Administration and use of β-catenin antagonists

Abstract

The present invention provides a method for administering a β-catenin peptide antagonist, and a method for treating solid tumors by administering a β-catenin peptide antagonist.

Description

[Background technology]

[0001] The Wnt / β-catenin signaling pathway regulates crucial cellular functions, including proliferation, differentiation, migration, and stem cell self-renewal, and plays a vital role in embryonic development and adult tissue homeostasis (Liu 2022). Dysregulation of this pathway is often accompanied by constitutive activation of β-catenin, an oncogenic transcription factor involved in the pathogenesis of many malignancies, and is associated with poor prognosis in many cancers (Shang 2017). Wnt / β-catenin signaling is also associated with the promotion of an immunosuppressive tumor microenvironment characterized by decreased chemokine expression, impaired dendritic cell recruitment, and subsequent reduction in tumor-infiltrating effector T cells (Ruiz de Galarreta 2019).

[0002] In normal human cells, β-catenin expression in the cytoplasm and nucleus is tightly regulated by the adenomatous polyposis (APC) complex, a protein complex that targets excess β-catenin for proteolytic degradation. Loss-of-function mutations in the components of the APC complex or gain-of-function mutations in β-catenin allow β-catenin to escape degradation and accumulate in the nucleus. In the nucleus, β-catenin binds to the transcription factor lymphoid enhancer factor / T cell factor (LEF / TCF) to induce the expression of oncogenes that promote cell survival, proliferation, and migration (Bugter 2021).

[0003] In cancer cells, the accumulation and retention of β-catenin in the nucleus is promoted by the B-cell CLL / lymphoma 9 protein (BCL9) (de la Roche 2008). BCL9 is a co-activator that shuttles β-catenin to the nucleus and acts as a scaffold for recruiting the enhancer protein Pygopus to the β-catenin transcription complex to enhance transcriptional activity (Mieszcanek 2019). BCL9 is overexpressed in many tumors and correlates with poor prognosis (Moor 2015, Vafaizadeh 2021, Wang 2019). Studies have shown that deletion of BCL9 / BCL9L inhibits tumor proliferation and growth with minimal impact on normal tissue homeostasis, suggesting that β-catenin antagonists may have significant antitumor effects while unlikely to cause on-target or off-tissue toxicity (Gay 2019).

[0004] Based on its crucial role in promoting malignant phenotypes and its inverse correlation with disease prognosis in multiple cancer types, β-catenin has been recognized as a potential therapeutic target for cancer treatment (Liu 2022). However, despite extensive ongoing research, β-catenin has traditionally been considered "undrug-proof," and there are currently no available drugs that target Wnt / β-catenin signaling.

[0005] Conventional approaches targeting the Wnt / β-catenin pathway for cancer treatment act upstream of β-catenin regulation or at the interaction site between β-catenin and LEF / TCF, resulting in nonspecific inhibition of the canonical Wnt pathway and on-target toxicity of bone (Zhong 2016) and intestine (Dotan 2020). Approaches that specifically inhibit the interaction between β-catenin and BCL9 are needed to avoid adverse effects such as on-target toxicity. [Overview of the project]

[0006] Some of the main embodiments of the present invention are summarized below. Additional embodiments are described in the sections on modes for carrying out the invention, examples, drawings, and claims. Each section of this disclosure is intended to be read in conjunction with the others. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present invention.

[0007] This disclosure provides a method for treating a solid tumor in a patient, comprising parenterally administering to the patient a pharmaceutical composition comprising an effective amount of a β-catenin peptide antagonist. Also provided are a parenterally administered pharmaceutical composition comprising an effective amount of a β-catenin peptide antagonist for use in treating a solid tumor in a patient.

[0008] In one embodiment, the solid tumor is selected from the group consisting of breast cancer, colorectal cancer, cholangiocarcinoma, hepatocellular carcinoma, melanoma, non-small cell lung cancer, ovarian cancer, endometrial cancer, pancreatic adenocarcinoma, and synovial sarcoma. In a particular embodiment, the patient has received prior treatment selected from the group consisting of chemotherapy, hormone-based therapy, radiotherapy, targeted therapy, immunotherapy, and combinations thereof. In a particular embodiment, the peptide antagonist is administered as a neoadjuvant, either alone or in combination. In one embodiment, the peptide antagonist is administered in combination with (i) bevacizumab and / or (ii) folinic acid, fluorouracil, and irinotecan (FOLFIRI). In one embodiment, the peptide antagonist is administered in combination with fluquintinib.

[0009] In some embodiments, the peptide antagonist comprises the D-amino acid sequence FRWLLRQLARLAQLAKLTPVKLTPV (SEQ ID NO: 1). In some embodiments, the peptide antagonist is administered to the patient at a dose of approximately 0.25 to 12 mg / kg.

[0010] One aspect of the present invention is a method for administering a β-catenin peptide antagonist, comprising parenterally administering a pharmaceutical composition containing the peptide antagonist, wherein the peptide antagonist comprises the D-amino acid sequence FRWLLRQLARLAQLAKLTPVKLTPV (SEQ ID NO: 1), and the peptide antagonist is administered at a dose of approximately 0.25 to 12 mg / kg. Also provided is a pharmaceutical composition containing a peptide antagonist for use in a method for parenterally administering a β-catenin peptide antagonist, wherein the peptide antagonist comprises the D-amino acid sequence FRWLLRQLARLAQLAKLTPVKLTPV (SEQ ID NO: 1), and the peptide antagonist is administered at a dose of approximately 0.25 to 12 mg / kg.

[0011] In the specific methods and compositions of the present invention, the peptide antagonist is administered in doses selected from about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 4 mg / kg, about 8 mg / kg, and about 12 mg / kg.

[0012] In some embodiments of the present invention, the peptide antagonist inhibits the binding of β-catenin to the B-cell CLL / lymphoma 9 protein (BCL9). In certain embodiments, the peptide antagonist contains an N-terminal octanoyl group. In certain embodiments, the peptide antagonist is ST316.

[0013] In certain embodiments, the pharmaceutical composition is administered intravenously, for example, by infusion. In embodiments in which the composition is administered by infusion, the duration of infusion may be, for example, about 30 to 180 minutes or about 60 to 90 minutes.

[0014] In some embodiments, the pharmaceutical composition is administered once a week or once every two weeks. In one embodiment, the pharmaceutical composition is administered for at least four weeks. [Brief explanation of the drawing]

[0015] [Figure 1]ST316 is shown to directly bind to β-catenin Arm1 with higher affinity than the transcriptional coactivator B cell CLL / lymphoma 9 protein (BCL9) in a fluorescence polarization assay. Results are plotted as normalized milli-P (mP) vs. β-catenin concentration. [Figure 2] ST316 is shown to inhibit the protein-protein interaction between β-catenin and BCL9 in an amplified luminescence proximity homogeneous assay (ALPHA). Both ST316 (circles) and BCL9 HD2 peptide (squares) inhibited the interaction between β-catenin and BCL9 with an IC50 of 1 μM. [Figure 3] A - B show attenuation of β-catenin nuclear localization by 3 μM or 5 μM ST316 in HCT116 colorectal cancer cells. YY1 and GAPDH were used as markers for the nuclear fraction and cytoplasmic fraction, respectively (A). [Figure 4] A - D show that ST316 enhances proteasomal degradation of BCL9 in Colo320 colorectal cancer cells. [Figure 5] A - B show that ST316 inhibits migration of HCT116 colorectal cancer cells across a semipermeable membrane in a 24-hour invasion assay compared to a control peptide. A shows cells after treatment with 1 μM negative control peptide (upper panel) or ST316 (lower panel). B shows the number of cells after treatment. [Figure 6] A - C show that ST316 decreases spontaneous adenoma formation in a mouse model of colorectal cancer. [Figure 7] Administration of ST316 (βCAP) is shown to inhibit tumor growth in a subcutaneous xenograft tumor model using HCT116 cells. N = 6 mice per group. [Figure 8] A - B show that administration of ST316 inhibits tumor growth (A) and decreases Axin2 expression (B) in a subcutaneous xenograft tumor model using 4T1-luc triple-negative breast cancer (TNBC) cells. N = 6 mice per group. [Figure 9]A and B show the time course of plasma concentrations of ST316 in cohorts 1 to 4 (0.5, 1, 2, or 4 mg / mL). [Figure 10A] This study demonstrates a reduction in the ST316-induced immunosuppressive polymorphonuclear (PMN) myeloid suppressor cell (MDSC) population in cancer patients. Peripheral blood samples were collected from patients with pancreatic ductal adenocarcinoma (PDAC) before (upper panel) and after (lower panel) administration of 4 mg / kg of ST316 quarterly for 3 weeks. The blood was processed to collect lymphocytes and myeloid cells, which were analyzed by flow cytometry. [Figure 10B] This study demonstrates a reduction in the ST316-induced immunosuppressive polymorphonuclear (PMN) myeloid suppressor cell (MDSC) population in cancer patients. Peripheral blood samples were collected from colorectal cancer (CRC) patients before (upper panel) and after (lower panel) administration of 4 mg / kg ST316 quarterly for 3 weeks. Blood was processed to collect lymphocytes and myeloid cells, which were analyzed by flow cytometry. [Figure 10C] This study shows a decrease in the ST316-induced immunosuppressive polymorphonuclear (PMN) myeloid suppressor cell (MDSC) population in cancer patients. PMN cells (CD3-, CD19-, CD14-, HLADRlow, CD11b+, and CD15+) were quantified as a percentage of myeloid cells (CD3-, CD19-). [Modes for carrying out the invention]

[0016] Unless otherwise indicated, the present invention may be implemented using prior art in the fields of pharmaceuticals, formulation science, protein chemistry, cell biology, cell culture, molecular biology, microbiology, recombinant DNA, immunology, clinical pharmacology, and clinical practice, which are within the scope of the art.

