Modified bcl9 mimetic peptides

Abstract

The present invention provides a BCL9 mimic peptide having a modified α-helical homologous domain-2 (HD2) region and optionally, a cell-penetrating region, a composition comprising the BCL9 mimic peptide, and a method for inhibiting the proliferation of neoplastic cells and / or promoting cytotoxicity using the BCL9 mimic peptide.

Description

Technology Field

[0001] 관련 출원들에 대한 교차-참조

[0002] This application claims U.S. provisional patent application No. 62 / 870,938 filed on July 5, 2019.

[0003] 서열 목록

[0004] The present application includes a list of sequences submitted electronically in ASCII format, the full text of which is incorporated into the references of this specification. The aforementioned ASCII copy was created on July 6, 2020, the filename is Sapience_004_WO1_SL.txt, and the size is 49,081 bytes.

[0005] 기술분야

[0006] The present invention relates to BCL9 mimic peptides having a modified α-helical homologous domain-2 (HD2) region and optionally, a cell-penetrating region, compositions comprising these BCL9 mimic peptides, and methods for inhibiting proliferation and / or promoting cytotoxicity of neoplastic cells using these BCL9 mimic peptides. Background Technology

[0007] 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 the normal cells from which these tumors originate (Zhan et al. (2017). BCL9 interacts with β-catenin through its α-helical homology domain-2 (HD2). Previous studies have shown that disruption of the BCL9 / β-catenin interaction using hydrocarbon-stapled BCL9 peptides inhibits the transcription of Wnt target genes that regulate tumor cell proliferation, migration, invasion, and metastatic potential (Takada et al., 2012; WO 2017 / 062518). The problem to be solved

[0008] Some of the key aspects of the present invention are summarized below. Additional aspects are described in the detailed description, embodiments, drawings, and claims of the present invention. Descriptions in each section of this specification are intended to be read in conjunction with other sections. Furthermore, the various embodiments described in each section of this disclosure may be combined in various other ways, and all such combinations are included within the scope of the present invention.

[0009] The present invention provides a BCL9 mimic peptide comprising a modified BCL9 α-helical homology domain-2 (HD2) region. In one embodiment, the present invention provides a BCL9 mimic 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 (sequence identification number: 1), wherein the variant is modified as follows at one or more positions of sequence identification number: 1: (i) E7 is substituted with R; (ii) R11 is substituted with E; (iii) S12 is substituted with A; (iv) Q14 is substituted with A or E; (v) T15 is substituted with A; (vi) D18 is substituted with A or R; (vii) I19 is substituted with L; (viii) R21 is substituted with E; (ix) M22 is substituted with A or L; (x) W, 1-Nal, or 2-Nal is added at position 25. The BCL9 mimic peptide may further include modifications, wherein F24 is substituted with W, 1-Nal, or 2-Nal and / or wherein 1 to 15 consecutive amino acids of sequence identification number: 1 are truncated starting from L1. In one embodiment, the modified BCL9 HD2 region comprises an amino acid sequence selected from the group consisting of: LSQEQLEHRERSLATLRAIQRMLF (sequence identification number: 3); LSQEQLRHREESLETLRRIQEMLF (sequence identification number: 4); LSQEQLEHRERALQALRAIQRALF (sequence identification number: 5); and ALQALRAIQRALF (sequence identification number: 6). A retro-inverso BCL9 mimic peptide containing a D-amino acid in an amino acid sequence reversed compared to the amino acid sequence described herein is also incorporated.

[0010] One embodiment of the present invention is a BCL9 mimic 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 (sequence identification number: 7), wherein the variant is modified as follows at one or more positions of sequence identification number: 7: (i) F1 is substituted with L or W; (ii) M3 is substituted with A, E, L, or V; (iii) R4 is substituted with O (ornithine); (iv) I6 is substituted with L; (v) D7 is substituted with A or E; (vi) R8 is substituted with A; (vii) T10 is substituted with A, K, Q, or R; (viii) Q11 is substituted with A, K, or R; and (ix) S13 is substituted with A. In a specific embodiment, the BCL9 mimic peptide further comprises a D-form or L-form W, F, R, 1-Nal, or 2-Nal at the N-terminus of the peptide.

[0011] In certain embodiments, the modified BCL9 HD2 region comprises a D-amino acid sequence selected from the group consisting of: FLMRQIDRLTQLA (Sequence Identification No.: 8); FLMRQLDRLTQLA (Sequence Identification No.: 9); FLARQLARLAQLA (Sequence Identification No.: 10); WLARQLARLAQLA (Sequence Identification No.: 11); WWLARQLARLAQLA (Sequence Identification No.: 12); FLMEQLRRLTELA (Sequence Identification No.: 13); FLAEQLRRLAELA (Sequence Identification No.: 14); WLAEQLRRLAELA (Sequence Identification No.: 15); WWLARQLERLAQLA (Sequence Identification No.: 16); 1-Nal-WLARQLARLRQLA (Sequence Identification No.: 17); FLLRQIDRLTQLA (Sequence Identification No.: 18); FLLRQLDRLTQLA (Sequence ID: 19); FLLRQLERLTQLA (Sequence ID: 20); WWLLRQLARLAQLA (Sequence ID: 102); 2-Nal-WLARQLARLAQLA (Sequence ID: 115); FWLARQLARLAQLA (Sequence ID: 116); WWLARQLARLRQLA (Sequence ID: 117); WFLARQLARLAQLA (Sequence ID: 118); WLLARQLARLAQLA (Sequence ID: 119); WWLERQLARLAQLA (Sequence ID: 120); WWLARQLARLQQLA (Sequence ID: 122); WWLARQLERLARLA (Sequence ID: 123); WWLARQLERLRRLA (Sequence ID: 124); WWLARQLARLKQLA (sequence identification number: 125); WWLARQLERLAKLA (sequence identification number: 126); WWLVRQLARLAQLA (sequence identification number: 127); and WWLAOQLAOLAQLA (sequence identification number: 140).

[0012] In certain embodiments, the BCL9 mimic peptide of the present invention comprises a modified BCL9 α-helical homology domain-2 (HD2) region of mixed chirality. In certain embodiments, said modified BCL9 HD2 region comprises an amino acid sequence selected from the group consisting of: (i) F D R L [WLARQLARLAQLA] D (Sequence identification number: 103); (ii) F D R L [WLVRQLARLAQLA] D (Sequence identification number: 104); (iii) F D W L [WLVRQLARLAQLA] D (Sequence identification number: 105); (iv) F D W L [WLARQLARLAALA] D (Sequence identification number: 106); (v) F D W L [WLARQLAALAQLA] D (Sequence identification number: 107); (vi) W L -[WLARQLARLAQLA] D (Sequence identification number: 108); (vii) W L -[WLARQLARLRQLA] D (Sequence identification number: 109); (viii) W L -[WLARQLERLRRLA] D (Sequence identification number: 110); (ix) W L -[WLARQLERLARLA] D (Sequence identification number: 111); (x) F L -[WLARQLARLAQLA] D (Sequence identification number: 112); (xi) R L -[WLARQLARLAQLA] D (Sequence identification number: 113); (xii) F D -W L -[WLARQLARLAQLA] D(Sequence identification number: 114); and W L -[WLVRQLARLAQLA] D (Sequence identification number: 141); where subscripts D and L indicate the chirality of the amino acids.

