Administration and use of CEBP-β antagonists

Abstract

Methods for administering peptide antagonists of CCAAT / enhancer protein β (C / EBPβ) and methods for treating solid tumors by administering peptide antagonists of C / EBPβ are provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Applications Nos. 63 / 041,986, 63 / 041,988, 63 / 041,989, 63 / 041,990, and 63 / 041,991, filed on 21 June 2020, and U.S. Provisional Application No. 63 / 172,560, filed on 8 April 2021, which are incorporated herein by reference in their entirety. [Background technology]

[0002] CCAAT enhancer-binding protein β (C / EBPβ) is a transcription factor involved in cellular processes such as differentiation, inflammation, cell survival, and metabolism. C / EBPβ, when upregulated or hyperactivated, promotes tumor survival and growth and inhibits differentiation in many different cancers, including breast cancer, glioblastoma, prostate cancer, and multiple myeloma (Homma 2006, Kim 2008, Pal 2009, Zahnow 2009, Aguilar-Morante 2011). Furthermore, C / EBPβ has been shown to be inversely correlated with disease prognosis and survival. Due to its role in regulating cell proliferation and differentiation (Lekstrom-Himes 1998; Lamb 2003) and increased expression or activation in many types of cancer (Oya 2003; Homma 2006; Zahnow 2009), C / EBPβ is considered a potential target for therapeutic intervention. However, some C / EBPβ inhibitors that were not effective due to the difficulty in partially targeting transcription factors had entered clinical development prior to the present invention.

[0003] Sequence List This application includes an electronically submitted sequence listing in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy was created on 18 June 2021, named Sapience_013_WO1_SL.txt, and has a size of 2,047 bytes. [Overview of the Initiative]

[0004] Some of the main aspects of the present invention are summarized below. Additional aspects are described in the detailed description of the invention, examples, drawings, and claims sections of this disclosure. The description in each section of this disclosure is 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 various different ways, and all such combinations are intended to be within the scope of the present invention.

[0005] This disclosure provides a method for treating solid tumors in patients, the method comprising parenterally administering to the patient a pharmaceutical composition containing an effective amount of a CCAAT enhancer-binding protein β (C / EBPβ) peptide antagonist. The disclosure also provides a pharmaceutical composition for parenteral administration containing an effective amount of a C / EBPβ peptide antagonist for use in treating solid tumors in patients. Preferably, the peptide antagonist is administered to the patient at a dose of about 0.5 to 16 mg / kg.

[0006] In one embodiment, the solid tumor is a melanoma, carcinoma, or sarcoma. In a specific embodiment, the patient is diagnosed with locally advanced / metastatic breast cancer (LA / MBC), melanoma, glioblastoma (GBM), or castration-resistant prostate cancer (CRPC).

[0007] In some cases, patients receive prior treatment selected from a group consisting of chemotherapy, hormone therapy, immunotherapy, targeted therapy, and combinations thereof. In one embodiment, the prior treatment is standard-of-care treatment for the patient's diagnosis.

[0008] In one embodiment, the peptide antagonist of C / EBPβ is the D-amino acid sequence VAEAREELERLEARLGQARGEL (Sequence ID). 1 ) includes. In one embodiment, the peptide antagonist is the amino acid sequence LEGRAQGLRAELRELEERAEAV (Sequence ID). 4 ) includes. The peptide antagonist may be a cell-permeable peptide. In certain embodiments, the peptide antagonist is ST101.

[0009] Certain methods of the present invention include methods of treating melanoma, HR pos LA / MBC, primary GBM, or CRPC, the method comprising administering to a patient by intravenous infusion a pharmaceutical composition comprising an effective amount of ST101. Also provided is a pharmaceutical composition for intravenous infusion comprising an effective amount of ST101 for use in the treatment of melanoma, HR pos LA / MBC, primary GBM, or CRPC in a patient.

[0010] In a preferred embodiment, the patient is a human patient.

[0011] In certain embodiments, the patient has received at least one line of treatment prior to administration of ST101.

[0012] In one embodiment, the patient has progressive / metastatic melanoma that has progressed after or during treatment with an immune checkpoint inhibitor. In one embodiment, the patient has melanoma comprising a BRAF mutation and the patient has received at least one line of targeted therapy.

[0013] In one embodiment, the GBM has recurred or progressed after pretreatment with maximal surgical resection, radiotherapy, and adjuvant chemotherapy with temozolomide or temozolomide in combination with radiotherapy.

[0014] In one embodiment, the patient has CRPC that has progressed after pretreatment with taxane, abiraterone, darolutamide, and / or enzalutamide / apalutamide.

[0015] In certain embodiments, the pharmaceutical composition is administered intravenously to the patient, for example, via infusion. In some embodiments, the total infusion period is from about 3**** minutes to about 360 minutes, or from about 60 minutes to about 360 minutes, or from about 60 minutes to about 180 minutes.

[0016] In one embodiment, the pharmaceutical composition is administered once a week. In another embodiment, the pharmaceutical composition is administered once every two weeks. The pharmaceutical composition can be administered for at least about three weeks or at least about four weeks.

[0017] Some embodiments of the present invention include the administration of one or more secondary agents. For example, the secondary agent is selected from the group consisting of antihistamines, leukotriene inhibitors, non-steroidal anti-inflammatory agents, acetaminophen, corticosteroids, antiemetics, intravenous saline, electrolytes, and combinations thereof. The one or more secondary agents can be administered to the subject simultaneously with, before, or after the administration of the pharmaceutical composition. In certain embodiments, an antihistamine and / or a leukotriene inhibitor is administered to the subject within about 48 hours before the administration of the pharmaceutical composition.

[0018] In certain embodiments, the pharmaceutical composition includes a buffering agent and a bulking agent. In certain embodiments, the pharmaceutical composition includes lactic acid and trehalose. The pH of the pharmaceutical composition can be, for example, about 3.0 to 8.0.

