Methods of administration and use of beta-catenin antagonists

Abstract

Methods of administering a peptide antagonist of beta-catenin and methods of treating solid tumors by administering a peptide antagonist of beta-catenin are provided.

Description

Background Technology

[0001] The Wnt / β-catenin signaling pathway regulates key cellular functions including proliferation, differentiation, migration, and stem cell renewal, and plays a crucial role in embryonic development and adult tissue homeostasis (Liu 2022). Dysregulation of this pathway often involves constitutive activation of β-catenin, an oncogenic transcription factor involved in the pathogenesis of many malignancies and 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 a reduction in subsequent tumor-infiltrating effector T cells (Ruiz de Galarreta 2019).

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

[0003] In cancer cells, B-cell CLL / lymphoma 9 protein (BCL9) promotes the accumulation and retention of β-catenin in the nucleus (de la Roche 2008). BCL9 is a coactivator that shuttles β-catenin into the nucleus and acts as a scaffold to recruit the enhancer protein Pygopus to the β-catenin transcription complex, thereby enhancing transcriptional activity (Mieszcanek 2019). BCL9 is overexpressed in many tumors and is associated with poor prognosis (Moor 2015; Vafaizadeh 2021; Wang 2019). Studies have shown that BCL9 / BCL9L deficiency inhibits tumor growth and proliferation with minimal impact on normal tissue homeostasis, suggesting that β-catenin antagonists may produce significant antitumor effects without causing on-target off-tissue toxicity (Gay 2019).

[0004] Based on the key role of β-catenin in promoting malignant phenotypes and its negative correlation with the prognosis of several cancer types, β-catenin has been considered a potential therapeutic target for cancer treatment (Liu 2022). However, despite extensive research, β-catenin is conventionally considered "undruggable," and there are currently no drugs available to target Wnt / β-catenin signaling.

[0005] Previous approaches targeting the Wnt / β-catenin pathway for cancer therapy act upstream of β-catenin regulation or at sites where β-catenin interacts with LEF / TCF, leading to nonspecific disruption of the canonical Wnt pathway and resulting in targeting toxicity in the bone (Zhong 2016) and intestine (Dotan 2020). A method is needed that specifically disrupts the interaction between β-catenin and BCL9 to avoid side effects such as targeting toxicity. Summary of the Invention

[0006] The main aspects of the invention are summarized below. Further aspects are described in the detailed description, embodiments, drawings, and claims sections of this disclosure. The descriptions in each section of this disclosure are intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in a variety of different ways, and all such combinations are intended to fall within the scope of the invention.

[0007] This disclosure provides a method for treating a patient with a solid tumor, the method comprising administering, parenterally, a pharmaceutical composition comprising an effective amount of a β-catenin peptide antagonist to the patient. A pharmaceutical composition comprising an effective amount of a β-catenin peptide antagonist is also provided for parenteral administration to treat a patient with a solid tumor.

[0008] In one implementation, 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 cancer, and synovial sarcoma. In some implementations, the patient has received prior treatment from the group consisting of: chemotherapy, hormone-based therapy, radiation therapy, targeted therapy, immunotherapy, and combinations thereof. In certain implementations, the peptide antagonist is administered as a neoadjuvant therapy as a single agent or in combination. In one implementation, the peptide antagonist is administered in combination with (i) bevacizumab and / or (ii) leucovorin, fluorouracil, and irinotecan (FOLFIRI). In one implementation, the peptide antagonist is administered in combination with fruquintinib.

[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 about 0.25-12 mg / kg.

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

[0011] In some methods and compositions of the present invention, the peptide antagonist is administered at 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.

[0012] In some aspects of the invention, the peptide antagonist disrupts the binding of β-catenin to B-cell CLL / lymphoma 9 protein (BCL9). In some embodiments, the peptide antagonist comprises an N-terminal octanoyl group. In a particular embodiment, the peptide antagonist is ST316.

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

[0014] In some embodiments, the pharmaceutical composition is administered once a week or every two weeks. In one embodiment, the pharmaceutical composition is administered for at least four weeks. Attached Figure Description

[0015] Figure 1 This study demonstrates that ST316 directly binds to β-catenin Arm1 with a greater affinity than the transcriptional coactivator B-cell CLL / lymphoma 9 protein (BCL9) in fluorescence polarization assays. The results are plotted as normalized milliP (mP) relative to β-catenin concentration.

[0016] Figure 2This study demonstrates that ST316 inhibits the protein-protein interaction between β-catenin and BCL9 in amplified luminescent proximity homogeneous assay (ALPHA). Both ST316 (circle) and the BCL9 HD2 peptide (square) inhibit the interaction between β-catenin and BCL9, with IC50 showing a significant inhibitory effect. 50 It is 1 μM.

[0017] Figure 3 A-3B shows the 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 nuclear and cytoplasmic fractions, respectively. Figure 3 A).

[0018] Figure 4 A-4D shows ST316 enhancing proteasome degradation of BCL9 in Colo320 colorectal cancer cells.

[0019] Figure 5 A-5B demonstrates that, compared to the control peptide, ST316 inhibits the migration of HCT116 colorectal cancer cells across the semipermeable membrane in a 24-hour invasion assay. Figure 5 A shows cells treated with either a 1 μM negative control peptide (top) or ST316 (bottom). Figure 5 B shows the cell count after treatment.

[0020] Figure 6 A-6C demonstrates that ST316 reduces spontaneous adenoma formation in a mouse model of colorectal cancer.

[0021] Figure 7 This study demonstrates that ST316 (βCAP) administration inhibits tumor growth in a subcutaneous xenograft tumor model using HCT116 cells. N = 6 mice / group.

[0022] Figure 8 A-8B illustrates the inhibition of tumor growth by ST316 administration in a subcutaneous xenograft tumor model using 4T1-luc triple-negative breast cancer (TNBC) cells. Figure 8 A) and reduced Axin2 expression ( Figure 8 B). N = 6 mice / group.

[0023] Figure 9 A-9B shows the ST316 plasma concentrations over time for cohorts 1-4 (0.5, 1, 2, or 4 mg / mL).

[0024] Figure 10A-10C shows a reduction in the population of immunosuppressive polymorphonuclear (PMN) myeloid-derived suppressor cells (MDSCs) in ST316-induced cancer patients. The reduction is observed before (top) and after (bottom) treatment with 4 mg / kg ST316 QW for 3 weeks in patients with pancreatic ductal adenocarcinoma (PDAC). Figure 10 A) or colorectal cancer (CRC) Figure 10 Peripheral blood samples were collected from patients (B). Blood was processed to collect lymphocytes and bone marrow cells, which were analyzed by flow cytometry. PMN cells (CD3-, CD19-, CD14-, HLADRlow, CD11b+, and CD15+) were quantified as a percentage of bone marrow cells (CD3-, CD19-). Figure 10 C). Detailed Implementation

[0025] Unless otherwise specified, the practice of this invention may employ conventional techniques of pharmaceutical, formulation science, protein chemistry, cell biology, cell culture, molecular biology, microbiology, recombinant DNA, immunology, clinical pharmacology, and clinical practice, all of which are within the scope of the art.

[0026] To facilitate a better understanding of the invention, certain terms are first defined. Further definitions are set forth throughout this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art related to this invention.

[0027] Any headings provided herein are not intended to limit the various aspects or embodiments of the invention, which can be obtained by referring to the entire specification. Therefore, the terms directly defined below are more fully defined through the entire specification.