[0013] In some embodiments, the BCL9 mimic peptide comprises a cell-penetrating region, wherein the BCL9 mimic peptide is a cell-penetrating peptide. In certain embodiments, the cell-penetrating region has an amino acid sequence selected from the group consisting of YGRKKRRQRRR (sequence identification number: 61) and VPTLK (sequence identification number: 32), or the cell-penetrating region has a D-amino acid sequence selected from the group consisting of RRRQRRKKRGY (sequence identification number: 73), KLTPV (sequence identification number: 74), PSDGRG (sequence identification number: 75), and OLTPV (sequence identification number: 143).

[0014] In some embodiments, the BCL9 mimic peptide comprises an N-terminal group selected from the group consisting of acetyl, naphthyl, octanoyl, phenyl, and isovaleryl, and / or the BCL9 mimic peptide comprises a C-terminal amide group.

[0015] In one aspect, the BCL9 mimic peptide of the present invention is intended for use in inhibiting the proliferation of neoplastic cells and / or promoting cytotoxicity.

[0016] Further aspects of the present invention provide a composition comprising the BCL9 mimic peptide of the present invention, e.g., a pharmaceutical composition; a kit comprising the BCL9 mimic peptide of the present invention; and a nucleic acid molecule encoding the BCL9 mimic peptide of the present invention.

[0017] The present invention further provides a method for inhibiting the proliferation of neoplastic cells and / or promoting cytotoxicity, the method comprising contacting the neoplastic cells with the BCL9 mimic peptide of the present invention. means of solving the problem

[0018] Unless otherwise indicated, the practice of the present invention will utilize conventional techniques in pharmaceuticals, formulation science, protein chemistry, cell biology, cell culture, molecular biology, microbiology, recombinant DNA, and immunology, all of which are within the scope of the art.

[0019] To facilitate a better understanding of the present invention, specific terms are defined first. Additional definitions are provided throughout this specification. Unless explicitly stated otherwise, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.

[0020] Any subject matter provided in this specification is not intended to limit the various aspects or embodiments of the invention, and the specification may be referenced in its entirety. Accordingly, the terms defined immediately below are all more fully defined by reference to the specification.

[0021] All documents cited herein are incorporated herein by reference in their entirety. Additionally, manufacturers' instructions or catalogs for products cited or mentioned herein are incorporated by reference. Any documents incorporated by reference in this specification or any teachings contained therein may be used in the practice of the present invention. Documents incorporated by reference in this text are not recognized as prior art.

[0022] I. definition

[0023] The phrases or terms in this specification are for illustrative purposes only and are not intended to be limiting, and should be interpreted by those skilled in the art in light of the teachings and guidelines.

[0024] As used in this specification and claims, it should be noted that the singular forms (“a,” “an,” and “the”) include plural concepts unless otherwise expressly stated. The terms singular indefinite article (“a,” “an”), “one or more,” and “at least one” are used interchangeably.

[0025] Furthermore, "and / or" shall be taken as a specific description of each specified feature or component. Thus, the term "and / or" used in a phrase, such as "A and / or B," is intended to imply "A and B," "A or B," "A" (sole), and "B" (sole). Similarly, the term "and / or" used in a phrase, such as "A, B, and / or C," is intended to imply 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 (sole); B (sole); and C (sole).

[0026] The language of "comprising" implies, where specific examples are described, similar specific examples otherwise described as "consisting of" and / or "consisting essentially of."

[0027] Units, prefixes, and symbols It is indicated in the International de Unites (SI) approval form. A numeric range includes a number defining the range, and any individual value provided herein may serve as an endpoint for a range containing other individual values ​​provided herein. For example, a series of values ​​such as 1, 2, 3, 8, 9, and 10 represents a numeric range of 1-10, 1-8, 3-9, etc. Likewise, a disclosed range is the beginning of each individual value included in that range. For example, the specified 5-10 range is also the beginning of 5, 6, 7, 8, 9, and 10.

[0028] The terms “polypeptide,” “peptide,” and “protein” are interchangeable herein and refer to a polymer of amino acids of any length. Such polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. Except as otherwise indicated, such as in the case of abbreviations for unconventional or non-natural amino acids presented herein, three-letter and one-letter abbreviations used in the art are used herein to denote amino acid residues. Amino acids are L-amino acids except those preceded by “D” or lowercase letters. Groups or strings of amino acid abbreviations are used to denote peptides. Unless specifically indicated otherwise, peptides are indicated with the left end as the N-terminus, and the sequence is described from the N-terminus toward the C-terminus.

[0029] Polypeptides, peptides, and proteins also include those modified naturally or synthetically, and may include other operations or modifications such as disulfide bonds, lactam bridges, glycosylation, lipidation, acetylation, acylation, amidation, phosphorylation, or conjugation with labeling components, or the addition of protecting groups. For example, polypeptides containing or composed of one or more analogs of amino acids (e.g., amino-isobutyric acid (Aib), non-natural amino acids such as naphthylalanine (Nal), etc.), and polypeptides containing or composed of D-amino acids, as well as polypeptides containing other modifications known in the art, are also included. The polypeptide may be in one or multiple salt forms. Preferred salt forms include acetate, chloride, or trifluoroacetate. In certain embodiments, the polypeptide may occur as a single chain, a covalent dimer, or a non-covalently bonded chain. Polypeptides may also be in a cyclic form. Cyclic polypeptides can be formed, for example, by the bridge linkage of free amino and free carboxyl groups. The formation of cyclic compounds can be achieved by treatment with a dehydrating agent and, if necessary, by providing a suitable protecting group. The reaction of an open chain (linear form) into a cyclic form may involve intramolecular cyclization. Cyclic polypeptides can be formed by other methods known in the art, for example, one or more lactam bridges, hydrogen bonding surrogates (Patgiri et al. 2008), Hydrocarbon Staple (Schafmeister) et al. 2000), Triazole Staple (Le Chevalier Isaad et al. 2009), or disulfide bridge (Wang et al. It can also be made by (2006). The bridges or staples can be spatially separated by lengths of, for example, 3, 4, 7, or 8 amino acids.

[0030] "Variant" refers to a polypeptide having one or more amino acid substitutions, deletions, and / or insertions when compared to a reference sequence. Deletions and insertions may be internal or and / or at one or more ends. Substitutions may include the replacement of one or more amino acids with similar or homologous amino acid(s) or dissimilar 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 to no effect on the overall net charge, polarity, or hydrophobicity of the protein. Some variants include non-conservative substitutions that alter the charge or polarity of the amino acid. Substitutions may be made in the L-form or D-form of the amino acid.

[0031] The "retro inverso" polypeptide has an inverted amino acid sequence compared to the original L-amino acid sequence and consists of D-amino acids (which invert the α-center chirality of the corresponding amino acid subunit) that help maintain a side chain topology similar to that of the original L-amino acid peptide.

[0032] As used herein, the term "conservative substitution" refers to the replacement of one or more amino acids with other biologically similar residues. Examples include the substitution of amino acid residues with similar characteristics, e.g., small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids, and aromatic amino acids. For additional information regarding phenotypic silenced substitutions in peptides and proteins, see, for example, Bowie et al. , ScienceSee 247:1306-1310 (1990). In the table below, conservative substitutions of amino acids are grouped by physicochemical properties; I: neutral and / or hydrophilic, II: acidic and amide, III: basic, IV: hydrophobic, V: aromatic, bulky amino acids.

[0033] Table I

[0034]

[0035] In the table below, conservative substitutions of amino acids are grouped by physicochemical properties; VI: Neutral or hydrophobic, VII: Acidic, VIII: Basic, IX: Polar, X: Aromatic.