[0019] In some embodiments of the present invention, the clearance of ST101 is 0.75 to 3.5 liters per hour. In some embodiments, the half-life (t 1 / 2 ) of ST101 is 10 to 70 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] [Figure 1] Shows the mechanism of action of ST101. C / EBPβ promotes tumor cell growth and survival and inhibits the differentiation of many cell types. ST101 interferes with the interaction between C / EBPβ and cofactors such as ATF5, removes oncogenic signals from cells, and results in selective tumor cell death. [Figure 2] Shows the modeled structure of ST101 (upper) bound to C / EBPβ (lower). This structure was generated using the crystal structure of ATF4 bound to C / EBPβ as a model in Molecular Operating Environment (MOE) (PDB ID 1ci6). [Figure 3] The circular dichroism (CD) spectroscopy is shown, demonstrating that ST101 interacts with C / EBPβ (panel A) but not with ATF5 (panel B). Individual spectra of ST101 (upper gray line, panels A and B), C / EBPβ (light gray line, panel A), and ATF5 (light gray line, panel B) are shown. The black line shows a comparison between the actual observations of ST101+C / EBPβ (panel A) or ST101+ATF5 (panel B) and the dashed line representing the average of the two individual spectra. [Figure 4] This paper presents a cell thermal shift assay demonstrating that ST101 associates with C / EBPβ in U251 human glioblastoma cells. Western blot (Panel A) and densitometry quantification (Panel B) of C / EBPβ / β-tubulin are shown. [Figure 5] The molar residue ellipticity (MRE) across the wavelength range indicates that C / EBPβ preferentially interacts with ST101 over ATF5. The lowest line at 220 nm in Panel A represents the ST101+C / EBPβ spectrum; the highest line in Panel B represents the ST101+ATF5 spectrum. The light gray lines represent the individual spectra of ATF5 (the upper line at 220 nm in Panel A) or C / EBPβ (the lower line at 220 nm in Panel B). The black lines show the actual spectrum of ST101+C / EBPβ incubated with ATF5, compared to the dashed lines representing the average of the ST101+C / EBPβ and ATF5 spectra (Panel A) or the average of the ST101+ATF5 and C / EBPβ spectra (Panel B). [Figure 6] This shows that ST101 interferes with the interaction between C / EBPβ and ATF5. Panel A shows the binding of ATF5 to C / EBPβ. Panel B shows the inhibition of ATF5 binding to C / EBPβ by ST101. [Figure 7]This demonstrates that ST101 crosses the blood-brain barrier in mice. The representative image shows ST101 identified by DAB (3,3-diaminobenzidine) staining of microvessels and glial cells (arrow, panel B), while staining is absent in the vehicle-treated control (panel A). [Figure 8] The status of the study described in Example 6 is shown below. PR = Partial response (at least a 30% reduction); SD = No change (less than a 30% reduction and a 20% increase). [Figure 9] The following shows the post-infusion ST101 plasma concentrations in patients from cohorts 1-4 for cycles 1 and 2, as described in Example 6. ST101 was administered at doses of 0.5 mg / kg (cohort 1), 1 mg / kg (cohort 2), 2 mg / kg (cohort 3), or 4 mg / kg (cohort 4). [Figure 10] The Cmax (Panel A) and AUC0-t (Panel B) of ST101 for individual patients in cohorts 1-4, as described in Example 6, are shown. ST101 was administered at doses of 0.5 mg / kg (cohort 1), 1 mg / kg (cohort 2), 2 mg / kg (cohort 3), or 4 mg / kg (cohort 4). [Figure 11] As described in Example 6, the ST101 plasma concentrations 4 hours after infusion in patients from cohorts 1-4 are shown. ST101 was administered at doses of 0.5 mg / kg (cohort 1), 1 mg / kg (cohort 2), 2 mg / kg (cohort 3), or 4 mg / kg (cohort 4). [Figure 12] The images show increased ST101 uptake in tumor biopsies with dose escalation of ST101 (Panels A-C). Biopsy samples taken from patients during cycle 2 of treatment were processed and immunoassayed for ST101 using rabbit polyclonal anti-ST101 antibody. The percentage of cells showing ST101 staining was measured by a committee-certified pathologist (Panel D). Scores were assigned based on the number of ST101-positive stained cells in the sample: 0 = negative; 1 = 1-10%; 2 = 11-25%; 3 = 26-50%; 4 = 51-75%; 5 = 76-100%. [Figure 13]This shows a reduction in tumor cell proliferation in patient biopsies after treatment with ST101. Biopsy samples taken from patients before treatment (screening) and after treatment cycle 2 were processed and immunoassayed for Ki67. Panel A images show staining of biopsy samples from patients in Cohort 3 before treatment (top image) and after treatment cycle 2 (bottom image). Higher Ki67 expression indicates greater tumor cell proliferation. The percentage of Ki67-stained cells from patients in each Cohort 1-3 was measured by a committee-certified pathologist (Panel B). Scores were assigned on a scale of 0-3 based on the intensity of the Ki67 signal: 0=negative; 1=mild; 2=moderate; 3=severe. [Modes for carrying out the invention]

[0021] Unless otherwise specified, the invention may be implemented using conventional techniques of pharmaceutical science, 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.

[0022] To make the present invention easier to understand, certain terms are defined first. Further definitions are provided throughout this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art relating to the present invention.

[0023] The headings provided herein are not intended to limit the various aspects or embodiments of the invention that can be obtained by referring to this entire specification. Therefore, the terms defined below are more fully defined by referring to this entire specification.

[0024] All references cited herein are incorporated herein by reference in their entirety. Furthermore, manufacturer's instructions or catalogs of products cited or mentioned herein are incorporated herein by reference. Any documents incorporated herein by reference, or any teachings therein, may be used in the practice of the present invention. Documents incorporated herein by reference are not considered prior art.

[0025] I. Definition The expressions and terms in this disclosure are for illustrative purposes only and not limiting; therefore, the terms and expressions in this specification should be interpreted by those skilled in the art in light of their teachings and guidance.

[0026] In this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless otherwise clearly indicated by the context. The terms “a” (or “an”), as well as the terms “one or more” and “at least one,” may be used interchangeably.

[0027] Furthermore, “and / or” is considered a specific disclosure of each of the two specified functions or components, in the presence or absence of the other. Thus, the term “and / or” as used in phrases such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0028] Whenever an embodiment is described using the word "includes," it is described using the terms "consist of" and / or "essentially consisting of" and otherwise similar embodiments are included.

[0029] Units, prefixes, and symbols are shown in the format approved by the Systeme International d'Unites (SI). Numerical ranges encompass the numbers that define the range, and any individual values ​​provided herein may function as endpoints of a range that includes other individual values ​​provided herein. For example, a set of values ​​such as 1, 2, 3, 8, 9, and 10 is also a disclosure of numerical ranges such as 1–10, 1–8, and 3–9. Similarly, a disclosed range is a disclosure of the individual values ​​(i.e., intermediate values) that are included in the range, including integers and fractions. For example, the stated range 5–10 is also a disclosure of 5, 6, 7, 8, 9, and 10 individually, as well as a disclosure of 5.2, 7.5, 8.7, etc.