[0028] All references cited in this disclosure are incorporated herein by reference in their entirety. Additionally, any manufacturer's descriptions or catalogues of any products referenced or mentioned herein are also incorporated by reference. Documents incorporated herein by reference, or any teachings therein, may be used in the practice of this invention. Documents incorporated herein by reference are not acknowledged as prior art.

[0029] I. Definition The wording or terminology used in this disclosure is for descriptive and not limiting purposes, and the terminology or terminology in this specification shall be interpreted by those skilled in the art based on the teachings and instructions.

[0030] Unless the context clearly specifies otherwise, the singular forms “a” and “the” as used in this specification and the appended claims include plural indicators. The terms “a” and “a or more” and “at least one” are used interchangeably.

[0031] Furthermore, "and / or" should be considered as each of the two specified features or components being, or not being, specifically disclosed with the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" is intended to include A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to include A, B and C; A, B or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0032] Whenever the word “comprising” is used to describe an implementation scheme, it includes other similar implementation schemes described in the terms “composed of” and / or “substantially composed of”.

[0033] Units, prefixes, and symbols are represented in a form acceptable in the International System of Units (SI). A numerical range includes the number that defines the range, and any single value provided herein can be used as an endpoint of a range that includes other single 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, 3-9, etc. Similarly, a disclosed range is a disclosure of each single value (i.e., intermediate value) contained within that range, including integers and fractions. For example, the stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10, as well as 5.2, 7.5, 8.7, etc.

[0034] Unless otherwise specified, the terms “at least” or “about” preceding a series of elements should be understood to refer to each element in the series. The term “about” depends on the context in which it is used. When preceding a numerical value, it typically includes ±10% of the listed value. For example, in one 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) can include 0.9% (w / v) to 11% (w / v).

[0035] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a polymer of amino acids of any length and its salts. Polymers may be linear or branched, may contain modified amino acids, and may be interspersed with non-amino acids. Unless otherwise specified, such as for abbreviations of uncommon or non-natural amino acids shown herein, three-letter and single-letter abbreviations as used in the art are used to denote amino acid residues. Amino acids are L-amino acids unless preceded by a “D” or a lowercase letter. Groups or strings of amino acid abbreviations are used to denote peptides. Unless specifically indicated, peptides are indicated by an N-terminus on the left, and the sequence is written from the N-terminus to the C-terminus.

[0036] The "reverse" peptide has an inverted amino acid sequence relative to the reference L-amino acid sequence and is composed of all D-amino acids (which reverse the chirality of the α-center of the amino acid subunit) to help maintain a side chain topology similar to the original L-amino acid peptide.

[0037] "Separated" molecules are molecules in forms not found in nature, including those that have been purified.

[0038] "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 specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair. The affinity of molecule X for its partner Y can generally be determined by the dissociation constant (K). D Affinity is expressed as a finite element. Affinity can be measured using methods commonly known in the art, including those described herein. Low-affinity binding pairs typically bind slowly and tend to dissociate easily, while high-affinity binding pairs typically bind quickly and tend to remain bound for longer periods.

[0039] The affinity or cohesion of a molecule to its binding partner can be determined experimentally using any suitable method known in the art, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g., KINEXA®, 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, eds. Paul, WE, 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 can vary if measured under different conditions (e.g., salt concentration, pH, temperature). Therefore, as known in the art, affinity and other binding parameters (e.g., KINEXA®, BIACORE™, or OCTET®) are determined using standard solutions of the binding partner and normalized buffers. D Or Kd, K on K off ) measurement.

[0040] "Active agents" are ingredients designed to provide biological activity. Active agents can associate with one or more other ingredients. Active agents that are peptides can also be called "active peptides".

[0041] The "effective amount" of an active agent is the amount sufficient to achieve the specifically specified purpose.

[0042] The term "pharmaceutical composition" refers to a formulation which is present in a form that allows the bioactivity of the active ingredient to be effective and does not contain any additional components that would have unacceptable toxicity to a subject to whom the composition will be administered. Such compositions may be sterile and may contain a pharmaceutically acceptable carrier, such as physiological saline. Suitable pharmaceutical compositions may contain one or more of the following: buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbates), stabilizers (e.g., polyols or amino acids), preservatives (e.g., sodium benzoate), and / or other conventional solubilizers or dispersants.

[0043] "Subject" or "individual" or "animal" or "patient" or "mammal" is any subject requiring diagnosis, prognosis, or treatment, particularly a mammalian subject. Mammal subjects include humans, livestock, farm animals, sport animals, and laboratory animals, including, for example, humans, non-human primates, canines, felines, pigs, cattle, horses, rodents (including rats and mice), rabbits, etc.

[0044] Terms such as “treating” or “alleviating” refer to therapeutic measures that cure, slow down, reduce or alleviate the symptoms of a diagnosed pathological condition or symptom and / or prevent the progression of a diagnosed pathological condition or symptom. In some implementations, a subject’s disease or symptom is considered successfully “treated” if the patient exhibits complete, partial, or temporary relief or elimination of at least one symptom or measurable bodily parameter associated with the disease or symptom.

[0045] "Control patients" are subjects who have not received the treatment of the present invention. "Control population" or "control patient population" is a group of subjects who have not received the treatment of the present invention. Subjects in control patients or control populations have the same disease or condition as subjects compared to them. For example, the clinical outcomes of cancer patients receiving the pharmaceutical composition or method of the present invention are compared to the mean (median) outcomes of subjects with the same type and / or stage of cancer who have not received the pharmaceutical composition or method of the present invention. In some embodiments, patients in control patients or control populations have received treatments different from the treatment of the present invention, such as standard care.

[0046] An "antagonist" is a substance that prevents, blocks, inhibits, neutralizes, or reduces the biological activity or action of another molecule, such as a receptor or ligand.

[0047] The terms “inhibition,” “blocking,” and “repression” are used interchangeably and refer to any statistically significant reduction in incidence or activity, including complete blockage of incidence or activity. For example, “inhibition” can refer to a reduction in activity or incidence of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. An “inhibitor” is a molecule, factor, or substance that produces a statistically significant reduction in the incidence or activity of a process, pathway, or molecule.

[0048] "Tumor cells" or "tumors" typically undergo some form of mutation / transformation compared to normal cells or tissues of the same type, resulting in abnormal growth. Tumors include irregular morphology and pathological proliferation. Tumor cells can be benign or malignant. Malignant tumors (i.e., cancers) differ from benign tumors in that they exhibit loss of cell differentiation and orientation, and possess invasive and metastatic properties.

[0049] A tumor or solid tumor is a cluster of tumor cells, such as cancer cells. The terms "advanced," "metastatic," and "advanced / metastatic" are used interchangeably to describe cancer in which malignant cells have migrated from the original tumor to another location in the patient's body (e.g., another organ).

[0050] Pharmacokinetics, or "PK," refers to the study of how a subject's body processes an administered substance. PK determines how a substance enters the bloodstream (absorption), how it disperses or is 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). The substance can be a drug, such as ST316. Pharmacokinetics can be evaluated using a variety of metrics, many of which are based on the amount of the substance in the body (e.g., in plasma) at different time points after administration.

[0051] The time following administration is measured from T0, which is the time at which a single dose of the substance is administered. In cases where administration of the pharmaceutical composition is paused and resumed once or more during the total infusion period, T0 is the start of the total infusion period.

[0052] "Total infusion duration" or "total infusion period" is the time from the start of administration of a single dose of the drug composition to the end of administration, and includes both the infusion period and the interruption period.