[0017] To make the present invention more easily understandable, certain terms are first defined. Further definitions are provided throughout this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the field to which the present invention relates.

[0018] No heading provided herein limits the various aspects or embodiments of the invention that can be obtained by referring to the entire specification. Therefore, the terms defined immediately below are more fully defined by referring to the entire specification.

[0019] All references cited herein are incorporated herein by reference in their entirety. Furthermore, any instructions or catalogs from any manufacturer relating to any product cited or mentioned herein are incorporated herein by reference. Any documents incorporated herein by reference, or any teachings contained herein, may also be used in the practice of the present invention. Documents incorporated herein by reference are not considered prior art.

[0020] I. Definition The technical terms and terminology used herein are for illustrative purposes only and not limiting; therefore, they should be interpretable to those skilled in the art in light of the teachings and guidance provided herein.

[0021] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. The terms “a” (or “an”) and “one or more” and “at least one” are interchangeable.

[0022] Furthermore, “and / or” is interpreted as a specific disclosure of each of two specific features or features, whether they include or do not include another component or feature. Thus, when the term “and / or” is used in a phrase such as “A and / or B,” it is intended to include A and B, A or B, A (alone), and B (alone). Similarly, when the term “and / or” is used in a phrase such as “A, B, and / or C,” it 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).

[0023] When an embodiment is described with the word “including,” it also includes similar embodiments otherwise described with the terms “consisting of” and / or “essentially consisting of.”

[0024] Units, prefixes, and symbols are shown in the form approved by the International System of Units (SI). Numerical ranges include the numbers that define the range, and any individual values ​​provided herein may function as endpoints of ranges that include 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 numerical ranges such as 1–10, 1–8, and 3–9. Similarly, a disclosed range is a disclosure of each individual value (i.e., an intermediate value) that falls within that range, including integers and fractions. For example, the stated range 5–10 discloses 5, 6, 7, 8, 9, and 10 individually, and also discloses 5.2, 7.5, 8.7, etc.

[0025] Unless otherwise indicated, the terms “at least” or “about” preceding a series of elements should be understood to refer to all elements in that series. The term “about” depends on the context in which it is used. When placed before a number, it generally includes ±10% of the stated value. For example, in a given context, a concentration of about 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of about 1% to 10% (w / v) may include 0.9% (w / v) to 11% (w / v).

[0026] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers of amino acids of any length and their salts. Polymers may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acids. Unless otherwise indicated, such as abbreviations for uncommon or unnatural amino acids as described herein, three-letter and one-letter abbreviations used in the art are used herein to represent amino acid residues. Amino acids are L-amino acids unless preceded by a “D” or in lowercase. Groups or strings of amino acid abbreviations are used to represent peptides. Unless otherwise indicated, peptides are shown with the N-terminus on the left, and sequences are described from the N-terminus toward the C-terminus.

[0027] "Retroinverso" peptides have an amino acid sequence that is the reverse of the reference L-amino acid sequence, and are composed entirely of D-amino acids (reversing the chirality of the α-center of the amino acid subunits), which helps maintain a side-chain topology similar to that of the original L-amino acid peptide.

[0028] An "isolated" molecule refers to a molecule that exists in a form not found in nature, including purified molecules.

[0029] "Binding affinity" generally refers to the sum of the non-covalent interactions between a single binding site of a molecule and its binding partner (e.g., a receptor and its ligand, an antibody and its antigen, or two monomers forming a dimer). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects the one-to-one interaction between the elements of a binding pair. The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (K). D ) can be expressed as. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity binding partners generally tend to bind slowly and dissociate quickly, while high-affinity binding partners generally tend to bind faster and maintain their binding for longer periods.

[0030] The affinity or binding activity of a molecule to its binding partner can be experimentally determined using any method known in the art, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or kinetics (e.g., KINEXA®, BIACORE®, or OCTET® analysis). Direct binding assays and competitive binding assays can be readily employed (see, for example, Berzofsky et al., “Antibody-Antigen Interactions,” in Fundamental Immunology, Paul, WEed., Raven Press: New York, NY (1984), Kuby, Immunology, WH Freeman and Company: New York, NY (1992)). The measured affinity of a particular binding pair interaction may vary when measured under different conditions (e.g., salt concentration, pH, temperature). Therefore, affinity and other binding parameters (e.g., K D Or Kd, K on , K off The measurement of the binding partner is performed using standardized solutions and standardized buffers of the binding partner, as is known in the art.

[0031] An "activator" is an ingredient intended to provide biological activity. An activator may be associated with one or more other ingredients. An activator that is a peptide may also be called an "active peptide."

[0032] An "effective dose" of an activator is an amount sufficient to accomplish the specific purpose stated.

[0033] The term "pharmaceutical composition" refers to a preparation that is in a form that can effectively exert the biological activity of an active ingredient and that does not contain any further ingredients that are unacceptably toxic to the subject to which the composition is administered. Such compositions may be sterile and may contain a pharmaceutically acceptable carrier such as physiological saline. A suitable pharmaceutical composition may contain one or more of the following: a buffer (e.g., acetic acid, phosphoric acid, or citrate buffer), a surfactant (e.g., polysorbate), a stabilizer (e.g., polyol or amino acid), a preservative (e.g., sodium benzoate), and / or other conventional solubilizers or dispersants.

[0034] "Subject," "individual," "animal," "patient," or "mammal" refers to any subject, particularly mammals, for whom diagnosis, prognosis, or treatment is desired. Mammal subjects include humans, non-human primates, canids, felines, pigs, bovines, equids, rodents including rats and mice, and rabbits, as well as domestic animals, livestock, sports animals, and laboratory animals.

[0035] Terms such as “to treat” or “to cure” or “to alleviate” or “to reduce” refer to therapeutic measures that cure, slow the progression of, reduce symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder. In a particular embodiment, if a patient exhibits whole-, partial or temporary relief or disappearance of at least one symptom or measurable physical parameter associated with the disease or disorder, the subject is considered to have been successfully “treated” for the disease or disorder.

[0036] A "control patient" is a subject who has not received treatment according to the present invention. A "control group" or "control patient group" is a group of subjects who have not received treatment according to the present invention. A control patient or subject in the control group has the same disease or disorder as the subject being compared to the control patient or control group. For example, the clinical outcome of a cancer patient receiving the pharmaceutical composition or method of the present invention is compared to the mean (median) outcome of a subject with the same type and / or stage of cancer who did not receive the pharmaceutical composition or method of the present invention. In some embodiments, patients in the control patient or control group receive treatment other than the treatment according to the present invention, e.g., standard treatment.

[0037] An "antagonist" is a substance that interferes with, blocks, inhibits, neutralizes, or reduces the biological activity or action of another molecule, such as a receptor or ligand.

[0038] The terms “inhibit,” “block,” and “suppress” are used interchangeably and refer to any statistically significant reduction in occurrence or activity, including 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% in activity or occurrence. An “inhibitor” is a molecule, factor, or substance that causes a statistically significant reduction in the occurrence or activity of a process, pathway, or molecule.

[0039] A "neoplastic cell" or "neoplasm" is typically a result of some form of mutation / transformation, leading to abnormal proliferation compared to normal cells or tissues of the same type. Neoplasms include morphological irregularities as well as pathological proliferation. Neoplastic cells can be benign or malignant. Malignant neoplasms, i.e., cancer, are distinguished from benign neoplasms by their loss of cell differentiation and orientation, and their invasive and metastatic characteristics.

[0040] A “tumor” or “solid tumor” is a mass of neoplastic cells, such as cancer cells. The terms “progressive,” “metastatic,” and “progressive / metastatic” are used interchangeably to describe cancer in which malignant cells have spread from the original tumor to another location in the patient's body, such as another organ.

[0041] Pharmacokinetics, or PK, refers to the study of how an administered substance is processed by the body. PK measurements include how a substance enters the bloodstream (absorption), how it is dispersed or distributed throughout the body's fluids and tissues (distribution), how it is recognized and transformed by the body (metabolism), and how it is removed from the body (excretion). This substance could be a drug, such as ST316. Pharmacokinetics can be evaluated using various indicators, many of which are calculated based on the amount of the substance in the body (e.g., in plasma) at various points in time after administration.

