRQLDRLTQLA (SEQ ID NO: 9), FLARQLARLAQLA (SEQ ID NO: 10), WLARQLARLAQLA (SEQ ID NO: 11), WWLARQLARLAQLA (SEQ ID NO: 12), FLMEQLRRLTELA (SEQ ID NO: 13), FLAEQLRRLAELA (SEQ ID NO: 14), WLAEQLRRLAELA (SEQ ID NO: 15), WWLARQLERLAQLA (SEQ ID NO: 16), 1 - Nal - WLARQLARLRQLA (SEQ ID NO: 17), FLLRQIDRLTQLA (SEQ ID NO: 18), FLLRQLDRLTQLA (SEQ ID NO: 19), FLLRQLERLTQLA (SEQ ID NO: 20), WWLLRQLARLAQLA (SEQ ID NO: 102), 2 - Nal - WLARQLARLAQLA (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 certain embodiments, the BCL9 mimetic peptide of the present invention comprises a modified BCL9 α - helical homology domain - 2 (HD2) region with mixed chirality. In certain embodiments, the modified BCL9 HD2 region is (i) F D R L [WLARQLARLAQLA] D (SEQ ID NO: 103), (ii) F D R L [WLVRQLARLAQLA] D (SEQ ID NO: 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 L -[WLVRQLARLAQLA] D comprising an amino acid sequence selected from the group consisting of (SEQ ID NO: 141), wherein the subscripts D and L represent the chirality of the amino acid.

[0009] In some embodiments, the BCL9 mimetic peptide comprises a cell membrane permeable region, and the BCL9 mimetic peptide is a cell membrane permeable peptide. In certain embodiments, 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).

[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

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 3C

Modes for Carrying Out the Invention

[0015] The practice of the present invention, unless otherwise indicated, will employ conventional techniques of pharmacy, pharmaceutics, protein chemistry, cell biology, cell culture, molecular biology, microbiology, recombinant DNA, and immunology within the skill of the art.

[0016] To more readily understand the present invention, some terms will first be defined. Further definitions are provided throughout this disclosure. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It has the same meaning as commonly understood.

[0017] Any headings provided herein are not generally intended to be limitations of the various aspects or embodiments of the invention that may be made by reference to this specification as a whole. Thus, the terms defined immediately below are more fully defined by reference to the entire specification.

[0018] All references cited in this disclosure are hereby incorporated by reference in their entirety. In addition, any manufacturer's instructions or catalogs for any products cited or mentioned in this specification are incorporated by reference. Any document incorporated by reference herein, or any teaching therein, may be used in the practice of the present invention. A document incorporated by reference herein does not admit that it is prior art.

[0019] I. Definitions The expressions or terms in this disclosure are for the purpose of explanation and not for limitation, such that they will be construed by those skilled in the art in light of the teachings and guidance of the terms or expressions in this specification.

[0020] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural meanings, unless the context clearly indicates otherwise. The terms "a" (or "an"), as well as the terms "one or more" and "at least one" may be used interchangeably.

[0021] Furthermore, "and / or" should be regarded as specifically disclosing each of the two specified features or components, regardless of the presence or absence of the other. Thus, the term "and / or" 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" used in the phrase "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 case where an embodiment is described using the word "comprising", other similar embodiments described using the words "consisting of" and / or "consisting essentially of" are included.

[0023] Units, prefixes, and symbols are expressed in their International System of Units (SI) approved forms. Numerical ranges include the numbers defining the range, and any individual value provided herein can serve as the endpoints of a range that includes other individual values provided herein. For example, a set of values, such as 1, 2, 3, 8, 9, and 10, is also a disclosure of ranges of numbers such as 1-10, 1-8, 3-9, etc. Similarly, the disclosed ranges are a disclosure of each individual value encompassed by the range. For example, the explicitly stated range of 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 a polymer of amino acids of any length. The polymer can be linear or branched, can include modified amino acids, and non-amino acids may interrupt. Unless otherwise indicated, 3-letter and 1-letter abbreviations as used in the art are used herein to represent amino acid residues, for example, as abbreviations for non-conventional or non-natural amino acids shown herein. Amino acids are L-amino acids, except when preceded by "D" or in lower case. Peptides are represented using a single block or string of amino acid abbreviations. Unless specifically indicated, peptides are shown with the N-terminus on the left, and the sequences are written from the N-terminus to the C-terminus.