[0030] Unless otherwise specified, the terms “at least” or “about” preceding a set of elements are understood to refer to all of those elements. The term “about” preceding a number includes ±10% of the stated value. For example, a concentration of about 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, about 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v).

[0031] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably and refer to polymers of amino acids of any length and their salts. The polymers may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acids. Unless otherwise indicated, the three-letter and one-letter abbreviations used in the art, for example, for uncommon or unnatural amino acid abbreviations described herein, are used to represent amino acid residues. Unless beginning with “D” or a lowercase letter, amino acids are L-amino acids. Groups or strings of amino acid abbreviations are used to represent peptides. Unless otherwise specified, peptides are shown with the N-terminus on the left, and the sequence is described from the N-terminus toward the C-terminus.

[0032] "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), making it easier to maintain a side-chain topology similar to that of the original L-amino acid peptide.

[0033] An "isolated" molecule is a molecule in a form not found in nature, including purified molecules.

[0034] "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 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 is generally expressed by the dissociation constant (K). D Affinity can be expressed by the following: Affinity can be measured by common methods known in the art, including the methods described herein. Low-affinity binding partners generally tend to bind slowly and dissociate easily, while high-affinity binding partners generally tend to bind faster and remain bound for longer.

[0035] The affinity or binding activity of a molecule to its binding partner can be experimentally measured using any suitable method known in the art, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or dynamics (e.g., KINEXA®, BIACORE®, or OCTET® analysis). Direct binding assays and competitive binding assays can be readily used (see, for example, Berzofsky et al., “Antibody-Antigen Interactions,” in Fundamental Immunology, Paul, WE, ed., Raven Press: New York, NY (1984); Kuby, Immunology, WH Freeman and Company: New York, NY (1992)). The affinity of a particular binding pair interaction to be measured may vary considerably when measured under different conditions (e.g., salt concentration, pH, temperature). Therefore, affinity and other binding parameters (e.g., K D Or Kd, K on , K off The measurement of ) is performed using standardized solutions and standardized buffers of the binding partner, as is well known in the art.

[0036] An "active agent" is a component intended to impart biological activity. Active agents can be used in combination with one or more other components. An active agent that is a peptide may be called an "active peptide."

[0037] The "effective dose" of an active agent is the amount sufficient to accomplish the specifically stated purpose.

[0038] The term "pharmaceutical composition" refers to a preparation that enables the biological activity of an active ingredient and does not contain additional components that are unacceptably toxic to the target population. Such compositions may be sterile and may contain a pharmaceutically acceptable carrier such as physiological saline. Suitable pharmaceutical compositions may contain buffers (e.g., acetates, phosphates, 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.

[0039] "Subject," "individual," "animal," "patient," or "mammal" refers to any subject, particularly mammalian subjects, for which diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, domesticated animals, livestock, athletic animals, and laboratory animals, such as humans, non-human primates, dogs, cats, pigs, cattle, horses, rodents including rats and mice, and rabbits.

[0040] Terms such as “to treat,” “to cure,” “to treat,” or “to alleviate,” or “to alleviate,” refer to therapeutic means that cure, slow down, reduce, and / or halt the progression of a diagnosed disease or disorder. In certain embodiments, a subject is “treated” for a disease or disorder if the patient exhibits overall, partial, or temporary relief or elimination of at least one symptom or measurable physical parameter associated with the disease or disorder.

[0041] A "control patient" is a subject who has not received treatment according to the present invention. A "control group" or "group of control patients" is a group of subjects who have not received treatment according to the present invention. Control patients or subjects in the control group have the same disease or disorder as the subjects being compared to the control patients or control group. For example, the clinical outcomes of cancer patients administered the pharmaceutical composition or method of the present invention are compared to the mean (median) outcomes of subjects with the same type of cancer who have not received the pharmaceutical composition or method of the present invention. In some embodiments, patients in the control patients or control group receive treatment other than the treatment according to the present invention, e.g., standard treatment.

[0042] An "antagonist" is a substance that prevents, blocks, inhibits, neutralizes, or reduces the biological activity or effect of another molecule, such as a receptor or ligand.

[0043] The terms “inhibit,” “block,” and “suppress” are used interchangeably and refer to a statistically significant reduction in occurrence or activity, including complete blockage 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 statistically significantly reduces the occurrence or activity of a process, pathway, or molecule.

[0044] A "neoplastic cell" or "neoplasm" is typically a morphological mutation / transformation that causes abnormal proliferation compared to normal cells or tissues of the same type. Neoplasms include morphological irregularities and pathological growth. Neoplastic cells can be benign or malignant. Malignant neoplasms, or cancers, are distinguished from benign neoplasms by their loss of cell differentiation and orientation and their invasive and metastatic characteristics.

[0045] A "tumor" or "solid tumor" is a mass of tumor cells, such as cancer cells. The terms "progression," "metastasis," and "progression / metastasis" are used interchangeably to describe cancer where malignant cells have spread from the original tumor to another location in the patient's body, such as another organ.

[0046] Pharmacokinetics, or PK, refers to the study of how an administered substance is processed by the target body. Determining PK involves how the substance enters the bloodstream (absorption), disperses or diffuses throughout body fluids and tissues (distribution), is recognized by the body, converted (metabolized), and removed from the body (excretion). The substance may be a drug, for example, ST101. Pharmacokinetics can be evaluated using various metrics, many of which are calculated based on the amount of the substance in the body (e.g., in plasma) at various time points after administration.

[0047] The time after administration is measured from T0, which is the time when a single administration of the substance is initiated. When the administration of the pharmaceutical composition is paused and restarted one or more times during the entire infusion period, T0 is the start of the entire infusion period.

[0048] The "total infusion time" or "total infusion period" is the time from the start to the end of a single administration of the pharmaceutical composition, including both the infusion period and the interruption period.

[0049] "C max " is a pharmacokinetic measurement criterion that refers to the peak plasma concentration of the substance after administration.

[0050] "T max " is a pharmacokinetic measurement criterion that refers to the time from the start of administration (T0) of the substance until C max is reached.