[0042] The time after administration is measured from T0, which is the time when the single dose of the substance is administered. If the administration of the pharmaceutical composition is interrupted and resumed one or more times during the entire infusion period, T0 is the start of the entire infusion period.

[0043] "Total infusion duration" or "total infusion period" refers to the time from the start of a single dose of the pharmaceutical composition to the end of administration, and includes both the infusion period and the interruption period.

[0044] "C max " is a pharmacokinetic indicator that refers to the highest plasma concentration of a substance after administration.

[0045] "T max "This means that after the start of administration of the substance (T0), C max This is a pharmacokinetic indicator that refers to the time it takes to reach a certain state.

[0046] "T last "Times of 100%" is a pharmacokinetic indicator that refers to the time when the last quantifiable concentration of a substance was measured.

[0047] 「AUC」 or 「area under the curve」 is a pharmacokinetic parameter that describes the change in the concentration of a substance in plasma over time. AUC can be calculated for different periods, such as from time zero to a specific time t (AUC t or AUC 0~t ), from time zero to infinity (AUC ∞ or AUC 0~∞ ), etc.

[0048] 「Elimination half-life」 or 「half-life」 or 「t 1 / 2 」 is a pharmacokinetic parameter that refers to the time required for the concentration of a substance to reach half of its original value.

[0049] 「Clearance」 is a pharmacokinetic parameter that refers to the volume of plasma from which a substance is removed per unit time.

[0050] 「V z 」 is a pharmacokinetic parameter that refers to the volume of distribution in the terminal phase.

[0051] II. Peptides and Compositions β-Catenin and Antagonist Peptides The Wnt / β-catenin pathway is a validated target for cancer treatment. Constitutive activation of the Wnt / β-catenin signaling pathway is involved in carcinogenesis, tumor progression, and poor prognosis in many cancers (Shang 2017). The complex formed by the interaction between β-catenin and its co-activator BCL9 drives cancer gene expression in multiple cancers through abnormal Wnt pathway signaling. Disruption of the β-catenin / BCL9 complex has been shown to suppress oncogenic Wnt / β-catenin transcription without affecting the homeostasis maintenance function of β-catenin (Takada 2012).

[0052] The interaction between β-catenin and BCL9 was previously considered a “drug-unavailable” target because small molecules cannot inhibit complex formation and antibodies cannot access the cytoplasm or nucleus to disrupt the interaction (Takada 2012). The peptide antagonism of β-catenin differs from these other approaches because it can specifically target the interaction between β-catenin and BCL9, exhibiting selective toxicity to tumor cells in which these proteins are overactivated, amplified, and / or mutated.

[0053] In some embodiments, the methods of the present invention involve treating patients with solid tumors with an effective amount of a β-catenin peptide antagonist. Preferably, the β-catenin peptide antagonist inhibits the interaction between β-catenin and BCL9. The β-catenin peptide antagonist can be rationally designed based, for example, on the native sequence of BCL9 modified to enhance the electrostatic interaction between the peptide antagonist and β-catenin. In particular, peptide antagonists for the interaction between β-catenin and BCL9 can be designed to interact with the first armadillo repeat (ARM-1) domain of β-catenin, which is derived from homology domain 2 (HD2) of BCL9 and is utilized by BCL9 but not by other β-catenin binding partners. WO2021 / 007158 provides numerous examples of such peptides.

[0054] The ability of peptides based on wild-type BCL9 to antagonize β-catenin activity, for example, to inhibit the interaction between β-catenin and BCL9, can be measured by the method described herein, e.g., Example 1. The cytotoxic activity of β-catenin peptide antagonists can be measured in vitro by known assays and / or in vivo using known tumor models. For example, WO2019 / 136125 and WO2021 / 007158 describe such assays and models.

[0055] β-catenin peptide antagonists may be cell-permeable peptides. In one embodiment, the peptide contains a cell-permeable domain. Numerous cell-permeable peptide sequences have been described and characterized in the literature (see WO2019 / 136125). In one embodiment, the peptide is a cyclic peptide. For example, cyclized peptides using hydrocarbon staples (Bernal 2007, Bird 2017) or other cyclization methods known in the art can enter cells via passive diffusion, endocytosis / endosomal escape, or other mechanisms (Dougherty 2019). Peptides may also be delivered to cells via mechanisms utilizing cell receptors, such as integrin-targeted RGD-like sequences. Alternatively, peptides may be delivered to cells encapsulated in vesicles such as exosomes or liposomes, or in micelles.

[0056] β-catenin peptide antagonists may have modified N-terminus and / or modified C-terminus. For example, peptide antagonists may optionally contain an N-terminal acetyl group and / or a C-terminal amide group. Other examples of optional N-terminal and / or C-terminal groups include linear or cyclic C2-C2 groups. 18 Examples include hydrophobic groups such as aliphatic or aromatic hydrocarbons, naphthyl groups, phenyl groups, octanoyl groups, and valeryl groups including isovaleryl groups. In some embodiments, the peptide antagonist includes a linker or spacer between the peptide and the hydrophobic group. Examples of such linkers or spacers include aminohexanoic acid, β-alanine, substituted alkyl groups, substituted cycloalkyl groups, and polyethylene glycol.

[0057] ST316 is a novel 25-amino acid peptide antagonist for the interaction between β-catenin and BCL9. ST316 is composed entirely of D-amino acids and is highly stable in the presence of proteases. Due to its resistance to protease degradation, ST316 has a long plasma half-life and should not be processed and presented by antigen-presenting cells, thereby avoiding stimulation of anti-drug antibody (ADA) responses.

[0058] ST316 consists of two domains: (i) a 15-amino acid N-terminal active domain derived from homology domain 2 (HD2) of BCL9, designed to interact with the first armadillo repeat (ARM-1) domain of β-catenin (a site utilized by BCL9 but not by other β-catenin binding partners); and (ii) a 10-amino acid C-terminal domain that enhances solubility and cell permeability. The D-amino acid sequence of ST316 is: [ka] The N-terminal active domain is shown in bold, and the C-terminal cell-permeable domain is shown in italics. ST316 also contains an N-terminal octanoyl group.

[0059] ST316 represents a first well-tolerated approach to drug-modifying the Wnt pathway by specifically binding to the N-terminus of β-catenin, in which case β-catenin interacts with BCL9, a cofactor that is not required for normal physiological function but is necessary for β-catenin's pathological, oncogenic, and immunosuppressive functions.

[0060] Composition and administration In certain embodiments, the present invention provides compositions comprising a β-catenin peptide antagonist such as ST316, for example, pharmaceutical compositions. The pharmaceutical compositions are suitable for parenteral administration. In one embodiment, ST316 may be in the form of a salt. Preferably, the composition comprises one or more carriers, diluents, excipients, or other additives. For example, the composition may comprise one or more bulking agents, one or more buffers, one or more pH adjusters, and / or one or more diluents.

[0061] Aspects of the present invention relate to a method for administering a β-catenin peptide antagonist to a subject. The β-catenin peptide antagonist is administered parenterally. Parenteral administration routes include intravenous (IV), intramuscular, intraperitoneal, intrathecal, and subcutaneous. In a preferred embodiment, a pharmaceutical composition comprising ST316 is administered by IV infusion. The pharmaceutical composition can be provided in an IV solution comprising, for example, physiological saline (0.9%), half-physical saline (0.45%), or 5% glucose aqueous solution (D5W). In one embodiment, the β-catenin peptide antagonist is provided in a vial as a sterile lyophilized solid and is redissolved, for example, in sterile water for injection (USP). The redissolved peptide can then be diluted for IV administration.

[0062] Beta-catenin peptide antagonists can be administered based on the patient's body weight. Beta-catenin peptide antagonists such as ST316 can be administered to patients in doses ranging from approximately 0.25 mg / kg to approximately 12 mg / kg. In certain embodiments, ST316 is administered in doses of approximately 0.25 mg / kg, approximately 0.5 mg / kg, approximately 0.75 mg / kg, approximately 1 mg / kg, approximately 1.5 mg / kg, approximately 2 mg / kg, approximately 2.5 mg / kg, approximately 3 mg / kg, approximately 3.5 mg / kg, approximately 4 mg / kg, approximately 4.5 mg / kg, approximately 5 mg / kg, approximately 5.5 mg / kg, approximately 6 mg / kg, approximately 6.5 mg / kg, approximately 7 mg / kg, approximately 7.5 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, approximately 10 mg / kg, approximately 11 mg / kg, or approximately 12 mg / kg. These amounts can also function as endpoints in the dose range, for example, approximately 0.75 mg / kg to approximately 7 mg / kg, or approximately 2 mg / kg to approximately 4 mg / kg.

[0063] Alternatively, β-catenin peptide antagonists can be administered in fixed doses. β-catenin peptide antagonists such as ST316 can be administered to patients in doses ranging from approximately 500 mg to approximately 1500 mg. In certain embodiments, ST316 is administered in doses of approximately 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, or 1500 mg. These amounts can also serve as endpoints to the administered dose range, for example, approximately 600 mg to 1100 mg, or approximately 750 mg to 900 mg.