[0025] Polypeptides, peptides, and proteins can include natural or synthetic modifications such as disulfide bonds, lactam cross-links, glycosylation, lipidation, acetylation, acylation, amidation, phosphorylation, or other manipulations or modifications such as conjugation with a labeling component or addition of a protecting group. Further, for example, polypeptides containing one or more analogs of amino acids (e.g., including amino-isobutyric acid (Aib), unnatural amino acids such as naphthylalanine (Nal), etc.), as well as polypeptides containing or consisting of D-amino acids and other modifications known in the art are also included. The polypeptide can be in one or more salt forms. Preferred salt forms include acetate, chloride, or trifluoroacetate. In certain embodiments, the polypeptide can exist as a single chain, a covalently linked dimer, or a non-covalently associated chain. The polypeptide may also be in a cyclic form. Cyclic polypeptides can be prepared, for example, by cross-linking a free amino acid and a free carboxyl group. The formation of the cyclic compound can be achieved by performing suitable protection and treating with a dehydrating agent as needed. The reaction from an open-chain (linear form) to a cyclic form can involve intramolecular cyclization. Cyclic polypeptides can also be prepared by other methods known in the art, such as using one or more lactam cross-links, hydrogen bond surrogates (Patgiri et al. 2008), hydrocarbon staples (Schafmeister et al. 2000), triazole staples (Le Chevalier Isaad et al. 2009), or disulfide cross-links (Wang et al. 2006). The spacing separating the cross-links or staples can be, for example, 3, 4, 7, or 8 amino acids.

[0026] The term "variant" refers to a polypeptide having one or more amino acid substitutions, deletions and / or insertions as compared to a reference sequence. Deletions and insertions can be internal and / or at one or more termini. Substitutions can include replacement of one or more amino acids by similar or identical amino acid(s) or non-similar amino acid(s). For example, some variants include 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 variants include non-conservative substitutions that change the charge or polarity of the amino acid. Substitutions can be by either L- or D-amino acids.

[0027] A "retro-inverso" polypeptide has an amino acid sequence that is reversed as compared to a native L-amino acid sequence and is composed of D-amino acids (where the α-center chirality of the amino acid subunit is reversed), which helps to 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 replacing one or more amino acids with another biologically similar residue. Examples include substitutions of amino acid residues having similar properties, such as substitutions of small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids and aromatic amino acids. For further information regarding substitutions that are phenotypically silent in peptides and proteins, see, for example, Bowie et.al., Science 247:1306-1310 (1990). In the following table, conservative substitutions of amino acids are classified by physicochemical properties, where I is neutral and / or hydrophilic, II is acidic and amide, III is basic, IV is hydrophobic, and V is aromatic bulky amino acid. [Table 1]

[0029] In the following table, conservative substitutions of amino acids are classified by physicochemical properties, where VI is neutral or hydrophobic, VII is acidic, VIII is basic, IX is polar, and X is aromatic. [Table 2]

[0030] Methods for identifying conservative 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. U.S.A. 94:412-417 (1997)).

[0031] The terms "identical" or "identity" percent with respect to two or more nucleic acids or polypeptides means that two or more sequences or subsequences are the same, or have a specified percentage of the same nucleotide or amino acid residues when compared and (optionally introducing gaps) aligned to obtain a maximum match without considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software that can be used to obtain an alignment of amino acid or nucleotide sequences are known in the art.

[0032] One such non-limiting example of a sequence alignment algorithm is described in Karlin et al., Proc. Natl. Acad. Sci., 87:2264-2268 (1990), which is It has been modified in Proc. Natl. 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 certain embodiments, gapped BLAST as described in Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997) can 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 certain embodiments, the percent identity of two nucleotide sequences is determined using the GAP program within 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 certain alternative embodiments, the percent identity of two amino acid sequences can be determined using the GAP program within 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, 5). Alternatively, in certain embodiments, the percent identity of a nucleotide or amino acid sequence is determined using the algorithm of Myers and Miller (CABIOS 4:11-17 (1989)). For example, the percent identity can be determined using the ALIGN program (version 2.0) and using the PAM120 residue table, a gap length penalty of 12 and a gap penalty of 4.One skilled in the art can determine appropriate parameters for maximum alignment by a specific alignment software. In certain embodiments, the default parameters of the alignment software are used. Other means for calculating identity include those 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" can 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. Polynucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerases. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. The above applies to any polynucleotide mentioned herein, including RNA and DNA.

[0034] An "isolated" molecule is in a form not found in nature and includes purified ones.