[0053] “C max "" refers to the pharmacokinetic measure of the peak plasma concentration of a substance after administration.

[0054] “T max"Refers to the time it takes for the substance to reach C after it is first applied (T0)" max Time-dependent pharmacokinetic measures.

[0055] “T last "" refers to the pharmacokinetic measure of the time when a substance reaches its final quantifiable concentration.

[0056] "AUC," or "area under the curve," is a pharmacokinetic measure describing the change in plasma concentration over time. It can be used for different time periods, such as from time zero to a specified time t. t or AUC 0-t From time zero to infinity (AUC) ∞ or AUC 0-∞ ) etc. to calculate AUC.

[0057] "Elimination half-life" or "half-life" or "t" ½ "Refers to the pharmacokinetic measure of the time required for a substance's concentration to reach half of its original value."

[0058] "Clearance rate" is a pharmacokinetic measure that refers to the volume of plasma cleared per unit time.

[0059] “V z "" refers to the pharmacokinetic measure of the volume of distribution during the terminal phase of metabolism.

[0060] II. Peptides and Compositions β-catenin and antagonist peptides The Wnt / β-catenin pathway is a validating target for cancer therapy. Constitutive activation of the Wnt / β-catenin signaling pathway is associated with oncogenicity, tumor progression, and poor prognosis in many malignancies (Shang 2017). The complex formed through the interaction between β-catenin and its coactivator BCL9 drives oncogene expression in various cancers due to aberrant Wnt pathway signaling. It has been shown that disruption of the β-catenin / BCL9 complex inhibits oncogenic Wnt / β-catenin transcription without affecting β-catenin homeostasis (Takada 2012).

[0061] The interaction between β-catenin and BCL9 was previously considered an "undrugable" target because small molecules could not inhibit complex formation and antibodies could not enter the cytoplasm or nucleus to disrupt the interaction (Takada 2012). Peptide antagonism of β-catenin differs from these other approaches because it can specifically target the interaction between β-catenin and BCL9 and exhibits selective toxicity to tumor cells in which these proteins are overactive, amplified, and / or mutated.

[0062] In some embodiments, the method of the present invention includes treating a patient with a solid tumor 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, for example, based on the native sequence of BCL9, to enhance the electrostatic interaction between the peptide antagonist and β-catenin. In particular, the peptide antagonist for the interaction between β-catenin and BCL9 can be derived from BCL9's homology domain 2 (HD2) and designed to interact with the first armadillo repeat (ARM-1) domain of β-catenin, which is a site utilized by BCL9 rather than other β-catenin binding partners. WO 2021 / 007158 provides numerous examples of such peptides.

[0063] The activity of β-catenin-based peptide antagonists, such as their ability to inhibit the interaction between β-catenin and BCL9, can be measured by methods described herein, for example, in 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, such assays and models are described in WO2019 / 136125 and WO 2021 / 007158.

[0064] β-catenin peptide antagonists can be cell-penetrating peptides. In one embodiment, the peptide contains a cell-penetrating domain. Numerous cell-penetrating peptide sequences are described and characterized in the literature (see WO 2019 / 136125). In one embodiment, the peptide is a cyclic peptide. Cyclic peptides can enter cells via passive diffusion, endocytosis / endosome escape, or other mechanisms, for example, using cyclopeptides (Bernal 2007; Bird 2017) or other cyclization methods known in the art (Dougherty 2019). Peptides can also be delivered to cells via mechanisms utilizing cell receptors (e.g., integrin targeting, RGD-like sequences). Alternatively, peptides can be encapsulated in vesicles such as exosomes or liposomes, or in micelles, and delivered to cells.

[0065] Peptide antagonists for β-catenin can have modified N-termini and / or modified C-termini. For example, the peptide antagonist may optionally contain an N-terminal acetyl group and / or a C-terminal amide group. Other examples of optional N-terminal and / or C-terminal groups include hydrophobic groups, such as linear or cyclic C2-C... 18Aliphatic or aromatic hydrocarbons, naphthyl, phenyl, octanoyl, and valeryl, including isovaleryl. In some embodiments, the peptide antagonist comprises a linker or spacer between the peptide and the hydrophobic group. Such linkers or spacers include, for example, aminocaproic acid, β-alanine, substituted alkyl groups, substituted cycloalkyl groups, and polyethylene glycol.

[0066] ST316 is a novel 25-amino acid peptide antagonist of the interaction between β-catenin and BCL9. ST316 is composed entirely of D-amino acids and is highly stable in the presence of proteolytic enzymes. 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, thus avoiding stimulation of anti-drug antibody (ADA) responses.

[0067] ST316 consists of two domains: (i) a 15-amino acid N-terminal active domain derived from BCL9 homologous domain 2 (HD2) and engineered to interact with the first armadillo repeat (ARM-1) domain of β-catenin, which is the site utilized by BCL9, rather than other β-catenin binding partners; and (ii) a 10-amino acid C-terminal domain that enhances solubility and cell penetration. The D-amino acid sequence of ST316 is: FRWLLRQLARLAQLAKLTPVKLTPV (SEQ ID NO: 1). The N-terminal active domain is shown in bold and the C-terminal cell penetration domain is shown in italics. ST316 also contains an N-terminal octanoyl group.

[0068] ST316 represents the first well-tolerated approach to drug research targeting the Wnt pathway by specifically binding to the n-terminus of β-catenin, in which β-catenin interacts with BCL9, a cofactor not essential for normal physiological function but necessary for the pathological, carcinogenic, and immunosuppressive functions of β-catenin.

[0069] Composition and application In some aspects, the present invention provides a composition, such as a pharmaceutical composition, comprising a β-catenin peptide antagonist, such as ST316. The pharmaceutical composition is suitable for parenteral administration. In one embodiment, ST316 may be in salt form. Preferably, the composition comprises one or more carriers, diluents, excipients, or other additives. For example, the composition may comprise one or more fillers, one or more buffers, one or more pH adjusters, and / or one or more diluents.

[0070] This invention relates to a method of administering a β-catenin peptide antagonist to a subject. The β-catenin peptide antagonist is administered parenterally. Parenterally administration routes include intravenous (IV), intramuscular, intraperitoneal, intrathecal, and subcutaneous. In a preferred embodiment, a pharmaceutical composition comprising ST316 is administered via IV infusion. The pharmaceutical composition may be provided, for example, in an IV solution comprising physiological saline (0.9%), semi-physiological saline (0.45%), or a 5% dextran aqueous solution (D5W). In one embodiment, the β-catenin peptide antagonist is a sterile lyophilized solid present in a vial for reconstitution, for example, with sterile water for injection (USP). The reconstituted peptide can then be diluted for IV administration.

[0071] β-catenin peptide antagonists can be administered based on the patient's weight. β-catenin peptide antagonists such as ST316 can be administered to patients at doses of about 0.25 mg / kg to about 12 mg / kg. In some embodiments, ST316 is administered at doses of about 0.25 mg / kg, about 0.5 mg / kg, about 0.75 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, or about 12 mg / kg. These amounts can also be used as endpoints of the range of doses to be administered, such as about 0.75 mg / kg to about 7 mg / kg, about 2 mg / kg to about 4 mg / kg, etc.

[0072] Alternatively, β-catenin peptide antagonists can be administered at a fixed dose. β-catenin peptide antagonists such as ST316 can be administered to patients at doses of about 500 mg to about 1500 mg. In some embodiments, ST316 is administered at doses of about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, or about 1500 mg. These amounts can also be used as endpoints of a range of doses to be administered, such as about 600 ng to about 1100 mg, about 750 mg to about 900 mg, etc.