[0064] In embodiments of the present invention, the pharmaceutical composition is administered to the subject by intravenous infusion, in which case the total infusion duration is approximately 360 minutes or less. In some embodiments, the total infusion duration is approximately 30 minutes to approximately 240 minutes. For example, the total infusion duration may be 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, or 180 minutes, or intermediate times such as 45 minutes or 100 minutes. In certain embodiments, the total infusion duration is approximately 60 minutes to approximately 90 minutes, or approximately 60 minutes to approximately 120 minutes, or approximately 90 minutes to approximately 120 minutes, or approximately 60 minutes to approximately 180 minutes.

[0065] In some embodiments, the injection of the pharmaceutical composition may be interrupted, that is, temporarily stopped and then resumed. The duration of the interruption may vary, for example, to about 15 minutes or less, or about 30 minutes or less, or about 1 hour or less, or about 2 hours or less, or about 3 hours or less, or about 4 hours or less.

[0066] In some embodiments, the pharmaceutical composition may be administered with one or more secondary agents intended to block histamine release, prevent or improve infusion-related reactions (IRRs), reduce fever or inflammation, and / or alleviate itching and / or urticaria. IRRs may include, for example, erythema, fever, chills / shivering, tachycardia, tachypnea, hypotension, and bronchospasm. One or more secondary agents may be administered simultaneously with, before, and / or after, the administration of the pharmaceutical composition. One or more secondary agents may be administered in a composition separate from the pharmaceutical composition, or in combination with the pharmaceutical composition. Furthermore, one or more secondary agents may be administered via the same route as the pharmaceutical composition, or via a different route (e.g., orally).

[0067] Examples of one or more secondary agents to be administered with a pharmaceutical composition include antihistamines (H1 antagonists, H2 antagonists, and mast cell degranulation inhibitors (e.g., acribastine, astemizole, azatadine, azelastine, bepotastine, bromopheniramine, bruflorin, cetirizine, chlorzoxazone, chlorpheniramine, cromolyn, cyproheptadine, desloratadine, dexbrompheniramine, diphenhydramine, doxantrozole, epinastine, etodoroxidine, famotidine, fexofenadine, forskolin, hydroxyzine, isoproterenol, ketotifen, levocetirizine, loratadine, rhodoxamide, mequitazine, methodyl Examples of such drugs include, but are not limited to, din, mizolastine, nedocromil, olopatadine, oxatomide, pemirolastine, pimecrolimus, pirbuterol, pizotifen, proxichromil, ranitidine, terfenadine, terbutaline); leukotriene inhibitors (e.g., montelukast, zafirlukast, zilote); nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., ibuprofen, naproxen, aspirin); acetaminophen / paracetamol; corticosteroids (e.g., hydrocortisone, dexamethasone, prednisone, prednisolone); antiemetics (e.g., prochlorperazine, ondansetron); and physiological saline and / or electrolytes. In a preferred embodiment, the secondary agent is an antihistamine such as chlorpheniramine or diphenhydramine. In certain embodiments, the secondary agent is selected from the group consisting of acetaminophen / paracetamol, H1 antagonists, H2 antagonists, montelukast, antiemetics, and combinations thereof.

[0068] In certain embodiments, the secondary agent is administered during the administration of the pharmaceutical composition, for example, during the infusion period. In certain embodiments, the secondary agent, such as an antihistamine, is administered to the subject before the administration of the pharmaceutical composition, for example, within about 7 days, or within about 6 days, or within about 5 days, or within about 4 days, or within about 72 hours, or about 48 hours, or about 24 hours, or about 8 to 12 hours, or about 6 to 8 hours, or about 4 to 6 hours, or about 2 to 4 hours, or about 1 to 2 hours, or within about 1 hour, or immediately before. In some embodiments, the secondary agent is administered within 24 hours after the administration of the pharmaceutical composition. For example, the secondary agent may be administered immediately after the completion of administration of the pharmaceutical composition, about 0.5 to 1 hour later, about 1 to 2 hours later, about 2 to 4 hours later, about 4 to 6 hours later, about 6 to 8 hours later, about 8 to 12 hours later, or about 24 hours later.

[0069] Secondary agents can be administered multiple times before, during, and / or after administration of the pharmaceutical composition. Combinations of secondary agents can be administered simultaneously or at different times. For example, an antihistamine may be administered before the pharmaceutical composition, and a corticosteroid after the pharmaceutical composition.

[0070] Peptide antagonists are typically administered to patients multiple times. β-catenin peptide antagonists can be administered once a week, once every two weeks, once every three weeks, once every four weeks, or a combination of these intervals. The total treatment duration may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

[0071] In one embodiment, administration of a β-catenin peptide antagonist may be performed once a week for a period of at least one, two, or three weeks (i.e., one, two, or three doses), six weeks (i.e., six doses), nine weeks, twelve weeks, three months, six months, nine months, or twelve months. In another embodiment, administration may be performed once every two weeks for a period of at least four weeks (i.e., two doses), eight weeks (i.e., four doses), twelve weeks, three months, six months, nine months, or twelve months. In some embodiments, a patient may receive a β-catenin peptide antagonist once a week for a period of at least one, two, three, six, nine, twelve weeks, three months, six months, nine months, or twelve months, followed by administration once every two weeks for a period of at least four weeks, eight, twelve weeks, three months, six months, nine months, or twelve months.

[0072] Neoadjuvant therapy can be administered to reduce the size or extent of a tumor, or to make subsequent treatment easier, more effective, or less invasive. In certain embodiments, β-catenin peptide antagonists can be administered as neoadjuvants, for example, before surgery and / or radiation therapy, either as monotherapy or in combination with a secondary agent.

[0073] The pharmacokinetics (PK) and pharmacodynamics of β-catenin antagonists can be evaluated by standard methods and as described in the examples. In some embodiments, the β-catenin antagonist is ST316.

[0074] III. Treatment method The subjects requiring treatment by the method of the present invention are patients diagnosed with solid tumors. For example, subjects may have unresectable, progressive and metastatic solid tumors. In one embodiment, the tumor has one or more abnormalities in the Wnt / β-catenin signaling pathway. In non-limiting examples, one or more abnormalities in the Wnt / β-catenin signaling pathway may include one or more mutations in one or more of the following: adenomatous polyposis (APC), APC membrane mobilization protein 1 (AMER1), exin 2, β-catenin, ring finger protein 43 (RNF43), and / or T cell factor 7 (TCF7).

[0075] In some embodiments, the subjects have locally advanced or metastatic breast cancer (BC), colorectal cancer (CRC), cholangiocarcinoma (CCA), endometrial cancer, hepatocellular carcinoma (HCC), melanoma, non-small cell lung cancer (NSCLC), ovarian cancer (OC), pancreatic adenocarcinoma (PDAC), or synovial sarcoma (SS). In certain embodiments, the disease is resistant to or intolerant to other available therapies that would affect survival. In certain embodiments of the present invention, administration of ST316 can result in inhibiting tumor growth, reducing tumor volume, or a combination of both.

[0076] In one embodiment, a patient is diagnosed with BC. More than 50% of BC tumors have been shown to have abnormalities in Wnt signaling (Lin 2000). Triple-negative breast cancer (TNBC) is a particularly aggressive form of BC, generally with a poorer prognosis compared to other BC subtypes, accounting for 10–15% of all BC cases (Won 2020). TNBC patients lack hormone receptors and human epidermal growth factor receptor 2 (HER2), and therefore do not respond to hormone-based and targeted therapies. Chemotherapy remains the standard treatment despite its limited benefits. Thus, the medical need for more effective drugs in this clinical context remains very high. In TNBC, mutations in the Wnt pathway are known to drive tumorigenesis and metastasis, leading to poor clinical outcomes and increasing the likelihood of developing metastases to the lungs and brain (Pohl 2017).

[0077] In one embodiment, the patient is diagnosed with CRC. CRC is one of the most diagnosed cancers globally, ranking second in women and third in men worldwide (Sung 2021). Approximately 95% of patients with metastatic CRC (mCRC) have microsatellite stable disease (MSS) and are ineligible for treatment with immune checkpoint inhibitors. This patient population relies on moderately effective chemotherapy and targeted therapy regimens, which represents a significant unmet medical need. More than 90% of CRC tumors have been shown to have abnormalities in Wnt signaling. These have been demonstrated to drive disease onset, progression, and metastasis (Nie 2020).

[0078] In one embodiment, the patient is diagnosed with CCA. CCA, also known as biliary tract cancer, refers to a heterogeneous group of gastrointestinal cancers that originate from the bile ducts, gallbladder, or ampulla of Vater. These cancers are often difficult to diagnose due to their anatomical location and the lack or nonspecificity of symptoms. Consequently, more than 75% of CCA patients present with progressive, unresectable disease (Lamarca 2014, Takahashi 2013). Progressive, unresectable CCA is an area of ​​unmet medical needs due to its highly aggressive nature, limited treatment options, and particularly poor prognosis after first-line treatment (generally gemcitabine and cisplatin combination) (Valle 2010). Clinical and preclinical studies suggest a crucial role for the activation of the Wnt / β-catenin signaling pathway in the induction and progression of CCA (Zhang 2020).