[0035] A "label" is a conjugate that binds directly or indirectly to a molecule to produce a "labeled" molecule. It is a detectable compound that can be modified. The label can be detectable itself (e.g., a radioisotope label or a fluorescent label), or indirectly, for example, by catalyzing a detectable chemical change of a substrate compound or composition (e.g., an enzyme label), or by other indirect detection means (e.g., biotinylation).

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

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

[0038] An "active agent" is a component intended to produce a biological activity. The active agent can be in association with one or more other components. An active agent that is a peptide may also be referred to as an "active peptide".

[0039] An "effective amount" of an active agent is an amount sufficient to effect a specifically indicated purpose.

[0040] The term "pharmaceutical composition" refers to a formulation that is in a form that enables the biological activity of the active ingredient to be effective and that contains no additional ingredients having unacceptable toxicity for the subject to which the composition is to be administered. Such compositions can be sterile and can contain a pharmaceutically acceptable carrier, such as physiological saline. Suitable pharmaceutical compositions can contain one or more of a buffer (e.g., an acetate, phosphate or citrate buffer), a surfactant (e.g., polysorbate), a stabilizer (e.g., a polyol or an amino acid), a preservative (e.g., sodium benzoate), and / or other conventional solubilizing or dispersing agents.

[0041] The terms "inhibit", "block" and "suppress" are used interchangeably and mean any statistically significant decrease in occurrence or activity, which includes completely blocking the occurrence or activity. For example, "inhibit" can mean a decrease in activity or occurrence of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. An "inhibitor" is a molecule, factor or substance that produces a statistically significant decrease in the occurrence or activity of a process, pathway or molecule.

[0042] "Neoplastic cells" or "neoplasms" typically result from some form of mutation / transformation and grow abnormally compared to normal cells or tissues of the same type. Neoplasms include morphological irregularities and pathological growth. Neoplastic cells can be benign or malignant. Malignant neoplasms, i.e., cancers, are distinguished from benign ones in that they show loss of cell differentiation and orientation and have the characteristics of invasion and metastasis.

[0043] II. BCL9 Mimetic Peptides and Compositions BCL9 Mimetic Peptides 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) 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 variant of the native BCL9 HD2 domain with a lactam bridge between residues 14 and 18. Previous studies have shown that an analog of ST-BC1 with a hydrocarbon bridge between residues 14 and 18 inhibits Wnt transcriptional activity in human colon cancer cells and exhibits antitumor activity in a mouse model (Takada et al. 2012). The inventors have discovered that a non-conservative linear variant of ST-BC1 induces cell death in neoplastic cells and reduces tumor volume in an animal model. The discovery that the BCL9-derived peptides of the present invention retain the ability to specifically target and kill neoplastic cells while having multiple non-conservative amino acid substitutions relative to the wild-type BCL9 HD2 region was not predictable prior to the present invention. Furthermore, the retro-inverso variant not only had activity but also had comparable activity compared to ST-BC1 and the linear "L" variant, which was also not predictable.

[0045] The present invention provides a BCL9 mimetic peptide having a modified BCL9 HD2 region and optionally a cell-permeable region. The BCL9 peptide of the present invention is a "mimetic" in the sense that it can interfere with or inhibit wild-type BCL9 activity in the 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 natural 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, such as axin, 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 assays 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 and 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 to 23 of SEQ ID NO: 1 and including at least one addition, deletion, or substitution compared to SEQ ID NO: 1. The modified BCL9 HD2 region can include, for example, the amino acid sequence shown in Table 1. The native 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 can be in a retro-inverso form and can have, for example, the D-amino acid sequence X1LX2X3QLX4X5LX6X7LA (SEQ ID NO: 142), where each amino acid at positions 1 to 13 is independently selected from those shown in Table 2. [Table 4]

[0048] In the D-amino acid HD2 domain sequence 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 can 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 can 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, when position -1 is R, position 1 is F or W, and / or position -2 is F, 1-Nal, 2-Nal, or W.

[0049] Specific examples of the retro-inverso BCL9 HD2 region are shown in Table 3. The retro-inverso sequence (SEQ ID NO: 7) of a part of the wild-type BCL9 HD2 region is shown as a reference point. The substitutions in SEQ ID NO: 7 are shown in underlined bold. [Table 5]

[0050] The modified BCL9 HD2 region can contain additional D-amino acids corresponding to the complete retro-inverso sequence of SEQ ID NO: 1. For example, the modified BCL9 HD2 region can have the D-amino acid sequence [Chemical formula] and can contain, where the substitutions and additions to the retro-inverso wild-type BCL9 HD2 sequence are shown in underlined bold.