[0036] Table II

[0037]

[0038] Methods for identifying conservative nucleotide and amino acid substitutions that do not affect protein function are well known in the art (e.g., 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)).

[0039] In the content of two or more nucleic acids or polypeptides, the term “identical” or “identical” percentage refers to two or more sequences or quasi-sequences having identical or a specific proportion of identical nucleotide or amino acid residues when compared and aligned for maximum correspondence (introducing gaps if necessary), without considering conservative amino acid substitutions as part of sequence identity. The identity percentage may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art.

[0040] One such non-restrictive example of a sequence alignment algorithm is Karlin et al. , Proc. Natl. Acad. Sci., Algorithm described in 87:2264-2268 (1990), Karlin et al. , Proc. Natl. Acad. Sci., Modified from 90:5873-5877 (1993), and integrated into NBLAST and XBLAST programs (Altschul et al. , Nucleic Acids Res., 25:3389-3402 (1991)) is an algorithm. In certain embodiments, Gapped BLAST is Altschul et al. , Nucleic Acids Res. 25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul et al. , Methods in Enzymology,As described in 266:460-480 (1996)), ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or Megalign (DNASTAR) is additionally available software that can be used to align sequences. In certain embodiments, the percentage of identity between two nucleotides 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 certain alternative embodiments, the Needleman and Wunsch algorithm is incorporated (J. Mol. Biol. (48):444-453 (1970)) The percentage of identity between two amino acid sequences can be determined using the GAP program of the GCG software package (e.g., using a BLOSUM 62 matrix or a 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 certain embodiments, the percentage of identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4:11-17 (1989)). For example, the percentage of identity can be determined using the ALIGN program (version 2.0) and PAM120 with a residue table, a gap length penalty of 12, and a gap penalty of 4. Appropriate parameters for maximum alignment by specific alignment software can be determined by those skilled in the art. In certain embodiments, default parameters of the alignment software are used. Other data that calculates identity is 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. The methods described in , 48:1073 (1988) are included.

[0041] As used herein, the term “polynucleotide” refers to a polymer of nucleotides of any length that may comprise one or more “nucleic acids,” “nucleic acid molecules,” or “nucleic acid sequences,” and includes DNA and RNA. This polynucleotide may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any material that may be incorporated into DNA or RNA polymerase. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and analogs thereof. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0042] An "isolated" molecule is a form of molecule not found in nature, including purified ones.

[0043] A "label" is a detectable compound that can be conjugated directly or indirectly to a molecule to produce a "labeled" molecule. The label is detectable on its own or ( 가령 , radioactive isotope label or fluorescent label), or, for example, can be detected indirectly by catalyzing a chemical change of a detectable substrate compound or composition (e.g., enzyme label), or by other indirect detection (e.g., biotinylation).

[0044] "Binding affinity" generally refers to the strength of the sum 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, two monomers forming a dimer, etc.). Unless otherwise noted, as used herein, "binding affinity" refers to the intrinsic binding affinity reflecting the 1:1 interaction between the components of a binding pair. The affinity of molecule X for its partner Y is given by the dissociation constant (K D It can be represented as ). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity partners generally bind to antigens slowly and tend to dissociate easily, whereas high-affinity partners generally bind faster and tend to maintain the binding for a longer period.

[0045] The affinity or binding strength (avidity) of the molecule for its binding partner may be determined by any suitable method known in the art, e.g., flow cytometry, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics ( 가령 It can be determined experimentally using KINEXA®, BIACORE™, or OCTET® assays. As well as direct binding assays, competitive binding assay formats are readily available. (e.g., Berzofsky et al. , “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, WE, ed., Raven Press: New York, NY (1984); Kuby, Immunology, see WH Freeman and Company: New York, NY (1992). The measured affinity of specific binding pair interactions may vary when measured under different conditions (e.g., salt concentration, pH, temperature). Therefore, affinity and other antigen-binding parameters (e.g., K D or Kd, K on , K off The measurement of ) is performed using a standardized solution of the binding partner and a standardized buffer known in the field.

[0046] "Activator" is a component intended to provide biological activity. The activator may be combined with one or more other components. An activator that is a peptide may also be referred to as an "active peptide."

[0047] The "effective amount" of the activator is an amount sufficient to perform the specifically stated purpose.

[0048] The term "pharmaceutical composition" refers to a formulation in a form intended to ensure that the biological activity of the active ingredient is effective, and does not include additional ingredients that impart unacceptable toxicity to the subject to whom the composition is administered. Such compositions may be sterilized and may include pharmaceutically acceptable carriers, such as physiological saline. Suitable pharmaceutical compositions include one or more buffers ( 가령 , acetate, phosphate, or citrate buffer), surfactant ( 가령, Polysorbate), stabilizer ( 가령, Polyols or amino acids), preservatives ( 가령, It may include sodium benzoate and / or other conventional solubilizers or dispersants.

[0049] The terms "inhibit," "block," and "inhibit" are used interchangeably and refer to a statistically significant reduction in occurrence or activity, including the complete blockade of occurrence or activity. For example, "inhibition" may refer to 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 statistically significantly reduces the occurrence or activity of a process, pathway, or molecule.

[0050] "Neoplastic tumor cells" or "neoplasms" have typically undergone some form of mutation or alteration resulting in abnormal growth compared to normal cells or tissues of the same form. Neoplasms involve pathological proliferation as well as morphological irregularities. Neoplastic tumor cells can be benign or malignant. Malignant neoplasms, or cancer, are distinguished from benign ones in that they exhibit a loss of cellular differentiation and orientation, and possess characteristics of invasion and metastasis.

[0051] II. BCL9 Mimic Peptide and Composition

[0052] BCL9

[0053] 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 domain, or "HD2 domain," of BCL9 is a 24-residue α-helical structure at amino acids 351–374 of BCL9 (sequence identification number: 1). The full amino acid sequence of wild-type human BCL9 is presented at NCBI accession number NP_004317.2.

[0054] Peptide ST-BC1 (sequence identification number: 2) is a cyclic variant having a lactam bridge between residues 14 and 18 in the intrinsic BCL9 HD2 domain. Previous studies have shown that ST-BC1 analogs with a hydrocarbon bridge between residues 14 and 18 inhibit Wnt transcriptional activity in human colon cancer cells and exhibit anti-tumor activity in mouse models (Takada et al. 2012). The inventors discovered that non-conservative and linear variants of ST-BC1 induce apoptosis in neoplastic cells and reduce tumor volume in animal models. The discovery that the BCL9-derived peptide of the present invention has multiple non-conservative amino acid substitutions for the wild-type BCL9 HD2 region, specifically targets neoplastic cells, and maintains the ability to kill them was not anticipated prior to the present invention. Furthermore, the retro-inverted variant not only exhibited activity but also had a level of activity comparable to that of ST-BC1 and linear "L" variants, which was also unpredictable.

[0055] The present invention provides a BCL9 mimetic peptide having a modified BCL9 HD2 region and optionally, a cell-penetrating region. The BCL9 peptides of the present invention are "mimetics," meaning that they can interfere with or inhibit wild-type BCL9 activity in cells to which they are introduced. More specifically, the BCL9 mimetic peptides 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 peptides can downregulate the expression of one or more members of the Wnt signaling pathway, e.g., axin, CD44, c-Myc, cyclin D1, LEF1, LGR5, sulfibin, and VEGF-A. In some embodiments, the BCL9 mimetic peptides can inhibit cell proliferation, angiogenesis, and / or cell migration. BCL9 activity is assessed using any assay known in the art, including the cell-apoptosis assay described herein (Kawamoto et al. It can be evaluated by WO 2009; WO 2017 / 062518).