[0079] In one embodiment, the patient is diagnosed with occidental osteoarthritis (OC). OC is the fifth leading cause of cancer death in women and the leading cause of death among gynecological cancers (ACS 2022). Two-thirds of OC patients are diagnosed at stages 3 and 4, which contributes to the relatively low 5-year survival rates for stage 3 and 4 tumors, 39% and 17%, respectively (NCCN 2022). Advanced OC is an area of ​​unmet medical need due to its highly aggressive nature, limited treatment options, and poor prognosis. Wnt activity has been shown to correlate with histological grade, epithelial-mesenchymal transition, chemotherapy resistance, and poor prognosis in OC (Teeuwssen 2019).

[0080] In one embodiment, the patient is diagnosed with endometrial cancer. Endometrial cancer is the most common gynecological cancer in developed industrial countries and is increasing in women of all ages, at least in part, due to the increasing incidence of obesity and associated hyperinsulinemia (Moore 2017, Parrish 2022). Older women have a higher risk of recurrence and a higher mortality rate (Moore 2017). Beta-catenin mutations are associated with worse outcomes in endometrial cancer patients, including increased recurrence rates and decreased survival rates (Parrish 2022).

[0081] The effectiveness of treatment can be evaluated by one or more known indicators. For example, patients treated with the method of the present invention may experience outcomes including increased overall survival, improved progression-free survival, improved duration of response, increased duration of remission, reduced risk of relapse, and / or improved tumor response to treatment with β-catenin peptide antagonists, compared to the same outcomes in patients who did not receive the method of the present invention, i.e., control patients. Outcomes in patients treated with the method of the present invention can be compared, for example, to the median outcome in a control patient population. The control patient population may be administered regimens selected from, for example, placebo, surgery, radiation therapy, chemotherapy, immunotherapy, hormone-based therapy, targeted therapy, and combinations thereof. The comparison can be statistically analyzed, for example, using the Wilcoxon signed-rank test or the Kaplan-Meier method.

[0082] In one embodiment, the outcomes of patients receiving a β-catenin peptide antagonist such as ST316 in combination with standard treatment on an optional basis are compared with the median outcomes of control patients receiving a placebo. In another embodiment, the outcomes of patients receiving a β-catenin peptide antagonist such as ST316 are compared with the median outcomes of control patients receiving standard treatment.

[0083] The tumor response to treatment can be evaluated, for example, by measuring tumor volume and / or tumor regression. The response to treatment is evaluated by comparing one or more efficacy indicators after the treatment regimen to baseline, for example, before treatment with ST316. The baseline evaluation is preferably performed within 24, 48, or 72 hours, or within 1, 2, 3, or 4 weeks, of the first treatment with a β-catenin peptide antagonist. In a preferred embodiment, the baseline evaluation is performed within one week of the first ST316 treatment.

[0084] "Tumor volume" refers to the total mass or total size of cancerous tissue in the patient's body. Tumor response can be evaluated by indicators including objective response rate (including partial and / or complete response), disease stabilization rate, disease control rate, duration of disease control, and duration of response. These parameters can be determined, for example, by the new guidelines for evaluating treatment efficacy in solid tumors (RECIST 1.1) (Eisenhauer 2009).

[0085] Objective response rates evaluate the reduction in tumor size, such as tumor diameter, which can be determined by clinical examination and / or imaging. If a patient has multiple tumors, tumor size can optionally be expressed as the average diameter of all tumors or as the sum of the diameters of all tumors. Superficial tumors can be measured clinically, for example, using calipers or by measurement with photographs and rulers. Imaging methods typically include computed tomography (CT) with contrast agents, X-ray, magnetic resonance imaging (MRI), and positron emission tomography (PET) (e.g., (18)F-fluorodeoxyglucose PET). In one preferred embodiment, CT is used to evaluate the tumor response, for example, in BC patients or melanoma patients. Accordingly, in one embodiment, the present invention provides a method for reducing tumor volume, i.e., tumor mass and / or tumor size, in a patient, comprising administering a β-catenin peptide antagonist (e.g., ST316) to the patient. The reduction in tumor volume is measured relative to baseline.

[0086] In certain embodiments, particularly those in which evaluation is performed according to RECIST 1.1, the disease control rate is defined as the best of the following levels of tumor response: complete response (CR), i.e., disappearance of the tumor(s); partial response (PR), i.e., reduction of 30% or more in tumor(s) size; stable disease (SD), i.e., a change in tumor size of less than 30% or less than 20%; or disease progression, i.e., an increase of 20% or more in tumor size and / or the appearance of new lesions. Patients treated by the methods of the present invention may experience CR, PR, or SD.

[0087] The duration of disease control is the length of time from achieving a response (CR or PR) or stable disease (SD) to disease progression. The duration of response is the length of time from achieving a response to disease progression, i.e., the period during which the tumor does not grow or expand, or the period until death. In patients receiving ST316 treatment, the duration of response may be, for example, at least 4, 6, 8, 10, or 12 weeks, at least 4, 6, 8, 10, 12, 16, 18, or 24 months, or at least 3, 4, or 5 years. Patients treated by the method of the present invention may experience an increase in the duration of disease control or an increase in the duration of response. Accordingly, in one embodiment, the present invention provides a method for extending the duration of disease control or the duration of response in a patient, comprising administering a β-catenin peptide antagonist (such as ST316) to the patient. The increase in the duration of disease control or the duration of response is measured against the median duration of disease control or the duration of response in the control population, respectively.

[0088] Survival time can be evaluated as overall survival, i.e., the length of time a patient is alive; progression-free survival, i.e., the length of time a patient is treated without disease progression or worsening; or event-free survival, i.e., the length of time a patient remains free from complications or adverse events such as relapse or disease progression. Survival time is calculated from the date treatment is initiated. Overall survival, median overall survival, progression-free survival, median progression-free survival, event-free survival, and median event-free survival can be calculated, for example, by Kaplan-Meier analysis based on the response to treatment.

[0089] Accordingly, in one embodiment, the present invention provides a method for extending overall survival in a patient, comprising administering a β-catenin peptide antagonist (such as ST316) to the patient. The increase in overall survival can be measured relative to the median overall survival in a control population. Alternatively, an increase in the percentage of patients surviving for a predetermined period (e.g., 6 months or 12 months) compared to the control population indicates an increase in overall survival.

[0090] In another embodiment, the present invention provides a method for extending progression-free survival in a patient, comprising administering a β-catenin peptide antagonist (such as ST316) to the patient. The increase in progression-free survival can be measured relative to the median progression-free survival in a control population. Alternatively, an increase in the percentage of patients who remain relapse-free for a predetermined period (e.g., 6 or 12 months) compared to a control population indicates an increase in progression-free survival.

[0091] In a further embodiment, the present invention provides a method for extending event-free survival in a patient, comprising administering a β-catenin peptide antagonist (such as ST316) to the patient. The increase in event-free survival is measured relative to the median event-free survival in a control population. Alternatively, an increase in the percentage of patients who are event-free for a predetermined period (e.g., 6 or 12 months) compared to a control population indicates an increase in event-free survival.

[0092] A patient has been successfully treated according to the method of the present invention if, after administration of a β-316 peptide antagonist (such as ST316), they experience or demonstrate at least one of the following outcomes: - Tumor (or at least one tumor if multiple tumors are present at baseline) is undetectable. - Tumor size reduced by at least approximately 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to baseline. - No significant increase in tumor size compared to baseline (e.g., less than 20% or less than 30%). -Optionally, if the patient group showed a significant extension of the duration of response compared to the median duration of response in the control patient group, -Optionally, if a significant extension of disease control duration is observed compared to the median disease control duration in the control patient population, -Optionally, a significant extension of progression-free survival compared to the median progression-free survival of the control patient population. - A significant extension of overall survival compared to the median overall survival of the control patient group, on an optional basis.

[0093] IV. Preparation method β-catenin peptide antagonists can be chemically synthesized, for example, using solid-phase peptide synthesis, liquid-phase peptide synthesis, or a combination of both. Optionally, they may also be synthesized as peptide fragments to be subsequently chemically or enzymatically linked.

[0094] Alternatively, β-catenin peptide antagonists can be expressed using recombinant methods. For example, the nucleic acid molecule encoding ST316 can be constructed chemically using an oligonucleotide synthesizer. The nucleic acid molecule can be designed based on the amino acid sequence of ST316 and the selection of codons favorable in the host cell that produces recombinant ST316. Nucleic acid molecules encoding β-catenin peptide antagonists (such as ST316) can be synthesized by applying standard methods.

[0095] Once prepared, the nucleic acid encoding the peptide can be inserted into an expression vector and operably ligated to an expression regulatory sequence appropriate for peptide expression in the desired host. To obtain high levels of peptide expression, the nucleic acid can be operably ligated or associated to functional transcriptional and translational expression regulatory sequences in a selected expression host.