[0073] In embodiments of the invention, the pharmaceutical composition is administered to the subject via intravenous infusion, wherein the total infusion duration does not exceed about 360 minutes. In some embodiments, the total infusion duration is from about 30 minutes to about 240 minutes. For example, the total infusion duration can be 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, or 180 minutes, or intermediate durations such as 45 minutes, 100 minutes, etc. In some embodiments, the total infusion duration is from about 60 minutes to about 90 minutes, or from about 60 minutes to about 120 minutes, or from about 90 minutes to about 120 minutes, or from about 60 minutes to about 180 minutes.

[0074] In some embodiments, the infusion of the pharmaceutical composition can be interrupted, i.e., it can be temporarily stopped and then resumed. The duration of the interruption can vary, for example, it can be 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.

[0075] In some embodiments, the pharmaceutical composition may be administered in conjunction with one or more secondary agents designed to block histamine release, prevent or mitigate infusion-related reactions (IRRs), reduce fever or inflammation, and / or relieve itching and / or urticaria. IRRs may include, for example, erythema, fever, chills / shivering, tachycardia, tachypnea, hypotension, and bronchospasm. The one or more secondary agents may be administered concurrently with, before, and / or after the pharmaceutical composition. The one or more secondary agents may be administered separately from the pharmaceutical composition or in combination with it. Additionally, the one or more secondary agents may be administered via the same route as the pharmaceutical composition or via a different route (e.g., orally).

[0076] Examples of one or more secondary agents administered with the pharmaceutical composition include, but are not limited to, antihistamines, including H1 antagonists, H2 antagonists, and mast cell degranulation inhibitors (e.g., acrivastine, astemizole, azatadine, azelastine, bepotastine, bromopheniramine, burfroline, cetirizine, chlorzoxazone, chlorpheniramine, cromoglycine, cyproheptadine, desloratadine, dexbrompheniramine). amine), diphenhydramine, doxantrozole, epinastine, etodroxizine, famotidine, fexofenadine, forskolin, hydroxyzine, isoproterenol, ketotifen, levocetirizine, loratadine, lodoxamide, mequitazine, methdilazine, mizolastine, nedocromil, olopatadine Targeted anti-inflammatory drugs (TADs), oxatomide, pemirolast, pimecrolimus, pirbuterol, pizothifen, proxicromil, ranitidine, terfenadine, terbutaline; leukotriene inhibitors (e.g., montelukast, zafirlukast, zileuton); nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., ibuprofen, naproxen, aspirin); acetaminophen / paracetamol;Corticosteroids (e.g., hydrocortisone, dexamethasone, prednisone, prednisolone); antinausea medications (e.g., prochlorperazine, ondansetron); and saline and / or electrolytes. In a preferred embodiment, the secondary agent is an antihistamine, such as chlorpheniramine or diphenhydramine. In a particular embodiment, the secondary agent is selected from the group consisting of acetaminophen / paracetamol, H1 antagonists and H2 antagonists, montelukast, antiemetics, and combinations thereof.

[0077] In some embodiments, a secondary agent is administered during administration of the pharmaceutical composition, such as during the infusion period. In some embodiments, a secondary agent, such as an antihistamine, is administered to the subject before administration of the pharmaceutical composition, such as approximately 7 days, 6 days, 5 days, 4 days, 72 hours, 48 ​​hours, 24 hours, 8-12 hours, 6-8 hours, 4-6 hours, 2-4 hours, 1-2 hours, 1 hour, or immediately before administration. In some embodiments, the secondary agent is administered within 24 hours after administration of the pharmaceutical composition. For example, the secondary agent may be applied immediately after the completion of the application of the pharmaceutical composition, approximately 0.5-1 hour after completion, approximately 1-2 hours after completion, approximately 2-4 hours after completion, approximately 4-6 hours after completion, approximately 6-8 hours after completion, approximately 8-12 hours after completion, or approximately 24 hours after completion.

[0078] Secondary agents can be administered multiple times before, during, and / or after the administration of the pharmaceutical composition. Combinations of secondary agents can be administered simultaneously or at different times. For example, an antihistamine can be administered before the administration of the pharmaceutical composition, and a corticosteroid can be administered after the administration of the pharmaceutical composition.

[0079] Peptide antagonists are typically administered to patients multiple times. β-catenin peptide antagonists can be administered weekly, every two weeks, every three weeks, every four weeks, or a combination of these intervals. The total duration of treatment can be at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve weeks, or at least three, four, five, six, seven, eight, nine, ten, eleven, or twelve months.

[0080] In one implementation, the β-catenin peptide antagonist may be administered weekly for a duration of at least one, two, or three weeks (i.e., one, two, or three administrations), six weeks (i.e., six administrations), nine weeks, twelve weeks, three months, six months, nine months, or twelve months. In another implementation, administration may be administered every two weeks for a duration of at least four weeks (i.e., two administrations), eight weeks (i.e., four administrations), twelve weeks, three months, six months, nine months, or twelve months. In some implementations, the β-catenin peptide antagonist may be administered to the patient weekly for at least one, two, three, six, nine, twelve weeks, three months, six months, nine months, or twelve months, followed by administration every two weeks for at least four, eight, twelve, three, six, nine, or twelve months.

[0081] Neoadjuvant therapy can be administered to reduce the size or extent of a tumor, or to make subsequent treatment easier, more effective, or less extensive. In certain embodiments, β-catenin peptide antagonists can be administered as neoadjuvant therapy, either as a single agent or in combination with secondary agents, prior to, for example, surgery and / or radiation.

[0082] The pharmacokinetics and pharmacodynamics of β-catenin antagonists can be evaluated using standard methods and as described in the examples. In some embodiments, the β-catenin antagonist is ST316.

[0083] III. Treatment Methods Subjects requiring treatment by the method of the present invention are patients diagnosed with solid tumors. For example, subjects may have advanced, unresectable, and metastatic solid tumors. In one embodiment, the tumor carries one or more abnormalities in the Wnt / β-catenin signaling pathway. As a non-limiting example, one or more abnormalities in the Wnt / β-catenin signaling pathway may include mutations in one or more of the following: Escherichia coli adenomatous polyposis (APC), APC membrane recruitment protein 1 (AMER1), Axin2, β-catenin, RING finger protein 43 (RNF43), and / or T cytokine 7 (TCF7).

[0084] In some embodiments, the subject has 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 cancer (PDAC), or synovial sarcoma (SS). In some embodiments, the disease is refractory to or intolerant to other available treatments that would affect survival. In a particular embodiment of the invention, administration of ST316 can inhibit tumor growth, reduce tumor volume, or a combination thereof.

[0085] In one implementation, the patient has been diagnosed with breast cancer (BC). More than 50% of BC tumors have been shown to carry abnormalities in Wnt signaling (Lin 2000). Triple-negative breast cancer (TNBC) is a particularly aggressive form of BC, generally exhibiting a poorer prognosis compared to other BC subtypes, and accounts for 10-15% of all BC cases (Won 2020). Due to the lack of hormone receptors and human epidermal growth factor receptor 2 (HER2), TNBC patients do not respond to hormone and targeted therapies. Chemotherapy remains the standard of care, although its benefits are limited. Therefore, the medical need for more effective agents remains very high in this clinical setting. 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 lung and brain metastases (Pohl 2017).