[0056] The "modified BCL9 HD2 region" is a sequence derived from a wild-type BCL9 HD2 region, and this sequence has at least one addition, deletion, or substitution compared to the wild-type BCL9 HD2 sequence. The modified BCL9 HD2 region preferably corresponds to positions 16–23 of sequence identification number: 1 and includes at least one addition, deletion, or substitution compared to sequence identification number: 1. The modified BCL9 HD2 region is, for example, Page 1 It may include the amino acid sequence shown in ). The above-mentioned unique BCL9 HD2 sequence (sequence identification number: 1) is shown for reference only. In sequence identification number: 1, substitutions are shown in bold with an underline.

[0057] Page 1

[0058]

[0059] The above modified BCL9 HD2 region may be in a retro-inverted form and may, for example, possess the D-amino acid sequence X1LX2X3QLX4X5LX6X7LA (sequence identification number: 142), wherein each amino acid at positions 1-13 is Page 2 It is independently selected from those shown in ).

[0060] Page 2

[0061]

[0062] In the D-amino acid HD2 domain sequence shown in Table 2, only one of position 4 or 8 may be alanine, that is, if position 4 is A, position 8 is not A, and the reverse is also true. The BCL9 HD2 region may optionally include a D-amino acid or an L-amino acid selected from the group consisting of F, 1-Nal, 2-Nal, R, and W at position -1. The BCL9 HD2 region may optionally further include a D-amino acid or an 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, position 1 is F or W, and / or position -2 is F, 1-Nal, 2-Nal, or W.

[0063] Specific examples of retro-inverted BCL9 HD2 regions are Page 3 Indicated in Figure 2. A retro-inverted sequence of part of the wild-type BCL9 HD2 region (sequence identification number: 7) is shown for reference. Substitutions in sequence identification number: 7 are shown in bold with an underline.

[0064] Page 3

[0065]

[0066] *1N = 1-Naphthylalanine; 2N = 2-Naphthylalanine; O = Ornithine

[0067] The modified BCL9 HD2 region may include an additional D-amino acid corresponding to the entire retro-inverted sequence of Sequence Identification No. 1. For example, the modified BCL9 HD2 region is a D-amino acid sequence WW L A RQ THERE RL A QL A It may include RERHELQEQSL (sequence identification number: 121), wherein substitutions and additions to the retro-inverted wild-type BCL9 HD2 sequence are indicated by a bold underline.

[0068] The modified BCL9 HD2 region may contain mixed chiral amino acids, so that in the peptide, one or more amino acids are in the L-form and one or more amino acids are in the D-form. For example, the L-peptide may contain one or more D-amino acids. Similarly, the retro-inverted D-peptide may contain one or more L-amino acids. In certain embodiments, the BCL9 HD2 region has a sequence selected from the group consisting of the following: (i) F D R L [ W L A RQ THERE RL A QL A ] D (Sequence identification number: 103); (ii) F D R L [ W L V RQ THERE RL A QL A ] D (Sequence identification number: 104); (iii) F D W L [ W L V RQ THERE RL A QL A ] D (Sequence identification number: 105); (iv) F D W L [ W L A RQ THERE RL AA L A ] D (Sequence identification number: 106); (v) F D W L [ W L A RQ LAA L A QL A ] D (Sequence identification number: 107); (vi) W L -[ W L A RQ THERE RL A QL A ] D (Sequence identification number: 108); (vii) W L -[ W L A RQ THERE RL R QL A ] D (Sequence identification number: 109); (viii) W L -[ W L A RQ THE RL RR L A ] D (Sequence identification number: 110); (ix) W L -[ W L A RQ THE RL AR L A ] D (Sequence identification number: 111); (x) F L -[ W L A RQ THERE RL A QL A ] D (Sequence identification number: 112); (xi) R L -[ W L A RQ THERE RL A QL A ] D (Sequence identification number: 113); (xii) F D - W L -[ W L A RQ THERE RL A QL A ] D (Sequence identification number: 114); and (xiii) W L -[ W L V RQ THERE RL A QL A ] D (Sequence identification number: 141); where D and L subscripts indicate the chirality of the corresponding amino acids, and where substitutions and additions to the retro-inverted wild-type BCL9 HD2 sequence (sequence identification number: 7) are indicated by bold underlines.

[0069] Variants of these sequences are also within the scope of the present invention. The BCL9 mimic peptide of the present invention may have an HD2 region of 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 respect to the sequence disclosed herein.

[0070] In embodiments where the above BCL9 mimic peptide comprises another active peptide, such as a cell-penetrating 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, through 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 derivatives thereof. After transporting the peptide into the cell, the linker may be cleaved. The modified BCL9 HD2 region directly linked to the active peptide by an amide bond may be referred to as a "fusion." The fusion may contain an amino acid linker sequence between the active peptide and the modified BCL9 HD2 region, as discussed in relation to the above active peptide. The active peptide may be linked to the N-terminus or C-terminus of the modified BCL9 HD2 region, or linked via a residue side chain. The active peptide and the modified BCL9 HD2 region may have the same or opposite chirality.

[0071] The cell-penetrating BCL9 mimic peptide of the present invention may comprise any combination of the cell-penetrating and modified BCL9 HD2 regions described herein. Examples of such peptides are Page 4 It is shown in, but is not limited to. The cell-penetrating region is indicated in italics. Peptide BCL-21 contains the intrinsic BCL9 HD2 sequence, which is inactive in inhibiting cell proliferation. Substitutions for the intrinsic BCL9 HD2 sequence are shown in bold with an underline.

[0072]

[0073] The retro-inverted form of the BCL9 mimic peptide of the present invention is also included. Exemplary embodiments of the cell-penetrating retro-inverted BCL9 mimic peptide are Page 5 It is represented in.

[0074] Page 5

[0075]

[0076] The cell-penetrating and RGD-like regions are indicated in italics. Substitutions and additions to the retro-inverted wild-type BCL9 HD2 sequence (sequence identification number: 7) are indicated by a bold underline. The present invention also includes active peptides different from the BCL9 HD2 regions shown in Table 5, such as peptides containing different cell-penetrating regions, and peptides containing the BCL9-HD2 regions shown in Table 5 without active peptides.

[0077] The BCL9 mimic peptide of the present invention may comprise mixed chiral amino acids, 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. Non-limiting examples of BCL9 mimic peptides having mixed chirality are Page 6 It is represented in.

[0078] Page 6

[0079]

[0080] The subscripts D and L indicate the chirality of the corresponding amino acids. The cell-penetrating region is indicated in italics. Substitutions and additions to the retro-inverted wild-type BCL9 HD2 sequence (sequence identification number: 7) are indicated by a bold underline. The present invention also includes active peptides different from the BCL9 HD2 regions shown in Table 6, such as peptides containing different cell-penetrating regions, and peptides containing the BCL9-HD2 region shown in Table 6 without active peptides.

[0081] The BCL9 mimic peptide of the present invention comprises a peptide 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 respect to the sequence disclosed herein.

[0082] The BCL9 mimic peptide of the present invention has any range of these lengths as an endpoint, for example, a range of 13 to 35 amino acids, 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.