[0051] The modified BCL9 HD2 region may contain amino acids with mixed chirality, such that one or more amino acids in the peptide are in the L form and one or more amino acids are in the D form. For example, an L-peptide may contain one or more D-amino acids. Similarly, a retro-inverso D-peptide may contain one or more L-amino acids. In certain embodiments, the BCL9 HD2 region is

Chemical formula

[0052] Variants of these sequences are also included within the scope of the present invention. The BCL9 mimetic peptides of the present invention may have an HD2 region with 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 to the sequences disclosed herein.

[0053] In embodiments where the BCL9 mimetic peptide comprises another active peptide, such as a cell membrane permeable region or an RGD-like sequence, the active peptide is operably linked to the modified BCL9 HD2 region. In some embodiments, the active peptide is covalently linked to the modified BCL9 HD2 region, for example, by 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 alkyl, substituted cycloalkyl, polyethylene glycol, and derivatives thereof. The linker is cleaved after the peptide is delivered into the cell It is possible. What has the active peptide directly linked to the modified BCL9 HD2 region by an amide bond may be called a "fusion body". The fusion body 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 a residue side chain. 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 include 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. Native BCL9 Substitutions to the native BCL9 HD2 sequence are shown in underlined bold. [Table 6]

[0055] Also included are retro-inverso forms of the BCL9 mimetic peptides. Exemplary embodiments of cell membrane permeable retro-inverso BCL9 mimetic peptides are shown in Table 5. [Table 7]

[0056] The cell membrane permeable and RGD-like regions are italicized. Substitutions and additions to the retro-inverso 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 different active peptides, such as different cell membrane permeable regions, and peptides containing the BCL9-HD2 region shown in Table 5 without an active peptide.

[0057] The BCL9 mimetic peptides of the present invention may contain amino acids with mixed chirality, where one or more amino acids in the peptide are in the L-form and one or more amino acids are in the D-form. Non-limiting examples of BCL9 mimetic peptides with mixed chirality are shown in Table 6. [Table 8]

[0058] The subscripts D and L represent the chirality of the amino acids. The cell membrane permeability region is in italics. Substitutions and additions to the retro-inverso wild-type BCL9 HD2 sequence (SEQ ID NO: 7) are shown in underlined bold. The present invention includes peptides containing the BCL9 HD2 region shown in Table 6 and different active peptides, such as different cell membrane permeability regions, and peptides containing the BCL9-HD2 region shown in Table 6 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 to the sequences disclosed herein.

[0060] The length of the BCL9 mimetic peptides 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 ranges having any of those lengths as endpoints, such as 13 - 35 amino acids.

[0061] The BCL9 mimetic peptide may have a modified N-terminus and / or a modified C-terminus. For example, the BCL9 mimetic peptide may optionally include 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 such as 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. Such linkers or spacers include, for example, aminohexanoic acid, beta-alanine, substituted alkyl, substituted cycloalkyl, and polyethylene glycol.

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

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

[0064] In one aspect, 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] Also provided herein are BCL9 mimetic peptides and compositions, and kits optionally including instructions for use, which are within the scope of the present invention. The kits may further contain at least one additional reagent and / or one or more additional active agents. The kits typically include a label indicating the intended use of the contents of the kit. In this regard, the term "label" includes any written or recorded matter provided on or with the kit, or otherwise associated with the kit.

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

[0067] Cell targeting The BCL9 mimetic peptides of the present invention can be introduced into target cells by methods known in the art. The introduction method selected can be determined, for example, according to the intended use.

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

[0069] The BCL9 mimetic peptides of the present invention can be delivered to cells via mechanisms that utilize cell receptors. Examples of such mechanisms include antibody-drug conjugates, chimeric antigen receptors, multiple antigen presentation (MAP) systems, and integrin targeting, RGD-like sequences. Examples of RGD-like sequences include GRGDS (SEQ ID NO: 28) and GRGDNP (SEQ ID NO: 29). The BCL9 mimetic peptides of the present invention can include one or more RGD-like sequences, e.g., 2, 3, 4, or 5 RGD-like sequences, linked as described herein or by any method known in the art. One or more RGD-like sequences (s) can be incorporated on the N-terminal side or the C-terminal side of the BCL9 HD2 region. Such RGD-like sequences can also be in a retro-inve rso form. One detailed example of a retro-inverso RGD-like sequence is PSDGRG (SEQ ID NO: 75). Alternatively, the BCL9 mimetic peptides can be encapsulated in vesicles, such as exosomes or liposomes, or micelles for delivery to cells. Another method for introducing the BCL9 mimetic peptides into cells is by cyclization, e.g., using hydrocarbon staples (Bernal et al. 2007, Bird et al. 2016), or other cyclization methods known in the art.