[0086] In one implementation, the patient has been diagnosed with CRC. CRC is one of the most frequently diagnosed cancers worldwide, ranking second among women and third among men globally (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, making it an area of ​​significant unmet medical need. Over 90% of CRC tumors have been shown to carry abnormalities in Wnt signaling. These have been shown to drive disease initiation, progression, and metastasis (Nie 2020).

[0087] In one implementation, the patient has been diagnosed with CCA. CCA, also known as biliary carcinoma, refers to a heterogeneous group of gastrointestinal cancers originating from the bile ducts, gallbladder, or ampulla of Fat. These cancers are often difficult to diagnose due to their anatomical location and the rarity or nonspecificity of their symptoms; consequently, >75% of patients with CCA have advanced, unresectable disease (Lamarca 2014; Takahashi 2013). Advanced, unresectable CCA represents an area of ​​unmet medical need due to its highly invasive nature, limited treatment options, and poor prognosis, especially after first-line therapy, typically with a combination of gemcitabine and cisplatin (Valle 2010). Clinical and preclinical studies have shown that activation of the Wnt / β-catenin signaling pathway plays a crucial role in the induction and progression of CCA (Zhang 2020).

[0088] In one implementation, the patient has been diagnosed with OC. OC is the fifth leading cause of cancer death in women and a leading cause of death among gynecological cancers (ACS 2022). Two-thirds of OC patients are diagnosed at stages 3 and 4, with relatively low 5-year survival rates of 39% and 17%, respectively, for stage 3 and 4 tumors (NCCN 2022). Advanced OC represents an area of ​​unmet medical need due to its highly invasive nature, limited treatment options, and poor prognosis. Wnt activity has been shown to be associated with histological grade of OC, epithelial-to-mesenchymal transition, chemotherapy resistance, and poor prognosis (Teeuwssen 2019).

[0089] In one implementation, the patient has been diagnosed with endometrial cancer. Endometrial cancer is the most common gynecological cancer in the industrialized world and is increasing in women of all ages, at least in part due to obesity and the resulting increased incidence of hyperinsulinemia (Moore 2017; Parrish 2022). Older women experience a higher risk of recurrence and a higher mortality rate (Moore 2017). Mutations in β-catenin are associated with poorer outcomes in patients with endometrial cancer, including higher recurrence rates and lower survival rates (Parrish 2022).

[0090] The efficacy of the treatment can be evaluated using one or more known methods. For example, compared to one or more identical outcomes in patients not treated with the methods of the present invention (i.e., control patients), patients treated with the methods of the present invention may experience outcomes including prolonged survival, improved progression-free survival, improved duration of response, longer remission, reduced risk of recurrence, and / or improved tumor response to treatment with a β-catenin peptide antagonist. For example, the outcomes of patients treated with the methods of the present invention can be compared to the median outcomes of a control patient population. The control patient population can be administered regimens selected from groups such as: placebo, surgery, radiation, chemotherapy, immunotherapy, hormone-based therapy, targeted therapy, and combinations thereof. Statistical analysis of the comparisons can be performed using, for example, the Wilcoxon signed-rank test or the Kaplan-Meier method.

[0091] In one implementation, the outcomes of patients receiving a β-catenin peptide antagonist such as ST316, optionally in combination with standard of care, are compared to the median outcomes of control patients receiving placebo.

[0092] Tumor response to treatment can be assessed, for example, by measuring tumor burden and / or tumor regression. Response to treatment is compared to one or more efficacy measures following a treatment regimen with a baseline (e.g., before treatment with ST316). Baseline assessment is preferably performed within 24, 48, or 72 hours before the first treatment with a β-catenin peptide antagonist, or within 1, 2, 3, or 4 weeks. In a preferred embodiment, baseline assessment is performed within one week before the first ST316 treatment.

[0093] "Tumor burden" is the total mass or size of cancerous tissue in a patient's body. Tumor response can be evaluated using measures including objective response rate (partial and / or complete response), disease stability, disease control rate, duration of disease control, and duration of response. These parameters can be determined, for example, using the Revised Standard for Evaluation of Solid Tumor Response (RECIST 1.1) (Eisenhauer 2009).

[0094] Objective response rate assesses a reduction in tumor size (e.g., tumor diameter), which can be determined by clinical examination and / or imaging. When a patient has multiple tumors, tumor size may 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 photography and ruler. Imaging methods include computed tomography (CT), typically using contrast agents; X-rays; magnetic resonance imaging (MRI); and positron emission tomography (PET), such as (18)F-fluorodeoxyglucose PET. In a preferred embodiment, CT is used to assess tumor response, for example, in patients with BC or melanoma. Thus, in one aspect, the present invention provides a method for reducing a patient's tumor burden (i.e., tumor mass and / or tumor size), the method comprising administering to the patient a β-catenin peptide antagonist, such as ST316. The reduction in tumor burden is measured relative to baseline.

[0095] In some implementations, particularly those evaluated by RECIST 1.1, the disease control rate defines the level of tumor response as optimal among: complete response (CR), where one or more tumors disappear; partial response (PR), where the size of one or more tumors decreases by at least 30%; stable disease (SD), where the change in tumor size is less than 30% or less than 20%; or disease progression, where the tumor size and / or new lesions increase by at least 20%. Patients treated by the methods of the present invention may experience CR, PR, or SD.

[0096] The duration of disease control is the length of time from achieving a response (CR or PR) or SD until disease progression. The duration of response is the length of time from achieving a response until disease progression, i.e., the period during which the tumor does not grow or spread or dies. The duration of response in patients treated with ST316 can 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 can experience an increase in the duration of disease control or an increase in the duration of response. Therefore, in one aspect, the present invention provides a method for increasing the duration of disease control or the duration of response in a patient, the method comprising administering to the patient a β-catenin peptide antagonist, such as ST316. The increase in the duration of disease control or the duration of response is measured relative to the median duration of disease control or the median duration of response in a control population, respectively.

[0097] Survival can be assessed as overall survival (OS), the length of time a patient survives; progression-free survival (PFS), the length of time a patient remains healthy without disease progression or worsening after treatment; or event-free survival (ESR), the length of time a patient remains healthy without complications or negative events such as relapse or disease progression. Survival is measured from the start of treatment. OS, median OS, PFS, median PFS, ESR, and median ESR can be calculated based on treatment response, for example, through Kaplan-Meier analysis.

[0098] Therefore, in one aspect, the present invention provides a method for increasing overall survival in a patient, the method comprising administering to the patient a peptide antagonist of β-catenin, such as ST316. The increase in overall survival can be measured relative to the median overall survival of a control group. Alternatively, an increase in the percentage of patients surviving for a given period of time (e.g., 6 months or 12 months) compared to a control group indicates an increase in overall survival.

[0099] In another aspect, the present invention provides a method for increasing progression-free survival in a patient, the method comprising administering to the patient a peptide antagonist of β-catenin, such as ST316. The increase in progression-free survival can be measured relative to the median progression-free survival of a control group. Alternatively, an increase in the percentage of patients who are relapse-free for a given time period (e.g., 6 months or 12 months) compared to a control group indicates an increase in progression-free survival.

[0100] In another aspect, the present invention provides a method for increasing event-free survival in a patient, the method comprising administering to the patient a peptide antagonist of β-catenin, such as ST316. The increase in event-free survival is measured relative to the median event-free survival of a control group. Alternatively, an increase in the percentage of patients who are event-free over a given time period (e.g., 6 months or 12 months) compared to a control group indicates an increase in event-free survival.