[0096] Those skilled in the art can utilize a wide variety of expression host / vector combinations. Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression regulatory sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids such as E. coli plasmids (pCR1, pBR322, pMB9 and their derivatives, etc.), plasmids with a broader host range (such as M13), and filamentous single-stranded DNA phages.

[0097] Suitable host cells include prokaryotes, yeasts, insects, or higher eukaryotic cells under the control of an appropriate promoter. Prokaryotes include Gram-negative or Gram-positive bacteria, such as E. coli or Bacillus species. Higher eukaryotic cells may be established or mammalian cell lines, 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. Cell-free translation systems can also be used.

[0098] Peptides can be purified using methods including, for example, reversed-phase high-performance liquid chromatography (RP-HPLC), multi-column countercurrent solvent gradient chromatography (MCSGP), hydrophobic interaction chromatography (HIC), and ion exchange chromatography. [Examples]

[0099] Embodiments of this disclosure can be further defined by reference to the following non-limiting embodiments. It will be apparent to those skilled in the art that many modifications to both materials and methods can be made without departing from the scope of this disclosure.

[0100] Example 1. ST316 binds to β-catenin and inhibits its interaction with BCL9. The inventors used a fluorescence polarization assay to measure the binding between ST316 conjugated to FITC and recombinant β-catenin. Full-length β-catenin at increased concentrations (0.24 nM to 2000 nM) was incubated with 12.5 nM ST316-FITC at room temperature for 1 hour. Fluorescence polarization was measured using a SpectraMax® plate reader. ST316 showed approximately 126.1 nM K D It binds to β-catenin Arm1 (Figure 1). The binding affinity between BCL9 and β-catenin is approximately 200 nM, indicating that ST316 binds to β-catenin with an affinity equal to or greater than that of the endogenous ligand.

[0101] The inventors used the ALPHA assay to quantify the interaction between BCL9 and β-catenin. The ALPHA assay was performed in an assay buffer consisting of 20 mM MES (pH 6.5), 60 mM NaCl, 0.01% BSA, 1 mM DTT, and 0.05% Tween-20. ST316 was serially diluted and added to a plate to a final concentration ranging from 50 μM to 6 nM, followed by the addition of 25 nM full-length recombinant β-catenin. The plate was incubated at room temperature for 1 hour. A C-terminally biotinylated BCL9 HD-2 peptide consisting of 26 amino acids was added to each well at a final concentration of 50 nM, followed by the addition of rabbit polyclonal anti-β-catenin antibody (Ab1605) (final concentration 50 ng / mL). Donor beads and acceptor beads (PerkinElmer AlphaScreen Streptavidin Donor Beads 6760007 and Protein A Acceptor Beads 6760136, respectively) were added to the mixture at a final concentration of 10 μg / mL. The plate was shielded from light and incubated at room temperature on a plate locker for 1 hour. The alpha signal was then measured using a SpectraMax® plate reader. The results showed that both ST316 and HD2 peptides produced approximately 1 μM IC50. 50 This demonstrated that it inhibits the interaction between BCL9 and β-catenin (Figure 2).

[0102] Example 2. In vitro effect of ST316 The inventors investigated the effect of ST316 on the nuclear localization of β-catenin in HCT116 cells. Briefly, cells were cultured in T25 culture flasks containing McCoy's 5A medium (containing 10% FBS and Mycozap) and treated with 3 μM ST316 or 5 μM ST316 by vehicle alone for 24 hours. To fractionate the cells, the cells were first collected and then incubated in a lysis buffer containing DTT and a protease inhibitor cocktail. Next, 10 IGEPAL CA-630 solution was added to the cells. The cells were centrifuged and the cytoplasmic fraction of the supernatant was collected. The remaining pellet was resuspended in an extraction buffer containing DTT and a protease inhibitor cocktail. The cells were centrifuged again and the nuclear supernatant was collected and analyzed by Western blotting (Figure 3A). Treatment with 5 μM ST316 significantly reduced the nuclear localization of β-catenin. Figure 3B shows the percentage of β-catenin in the nucleus and cytoplasm in Western blot images quantified by ImageJ analysis software.

[0103] To investigate the effect of ST316 on proteasomal degradation of BCL9, Colo320 colorectal cancer cells were incubated with ST316 for 24 hours, with or without the proteasomal inhibitor MG132. No effect on cell viability was observed. Protein expression was analyzed using the Jess protein detection system. The results are shown in Figures 4A-4D. Similar results were observed in HCT116 cells.

[0104] Example 3. ST316 exhibits an inhibitory effect on cancer cell invasion in vitro. The inventors used an in vitro Boyden chamber assay to investigate the inhibitory effect of ST316 on the invasion of newly generated cells. HCT116 colorectal cancer cells (2 × 10⁶ 5Cells were equilibrated for 2 hours in serum-free cell culture medium within a Matrigel-coated infiltration plate insert. Subsequently, complete growth medium containing 10% fetal bovine serum as a chemoattractant was added to the bottom well of each insert. The cells were then exposed to 1 μM ST316 or a negative control peptide for 24 hours. Under these conditions, no significant effect on cell viability was observed.

[0105] For analysis, the culture medium in the insert was aspirated and the upper layer of the membrane was removed. After fixation with MeOH and staining with crystal violet, the membrane was detached and placed on a glass slide. Images of the membrane containing the infiltrated cells were acquired at 20x magnification using a Nikon Digital Sight 1000 Microscope and a Nikon Eclipse T2S microscope. Cell counts were manually determined using ImageJ. Data represent the mean and standard error of three biological replicates, including at least three representative fields of view. The results are shown in Figures 5A-5B.

[0106] Example 4. Administration of ST316 inhibits tumor growth in vivo. The present inventors have identified an APC that has a mutation in the APC necessary for regulating β-catenin concentration. min / + The effect of ST316 on tumor formation in mice was investigated. APC min / + Mice are prone to developing cachexia and intestinal adenomas. (6-week-old female APC) min / + Mice were administered ST316 at a dose of 10 mg / kg once a week by subcutaneous injection (n=3 in each group). Mice received a total of 10 doses. Body weight was measured three times a week during the study period. After 16 weeks, the mice were euthanized, plasma was collected for cytokine analysis, and the small intestine was collected for adenoma quantification. ST316 treatment reduced adenoma formation by 55% compared to mice that did not receive ST316 treatment (Figure 6A), prevented the development of cachexia (Figure 6B), and increased the expression of pro-inflammatory cytokines CCL2 / CCL4 in the serum (Figure 6C).

[0107] Furthermore, the inventors investigated the effect of ST316 on tumor volume in a subcutaneous tumor model using colorectal cancer cells. Briefly, HCT116 cells (5 × 10) suspended in Matrigel in a 1:1 ratio... 5 ST316 was transplanted into the axilla of female NOD / SCID mice by subcutaneous injection. ST316 was administered at a dose of 5 mg / kg three times a week by subcutaneous injection for three weeks. The administration was performed when the average starting tumor volume was approximately 200 mm². 3 Treatment was initiated 12 days after tumor inoculation. Tumor volume was monitored three times a week. ST316 treatment inhibited tumor growth by 99% compared to the vehicle or control peptide (Figure 7).

[0108] The inventors also investigated the effect of ST316 on tumor volume in a subcutaneous tumor model using triple-negative breast cancer cells. Briefly, 4T1-luc cells (5 × 10⁶) suspended in Matrigel in a 1:1 ratio... 5 ST316 was transplanted into the axilla of female NOD / SCID mice by subcutaneous injection. ST316 was administered at a dose of 5 mg / kg once a week by subcutaneous injection for 8 weeks. Administration was performed when the average starting tumor volume was approximately 100 mm. 3 The treatment was initiated on day 6 after tumor inoculation. Tumor volume was monitored three times a week. ST316 treatment inhibited tumor growth by 84% compared to the control peptide (Figure 8A). Tumors were collected on day 60, and Axin2 expression was analyzed by quantitative PCR (n=3 in each group). The results are shown in Figure 8B.

[0109] Example 5. ST316 is safe and well-tolerated in animal models. Safety pharmacology The potential pharmacological effects of ST316 on the cardiovascular system (miniature pigs), respiratory system (rats and miniature pigs), and central nervous system (CNS) (rats and miniature pigs) were investigated in a 28 / 29-day GLP toxicity study. No ST316-related effects were observed on body weight, food intake, ophthalmic examination, respiratory system, or CNS in rats or miniature pigs. Furthermore, no ST316-related effects were observed in the Functional Observational Balance (FOB) in rats, and no ST316-related effects were observed in electrocardiogram (ECG) rhythm, morphology, or quantitative measurements of QRS, RR, PR, QT, and QTcF intervals in miniature pigs.

[0110] ADA formation was not detected in a 10-week non-GLP toxicity study using C57BL / 6 mice with weekly administration, or in a 4-week GLP toxicity study using miniature pigs with weekly administration (5 doses). In addition, the eligibility assay in the 4-week GLP toxicity study using rats with weekly administration (5 doses) was within a 5% error range. One positive sample was observed in the GLP miniature pig study (96 samples total), and two positive samples (1%) (198 samples total) were observed in the GLP rat study at a 10 mg / kg dose level derived from control animals not exposed to ST316.