[0051] "T last " is a pharmacokinetic measurement criterion that refers to the time of the last quantifiable concentration of the substance.

[0052] "AUC" or "area under the curve" is a pharmacokinetic measurement criterion that represents the variation in the concentration of the substance in plasma as a function of time. AUC can be calculated for various periods, for example, from time zero to a specified time t (AUC t or AUC 0-t ), from time zero to infinity (AUC ∞ or AUC 0-∞ ), etc.

[0053] "Elimination half-life" or "half-life" or "t 1 / 2 " is a pharmacokinetic measurement criterion that refers to the time required for the concentration of the substance to reach half of its original value.

[0054] "Clearance" is a pharmacokinetic measurement criterion that refers to the volume of plasma from which the substance is removed per unit time.

[0055] "V z " is a pharmacokinetic measurement criterion that refers to the volume of distribution at the end stage.

[0056] II. Peptides and Compositions C / EBPβ Transcription factors (TFs) account for approximately 20% of all known oncogenes and are involved in many human cancers (Lambert 2018). Nevertheless, TFs have historically been considered "unmanageable" targets because their activity cannot be affected by small molecules, or they cannot reach cells by large molecules (Yan 2013; Bushweller 2019). TFs typically dimerize via α-helix basic leucine zipper (bZIP) domains, consolidating their DNA-binding domains to enable efficient interaction with key consensus sequences within DNA (Asada 2011; Potapov 2015). Disrupting these TF protein-protein interactions (PPIs) could be a powerful approach to delivering drugs to this elusive class of targets. Recently, peptides have emerged as a therapeutic class capable of targeting and disrupting TF interactions with high affinity and specificity, and experimental antagonism has been demonstrated against several previously "unmanageable" targets (Takada 2012; Walensky 2014; Bruzzoni-Giovanelli 2018; Lathbridge 2018; Demma 2019; Lathbridge 2019).

[0057] C / EBPβ is a primary target for developing peptide antagonists because it relies on basic leucine zipper interactions with cofactors. To bind to DNA and transactivate gene expression, C / EBPβ dimerizes with its binding partner via interactions between their bZIP domains. In addition to homodimerization, C / EBPβ heterodimerizes with bZIP-containing transcription factors, such as Jun / Fos, C / EBPγ (Huggins 2013), δ-interacting protein A (Bezy 2005), and the CREB / ATF family (Zhao 2014).

[0058] Activated transcription factor 5 (ATF5) is a CREB / ATF factor that has been shown to bind to and activate C / EBPβ in HEK293 and HCT116 cancer cells, leading to transactivation of the pro-survival phenotype (Zhang 2015). ATF5 is highly expressed in many cancers, including gliomas, and contributes to the oncogenic phenotype by promoting the overexpression of Bcl-2 family proteins and survivin, but is hardly observed in differentiated cell types (Sheng 2010). In glioma and other tumor cells, overexpression of the truncated bZIP domain of ATF5, which lacks the DNA-binding domain, induced cytotoxicity in cancer cells (Angelastro 2006); similar results were obtained when peptides containing the truncated bZIP domain were administered (Cates 2016; Karpel-Massler 2016).

[0059] C / EBPβ antagonist peptide In some embodiments, the method of the present invention involves treating a patient with a solid tumor with an effective amount of a C / EBPβ peptide antagonist. In one embodiment, the C / EBPβ peptide antagonist comprises the D-amino acid sequence VAEAREELERLEARLGQARGEL (SEQ ID NO: 1), which is a retroinversovarian of the wild-type ATF5 bZIP domain. The C / EBPβ peptide antagonist can be designed, for example, as described in Example 1.

[0060] The C / EBPβ peptide antagonist may be a cell-permeable peptide. 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 2016) or other cyclization methods known in the art can enter cells by passive diffusion, endocytosis / endosomal escape, or other mechanisms (Dougherty 2019). The peptide may also be delivered to cells via mechanisms utilizing RGD-like sequences that target cell receptors, such as integrins. Alternatively, the peptide may be encapsulated and delivered to cells in vesicles such as exosomes or liposomes, or in micelles.

[0061] The ability of peptides based on the natural ATF5 bZIP domain to antagonistize the activity of C / EBPβ can be measured by the method described herein, for example, in Example 2. The cytotoxicity of C / EBPβ peptide antagonists can be measured in vitro by known assays and / or in vivo using known tumor models; for example, WO2019 / 136125 describes such assays and models.

[0062] ST101 is a D-amino acid peptide that exhibits potent antitumor activity and resistance to proteolysis both in vitro and in vivo. In particular, the cytotoxicity of ST101 has been previously demonstrated in HL60 (promyelocytic leukemia), AML14 (acute myeloid leukemia), SET2 (megakaryoblastic leukemia), A375 (melanoma), MCF7 (breast cancer), U87 (glioblastoma), U251 (glioblastoma), DU145 (prostate cancer), A549 (lung cancer), peripheral blood mononuclear cells (PBMCs), and bone marrow mononuclear cells (BMMCs) (see WO2019 / 136125). Furthermore, subcutaneous administration of ST101 to xenograft mouse models using A375, HL60, MCF7, and U251 cells resulted in a significant reduction in tumor volume (see WO2019 / 136125).

[0063] ST101 consists of a modified domain based on the ATF5 bZIP domain and the Antennapedia penetratin domain to enable cell permeability. The D-amino acid sequence of ST101 is VAEAREELERLEARLGQARGELKKWKMRRNQFWLKLQR (SEQ ID NO: 2), and the cell permeability region is shown in italics. As shown herein, ST101 promotes the cytotoxic activity of tumor cells by disrupting the association of C / EBPβ with anti-apoptotic transcription factors (Figure 1; Example 2).

[0064] Composition and administration In certain embodiments, the present invention provides a pharmaceutical composition comprising a C / EBPβ peptide antagonist such as ST101. In one embodiment, ST101 may be in the form of a salt, such as an acetate. Preferably, the composition comprises one or more carriers, diluents, excipients, or other additives. For example, the composition may comprise one or more bulking agents (e.g., dextran 40, glycine, lactose, mannitol, trehalose), one or more buffering agents (e.g., acetate, citrate, histidine, lactate, phosphate, Tris), one or more pH adjusters (e.g., hydrochloric acid, acetic acid, nitric acid, potassium hydroxide, sodium hydroxide), and / or one or more diluents (e.g., water, physiological saline). The pH of the composition is preferably about 3.0 to 8.0. In one embodiment, the pH is about 3.5 to 6.5, or about 5.0 to 7.5.