[0101] A patient is considered successfully treated according to the method of the invention if they experience or exhibit at least one of the following outcomes after administration of a β-316 peptide antagonist such as ST316: - No tumor detected (or, in cases where multiple tumors are present at baseline, at least one tumor cannot be detected); - Compared to baseline, the tumor size decreased by at least approximately 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%; - The tumor size did not increase significantly compared to baseline (e.g., an increase of less than 20% or less than 30%); - Optionally, the duration of response was significantly increased compared with the median duration of response in the control patient population; - Optionally, the duration of disease control was significantly increased compared with the median duration of disease control in the control patient population; - Optionally, progression-free survival was significantly increased compared with the median progression-free survival in the control patient population; - Optionally, overall survival was significantly increased compared to the median overall survival of the control patient population.

[0102] IV. Preparation Method β-catenin peptide antagonists can be chemically synthesized, for example, using solid-phase peptide synthesis, solution-phase peptide synthesis, or a combination of both. The synthesis can optionally proceed as a subsequent chemical or enzymatic combination of peptide fragments.

[0103] Alternatively, peptide antagonists of β-catenin can be expressed using recombinant methods. For example, nucleic acid molecules encoding ST316 can be constructed chemically using an oligonucleotide synthesizer. The nucleic acid molecules can be designed based on the amino acid sequence of ST316 and the selection of those codons favorable in the host cell that produces recombinant ST316. Standard methods can be applied to synthesize nucleic acid molecules encoding peptide antagonists of β-catenin, such as ST316.

[0104] Once prepared, the nucleic acid encoding the peptide can be inserted into the expression vector and operatively linked to an expression control sequence suitable for expressing the peptide in the desired host. To obtain high expression levels of the peptide, the nucleic acid can be operatively linked or associated with transcriptional and translational expression control sequences that are functional in the selected expression host.

[0105] A variety of expression host / vector combinations can be employed by anyone skilled in the art. Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli (E. coli), including pCR1, pBR322, pMB9, and their derivatives, plasmids with a wider host range, such as M13, and filamentous single-stranded DNA bacteriophages.

[0106] Suitable host cells include prokaryotes, yeast, insects, or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include Gram-negative or Gram-positive organisms such as *Escherichia coli* or bacilli. Higher eukaryotic or mammalian-derived cell lines can be established; examples 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.

[0107] Peptides can be purified using methods including, for example, reversed-phase high-performance liquid chromatography (RP-HPLC), multi-column countercurrent solvent gradient purification (MCSGP), hydrophobic interaction chromatography (HIC), and ion exchange chromatography.

[0108] Example The embodiments of this disclosure can be further defined by referring to the following non-limiting examples. It will be apparent to those skilled in the art that many modifications can be made to both the materials and methods without departing from the scope of this disclosure.

[0109] Example 1. ST316 binds to β-catenin and disrupts its interaction with BCL9. We used fluorescence polarization assays to measure the binding between FITC-conjugated ST316 and recombinant β-catenin. Incremental concentrations of full-length β-catenin (0.24 nM–2000 nM) were incubated with 12.5 nM ST316-FITC for 1 hour at room temperature. Fluorescence polarization was measured using a SpectraMax® microplate reader. ST316 showed a polarization at approximately 126.1 nM K. D Binding to β-catenin Arm1 ( Figure 1 BCL9 has a binding affinity of approximately 200 nM for β-catenin, indicating that ST316 binds to β-catenin with an affinity equal to or greater than that of the endogenous ligand.

[0110] We 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 MME S (pH 6.5), 60 mM NaCl, 0.01% BSA, 1 mM DTT, and 0.05% Tween-20. ST316 was serially diluted to final concentrations ranging from 50 μM to 6 nM and added to the plate, followed by the addition of 25 nM of full-length recombinant β-catenin. The plate was incubated at room temperature for 1 hour. A 26-amino acid C-terminal biotinylated BCL9 HD-2 peptide 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 and acceptor beads (PerkinElmer AlphaScreen streptavidin donor bead 6760007 and protein A acceptor bead 6760136, respectively) were added to the mixture at a final concentration of 10 μg / mL, and the plate was incubated in the dark on a shaker at room temperature for 1 hour. The α signal was then measured using a SpectraMax® microplate reader. The results showed that both ST316 and HD2 peptide inhibited the interaction between BCL9 and β-catenin, with an IC50 of [missing value]. 50 Approximately 1 μM ( Figure 2 ).

[0111] Example 2. In vitro effects of ST316 We examined the effect of ST316 on the nuclear localization of β-catenin in HCT116 cells. Briefly, cells were cultured in T25 flasks in McCoy 5A medium (with 10% FBS and Mycozap) and treated with medium only, 3 μM ST316, or 5 μM ST316 for 24 hours. Cells were fractionated by first harvesting them and then incubating them in lysis buffer containing a mixture of DTT and protease inhibitors. Next, 10 μL of IGEPAL CA-630 solution was added to the cells. Cells were centrifuged to collect the supernatant cytoplasmic fraction. The remaining cell clusters were resuspended in extraction buffer containing a mixture of DTT and protease inhibitors. Cells were centrifuged and the nuclear supernatant was collected and analyzed by Western blotting. Figure 3 A). Treatment with 5 μM ST316 significantly reduced the nuclear localization of β-catenin. Figure 3 B indicates the percentage of β-catenin in the nucleus and cytoplasm of a protein blot image, quantified using ImageJ analysis software.

[0112] To examine the effect of ST316 on the proteasome degradation of BCL9, Colo320 colorectal cancer cells were incubated with ST316 for 24 hours with or without the proteasome inhibitor MG132. No effect on cell viability was observed. Protein expression was analyzed using the Jess protein detection system. Results are in... Figure 4 The results are shown in A-4D. Similar results were observed in HCT116 cells.

[0113] Example 3. ST316 exhibits inhibitory effects on cancer cell invasion in vitro. We used an in vitro Boyden chamber assay to investigate the inhibitory effect of ST316 on tumor cell invasion. HCT116 colorectal cancer cells (2 × 10⁻⁶) were used. 5 Cells were equilibrated in serum-free cell culture medium for 2 hours in matrix gel-coated invasive plate inserts, after which complete growth medium supplemented with 10% fetal bovine serum was added to the bottom wells of each insert as a chemical inducer. Cells were then exposed to 1 μM ST316 or a negative control peptide for 24 hours. No significant effect on cell viability was observed under these conditions.

[0114] For analysis, the culture medium was aspirated from the insert, and the upper layer of the membrane was removed. After fixation with MeOH and staining with crystal violet, the membrane was removed and mounted on a slide. Images of the membrane with invading cells were acquired at 20x magnification using a Nikon Digital Sight 1000 microscope on a Nikon Eclipse T2S microscope. Cell counts were determined manually using ImageJ. Data represent the mean and standard error of three biological replicates containing at least three representative fields of view. Results are presented in... Figure 5 As shown in A-5B.

[0115] Example 4. Inhibition of tumor growth in vivo by administration of ST316 We inspected the ST316 to APC pair. min / + The effect on tumor formation in mice carrying a mutation in APC essential for regulating β-catenin concentration. min / + Mice are prone to cachexia and develop intestinal adenomas. This was observed in six-week-old female APCs. min / + Mice were administered ST316 subcutaneously once weekly at a dose of 10 mg / kg (n = 3 per group). Mice received a total of 10 doses. Body weight was measured three times weekly throughout the study period. After week 16, mice were euthanized, plasma was collected for cytokine analysis, and small intestine was collected for adenoma quantification. Compared to mice not treated with ST316, ST316 treatment resulted in a 55% reduction in adenoma formation. Figure 6 A), to prevent the onset of cachexia ( Figure 6 B), increases serum pro-inflammatory CCL2 / CCL4 cytokine expression ( Figure 6 C).