[0111] Tolerance In GLP toxicity studies, findings in both rats and miniature pigs were largely limited to the injection site. No findings clearly related to ST316 in animals administered ST316 via catheter were considered harmful. No pathological findings related to the test substance were observed in miniature pigs.

[0112] Evaluation of false allergic reactions The dose-dependent response (DLT) associated with ST316 exposure in MTD studies was a dose-dependent pseudoallergic infusion-related response (IRR) observed in mice, rats, and miniature pigs. Clinical signs of pseudoallergic reactions in mice may include decreased body temperature, decreased activity, and piloerection. Nonclinical toxicity studies of intravenously administered ST316 were characterized by 29-day GLP toxicity studies in rats and 28-day GLP toxicity studies in miniature pigs, which were supported by same-species maximum tolerated dose (MTD) and 7-day repeated dose-setting (DRF) studies.

[0113] Toxicity studies to characterize the mechanism of pseudoallergic IRR in ST316 demonstrated that the ST316 response is mediated by mast cell activation and histamine release, and is mitigated by prior treatment with antihistamines (ranitidine and pyriramine) and / or leukotriene receptor antagonists (montelukast).

[0114] Example 6: Administration of ST316 to patients with solid tumors An open-label, two-part Phase I-II trial is being conducted to determine the safety, tolerability, pharmacokinetics (PK), pharmacodynamics, and proof-of-concept efficacy of intravenously administered ST316 in subjects with specific advanced solid tumors likely to have abnormalities in the Wnt / β-catenin signaling pathway. Abnormalities in the Wnt / β-catenin pathway are a suitable target due to their impact on carcinogenesis, tumor progression, and prognosis of the tumor types selected in this trial, as well as their effects on the immune components of the tumor microenvironment. Furthermore, non-clinical studies have demonstrated ST316-specific antagonistic activity of β-catenin and potent in vitro / in vivo activity in breast and colorectal cancer models. This trial utilizes a novel peptide drug with a new mechanism of action in subjects that have exhausted standard therapies. This trial consists of two phases: a Phase I dose-escalation / regime exploration phase and a Phase II expansion phase.

[0115] Dose escalation phase The dose escalation phase employs a standard approach to evaluate safety, tolerability, and the selection of the recommended Phase II dose in a wide range of advanced, unresectable and metastatic solid tumors, including subjects diagnosed with locally advanced or metastatic BC, CCA, CRC, endometrial cancer, HCC, melanoma, NSCLC, OC, PDAC, or SS, where the disease is refractory or intolerant to all available therapies that would affect survival. Table 1 shows the study design for the dose escalation phase. [Table 1]

[0116] ST316 is administered intravenously. The dose-escalation cohort will be recruited using a standard 3+3 design. The infusion duration for the initial dose will be 60–180 minutes for all subjects, and this infusion duration may be modified in subsequent doses, at least partially based on the presence or absence of IRR.

[0117] The dose cohorts consist of 0.5, 1, 2, 4, 8, and 12 mg / kg once weekly (QW), with optional dose cohorts of 0.25, 0.75, 1.5, 3, and 6 mg / kg, as well as a fixed dose. One treatment cycle consists of 21 days of once-weekly administration.

[0118] Treatment will continue until disease progression, withdrawal of consent, or lack of clinical benefit as determined by the treating physician. Patients may continue treatment after progression if they are clinically stable. If subsequent imaging shows a decrease or stabilization of tumor volume compared to the initial scan showing PD, treatment will continue as planned. If subsequent imaging confirms PD, treatment will be discontinued. When determining whether tumor volume has increased, stabilized, or decreased, the principal investigator will consider all target and non-target lesions.

[0119] The National Cancer Institute's Common Terminology Criteria for Adverse Events (CTCAE) v5.0 will be used to grade adverse events. Safety assessments will include adverse events, serious adverse events, physical examination, vital signs measurement, Eastern Cooperative Oncology Group (ECOG) status, clinical safety laboratory assessments (hematology, serology, and liver function panels, coagulation, and urinalysis), and electrocardiograms. All subjects will be followed up for safety for at least 30 days after the last dose of ST316.

[0120] To mitigate potential IRR, subjects should be pretreated with (i) daily montelukast (10 mg orally) from at least two days before the first dose of ST316 until the day of administration, and (ii) with both H1 and H2 antagonists at least 30 minutes before the start of infusion on the day of ST316 administration. When using oral antihistamines, the expected C max It is administered early based on the following criteria. Preferred H1 / H2 antagonists include famotidine (20 mg orally or 20-40 mg intravenously), as well as chlorphenamine (10 mg intravenously / oral) or diphenhydramine (50 mg orally / intravenously).

[0121] The subjects are monitored for IRR 24 hours after the first ST316 infusion, 4 hours after the second infusion, and 2 hours after subsequent infusions. Treatment of IRR includes, for example, pausing and slowing down infusions, and / or administering antihistamines such as diphenhydramine, leukotriene receptor antagonists, acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs), intravenous fluids, antiemetics, oxygen, bronchodilators, and corticosteroids if other means are insufficient. Table 2 shows a non-limiting list of drugs for treating IRR. [Table 2]

[0122] Available safety data, as well as PK, PD, and efficacy data, are considered for selecting the maximum tolerated dose and recommended Phase II dose, and, where applicable, for the possibility of modeling a fixed-dose cohort(s). Once decisions regarding the recommended Phase II dose and regimen are made, the trial proceeds to the expansion phase.

[0123] Effectiveness Patients in cohorts 1–4 were receiving treatment and underwent initial evaluations, as summarized in Table 3. The results showed two CRC patients with stable disease (SD), one for over 4 months and the other for over 6 months. [Table 3]

[0124] safety The Wnt pathway, which ultimately signals via β-catenin, plays a crucial role in healthy individuals. β-catenin is an active transcription factor that regulates specific stem cell populations and organ regeneration. Given the importance of the Wnt pathway to many cancers, numerous clinical attempts have been made to target this pathway despite "on-target off-tissue" concerns. Past attempts have included the use of pan-Wnt inhibitors (Kahn 2014, Shaw 2019) that suffer from on-target toxicity to gastrointestinal stem cells and bone, inhibition of Wnt pathway members such as Frizzled, Tankyrase, Dickkopf-1, and Porcupine, and disruption of the β-catenin transcription complex by targeting T cell factors / lymphatic enhancer factors. All of these approaches have resulted in toxicity.

[0125] Notably, safety data from cohorts 1-4 revealed no dose-limiting toxicities, infusion-related reactions, or serious adverse events associated with ST316.

[0126] Pharmacokinetics and pharmacodynamics Pharmacokinetic (PK) data from patients in the first four cohorts showed relatively linear PK parameters, with no significant increase in accumulation or clearance over time. AUC of Cohort 2 (0-168) The biological activity threshold was exceeded as predicted by the modeling. The results are shown in Table 4 and Figures 9A-9B. [Table 4]

[0127] Analysis of immune cell populations in blood samples taken from patients before and after ST316 administration revealed a clear pharmacodynamic effect of ST316 on the WNT / β-catenin pathway. ST316 treatment resulted in a reduction in the immunosuppressive polymorphonuclear leukocyte (PMN) myeloid suppressor cell (MDSC) population (Figures 10A-10C). PMN-MDSCs exhibited a CD3-CD19-CD14-HLADRlowCD11b+CD15+ phenotype.

[0128] In summary, the clinical data demonstrate that the administered dose of ST316 is pharmacologically appropriate, that ST316 is safe, reaches its target, and has a positive impact on patients' diseases.

[0129] Expansion phase The expansion phase will consist of three colorectal cancer cohorts. Cohort 1 will enroll 15 patients receiving ST316 monotherapy. If one or more patients meet the efficacy criteria, defined as stable disease (SD) for more than 4 months, or any response, partial response (PR), or complete response (CR), an additional 15 patients will be recruited for Cohort 1. If Cohort 1 proves safe, Cohorts 2 and 3 will be recruited.

[0130] In Cohort 2, 15 patients will be recruited for second-line treatment and treated with ST316 in combination with bevacizumab and FOLFIRI. If two or more patients demonstrate a response (PR or CR) and / or a progression-free survival (PFS) of more than 7 months, additional patients may be recruited.

[0131] In Cohort 3, 15 patients with up to four prior treatments will be recruited and treated with ST316 in combination with fluquintinib. If one or more patients demonstrate a response (PR or CR) and / or a progression-free survival (PFS) of more than 4 months, additional patients may be recruited.

[0132] Based on efficacy signals during dose escalation and expansion phases, additional tumor types (e.g., endometrial cancer, melanoma, TNBC, ovarian cancer) and combination therapies may be added during the expansion phase.

[0133] Pharmacokinetic evaluation The pharmacokinetic (PK) of ST316 in plasma is evaluated using standard methods. AUC t , C max t 1 / 2 AUC ∞ , and t max A complete plasma PK profile, including the data, will be obtained and analyzed using a non-compartmental method for the entire population during the dose escalation phase. Blood samples will be collected pre-administration, at the end of infusion, and at various post-infusion time points during the first cycle, and then at a less frequent rate thereafter.