[0065] In some embodiments, the pharmaceutical composition includes a bulking agent such as trehalose, the amount of which is in a specific ratio to the amount of ST101, for example, a ratio of bulking agent:ST101 of about 6:1 to about 2:1 by weight.

[0066] Aspects of the present invention relate to a method for administering a C / EBPb peptide antagonist to a target. The C / EBPβ peptide antagonist is administered parenterally. Parenteral administration routes include intravenous (IV), intramuscular, intraperitoneal, subarachnoid, and subcutaneous. In a preferred embodiment, a pharmaceutical composition comprising ST101 is administered by IV infusion. The pharmaceutical composition may be provided as an IV solution containing, for example, physiological saline (0.9%), semi-physiological saline (0.45%), or a 5% dextrose aqueous solution (D5W).

[0067] C / EBPβ peptide antagonists are administered based on the patient's body weight. C / EBPβ peptide antagonists such as ST101 can be administered to a patient in doses ranging from approximately 0.5 mg / kg to approximately 16 mg / kg. In certain embodiments, ST101 is administered in doses of 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 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 10 mg / kg, approximately 12 mg / kg, approximately 14 mg / kg, or approximately 16 mg / kg. These amounts also serve as endpoints in the range of doses to be administered, for example, approximately 0.5 mg / kg to approximately 8 mg / kg, approximately 2 mg / kg to approximately 4 mg / kg, etc.

[0068] In embodiments of the present invention, the pharmaceutical composition is administered to the subject by intravenous infusion, in which case the total infusion time is approximately 360 minutes or less. In some embodiments, the total infusion time is approximately 30 minutes to approximately 240 minutes. For example, the total infusion time 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 time 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.

[0069] In some embodiments, the injection of the pharmaceutical composition may be interrupted, that is, temporarily stopped and then resumed. The interruption time 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.

[0070] In some embodiments, the pharmaceutical composition may be administered together 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. One or more secondary agents may be administered simultaneously with the administration of the pharmaceutical composition, before and / or after administration. 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).

[0071] Examples of one or more secondary agents to be administered together with the pharmaceutical composition include antihistamines containing H1 antagonists and H2 antagonists, and mast cell degranulation inhibitors (e.g., acribastine, astemizole, azatadine, azelastine, bepotastine, bromopheniramine, valfororin, cetirizine, chlorzoxazone, chlorpheniramine, cromolyn, cyproheptadine, desloratadine, dexbromphenylamine, diphenhydramine, doxantrozole, epinastine, etodoroxidine, famotidine, fexofenadine, forskolin, hydroxyzine, isoproterenol, ketotifen, levocetirizine, loratadine, rhodoxamide, mequitazine, me Examples of drugs include, but are not limited to: todilazine, mizolastine, nedocromil, olopatadine, oxatomide, pemirolast, 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 one 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.

[0072] In certain embodiments, the secondary agent is administered during the administration of the pharmaceutical composition, such as 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 within about 48 hours, or within about 24 hours, or within about 8 to 12 hours, or within about 6 to 8 hours, or within about 4 to 6 hours, or within about 2 to 4 hours, or within 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 end 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.

[0073] 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 can be administered before the pharmaceutical composition, and a corticosteroid can be administered after the pharmaceutical composition.

[0074] In one embodiment, the C / EBPβ peptide antagonist may be administered once a week for at least 3 weeks (i.e., 3 doses), 6 weeks (i.e., 6 doses), 9 weeks, 12 weeks, 3 months, 6 months, 9 months, or 12 months. In another embodiment, administration may be given every two weeks for at least 4 weeks (i.e., 2 doses), 8 weeks (i.e., 4 doses), 12 weeks, 3 months, 6 months, 9 months, or 12 months. In some embodiments, the patient may be administered the C / EBPβ peptide antagonist once a week for at least 3 weeks, 6 weeks, 9 weeks, 12 weeks, 3 months, 6 months, 9 months, or 12 months, followed by administration every two weeks for at least 4 weeks, 8 weeks, 12 weeks, 3 months, 6 months, 9 months, or 12 months.

[0075] The pharmacokinetics (PK) of the C / EBPβ antagonist can be evaluated by standard methods as described in the examples. In some embodiments, the C / EBPβ antagonist is ST101. The clearance of ST101 may be approximately 0.75 L / hour to approximately 3.5 L / hour or approximately 1 L / hour to approximately 3 L / hour or approximately 1 L / hour to approximately 2.5 L / hour. The half-life of ST101 is (t 1 / 2 ) could be approximately 10 to 70 hours, or approximately 20 to 60 hours, or approximately 25 to 45 hours.

[0076] In some embodiments, PK is dose-proportional. For example, in a patient administered 0.5 mg / kg of ST101, C max This can be approximately 1,000 ng / mL to approximately 2,000 ng / mL, or approximately 1,300 ng / mL to approximately 1,800 ng / mL, or approximately 1,370 ng / mL to approximately 1,770 ng / mL. In patients administered 1.0 mg / kg of ST101, C max This can be approximately 2,500 ng / mL to approximately 5,500 ng / mL, or approximately 2,670 ng / mL to approximately 5,300 ng / mL, or approximately 2,840 ng / mL to approximately 5,110 ng / mL. In patients administered 2.0 mg / kg of ST101, C max This can range from approximately 8,000 ng / mL to approximately 13,000 ng / mL, or approximately 8,500 ng / mL to approximately 12,500 ng / mL, or approximately 9,000 ng / mL to approximately 11,900 ng / mL. In patients administered 4.0 mg / kg of ST101, C max This could be approximately 7,000 ng / mL to approximately 35,000 ng / mL, or approximately 7,650 ng / mL to approximately 32,900 ng / mL, or approximately 9,150 ng / mL to approximately 30,000 ng / mL, or approximately 15,000 ng / mL to approximately 28,000 ng / mL.