[0070] Certain BCL9 mimetic peptides of the present invention include a cell membrane permeable domain or a cell penetrating peptide (CPP). The terms "cell membrane permeable domain", "cell membrane permeable region" and "cell penetrating 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, the CPP is 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 includes ranges 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 cargos, such as polypeptides, polynucleotides, and nanoparticles, across the cell membrane and the BBB. The translocation can be by endocytosis via a translocation route or energy - independent (e.g., not by endocytosis). Numerous CPPs have been described and characterized in the literature (see, e.g., Handbook of Cell - Penetrating Peptides (2d ed. Ulo Langel ed., 2007), Herve et al. 2008, Heitz et al. 2009, Munyendo et al. 2012, Zou et al. 2013, Krautwald et al. 2016). A curated database of CPPs is maintained at crdd.osdd.net / raghava / cppsite (Gautam et al. 2012).

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

[0073] Non-limiting examples of CPPs suitable for use in the present invention include peptides derived from proteins, such as those derived from the Drosophila antennapedia transcription factor (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 transactivator of transcription (Tat) (Vives et al. 1997, Hallbrink et al. 2001), those derived from rabies virus glycoprotein (RVG) (Kumar et al. 2007), those derived from herpes simplex virus VP22 (Elliott e t al. 1997), those derived from antimicrobial protegrin 1 (SynB) (Rousselle et al. 2001), those derived from the rat insulin 1 gene enhancer protein (pIS1) (Kilk et al. 2001, Magzoub et al. 2001), those derived from mouse vascular endothelial cadherin (pVEC) (Elmquist et Those derived from, for example, calcitonin gene-related peptide (CGRP) (Kangawa et al. 2001), human calcitonin (hCT) (Schmidt et al. 1998), and fibroblast growth factor 4 (FGF4) (Jo et al. 2005) can be mentioned. Suitable CPPs for use in the present invention also include synthetic and chimeric peptides, for example, transportan (TP) and its derivatives (Pooga et al. 1998, Soomets et al. 2000), membrane translocation sequences (MTS) (Brodsky et al. 1998, Lindgren et al. 2000, Zhao et al. 2001), such as the MPS peptide (also known as a fusion sequence-based peptide or FBP) (Chaloin et al. 1998), sequence signal-based peptide (SBP) (Chaloin et al. 1997), model amphiphilic peptide (MAP) (Oehlke et al. 1998, Scheller et al. 1999, Hallbrink et al. 2001), translocation peptide 2 (TP2) (Cruz et al. 2013), MPG (Morris et al. 1997, Kwon et al. 2009), Pep-1 (Morris et al. 2001, Munoz-Morris et al. 2007), and polyarginine (for example, R7-R 12 (SEQ ID NO: 144)) (Mitchell et al. 2000, Wender et al. 2000, Futaki et al. 2001, Suzuki et al. 2002) can also be mentioned. Representative sequences are shown in Table 7 without limitation.

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 an inverse sequence and / or inverse chirality, such as those shown in Table 7 and / or known in the art. For example, retro-inverso forms of CPP (inverse sequence and inverse chirality) are suitable for use in the present invention. Examples of retro-inverso CPPs include those having the D-amino acid sequences KKWKMRRNQFWIKIQR (SEQ ID No. 71), KKWKMRRNQFWLKLQR (SEQ ID No. 72), RRRQRRKKRGY (SEQ ID No. 73), KLTPV (SEQ ID No. 74) or OLTPV (SEQ ID No. 143). Variants of these sequences having one or more amino acid additions, deletions and / or substitutions that retain the ability to cross cell membranes and / or the BBB are also suitable for use in the present invention. The BCL9 mimetic peptides of the present invention may comprise 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 to the exemplary sequences shown in Table 7. The effect of amino acid addition(s), deletion(s) and / or substitution(s) on the ability of CPP to mediate cell membrane permeation can be tested using methods known in the art.

[0075] III. Preparation Methods The BCL9 mimetic peptides of the present invention can be chemically synthesized using, for example, solid-phase peptide synthesis or solution-phase peptide synthesis, or can be expressed using recombinant methods. Synthesis or expression can be effected as fragments of the peptide, which can then be chemically or enzymatically ligated.