[0116] Additionally, we examined the effect of ST316 on tumor volume using a subcutaneous tumor model of colorectal cancer cells. Briefly, HCT116 cells (5 × 10⁻¹) suspended 1:1 in matrix gel were used. 5 ST316 was implanted subcutaneously into the axilla of female NOD / SCID mice. ST316 was administered subcutaneously three times weekly for three weeks at a dose of 5 mg / kg. Administration began on day 12 post-tumor inoculation, with an average initial tumor volume of approximately 200 mm. 3 Tumor volume was monitored three times a week. ST316 treatment resulted in a 99% inhibition of tumor growth compared to the mediator or control peptide. Figure 7 ).

[0117] We also examined the effect of ST316 on tumor volume in a subcutaneous tumor model using triple-negative breast cancer cells. Briefly, 4T1-luc cells (5 × 10⁻¹) suspended 1:1 in matrix gel were used. 5 ST316 was implanted subcutaneously into the axilla of female NOD / SCID mice. ST316 was administered subcutaneously once weekly for 8 weeks at a dose of 5 mg / kg. Administration began on day 6 post-tumor inoculation, with an average initial tumor volume of approximately 100 mm. 3 Tumor volume was monitored three times a week. ST316 treatment resulted in an 84% inhibition of tumor growth compared to the control peptide. Figure 8 A). Tumors were collected on day 60 and Axin2 expression was analyzed by quantitative PCR (n = 3 animals / group). Results were... Figure 8 As shown in B.

[0118] Example 5. ST316 is safe and well-tolerated in animal models. Safety Pharmacology The potential pharmacological effects of ST316 on the cardiovascular (miniature pig), respiratory (rat and miniature pig), and central nervous system (CNS) (rat and miniature pig) systems were investigated in a 28 / 29-day GLP toxicology study. No ST316-related effects on body weight, food intake, ophthalmic examination, respiration, or CNS were observed in rats or miniature pigs. Furthermore, in rats, there were no ST316-related effects on the functional observation battery (FOB), and in miniature pigs, there were no ST316-related effects on quantitative measurements of electrocardiogram (ECG) rhythm, morphology, or QRS, RR, PR, QT, or QTcF intervals.

[0119] No ADA formation was detected in non-GLP toxicology studies in C57BL / 6 mice after 10 weeks of weekly administration or in GLP toxicology studies in miniature pigs after 4 weeks (5 doses) of weekly administration. In rats, ADA formation was within the 5% error rate of a qualified assay in GLP toxicology studies after 4 weeks (5 doses) of weekly administration. One positive sample was observed in the GLP miniature pig study (out of a total of 96 samples) and two positive samples (1%) (from control animals not exposed to ST316) were observed at a dose level of 10 mg / kg in the GLP rat study (out of a total of 198 samples).

[0120] Tolerance The findings of GLP toxicology studies were mostly limited to injection sites in both rats and miniature pigs. In animals receiving ST316 via catheter, no observations clearly related to ST316 were considered adverse. In miniature pigs, no pathological findings related to the test substance were observed.

[0121] Assessment of pseudohypersensory reactions DLT associated with ST316 exposure in the MTD study was a dose-dependent pseudo-anaphylactic infusion-related response (IRR) observed in mice, rats, and miniature pigs. Clinical signs of pseudo-anaphylactic response in mice may include decreased body temperature, reduced activity, and prickly prickling. Non-clinical toxicology of IV-administered ST316 was characterized in a 29-day GLP toxicology study in rats and a 28-day GLP toxicology study in miniature pigs, supported by maximum tolerated dose (MTD) and 7-day repeat dose range discovery (DRF) studies in the same species.

[0122] Toxicological studies characterizing the mechanism of ST316 pseudoallergic IRR demonstrated that the ST316 response is mediated by mast cell activation and histamine release, and is mitigated by pretreatment with antihistamines (ranitidine and piracetam) and / or leukotriene receptor antagonists (montelukast).

[0123] Example 6: Administration of ST316 to a patient with a solid tumor An open-label, two-part, phase 1-2 study was conducted to determine the safety, tolerability, pharmacokinetics (PK), pharmacodynamics, and proof-of-concept efficacy of intravenous ST316 in selected advanced solid tumors potentially carrying abnormalities in the Wnt / β-catenin signaling pathway. These abnormalities are appropriate targets due to their impact on oncogenicity, tumor progression, and prognosis in the selected tumor types, as well as their influence on the immune components of the tumor microenvironment. Furthermore, non-clinical studies have demonstrated ST316's specific antagonism of β-catenin and its effective in vitro / in vivo activity in breast and colon cancer models. This study utilized a novel peptide drug with a novel mechanism of action in subjects who had exhausted standard of care. The study consisted of two phases: a phase 1 dose escalation / protocol exploration phase and a phase 2 expansion phase.

[0124] Dosage escalation phase The dose-escalation phase employed standard methods to evaluate the safety, tolerability, and recommended two-stage dose selection for a wide range of advanced unresectable and metastatic solid tumors. Subjects diagnosed with locally advanced or metastatic BC, CCA, CRC, endometrial cancer, HCC, melanoma, NSCLC, OC, PDAC, or SS whose disease is refractory to or intolerant of all available treatments that would affect survival were included. Table 1 illustrates the study design for the dose-escalation phase.

[0125] Table 1. Overview of Phase 1 Dosage Elevation

[0126] ST316 was administered intravenously. A standard 3+3 design was used to recruit a dose-escalation cohort. For all subjects, the initial dose infusion duration was 60–180 minutes, which could be varied in subsequent doses at least in part based on the presence or absence of IRR.

[0127] Dosage cohorts were 0.5, 1, 2, 4, 8, and 12 mg / kg once weekly (QW), optionally with dose cohorts of 0.25, 0.75, 1.5, 3, and 6 mg / kg, as well as a flat dose. One treatment cycle consisted of 21-day QWs.

[0128] Subjects continue treatment until disease progression, withdrawal of consent, or lack of clinical benefit as determined by the treating physician. If a patient is clinically stable, treatment may continue after progression. If repeat imaging shows a reduced or stable tumor burden compared to the initial scan demonstrating disease progression (PD), treatment continues as planned. If repeat imaging confirms PD, treatment is discontinued. In determining whether the tumor burden is increased, stable, or reduced, investigators consider all target lesions as well as non-target lesions.

[0129] Toxicity was graded using the National Cancer Institute Common Terminology Standard for Adverse Events (CTCAE), v5.0. Safety assessments included adverse events, serious adverse events, physical examination, vital sign measurements, Eastern Cooperative Oncology Group (ECOG) status, clinical safety laboratory evaluations (hematology, serum chemistry and hepatic panel, coagulation and urinalysis), and electrocardiogram. All subjects were followed up for safety at least 30 days after the last dose of ST316.