[0077] III. Treatment method The subjects requiring treatment by the method of the present invention are patients diagnosed with a solid tumor. For example, a subject may have a locally advanced solid tumor or a metastatic, inoperable tumor. In some embodiments, the subject has a melanoma, carcinoma, or sarcoma. In one embodiment, the melanoma is a cutaneous melanoma or a mucosal melanoma. In one embodiment, the carcinoma is an adenocarcinoma such as bladder adenocarcinoma, colorectal adenocarcinoma, pancreatic adenocarcinoma, gastric adenocarcinoma / signet-ring cell carcinoma, or small intestinal adenocarcinoma. In one embodiment, the sarcoma is an abdominal sarcoma or myofibroblastic sarcoma. In certain embodiments of the present invention, administration of ST101 allows for the inhibition of tumor growth, a reduction in tumor volume, or a combination thereof.

[0078] In one embodiment, the patient has locally advanced / metastatic breast cancer (LA / MBC). In one embodiment, the LA / MBC is hormone receptor-positive (HR pos In one embodiment, the patient has LA / MBC that has progressed after receiving one or two prior hormonal therapies. In one embodiment, the patient has received prior targeted therapy, e.g., cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitors, or mammalian target of rapamycin (mTOR) inhibitors, or chemotherapy, or a combination thereof.

[0079] In one embodiment, the patient is diagnosed with melanoma. In one embodiment, the patient has advanced melanoma, particularly advanced / metastatic melanoma, after receiving one or two prior treatments for melanoma.

[0080] In one embodiment, the patient has melanoma that has progressed after / during treatment with an immune checkpoint inhibitor (CPI) or a combination of CPIs. Immune CPIs include, for example, CTLA-4 inhibitors such as ipilimumab and tremelimumab; PD-1 inhibitors such as semiprimab, nivolumab, pembrolizumab, pidilizumab, and spartalizumab; PD-L1 inhibitors such as atezolizumab, avelumab, and durvalumab; LAG-3 inhibitors such as reratrimab; TIGIT inhibitors such as tilagormab; and Toll-like receptor 9 (TLR9) targeting agents such as TLR9 agonists.

[0081] In one embodiment, the patient has a BRAF mutation disorder and is receiving at least one targeted therapy, such as a BRAF inhibitor and / or MEK inhibitor, e.g., vemurafenib, dabrafenib, encorafenib, trametinib, cobimetinib, binimetinib, or a combination thereof.

[0082] In one embodiment, the patient is diagnosed with glioblastoma (GBM). In one embodiment, the GBM has relapsed or progressed after prior treatment. Prior treatments include, for example, surgery, radiation, chemotherapy, targeted therapy, electric field therapy, and combinations thereof. Examples of chemotherapy compounds include temozolomide, carmustine, and lomustine. Examples of targeted therapies include bevacizumab.

[0083] In one embodiment, GBM is primary (novel) GBM that has relapsed or progressed (according to modified RANO criteria) after a single standard treatment regimen. “Standard treatment regimen” is defined as maximum surgical resection, radiotherapy, and temozolomide or temozolomide adjuvant chemotherapy in combination with radiotherapy. In one embodiment, the patient is receiving tumor field therapy as an adjuvant to first-line treatment.

[0084] In one embodiment, the patient is diagnosed with prostate cancer. In one embodiment, the prostate cancer is castration-resistant prostate cancer (CRPC). Castration-resistant prostate cancer (CRPC) is defined by disease progression despite androgen depletion therapy, for example, according to the Prostate Cancer Clinical Trials Working Group 3 (PCWG3) criteria (Scher 2016). In some embodiments, the patient has CRPC that has progressed after treatment selected from the group consisting of: chemotherapy such as taxanes including docetaxel and cabazitaxel, in combination with corticosteroids such as prednisone or prednisolone; immunotherapy including ciproisel-T; hormonal therapy including abiraterone, enzalutamide, dalalutamide, and apalutamide; and combinations thereof. In one embodiment, the patient has CRPC that has progressed after prior treatment with taxanes, abiraterone, dalalutamide, and / or enzalutamide / apalutamide.

[0085] The effectiveness of treatment can be evaluated by one or more known measures. For example, patients treated with the method of the present invention may experience extended survival, improved progression-free survival, improved duration of response, extended duration of remission, reduced risk of relapse, and / or improved tumor reduction effect compared to treatment with a C / EBPβ peptide antagonist, compared to the same outcomes in patients who have not received the method of the present invention, i.e., control patients. The outcomes of patients treated with the method of the present invention can be compared, for example, to the median outcome of a control patient population. The control patient population may be administered regimens selected from, for example, placebo, surgery, radiotherapy, chemotherapy, immunotherapy, hormonal 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.

[0086] In one embodiment, the outcomes of patients receiving a C / EBPβ peptide antagonist, such as ST101, in combination with standard treatment on an optional basis are compared to the median outcomes of control patients receiving a placebo. In another embodiment, the outcomes of patients receiving a C / EBPβ peptide antagonist, such as ST101, are compared to the median outcomes of control patients receiving standard treatment.

[0087] In one embodiment, the outcomes of LA / MBC patients receiving ST101 in combination with chemotherapy on an optional basis are compared with the median outcomes of LA / MBC patients receiving chemotherapy. In another embodiment, the outcomes of LA / MBC patients receiving ST101 in combination with hormonal therapy on an optional basis are compared with the median outcomes of LA / MBC patients receiving hormonal therapy. In yet another embodiment, the outcomes of LA / MBC patients being administered ST101 are compared with the median outcomes of LA / MBC patients receiving targeted therapy.

[0088] In one embodiment, the outcomes of melanoma patients receiving ST101 in combination with chemotherapy (optional) are compared to the median outcomes of melanoma patients receiving chemotherapy. In another embodiment, the outcomes of melanoma patients receiving ST101 in combination with immunotherapy (optional) are compared to the median outcomes of melanoma patients receiving immunotherapy. In yet another embodiment, the outcomes of melanoma patients receiving ST101 in combination with targeted therapy (optional) are compared to the median outcomes of melanoma patients receiving targeted therapy.

[0089] In one embodiment, the outcomes of GBM patients receiving ST101 in combination with one or more of the following treatments (optional): surgery, radiation, chemotherapy, and targeted therapy, are compared to the median outcomes of GBM patients receiving surgery, radiation, chemotherapy, targeted therapy, or a combination thereof. In another embodiment, the outcomes of GBM patients receiving ST101 in combination with a standard treatment regimen (optional): are compared to the median outcomes of GBM patients receiving a standard treatment regimen.