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

[0077] Once prepared, the nucleic acid encoding a particular BCL9 mimetic peptide can be inserted into an expression vector and operably linked to expression control sequences suitable for expression of the peptide in the desired host. To obtain high expression levels of the BCL9 mimetic peptide, the nucleic acid can be operably linked or associated with transcriptional and translational expression control sequences that function in the selected expression host.

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

[0079] Suitable host cells include prokaryotes, yeast, 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 cell lines obtained from mammals, examples of which include 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 translocation system can also be employed.

Examples

[0080] Embodiments of the present 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 can be made to both materials and methods without departing from the scope of the present disclosure.

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

Table 10

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

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

[0084] Cell viability was quantified by spectrophotometry using a Roche MTT cell proliferation assay kit according to the manufacturer's instructions. Briefly, the cells were washed with PBS and incubated for 4 hours at 37 °C in fresh MEM medium containing 10% FBS, 1% penicillin / streptomycin, 1% non-essential amino acids, and 10 μL of MTT reagent. 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 OD570nm relative to OD650nm. Absorbance is proportional to the number of live cells. The percentage of relative absorbance compared to the untreated control was quantified and expressed as % cell viability.

[0085] Rather than BCL-21 with the native BCL9 HD2 domain sequence, the modified BCL9 peptide of the present invention demonstrated equal or greater anti-proliferative activity compared to peptide ST-BC1 in MCF7 breast cancer cells (EC 50 value < 10 μM) (Figure 1). In a functional assay, EC 50 is the concentration that reduces the biological response by 50% of its maximum value. For the BLC9 peptide, EC 50 is measured as the concentration that reduces cell viability by 50% of its maximum value. EC 50 can be calculated by any number of means known in the art.

[0086] Example 2. Retro-inverso BCL9 peptide exhibits anti-proliferative activity in cancer cells in vitro The inventors prepared a retro-inverso peptide: D-amino acid sequence [Chemical formula] BCL-26 having [chemical formula], and D-amino acid sequence [Chemical formula] BCL-27 having [chemical formula]. The cell-permeable region derived from the Bax inhibitory peptide is shown in italics. In BCL-26, the sequence of the HD2 domain is shortened by only 11 amino acids and is substituted at two positions compared to the native sequence. In BCL-27, the sequence of the HD2 domain is shortened by only 11 amino acids and is substituted at six positions compared to the native sequence and contains an additional N-terminal tryptophan.

[0087] The inventors examined the cytotoxicity of the peptides BCL-26 and BCL-27 using an assay in HL60 promyelocytic leukemia cells. The density of HL60 PML suspension cells in 150 μL of RPMI + 1.5% fetal bovine serum (FBS) placed in a 96-well dish was 3.5×10 3Cells / wells were determined. Reconstituted BCL-26 or BCL-27 at a concentration of 10 mg / mL with 20 mM His, pH 7.5 was added to each well in a volume of 50 μL to obtain 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 show green fluorescence, dead cells show red and green fluorescence, and live cells show little or no fluorescence. Cells were selected for analysis based on forward scatter (FSC) versus side scatter (SSC) and analyzed by a BD Accuri C6 Plus flow cytometer (Ex = 488 nm, Em = 530 nm) using an FITC signal detector to detect Annexin V-FITC binding and PI staining was analyzed by a phycoerythrin emission signal detector. The percentages of 低 Annexin V 低 and PI were quantified and expressed as viability %. The retro-inverso peptide had activity comparable to that of the standard peptide tested in Example 1 (Figures 2A - 2B).

[0088] The inventors examined the cytotoxicity of further retro-inverso and mixed chirality BCL9 mimetic peptides using the above assay in HL60 cells. The results are shown in Table 9.

Table 11-1

Table 11-2

[0089] The subscript letters D and L represent the chirality of the amino acids. Substitutions and additions when compared to the retro-inverso wild-type BCL9 HD2 sequence (SEQ ID NO: 7) are shown in underlined bold. The cell membrane permeability and RGD-like regions are in italics. As expected, BCL-12 showed no cytotoxic activity as it lacks the sequence for cell membrane permeation.

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

[0091] The lack of cytotoxic activity by BCL-138 indicates the necessity that at least one of the two N-terminal amino acids of the HD2 domain is hydrophobic. (The activity of BCL-91b is to be compared with the activity of BCL-138.)