[0083] The above BCL9 mimic peptide may have a modified N-terminus and / or a modified C-terminus. For example, the BCL9 mimic 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 Aliphatic or aromatic hydrocarbons, naphthyl groups, phenyl groups, octanoyl groups, and valery groups including isovaleryl groups are incorporated. In some embodiments, the BCL9 mimic peptide comprises 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.

[0084] The BCL9 mimic peptide of the present invention may optionally be cyclic. For example, the BCL9 mimic peptide of the present invention may contain one or more lactam bridges. The lactam bridges are preferably formed between side chains spaced apart by three, four, seven, or eight amino acid lengths (e.g., BxxB, BxxxB, BxxxxxxB, BxxxxxxxB), but are not necessarily required. The lactam bridges may be formed, for example, between the side chains of Asp or Glu and Lys or Orn. ​​Amino acid substitutions may be made at the lactam bridge sites to promote linkage.

[0085] The BCL9 mimic peptide of the present invention may optionally include one or more epitopes and / or affinity tags, for example, for purification or detection. Non-limiting examples of such tags include FLAG, HA, His, Myc, GST, and similar ones. The BCL9 mimic peptide of the present invention may optionally include one or more labels.

[0086] In certain aspects, the present invention relates to a composition, 가령 , a pharmaceutical composition comprising a BCL9 mimic peptide of the present invention and optionally further comprising one or more carriers, diluents, excipients, or other additives.

[0087] A kit comprising the BCL9 mimic peptide and composition provided herein, optionally including instructions for use, is also within the scope of the present invention. The kit may further comprise at least one additional reagent and / or one or more additional active agents. The kit typically includes a label specifying the intended use of the kit contents. In this context, the term “label” includes any described or recorded material on or supplied with the kit, or otherwise accompanying the kit.

[0088] The BCL9 mimic peptide of the present invention can be used to inhibit proliferation and / or promote cytotoxicity in neoplastic tumor cells. Proliferation and cytotoxicity can be measured by known assays, including the apoptosis verification described herein.

[0089] 세포 표적화

[0090] The BCL9 mimic peptide of the present invention can be introduced into target cells by methods known in the art. The introduction method may vary, for example, depending on the intended application.

[0091] In some cases, DNA or RNA encoding the BCL9 mimic peptide may be delivered into target cells and expressed therein. Delivery may be carried out via any suitable vector depending on the application. Examples of vectors include plasmids, cosmids, phages, bacterial, yeast, and viral vectors, and vectors prepared from retroviruses, such as lentiviruses, adenoviruses, adeno-associated viruses, and enveloped pseudotyped viruses. The vector may be introduced into cells using, for example, nanoparticles, hydrodynamic delivery, electroporation, sonoporation, calcium phosphate precipitation, or cationic polymers such as DEAE-dextran. The vector may be encapsulated in liposomes or complexed with lipids associated with cationic condensers.

[0092] The BCL9 mimic peptide of the present invention can be delivered into a cell via a mechanism utilizing a cellular response. Examples of such mechanisms include antibody-drug conjugates, chimeric antigen receptors, multiple antigen presentation (MAP) systems, and integrin-targeting and RGD-like sequences. Examples of RGD-like sequences include GRGDS (Sequence Identification No.: 28) and GRGDNP (Sequence Identification No.: 29). The BCL9 mimic peptide of the present invention may comprise one or more RGD-like sequences, such as two, three, four, or five RGD-like sequences, linked by any method described herein or known in the art. The one or more RGD-like sequence(s) may be incorporated into the N-terminal or C-terminal side of the BCL9 HD2 region. These RGD-like sequences can be retro-inverted independently of each other and independently of the BCL9 HD2 region. One specific example of a retro-inverted RGD-like sequence is PSDGRG (sequence identification number: 75). Alternatively, BCL9 mimic peptides can be encapsulated in vesicles or micelles, such as exosomes or liposomes, and delivered to cells. Another method of introducing BCL9 mimic peptides into cells is, for example, cyclization via hydrocarbon staples (Bernal et al. 2007; Bird et al. 2016) or by other cyclization methods known in the field.

[0093] A specific BCL9 mimic peptide of the present invention comprises a cell-penetrating domain or a cell-penetrating peptide (CPP). The terms "cell-penetrating domain," "cell-penetrating region," and "cell-penetrating peptide" are used interchangeably herein.

[0094] CPPs are short peptides (typically about 6-40 amino acids) that can cross cell membranes. Many CPPs can cross the blood-brain barrier (BBB). In some embodiments, the CPP is an amino acid of length 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, including a range of 10-30 amino acids, as an endpoint. CPPs possess the ability to transport covalently or non-covalently linked molecular cargo, such as polypeptides, polynucleotides, and nanoparticles, across cell membranes and the BBB. Translocation can be endocytotic via translocation or energy-independent (i.e., non-endocytotic). Numerous CPPs have been described and characterized in the literature ( 가령 , Handbook of Cell-Penetrating Peptides (2d ed. Ulo Langel ed., 2007); et al. 2008; Heitz et al. 2009; Munyendo et al. 2012; Zou et al. 2013; Krautwald et al. (Refer to 2016). A curated database of CPP is maintained at crdd.osdd.net / raghava / cppsite (Gautam et al. 2012).

[0095] Peptides referred to as nuclear localization sequences (NLSs) are a subset of CPPs. Classical NLSs contain one (single molecule) or two (bimolecular) regions of basic amino acids. The consensus sequences of classical single-molecule and bimolecular NLSs are, in turn, K(K / R)X(K / R) (sequence identification number: 30) and (K / R)(K / R)X 10-12 (K / R) 3 / 5(Sequence identification number: 31), where 3 / 5 indicates that at least 3 of the 5 consecutive amino acids are lysine or arginine (Kosugi et al. 2009). The NLS sequence of SV40 giant T antigen, PKKKRKV (sequence identification number: 57), is an example of a classic single-molecule NLS, while the NLS sequence of nuclear plasmin, KRPAATKKAGQAKKK (sequence identification number: 44), is an example of a classic bimolecule NLS (Lange et al. 2007; Kosugi et al. 2009). There are also numerous non-classical NLSs, such as the ribonucleoprotein (RNP) hnRNP A1, hnRNP K, and U snRNP (Mattaj et al. 1998).

[0096] Non-limiting examples of CPPs suitable for use in the present invention include proteins, such as, 초파리(Drosophila) Antennapedia transcription factors (penetratin and its derivatives RL-16 and EB1) (Derossi et al. 1998; et al. 2000; Lundberg et al. 2007; Alves et al. 2008); Trans-activator of HIV-1 transcription (Tat) ( et al. 1997; et al. 2001); Rabies virus glycoprotein (RVG) (Kumar et al. 2007); Herpes simplex virus VP22 (Elliott et al. 1997); Antimicrobial protegreen 1 (SynB) (Rousselle et al. 2001), rat insulin 1 gene enhancer protein (pIS1) (Kilk et al. 2001; Magzoub et al. 2001); Murine vascular endothelium cadharin (pVEC) (Elmquist et al.2001); Human calcitonin (hCT) (Schmidt et al. 1998); and fibroblast growth factor 4 (FGF4) (Jo et al. It is a peptide derived from a protein such as (2005). CPPs suitable for use in the present invention include synthetic and chimeric peptides, such as Transportan (TP) and its derivatives (Pooga et al. 1998; Soomets et al. 2000); translocating sequences (MTSs) (Brodsky et al. 1998; Lindgren et al. 2000; Zhao et al. 2001), for example, MPS peptide (also known as fusion sequence-based peptide or FBP) (Chaloin et al. 1998); Sequence signal-based peptide (SBP) (Chaloin et al. 1997); Model amphoteric peptide (MAP) (Oehlke et al. 1998; Scheller et al. 1999; et al. 2001), translocating peptide 2 (TP2) (Cruz et al. 2013), MPG (Morris et al. 1997; Kwon et al. 2009), Pep-1 (Morris et al. 2001; -Morris et al. 2007), and poly-arginine ( 가령 , R7-R 12 ) (Sequence identification number: 144) (Mitchell et al. 2000; Wender et al. 2000; Futaki et al. 2001; Suzuki et al. It also includes 2002). A representative, however, non-restrictive sequence is Page 7 It is represented in.