[0134] Pharmacodynamic evaluation Blood and tumor samples will be collected for pharmacodynamic evaluation. This may include circulating tumor DNA analysis to examine mechanistically related genetic abnormalities and changes over time, as well as baseline and intra-treatment tumor analysis using quantitative reverse transcription polymerase chain reaction, ribonucleic acid sequencing, NanoString analysis, and immunohistochemistry.

[0135] Relevant disease markers, such as tumor biomarkers, will be collected at the time of screening and during the study period. At the time of screening, all subjects will provide a core biopsy or excisional biopsy taken after the last dose of previous systemic therapy and before enrollment. In subjects who have undergone screening biopsies, if possible and if tumor remains, a post-treatment biopsy of the same lesion will be performed.

[0136] Effectiveness evaluation Tumor response, disease control rate (DCR), duration of response (DOR), and / or progression-free survival (PFS) will be evaluated using RECIST 1.1 (Eisenhauer 2009). PFS is defined as the time from the first dose of the study treatment to the first recorded disease progression or death. DOR is defined as the time from the first observed response to the first recorded disease progression or death.

[0137] To evaluate tumor response, either CT or MRI will be used, with CT being the preferred imaging technique. The same methodology will be used for all evaluations at baseline and throughout the study. All affected organs will be recorded at baseline as either target or non-target lesions according to RECIST 1.1 and followed up throughout the study period.

[0138] Radiographic evaluation of complete response (CR) or partial response (PR) requires confirmatory imaging at least four weeks after the initial assessment of response. To expand the extended cohort, significant clinical benefit may be used instead of radiographically defined response.

[0139] References American Cancer Society (ACS). Key Statistics for Ovarian Cancer. Web page accessed 16 November 2022. Bernal F, et al. Reactivation of the p53 Tumor Suppressor Pathway by a Stapled p53 Peptide. J. Am. Chem. Soc. 2007;129:2456-2457. Bird GH, et al. Biophysical Determinants for Cellular Uptake of Hydrocarbon-Stapled Peptide Helices. Nat. Chem. Biol. 2017;12:845-852. Bugter JM, et al. Mutations and mechanisms of WNT pathway tumour suppressors in cancer. Nat Rev Cancer. 2021 Jan;21(1):5-21. de la Roche M, et al. The function of BCL9 in Wnt / beta-catenin signaling and colorectal cancer cells. BMC Cancer. 2008 Jul 15;8:199. Dotan E, et al. Phase Ib Study of Wnt Inhibitor Ipafricept with Gemcitabine and nab-paclitaxel in Patients with Previously Untreated Stage IV Pancreatic Cancer. Clin Cancer Res. 2020 Oct 15;26(20):5348-5357. Dougherty PG, et al. Understanding Cell Penetration of Cyclic Peptides. Chem. Rev. 2019;119(17):10241-10287. Eisenhauer EA, et al. New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur J Cancer 2009;45(2):228-247. Gay DM, et al. Loss of BCL9 / 9l suppresses Wnt driven tumourigenesis in models that recapitulate human cancer. Nat Commun. 2019 Feb 13;10(1):723. Kahn M, Can we safely target the WNT pathway? Nat. Rev. Drug Discov. 2014 Jul;13(7):513-532. Lamarca A, et al. Second-line chemotherapy in advanced biliary cancer: a systematic review. Annals of oncology. 2014 Dec 1;25(12):2328-38. doi: 10.1093 / annonc / mdu162. Lin SY, et al. Beta-catenin, a novel prognostic marker for breast cancer: its roles in cyclin D1 expression and cancer progression. Proc Natl Acad Sci U S A. 2000 Apr 11;97(8):4262-6. Liu J, et al. Wnt / β-catenin signalling: function, biological mechanisms, and therapeutic opportunities. Signal Transduct Target Ther. 2022 Jan 3;7(1):3. Mieszczanek J, et al. Bcl9 and Pygo synergise downstream of Apc to effect intestinal neoplasia in FAP mouse models. Nat Commun. 2019 Feb 13;10(1):724. Moor AE, et al. BCL9 / 9L-β-catenin Signaling is Associated With Poor Outcome in Colorectal Cancer. EBioMedicine. 2015 Oct 30;2(12):1932-43. Moore K, et al. Endometrial Cancer: Is This a New Disease? Am. Soc. Clin. Oncol. Educational Book. 2017 435-442. National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines in Oncology (NCCN guidelines) with NCCN Evidence Blocks - Ovarian Cancer 2022. Nie X, et al. Emerging roles of Wnt ligands in human colorectal cancer. Frontiers in oncology. 2020 Aug 14;10:1341. doi: 10.3389 / fonc.2020.01341 Parrish ML, et al. Mechanisms of mutant β-catenin in endometrial cancer progression. Frontiers Oncol. 2022 Sep. 29;12:1009345. Pohl, SG, et al. Wnt signaling in triple-negative breast cancer. Oncogenesis. 2017;6:e310. doi: 10.1038 / oncsis.2017.14. Ruiz de Galarreta M, et al. β-Catenin Activation Promotes Immune Escape and Resistance to Anti-PD-1 Therapy in Hepatocellular Carcinoma. Cancer Discov 2019;9(8):1124-41. doi: 10.1158 / 2159-8290.CD-19-0074. Shang S, et al. The regulation of β-catenin activity and function in cancer: therapeutic opportunities. Oncotarget 2017;8(20):33972-33989. Shaw HV, et al. Targeting the Wnt signalling pathway in cancer: prospects and perils. Swiss Medical Weekly 2019;149:w20129. Simon R. Optimal two-stage designs for phase 2 clinical trials. Controlled Clinical Trials 1989;10:1-10. Sung H, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021 May;71(3):209-249. doi: 10.3322 / caac.21660. Epub 2021 Feb 4. PMID: 33538338. Takada K, et al. Targeted disruption of the BCL9 / β-catenin complex inhibits oncogenic Wnt signaling. Sci Transl Med. 2012 Aug 22;4(148). Takahashi Y, et al. Surgical treatment of perihilar cholangiocarcinoma in octogenarians: a single center experience. J Hepatobiliary Pancreat Sci. 2013 Mar;20(3):324-31. 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[0140] The present invention is further described by the following claims.

Claims

1. A method for treating a solid tumor in a patient, comprising parenterally administering to the patient a pharmaceutical composition comprising an effective amount of a β-catenin peptide antagonist.

2. The method according to claim 1, wherein the solid tumor is selected from the group consisting of breast cancer, colorectal cancer, bile duct cancer, endometrial cancer, hepatocellular carcinoma, melanoma, non-small cell lung cancer, ovarian cancer, pancreatic adenocarcinoma, and synovial sarcoma.

3. The method according to claim 1, wherein the patient has received prior treatment selected from the group consisting of chemotherapy, hormone-based therapy, radiotherapy, targeted therapy, and combinations thereof.

4. The method according to claim 1, wherein the peptide antagonist is administered as a neoadjuvant.

5. The method according to claim 1, wherein the peptide antagonist comprises the D-amino acid sequence FRWLRQLARLAQLAKLTPVKLTPV (SEQ ID NO: 1).

6. The method according to claim 1, wherein the peptide antagonist is administered to the patient at a dose of about 0.25 to 12 mg / kg.

7. A method for administering a β-catenin peptide antagonist to a subject, comprising administering a pharmaceutical composition containing the peptide antagonist to the subject parenterally, The peptide antagonist comprises the D-amino acid sequence FRWLLRQLARLAQLAKLTPVKLTPV (SEQ ID NO: 1), The method wherein the peptide antagonist is administered in a dose of approximately 0.25 to 12 mg / kg.

8. The method according to claim 1, wherein the peptide antagonist is administered in a dose selected from about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 4 mg / kg, about 8 mg / kg, and about 12 mg / kg.

9. The method according to claim 1, wherein the peptide antagonist inhibits the binding of β-catenin to B cell CLL / lymphoma 9 protein (BCL9).

10. The method according to claim 1, wherein the peptide antagonist comprises an N-terminal octanoyl group.

11. The method according to claim 1, wherein the peptide antagonist is ST316.

12. The method according to claim 1, wherein the pharmaceutical composition is administered intravenously.

13. The method or composition according to claim 12, wherein the pharmaceutical composition is administered by injection.

14. The method or composition according to claim 13, wherein the injection duration is approximately 30 to 180 minutes.

15. The method or composition according to claim 14, wherein the injection duration is approximately 60 to 90 minutes.

16. The method according to claim 1, wherein the pharmaceutical composition is administered once a week.

17. The method according to claim 1, wherein the pharmaceutical composition is administered once every two weeks.

18. The method according to claim 1, wherein the pharmaceutical composition is administered for at least four weeks.

19. The method according to claim 1, further comprising administering bevacizumab, folinic acid, fluorouracil, and irinotecan (FOLFIRI) to the patient.

20. The method according to claim 1, further comprising administering fluquintinib to the patient.

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