[0092] Example 3. Retro-inverso BCL9 peptide exhibits anti-tumor activity in vivo In this experiment, the inventors examined the effect of peptide BCL-26 on tumor volume in an MCF7 subcutaneous tumor model. Briefly, 2×10 6 MCF7 breast cancer cells suspended 1:1 in Matrigel were transplanted by subcutaneous injection into the axilla of NU / J mice. Peptide BCL-26 was administered by subcutaneous injection at a dose of 12.5 mg / kg three times a week for 3 weeks. The dosing started at an average starting tumor volume of about 25 mm 3 2 days after tumor inoculation. Peptide BCL-26 significantly decreased the tumor volume compared to the vehicle (Figure 3 A).

[0093] The inventors also examined the effect 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 3 weeks. The dosing started at an average tumor volume of about 470 mm 3It started with the average starting tumor volume. Peptide BCL-87 significantly reduced the tumor volume compared to the vehicle (Figure 3B).

[0094] The inventors further examined the effects of different concentrations of the 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 three times a week for 3 weeks at doses of 1 mg / kg or 5 mg / kg. The dosing started on the 14th day after tumor inoculation at an average starting tumor volume of approximately 340 mm 3 It started with the average starting tumor volume. Both peptides significantly reduced the tumor volume compared to the vehicle and the peptide control 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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U.S.A. 97:13003-13008(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 will be further described in the following claims.

Claims

**Claim 1** A BCL9 mimetic peptide comprising a modified BCL9 α - helical homology domain - 2 (HD2) region, wherein the modified BCL9 HD2 region is i. WWLARQLARLAQLA (SEQ ID NO: 12), ii. FLMRQIDRLTQLA (SEQ ID NO: 8), iii. FLMRQLDRLTQLA (SEQ ID NO: 9), iv. FLARQLARLAQLA (SEQ ID NO: 10), v. WLARQLARLAQLA (SEQ ID NO: 11), vi. FLMEQLRRLTELA (SEQ ID NO: 13), vii. FLAEQLRRLAELA (SEQ ID NO: 14), viii. WLAEQLRRLAELA (SEQ ID NO: 15), ix. WWLARQLERLAQLA (SEQ ID NO: 16), x. 1 - Nal - WLARQLARLRQLA (SEQ ID NO: 17), xi. FLLRQIDRLTQLA (SEQ ID NO: 18), xii. FLLRQLDRLTQLA (SEQ ID NO: 19), xiii. FLLRQLERLTQLA (SEQ ID NO: 20), xiv. WWLLRQLARLAQLA (SEQ ID NO: 102), xv. 2 - Nal - WLARQLARLAQLA (SEQ ID NO: 115), xvi. FWLAARQLARLAQLA (SEQ ID NO: 116), xvii. WWLARQLARLRQLA (SEQ ID NO: 117), xviii. WFLAARQLARLAQLA (SEQ ID NO: 118), xix. WLLARQLARLAQLA (SEQ ID NO: 119), xx. WWLERQLARLAQLA (SEQ ID NO: 120), xxi. WWLARQLARLQQLA (SEQ ID NO: 122), xxii. WWLARQLERLARLA (SEQ ID NO: 123), xxiii. WWLARQLERLRRLA (SEQ ID NO: 124), xxiv. WWLARQLARLKQLA (SEQ ID NO: 125), xxv. WWLARQLERLAKLA (SEQ ID NO: 126), xxvi. WWLVRQLARLAQLA (SEQ ID NO: 127), and xxvii. WWLAOQLAOLAQLA (SEQ ID NO: 140) The BCL9 mimetic peptide comprising a D - amino acid sequence selected from the group consisting of **Claim 2** A BCL9 mimetic peptide comprising a modified BCL9 α - helical homology domain - 2 (HD2) region, wherein the modified BCL9 HD2 region is i.F Ｄ R Ｌ [WLARQLARLAQLA] Ｄ (SEQ ID NO: 103), ii. F Ｄ R Ｌ [WLVRQLARLAQLA] Ｄ (SEQ ID NO: 104), iii. F Ｄ W Ｌ [WLVRQLARLALQLA] Ｄ (SEQ ID NO: 105), iv. F Ｄ W Ｌ [WLARQLARLAAALA] 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 -[WLARQLERLARLAR] 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 -[WLARQLARLARQLA] Ｄ (SEQ ID NO: 114), and xiii. W Ｌ -[WLVRQLARLAQLA] Ｄ (SEQ ID NO: 141) 〔wherein the subscripts D and L represent the chirality of the amino acid〕 The BCL9 mimetic peptide comprising an amino acid sequence selected from the group consisting of **Claim 3** 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 as follows at one or more positions of SEQ ID NO: 7: i. Replace F1 with L or W; ii. Replace M3 with A, E, L or V; iii. Replace R4 with O; iv. Replace I6 with L; v. Replace D7 with A or E; vi. Replace R8 with A; vii. Replace T10 with A, K, Q or R; viii. Replace Q11 with A, K or R; ix. Replace S13 with A, such that the BCL9 mimetic peptide has been modified.