[0097] Page 7

[0098]

[0099]

[0100] Because the function of CPPs depends on their physical properties rather than sequence-specific interactions, these Page 7 It may have reverse sequences and / or reverse chirality as provided and / or known in the art. For example, retro-reversed forms of the above CPPs (reverse sequences and reverse chirality) are suitable for use in the present invention. Examples of retro-reversed CPPs include those having the D-amino acid sequences KKWKMRRNQFWIKIQR (Sequence Identification No.: 71), KKWKMRRNQFWLKLQR (Sequence Identification No.: 72), RRRQRRKKRGY (Sequence Identification No.: 73), KLTPV (Sequence Identification No.: 74), or OLTPV (Sequence Identification No.: 143). Variants of these sequences having one or more amino acid additions, deletions, and / or substitutions, while retaining the ability to cross the cell membrane and / or BBB, are also suitable for use in the present invention. The BCL9 mimic peptide of the present invention Page 7 It also includes a cell-penetrating 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 respect to the exemplary sequence provided. The effect of amino acid addition(s), deletion(s), and / or substitution(s) on the ability of CPP to mediate cell permeability can be tested using methods known in the art.

[0101] III. Manufacturing method

[0102] The BCL9 mimic peptides of the present invention can be chemically synthesized, for example, using solid-phase peptide synthesis or solution-method peptide synthesis, or expressed using a recombinant method. Synthesis or expression may produce fragments of peptides that are subsequently chemically or enzymatically combined.

[0103] Accordingly, the present invention also provides a nucleic acid molecule encoding a BCL9 mimic peptide. Such nucleic acids can be constructed by chemical synthesis using an oligonucleotide synthesizer. The nucleic acid molecule of the present invention can be designed based on the amino acid sequence of the desired BCL9 mimic peptide and the selection of codons preferred in the host cell where the recombinant BCL9 mimic peptide is to be produced. Standard methods may be applied to synthesize the nucleic acid molecule encoding the BCL9 mimic peptide of interest.

[0104] Once prepared, a nucleic acid encoding a specific BCL9 mimic peptide can be inserted into an expression vector and operably linked to an expression control sequence suitable for the expression of the peptide in a desired host. To obtain high expression levels of the BCL9 mimic peptide, the nucleic acid can be operably linked or associated with transcription and translation expression control sequences that function in a selected expression host.

[0105] A wide range of expression host / vector combinations may be used by any person known in the art. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include Escherichia coli, including pCR1, pBR322, pMB9, and their derivatives ( E. coli)It includes known bacterial plasmids such as plasmids of origin, broader host range plasmids such as M13, and filamentous single-stranded DNA phages.

[0106] 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, for example, E. coli ( E. coli) Or Bacilli are involved. Higher-order eukaryotic or mammalian cell lines can be established, 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. Cell-free detoxification systems can also be used. Effects of the invention

[0107] The present invention provides a BCL9 mimic peptide having a modified α-helical homologous domain-2 (HD2) region and optionally, a cell-penetrating region, a composition comprising the BCL9 mimic peptide, and a method for inhibiting the proliferation of neoplastic cells and / or promoting cytotoxicity using the BCL9 mimic peptide. Brief explanation of the drawing

[0108] Fig. 1 The modified BCL9 mimic peptide of the present invention is β - It demonstrates that it antagonizes catenin and exerts anti-proliferative activity in cultured MCF7 breast cancer cells. Fig. 2A-2B This shows that the retro-reversed BCL9 mimic peptide of the present invention exerts anti-proliferative activity in cultured MCF7 breast cancer cells. Peptide BCL-26 ( Fig. 2A ) and peptide BCL-27 ( Fig. 2B Represents cytotoxicity data for ). Figs. 3A-3CThis shows that the retro-inverted BCL9 mimic peptide of the present invention exhibits anti-tumor activity in an MCF7 breast cancer mouse model. Data points represent mean ± SEM. BCL-26 (12.5 mg / kg) was administered to nude mice at 2 days post-tumor inoculation ( Fig. 3A ; p<0.0001). BCL-87 (5 mg / kg) was administered on day 21 post-vaccination ( Fig. 3B; p<0.005). BCL-87 and BCL-27 at two concentrations (1 mg / kg and 5 mg / kg) were administered on day 14 post-inoculation ( Fig. 3C ; p≤0.0004 for the control group for all test peptides). Specific details for implementing the invention

[0109] Specific embodiments of the present invention may be further defined by reference to the following non-limiting embodiments of the method of the present invention. It will be apparent to those skilled in the art that many variations of both the material and the method may be made without departing from the scope of this specification.

[0111] Example 1: BCL9 peptide exhibits anti-proliferative activity in cancer cells in vitro.

[0112] We generated a panel of peptides containing a cell-penetrating region and a BCL9 HD2 domain, and compared their activity with the previously described cyclic peptide (Takada et al. 2012), ST-BC1. The BCL9 peptide discussed in this example is Table 8 It is summarized in.

[0113] Table 8

[0114]

[0115] ST-BC1 contains a lactam bridge between the underlined and bolded residues. The other five peptides are linear and have a TAT cell-penetrating domain indicated in italics. Peptide BCL-21 contains an intrinsic HD2 domain, while each of peptides BCL-22-25 has an amino acid substitution in the intrinsic BCL9 HD2 domain. In addition to the substitution, peptide BCL-25 also possesses an HD2 domain that is shortened compared to the intrinsic sequence.

[0116] MCF-7 breast cancer cells were cultured at 2.5 x 10⁶ per well in 150 μL of MEM medium + 10% fetal bovine serum (FBS) in 96-well culture dishes. 4 The cell density was set. Lyophilized BCL9 peptide was reconstituted to a concentration of 10 mg / mL in 270 mM trehalose buffer and added to each well in a volume of 50 μL so that the final concentration range was 2.5–40 μM. Cells were incubated with BCL9 peptide at 37°C for 96 hours.

[0117] In accordance with the manufacturer's instructions, cell viability was quantified spectrophotometrically using the Roche MTT cell proliferation assay kit. Briefly, cells were washed with PBS and incubated in fresh MEM medium at 37°C for 4 hours with 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 in 5% carbon dioxide. Absorbance was measured at OD 570 nm, and the reference was measured at OD 650 nm. The degree of absorption is proportional to the number of living cells. The percentage of absorbance relative to the untreated control was quantified and presented as cell viability %.

[0118] The modified BCL9 peptide of the present invention has been proven to have equivalent or greater anti-proliferative activity in MCF7 breast cancer cells compared to peptide ST-BC1, but BCL-21, which does not have the intrinsic BCL9 HD2 domain sequence, has not (EC 50 Value <10 μM) Fig. 1 ). In functional testing, EC 50 is the concentration at which the biological response decreases to 50% of the maximum response. In the case of the BLC9 peptide, EC 50 Cell viability is measured at a concentration where it is reduced by 50% from its maximum value. EC 50 It can be produced by any means known in the field.