[0090] In one embodiment, the outcomes of prostate cancer patients receiving ST101 in combination with chemotherapy (optional) are compared to the median outcomes of prostate cancer patients receiving chemotherapy. In another embodiment, the outcomes of prostate cancer patients receiving ST101 in combination with immunotherapy (optional) are compared to the median outcomes of prostate cancer patients receiving immunotherapy. In another embodiment, the outcomes of prostate cancer patients receiving ST101 in combination with hormonal therapy (optional) are compared to the median outcomes of prostate cancer patients receiving hormonal therapy. In yet another embodiment, the outcomes of prostate cancer patients receiving ST101 in combination with taxanes, abiraterone, dalalutamide, and / or enzalutamide / apalutamide (optional) are compared to the median outcomes of prostate cancer patients receiving taxanes, abiraterone, dalalutamide, and / or enzalutamide / apalutamide.

[0091] The response to treatment is assessed by comparing one or more measures of efficacy after the treatment regimen to, for example, baseline before treatment with ST101. Baseline assessment is preferably performed within 24, 48, or 72 hours or within 1, 2, 3, or 4 weeks prior to the first treatment with the C / EBPβ peptide antagonist. In a preferred embodiment, baseline assessment is performed within 24 hours prior to the first ST101 treatment.

[0092] In embodiments where the subjects are prostate cancer patients, baseline and response to treatment can be measured by evaluation of blood-based biomarkers, e.g., testosterone, prostate-specific antigen (PSA), and serum cytokine imaging, e.g., computed tomography (CT) including contrast-enhanced CT, or magnetic resonance imaging (MRI) including cross-sectional MRI; biopsy / histological analysis; and patient-reported factors, e.g., pain, analgesic use, physical function, and quality of life. These parameters can be measured, for example, according to the PCWG3 guidelines (Scher 2016).

[0093] The PCWG3 paradigm defines two general categories of treatment response: (1) the extent to which treatment controls, alleviates, or eliminates the symptoms of the disease that were present at baseline; and (2) the extent to which treatment can prevent or delay the onset of the disease in the future (Scher 2016; Scher 2011). Accordingly, in one aspect, the present invention provides a method for controlling, alleviating, or eliminating at least one sign of prostate cancer in patients, including CRPC patients, comprising administering a C / EBPβ peptide antagonist such as ST101 to the patient. In another aspect, the present invention provides a method for preventing or delaying at least one sign of prostate cancer in patients, including CRPC patients, comprising administering a C / EBPβ peptide antagonist such as ST101 to the patient. In one embodiment, signs of prostate cancer are prevented or delayed for 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. The prevention or delay period is measured in comparison to the median prevention or delay period in the control population. Signs of prostate cancer include, for example, tumor volume, new metastatic lesions, elevated PSA levels, bone conditions including fracture incidence, pain, use of analgesics including opioids, and a decline in quality of life (Scher 2016; Scher 2011).

[0094] "Tumor volume" refers to the total mass or total size of cancerous tissue in the patient's body. Tumor reduction can be evaluated by measures including response rate, disease control rate, and duration of response. These parameters vary depending on the type of tumor and can be determined, for example, by the criteria for evaluating the response to solid tumors (RECIST 1.1) (Eisenhauer 2009), modified response assessment in neuro-oncology (mRANO) (Ellingson 2017), or the PCWG3 guidelines (Scher 2016).

[0095] The response rate evaluates the reduction in tumor size. For example, tumor diameter 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 photographic and ruler measurements. Imaging methods typically include computed tomography (CT) with contrast agents; X-ray; magnetic resonance imaging (MRI); and positron emission tomography (PET), such as (18)F-fluorodeoxyglucose PET. In one preferred embodiment, CT is used to evaluate the tumor reduction effect in LA / MBC patients or melanoma patients. In another preferred embodiment, MRI, such as gadolinium-enhanced MRI, is used to evaluate the tumor reduction effect in GBM patients, for example. Thus, in one aspect, the present invention provides a method for reducing tumor volume, i.e., tumor mass and / or tumor size, comprising administering a C / EBPβ peptide antagonist such as ST101 to a patient. The reduction in tumor volume is measured compared to baseline.

[0096] In certain embodiments, particularly those where the evaluation is based on RECIST 1.1, disease control rate is defined as the level of tumor reduction effect as follows: complete response (CR) (disappearance of tumor(s)); partial response (PR) (at least 30% reduction in tumor(s) size); unchanged (no change in tumor(s) size); or disease progression (at least 20% increase in tumor size and / or new lesions).

[0097] In some embodiments, disease control rates define the level of tumor reduction as complete response (CR), partial response (PR), disease progression (PD), or stable disease (SD). Table I lists the modified RANO guidelines for these disease states, based on changes in tumor measurements compared to baseline, lasting at least four weeks. Additional parameters and details are described in Ellingson 2017. [Table 1]

[0098] The duration of response is the length of time from the achievement of a response to disease progression, i.e., the period during which the tumor does not grow, metastasize, or cause death. In patients receiving ST101 treatment, the duration of response is, 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. Accordingly, in one embodiment, the present invention provides a method for extending the duration of response in a patient, comprising administering a C / EBPβ peptide antagonist such as ST101 to the patient. The increase in the duration of response is measured by comparison with the median duration of response in the control population.

[0099] Survival can be evaluated as overall survival, i.e., the period of time a patient is alive, or as progression-free survival, i.e., the period of time a patient is treated without disease progression or worsening. Survival can be measured from the date of diagnosis or the date of initiation of treatment. Overall survival, median overall survival, progression-free survival, and median progression-free survival can be calculated, for example, by Kaplan-Meier analysis based on the response to treatment. Accordingly, in one embodiment, the present invention provides a method for extending a patient's overall survival, comprising administering a C / EBPβ peptide antagonist such as ST101 to the patient. The increase in overall survival is measured in comparison to the median overall survival of the control population. In another embodiment, the present invention provides a method for extending a patient's progression-free survival, comprising administering a C / EBPβ peptide antagonist such as ST101 to the patient. The increase in progression-free survival is measured in comparison to the median progression-free survival of the control population.

[0100] If a patient experiences or exhibits at least one of the following outcomes after administration of a C / EBPβ peptide antagonist such as ST101, the patient is successfully treated according to the method of the present invention: - Tumor undetectable (or, if multiple tumors are present at baseline, at least one tumor); - Tumor size is reduced by at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to baseline; - Tumor size does not show a significant increase compared to baseline (e.g., less than 20%); - A significant extension of the duration of response compared to the median duration of response in the control patient population, as selected by the patient group. - A significant increase in progression-free survival compared to the median progression-free survival of the control patient population, as determined by the patient's choice; - A significant increase in overall survival compared to the median overall survival of the control patient group, on an optional basis.