4. The BCL9 mimetic peptide according to claim 3, further comprising W, F, R, 1-Nal or 2-Nal at the N-terminus of the peptide, which is either in the D-form or the L-form.

5. A BCL9 mimetic peptide comprising a modified BCL9 α-helical homology domain-2 (HD2) region, wherein the modified BCL9 HD2 region comprises a variant of the amino acid sequence LSQEQLERERSLQTLRDIQRMLF (SEQ ID NO: 1), and the variant is as follows at one or more positions of SEQ ID NO: 1: i. Replace E7 with R; ii. Replace R11 with E; iii. Replace S12 with A; iv. Replace Q14 with A or E; v. Replace T15 with A; vi. Replace D18 with A or R; vii. Replace I19 with L; viii. Replace R21 with E; ix. Replace M22 with A or L; x. Add W, 1-Nal or 2-Nal at position 25, such that the BCL9 mimetic peptide has been modified.

6. The BCL9 mimetic peptide according to claim 5, wherein F24 is replaced with W, 1-Nal or 2-Nal.

7. The BCL9 mimetic peptide according to claim 5 or claim 6, wherein the 1-15 consecutive amino acids starting from L1 of SEQ ID NO: 1 are cleaved.

8. The BCL9 mimetic peptide according to any one of claims 5 to 7, wherein the peptide comprises D-amino acids in an amino acid sequence that is reversed compared to the amino acid sequence described in any preceding claim.

9. A BCL9 mimetic peptide comprising a modified BCL9 α-helical homology domain-2 (HD2) region, wherein the modified BCL9 HD2 region is i. LSQEQLERERSLATLRAIQRMLF (SEQ ID NO: 3), ii. LSQEQLRHERESLETLRRIQEMLF (SEQ ID NO: 4), iii. LSQEQLERERALLRALRAIQRALF (SEQ ID NO: 5), and iv. ALRALRAIQRALF (SEQ ID NO: 6) The BCL9 mimetic peptide comprising an amino acid sequence selected from the group consisting of **Claim 10** The BCL9 mimetic peptide according to any preceding claim, further comprising a cell membrane permeability region, wherein the BCL9 mimetic peptide is a cell membrane permeable peptide. **Claim 11** The cell membrane permeability 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 permeability 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 cell membrane permeable BCL9 mimetic peptide according to claim 10. **Claim 12** The BCL9 mimetic peptide according to any preceding claim, wherein the peptide comprises an N-terminal group selected from the group consisting of acetyl, naphthyl, octanoyl, phenyl and isovaleryl. **Claim 13** The BCL9 mimetic peptide according to any preceding claim, wherein the peptide comprises a C-terminal amide group. **Claim 14** A composition comprising the BCL9 mimetic peptide according to any preceding claim. **Claim 15** The composition according to claim 14, which is a pharmaceutical composition. **Claim 16** A kit comprising the BCL9 mimetic peptide according to any one of claims 1 to 13 or the composition according to claim 14 or claim 15. **Claim 17** A nucleic acid molecule encoding the BCL9 mimetic peptide according to any one of claims 1 to 13. **Claim 18** A method for promoting cytotoxicity in neoplastic cells, comprising contacting the neoplastic cells with the BCL9 mimetic peptide according to any one of claims 1 to 13 or the composition according to claim 14 or claim 15 The method as described above. **Claim 19** A method for inhibiting the growth of neoplastic cells, comprising contacting the neoplastic cells with the BCL9 mimetic peptide according to any one of claims 1 to 13 or the composition according to claim 14 or claim 15 The method comprising the above.

20. The BCL9 mimetic peptide according to any one of claims 1 to 13 or the composition according to claim 14 or claim 15 for use in promoting cytotoxicity in neoplastic cells.

21. The BCL9 mimetic peptide according to any one of claims 1 to 13 or the composition according to claim 14 or claim 15 for use in inhibiting the growth of neoplastic cells.

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