[0120] Example 2: Retro-inverted BCL9 peptide exhibits anti-proliferative activity in cancer cells in vitro.

[0121] We have the following retro-inverted peptide: D-amino acid sequence FLMRQLDRLTQLA KLTPV BCL-26 having (sequence identification number: 26), and D-amino acid sequence WWLARQLARLAQLA KLTPV BCL-27 with sequence identification number: 27 was prepared. Cell-penetrating regions derived from Bax-inhibitory peptides are indicated in italics. In the case of BCL-26, the sequence of the HD2 domain is shortened by 11 amino acids and substituted at 2 positions compared to the original sequence. In the case of BCL-27, the sequence of the HD2 domain is shortened by 11 amino acids and substituted at 6 positions compared to the original sequence, and additionally contains N-terminal tryptophan compared to the original sequence.

[0122] We investigated the cytotoxicity of peptides BCL-26 and BCL-27 in HL60 pre-myeloid leukemia cells using an assay. HL60 PML suspension cells were cultured at 3.5 x 10⁶ per well in 150 μL of RPMI + 1.5% fetal bovine serum (FBS) in 96-well culture dishes. 3Cell density was set. Peptides BCL-26 or BCL-27, reconstituted at a concentration of 10 mg / mL in 20 mM His, pH 7.5, were added to each well in a volume of 50 μL to achieve a final concentration range of 0–80 μM. Cells were incubated with the peptides 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 living cells exhibit no or minimal fluorescence. Forward scattering (FSC) contrast. Cells were selected by side scattering (SSC)-based analysis and analyzed by a BD Accuri C6 Plus flow cytometer to detect Annexin V-FITC binding (Ex = 488 nm; Em = 530 nm) using a FITC signal detector and PI staining with a phycoerythrin emission signal detector. Annexin V 낮음(low) and PI 낮음(low) The percentage was quantified and expressed as viability %. The retro-inverted peptide had activity comparable to the standard peptide tested in Example 1 ( Fig. 2A-2B ).

[0123] Using the assay described above, we investigated the cytotoxicity of additional retro-reversed and mixed chiral BCL9-mimicking peptides in HL60 cells. The results were Table 9 It is represented in.

[0124] Table 9

[0125]

[0126]

[0127] +++ is EC 50 < 2 μM indicates; ++ is EC 50 = represents 2–10 μM; + is EC 50 > 10 μM indicates; NA indicates no activity.

[0128] The subscripts D and L indicate the chirality of the corresponding amino acids. Substitutions and additions to the retro-inverted wild-type BCL9 HD2 sequence (sequence identification number: 7) are indicated by a bold underline. Cell-penetrating and RGD-like regions are indicated in italics. As expected, BCL-12 did not exhibit cytotoxic activity because it lacked a sequence for cell penetration.

[0129] The lack of cytotoxic activity by BCL-134 demonstrates a requirement for a positively charged amino acid at at least one of position 4 or position 8 of the HD2 domain compared to Sequence Identification No. 7. (Compare the activity of BCL-133 with the activity of BCL-134.)

[0130] The lack of cytotoxic activity by BCL-138 demonstrates the requirement that at least one of the two N-terminal amino acids of the HD2 domain must be hydrophobic. (Compare the activity of BCL-91b with that of BCL-138.)

[0132] Example 3: Retro-inverted BCL9 peptide exhibits in vitro anti-tumor activity.

[0133] In this experiment, we investigated the effect of peptide BCL-26 on tumor volume in an MCF7 subcutaneous tumor model. Briefly, 2 x 10 6MCF7 breast cancer cells were suspended in Matrigel at a 1:1 ratio and injected subcutaneously into the axilla of NU / J mice. Peptide BCL-26 was administered subcutaneously three times a week for three weeks at a dose of 12.5 mg / kg. Administration began on day 2 after tumor inoculation, and the starting mean tumor volume range was approximately 25 mm. 3 was. Peptide BCL-26 significantly reduced tumor volume compared to the vehicle ( Fig. 3A ).

[0134] We also investigated the effects of a BCL9 mimic peptide in established tumors. MCF7 cells were transplanted as described above. Peptide BCL-87 was administered via subcutaneous injection three times a week for three weeks at a dose of 5 mg / kg. Administration began on day 21 after tumor inoculation, and the starting mean tumor volume range was approximately 470 mm². 3 was. Peptide BCL-87 significantly reduced tumor volume compared to the vehicle ( Fig. 3B ).

[0135] We further investigated the effects of different concentrations of mimic peptides in established tumors. MCF7 cells were transplanted as described above. Peptides BCL-27 and BCL-87 were administered via subcutaneous injection three times a week for three weeks at doses of 1 mg / kg or 5 mg / kg. Administration began on day 14 after tumor inoculation, and the starting mean tumor volume range was approximately 340 mm². 3 Both peptides significantly reduced tumor volume compared to the vehicle, the peptide control, and BCL-134 ( Fig. 3C ).

[0137] Reference materials

[0138] 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).

[0139] Bernal F, et al. Reactivation of the p53 Tumor Suppressor Pathway by a Stapled p53 Peptide. J. Am. Chem. Soc. 129:2456-2457 (2007).

[0140] Bird GH, et al. Biophysical Determinants for Cellular Uptake of Hydrocarbon-Stapled Peptide Helices. Nat. Chem. Biol. 12:845-852 (2017).

[0141] Brodsky JL, et al. Translocation of proteins across the endoplasmic reticulum membrane. Int. Rev. Cyt. 178:277-328 (1998).

[0142] Chaloin L, et al. Conformations of primary amphipathic carrier peptides in membrane mimicking environments. Biochem. 36:11179-11187 (1997).

[0143] Chaloin L, et al. Design of carrier peptide-oligonucleotide conjugates with rapid membrane translocation and nuclear localization properties. Biochem. Biophys. Res. Commun.243:601-608 (1998).

[0144] Cruz J, et al. A membrane-translocating peptide penetrates into bilayers without significant bilayer perturbations. Biophys. J. 104:2419-2428 (2013).

[0145] Derossi D, et al. Trojan peptides: the penetratin system for intracellular delivery. Trends Cell Biol. 8:84-87 (1998).

[0146] Elliott G, et al. Intercellular trafficking and protein delivery by a Herpesvirus structural protein. Cell 88:223-233 (1997).

[0147] Elmquist A, et al. VE-cadherin-derived cell-penetrating peptide, pVEC, with carrier functions. Exp. Cell Res. 269:237-244 (2001).

[0148] Futaki S, et al. Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J. Biol. Chem. 276:5836-5840 (2001).

[0149] Gautam A, et al. CPPsite: a curated database of cell penetrating peptides. Database doi:10.1093 / database / bas015 (2012).

[0150] M, et al. Cargo delivery kinetics of cell-penetrating peptides. Biochim. Biophys. Acta 1515:101-109 (2001).

[0151] Heitz F, et al. Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics. Brit. J. Pharmacol. 157:195-206 (2009).

[0152] F, et al. CNS delivery via adsorptive transcytosis. AAPS J. 10:455-472 (2008).

[0153] Jo, D, et al. Intracellular protein therapy with SOCS3 inhibits inflammation and apoptosis. Nat. Med . 11:892-898 (2005).