[0130] To mitigate potential IRR, subjects were pretreated with the following before the first dose of ST316: (i) daily montelukast (10 mg PO) starting at least 2 days prior to ST316 administration and on the day of administration; and (ii) both H1 and H2 antagonists at least 30 minutes before the start of infusion on the day of ST316 administration. If oral antihistamines were used, C was determined according to their predicted C. max Early administration. Preferred H1 / H2 antagonists include famotidine (20 mg PO or 20-40 mg IV) plus chlorpheniramine (10 mg IV / PO) or diphenhydramine (50 mg PO / IV).

[0131] IRR was monitored in subjects 24 hours after the first ST316 administration, 4 hours after the second infusion, and 2 hours after subsequent infusions. Treatment of IRR included, for example, pausing and slowing the infusion rate, and / or administering antihistamines such as diphenhydramine, leukotriene receptor antagonists, acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs), IV fluids, antiemetics, oxygen, bronchodilators, and corticosteroids (if other measures were insufficient). Table 2 provides non-limiting examples of agents used to treat IRR.

[0132] Table 2. Medications for treating IRR

[0133] The maximum tolerated dose and recommended two-stage dose are selected by considering available safety data, as well as PK, PD, and efficacy data, and one or more flat-dose cohorts are potentially modeled (if applicable). Once the recommended two-stage dose and regimen are determined, the study proceeds to the expansion phase.

[0134] effect Patients in cohorts 1-4 were receiving treatment and had undergone their first assessment, as summarized in Table 3. Results showed that two CRC patients had stable disease, one for more than 4 months and the other for more than 6 months.

[0135] Table 3. Summary of Phase 1 Dosage Elevation

[0136] Security The Wnt pathway, which ultimately signals via β-catenin, plays a crucial role in healthy individuals. β-catenin is an active transcription factor that controls the regeneration of certain stem cell populations and organs. Given that the Wnt pathway is also critical in many cancers, numerous clinical attempts have been made to investigate drugs targeting this pathway, despite concerns about “extra-tissue targeting.” Previous attempts have included: pan-Wnt inhibitors, which exhibit targeted toxicity to gastrointestinal stem cells and bone (Kahn 2014; Shaw 2019); inhibition of Wnt pathway members such as frizzled, tankyrase, Dickkopf-1, and Porcupine; and disruption of the β-catenin transcription complex by targeting T cytokines / lymphocyte-enhancing factors. All of these approaches have resulted in toxicity.

[0137] Notably, safety data from cohorts 1–4 revealed no dose-limiting toxicities, no infusion-related reactions, and no ST316-related serious adverse events.

[0138] Pharmacokinetics and Pharmacodynamics Pharmacokinetic (PK) data from the first four patient cohorts showed relatively linear PK parameters with no significant accumulation or enhanced clearance over time. AUC in cohort 2... (0-168) The bioactivity threshold predicted by the self-modeling was exceeded. Results are shown in Table 4 and... Figure 9 As shown in A-9B.

[0139] Table 4. Summary of Pharmacokinetics

[0140] The immune cell populations of blood samples taken from patients before and after ST316 exposure were analyzed, demonstrating a clear pharmacodynamic effect of ST316 on the WNT / β-catenin pathway. ST316 treatment resulted in a decrease in the population of immunosuppressive polymorphonuclear (PMN) myeloid-derived suppressor cells (MDSCs). Figure 10 A-10C). PMN-MDSC exhibits the CD3-CD19-CD14-HLADRlowCD11b+CD15+ phenotype.

[0141] In conclusion, clinical data demonstrate that the delivery dose of ST316 is pharmacologically sound, and that ST316 is safe, hits the target, and has a positive effect on the patient's disease.

[0142] Expansion phase The expansion phase consists of three colorectal cancer cohorts. Fifteen patients will be recruited in cohort 1, which will use ST316 monotherapy. An additional 15 patients will be recruited in cohort 1 if one or more patients meet the following criteria for positive efficacy: stable disease (SD) for more than four months; or any response, partial response (PD), or complete response (CR). If cohort 1 is safe, cohorts 2 and 3 will be recruited.

[0143] Cohort 2 will enroll 15 second-line patients to receive ST 316 in combination with bevacizumab and FOLFIRI. Additional patients may be enrolled if two or more subjects achieve a response (PR or CR) and / or progression-free survival (PFS) lasting more than seven months.

[0144] Cohort 3 will recruit 15 patients who have received up to four lines of prior therapy to receive ST 316 in combination with fruquintinib. Additional patients may be recruited if one or more subjects achieve a response (PR or CR) and / or progression-free survival (PFS) lasting more than four months.

[0145] Based on efficacy signals during dose escalation and expansion phases, additional tumor types (e.g., endometrial cancer, melanoma, TNBC, ovarian cancer) and combinations can be added during the expansion phase.

[0146] Pharmacokinetic assessment ST316 PK in plasma was assessed using standard methods. During dose escalation, whole plasma PK profiles, including AUCt, Cmax, t½, AUC∞, and tmax, were obtained for all subjects and analyzed using non-compartmental methods. Blood samples were drawn at different time points before administration, at the end of the infusion, and after the first cycle, with the frequency of draws decreasing thereafter.

[0147] Pharmacodynamic assessment Blood and tumor samples are collected for pharmacodynamic evaluation, which may include circulating tumor DNA analysis for mechanism-related genetic abnormalities and changes over time, as well as analysis of tumors at baseline and during treatment using quantitative reverse transcriptase-polymerase chain reaction, RNA sequencing, NanoString analysis, and immunohistochemistry.

[0148] Relevant disease biomarkers, such as tumor biomarkers, were collected during screening and the study. At screening, all subjects underwent a single biopsy (either puncture or excision) following the last dose of prior systemic therapy and prior to recruitment. If feasible and if the tumor persisted in subjects who underwent screening biopsies, a post-treatment biopsy of the same lesion was performed.

[0149] Efficacy evaluation RECIST 1.1 (Eisenhauer 2009) was used to assess tumor response, disease control rate (DCR), duration of response (DOR), and / or progression-free survival (PFS). PFS was determined by the time from the first administration of study treatment to the first recorded disease progression or death. DOR was determined by the time from the first observed response to the first recorded disease progression or death.

[0150] Tumor response was assessed using computed tomography (CT) or MRI, with CT being the preferred imaging technique. The same methodology was used for all assessments at baseline and throughout the study. All affected organs were recorded as target or non-target lesions at baseline, in accordance with RECIST 1.1, and were followed up throughout the study.

[0151] Radiological assessment of complete response (CR) or partial response (PR) requires confirmatory imaging at least 4 weeks after the initial assessment observing a response. Significant clinical benefit may be used in place of radiologically defined response to expand the extended cohort.

[0152] References *** The invention is further described by way of the appended claims.

Claims

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

2. The method of 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 cancer, and synovial sarcoma.

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

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

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

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

7. A method of administering a β-catenin peptide antagonist to a subject, the method comprising administering a pharmaceutical composition comprising the peptide antagonist parenterically to the subject. 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 0.25-12 mg / kg.

8. The method of claim 1, wherein the peptide antagonist is administered at 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 of claim 1, wherein the peptide antagonist disrupts the binding of β-catenin to B-cell CLL / lymphoma 9 protein (BCL9).

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

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

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

13. The method or composition of claim 12, wherein the pharmaceutical composition is administered by infusion.

14. The method or composition of claim 13, wherein the infusion duration is about 30-180 minutes.

15. The method or composition of claim 14, wherein the infusion duration is about 60-90 minutes.

16. The method of claim 1, wherein the pharmaceutical composition is administered once weekly.

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

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

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

20. The method of claim 1, further comprising administering fruquintinib to the patient.

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