[0154] Kawamoto SA, et al. Analysis of the Interaction of BCL9 with β-Catenin and Development of Fluorescence Polarization and Surface Plasmon Resonance Binding Assays for this Interaction. Biochem. 48:9534-9541 (2009).

[0155] Kilk K, et al.Cellular internalization of a cargo complex with a novel peptide derived from the third helix of the islet-1 homeodomain. Comparison with the penetratin peptide. Bioconjug. Chem. 12:911-916 (2001).

[0156] Klein JS, et al. Design and characterization of structured protein linkers with differing flexibilities. Protein Eng. Des. Sel. 27:325-330 (2014).

[0157] Kosugi S, et al. Six Classes of Nuclear Localization Signals Specific to Different Binding Grooves of Importin α. J. Biol. Chem. 284:478-485 (2009).

[0158] Krautwald S, et al. Inhibition of regulated cell death by cell-penetrating peptides. Cell. Mol. Life Sci. 73:2269-2284 (2016).

[0159] Kumar P, et al. Transvascular delivery of small interfering RNA to the central nervous system. Nature 448:39-43 (2007).

[0160] Kwon S-J, et al.Transduction of the MPG-tagged fusion protein into mammalian cells and oocytes depends on amiloride-sensitive endocytic pathway. BMC Biotechnol. 9:73-84 (2009).

[0161] Lange A, et al. Classical Nuclear Localization Signals: Definition, Function, and Interaction with Importin α. J. Biol. Chem. 282:5101-5105 (2007).

[0162] Le Chevalier Isaad A, et al. Side chain-to-side chain cyclization by click reaction. J. Pept. Sci. 15:451-454 (2009).

[0163] Lindgren M, et al. Cell-penetrating peptides. Trends Pharmacol. Sci. 21:99-103 (2000).

[0164] Lundberg P, et al. Delivery of short interfering RNA using endosomolytic cell-penetrating peptides. FASEB J. 21:2664-2671 (2007).

[0165] Magzoub M, et al. Interaction and structure induction of cell-penetrating peptides in the presence of phospholipid vesicles. Biochim. Biophys. Acta 1512:77-89 (2001).

[0166] Mattaj IW, et al.Nucleocytoplasmic Transport: The Soluble Phase. Ann. Rev. Biochem. 67:265-306 (1998).

[0167] Mitchell DJ, et al. Polyarginine enters cells more efficiently than other polycationic homopolymers. J. Pept. Res. 56:318-325 (2000).

[0168] Morris MC, et al. A new peptide vector for efficient delivery of oligonucleotides into mammalian cells. Nucleic Acids Res. 25:2730-2736 (1997).

[0169] Morris MC, et al. A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat. Biotechnol. 19:1173-1176 (2001).

[0170] -Morris MA et al. The peptide carrier Pep-1 forms biologically efficient nanoparticle complexes. Biochem. Biophys. Res. Commun. 355:877-882 (2007).

[0171] Munyendo WLL, et al. Cell penetrating peptides in the delivery of biopharmaceuticals. Biomolecules 2:187-202 (2012).

[0172] Oehlke J, et al.Cellular uptake of an alpha-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically. Biochim. Biophys. Acta 1414:127-139 (1998).

[0173] Patgiri A, et al. A hydrogen bond surrogate approach for stabilization of short peptide sequences in alpha helical conformation. Acc. Chem. Res. 41:1289-1300 (2008).

[0174] Pooga M, et al. Cell penetration by transportan. FASEB J. 12:67-77 (1998).

[0175] Rousselle C, et al. Enhanced delivery of doxorubicin into the brain via a peptide-vector-mediated strategy: saturation kinetics and specificity. J. Pharmacol. Exp. Ther. 296:124-131 (2001).

[0176] Sampietro J, et al. Crystal Structure of a β-Catenin / BCL9 / Tcf4 Complex. Molecule 24:293-300 (2006).

[0177] Schafmeister CE, et al. An All-Hydrocarbon Cross-Linking System for Enhancing the Helicity and Metabolic Stability of Peptides. J. Am. Chem. Soc.122:5891-5892 (2000).

[0178] Scheller A, et al. Structural requirements for cellular uptake of alpha-helical amphipathic peptides. J. Peptide Sci. 5:185-194 (1999).

[0179] Schmidt MC, et al. Translocation of human calcitonin in respiratory nasal epithelium is associated with self assembly in lipid membrane. Biochem. 37:16582-16590 (1998).

[0181] Soomets U, et al. Deletion analogues of transportan. Biochemistry. Biophysics Acta 1467:165-176 (2000).

[0182] Suzuki T, et al. Possible existence of common internalization mechanisms among arginine-rich peptides. J. Biol. Chem. 277:2437-2443 (2002).

[0183] Takada K, et al. Targeted Disruption of the BCL9 / β-catenin Complex Inhibits Oncogenic Wnt Signaling. Sci. Transl. Med. 4:148ra117 (2012).

[0184] PEG, et al. The Antennapedia peptide penetratin translocates across lipid bilayers - the first direct observation. FEBS Lett.482:265-68 (2000).

[0185] E, et al. A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 272:16010??16017 (1997).

[0186] Wang X-Y, et al. Synthesis of small cyclic peptides containing the disulfide bond. ARKIVOC xi:148-154 (2006).

[0187] Wender PA, et al. The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc. Natl. Acad. Sci. USA 97:13003-13008 (2000).

[0188] Zhan T, et al. Wnt signaling in cancer. Oncogene 36:1461-1473 (2017).

[0189] Zhao Y, et al. Chemical engineering of cell penetrating antibodies. J. Immunol. Methods 254:137-45 (2001).

[0190] Zou LL, et al. Cell-penetrating peptide-mediated therapeutic molecule delivery into the central nervous system. Curr. Neuropharmacol.11:197-208 (2013).

Claims

Claim 1 A BCL9 mimic peptide comprising a modified BCL9 α-helical homology domain-2 (HD2) region and a cell-penetrating region, wherein the modified BCL9 HD2 region is a D-amino acid sequence consisting of variants of the D-amino acid sequence FLMRQIDRLTQLS (sequence identification number: 7), and the variants are modified at each position of sequence identification number: 7 as follows: i. F1 is substituted with W; ii. M3 is replaced with L; iii. I6 is replaced with L; iv. D7 is replaced with A;v. T10 is replaced with A; vi. BCL9 mimic peptide in which S13 is substituted with A, and the cell-penetrating domain contains the D-amino acid sequence KLTPV (sequence identification number: 74). Claim 2 A BCL9 mimic peptide according to claim 1, further comprising F of the D form at position -2 of sequence identification number: 7 and R of the D form at position -1 of sequence identification number:

7. Claim 3 A BCL9 mimic peptide according to claim 1 or 2, wherein the peptide comprises an octanoyl N-terminal group. Claim 4 A BCL9 mimic peptide according to any one of claims 1 to 3, wherein the peptide comprises a C-terminal amide group. Claim 5 A method in vitro for promoting cytotoxicity within neoplasm cells, comprising the step of contacting a neoplasm cell with a BCL9 mimic peptide according to any one of claims 1 to 4 or a composition comprising said BCL9 mimic peptide. Claim 6 A method in vitro for inhibiting the proliferation of neoplastic cells, comprising the step of contacting a neoplastic cell with a BCL9 mimic peptide according to any one of claims 1 to 4 or a composition comprising said BCL9 mimic peptide.

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