[0101] IV. Preparation method The C / EBPβ peptide antagonist can be chemically synthesized, for example, by solid-phase peptide synthesis, liquid-phase peptide synthesis, or a combination of both. Optionally, it may also be synthesized as a peptide fragment to be subsequently chemically or enzymatically attached.

[0102] Alternatively, C / EBPβ peptide antagonists can be expressed using recombinant methods. For example, a nucleic acid molecule encoding ST101 can be constructed by chemical synthesis using an oligonucleotide synthesizer. The nucleic acid molecule can be designed based on the amino acid sequence of ST101 and the selection of codons preferred by the host cell producing recombinant ST101. Nucleic acid molecules encoding C / EBPβ peptide antagonists such as ST101 can be synthesized by applying standard methods.

[0103] 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 bound to transcriptional and translational expression regulatory sequences that function in the selected expression host.

[0104] 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 containing pCR1, pBR322, pMB9, and their derivatives, plasmids with a broader host range such as M13, and filamentous single-stranded DNA phages.

[0105] Suitable host cells include prokaryotes, yeasts, insects, or higher eukaryotes under the control of appropriate promoters. Prokaryotes include Gram-negative or Gram-positive bacteria such as E. coli or bacilli. Higher eukaryotic cells or mammalian 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 are also available.

[0106] For example, peptides can be purified using methods including reversed-phase high-performance liquid chromatography (RP-HPLC), multi-column counterflow solvent gradient purification (MCSGP), and ion exchange chromatography. [Examples]

[0107] Embodiments of the present disclosure may 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 implemented without departing from the scope of the present disclosure.

[0108] Example 1. Design of a C / EBPβ peptide antagonist The initial design of the precursor peptide, which includes the 22-amino acid portion LEGECQGLEARNRELKERAESV (SEQ ID NO: 3) of the natural ATF5 bZIP domain and a 16-amino acid penetratin domain, resulted in dose-dependent cytotoxicity of HL-60 promyelocytic leukemia cells. 50 The value was 17.4 ± 0.5 μM. We hypothesized that the potency would be enhanced by modifications designed to strengthen the electrostatic interaction between the peptide and C / EBPβ. We synthesized a panel of 46 rationally designed peptides to examine the molecular packing of non-leucine amino acids of peptide antagonists and the C / EBPβ bZIP domain, and screened them for cytotoxic activity. The "winner" peptide exhibiting the greatest cytotoxicity was identified at 5.5 ± 0.5 μM EC 50 The value resulted in a 3.2-fold increase in potency compared to the parent peptide, demonstrating that the introduced rational changes improve the in vitro peptide antitumor activity. The "winner" peptide contains the amino acid sequence LEGRAQGLRAELRELEERAEAV (SEQ ID NO: 4) and a bZIP domain sequence with a penetratin domain.

[0109] Because L-amino acid peptides typically lack stability in biological matrices, limiting their pharmacokinetics and thus their therapeutic potential, we generated an optimized whole-D-amino acid peptide version of the ATF5 bZIP domain, VAEAREELERLEARLGQARGEL (SEQ ID NO: 1) (which also contains a penetratin domain). This peptide, ST101, showed resistance to proteolysis when incubated at 37°C for 17 hours in the presence of pepsin and trypsin, whereas the L-enantiomer peptide was 100% degraded within 5 minutes. The minimal loss of ST101 observed in untreated controls was attributed to the common peptide chemical degradation that occurs under these conditions.

[0110] In the cytotoxicity test of ST101, 4.9 ± 0.2 μM EC was detected in HL-60 promyelocytic leukemia cells. 50This revealed that the D-amino acid variant exhibited activity comparable to that of the L-enantiomer peptide with the same amino acid sequence, and showed significantly improved stability while retaining antitumor activity.

[0111] The interaction between ST101 and the C / EBPβ bZIP domain is related to the interaction between C / EBPβ and the CREB / ATF family cofactor ATF4. 40 The model was based on the crystal structure of ST101. ST101 was assumed to be helical in solution. The model of ST101 bound to C / EBPβ was generated using Molecular Operating Environment (MOE) software by constraining the ST101 binding sequence to an α-helix structure, overlaying it with a portion of ATF4 that shares the highest sequence similarity, and minimizing it to favor side-chain interactions. This model shows that ST101 is likely to interact primarily via hydrophobic contacts, with several charge-to-charge interactions (ST101 E12~C / EBPβ R279 and ST101 E21~C / EBPβ K267), and cation-pi interactions (ST101 R5~C / EBPβ F282) (Figure 2).

[0112] Example 2. ST101 interacts with C / EBPβ but not with ATF5. Circular dichroism (CD) spectroscopy was used to demonstrate that ST101 interacts in silico with C / EBPβ. In CD spectroscopy, when peptides / proteins interact, the observed spectrum of the bound sample differs from the average of the individual spectra of each component. CD was performed using an Applied Photophysics Chirascan CD instrument (Leatherhead, UK) with 200 μl of sample in a CD cell with a path length of 1 mm. The sample contained a total peptide concentration of 150 μM (i.e., 75 μM per peptide) in equimolar concentrations of heterodimer solution and was suspended in 10 mM potassium phosphate and 100 mM potassium fluoride at pH 7.0 before analysis. The CD spectrum of the sample was scanned in 1 nm increments from 260 nm to 200 nm, with an average scanning time of 0.5 seconds at each wavelength. Three scans were averaged at 20°C.

[0113] ST101, when combined with a peptide derived from the bZIP domain of C / EBPβ, produced a spectrum significantly different from the mean of the individual spectra, indicating a structural interaction between the two components (Figure 3, Panel A). In contrast, the observed spectrum of ST101 combined with a peptide derived from the bZIP domain of ATF5 did not deviate from the mean of the individual spectra of the two peptides (Figure 3, Panel B), indicating that these peptides do not interact under these conditions. These data indicate that ST101 specifically interacts with C / EBPβ.

[0114] The interaction between ST101 and C / EBPβ was further demonstrated in a biological setting of U251 human glioblastoma cells using a cell thermal shift assay (CETSA). Cells were exposed to a vehicle or 10 μM ST101 for 1 hour. Lysates were prepared from the cells and subsequently subjected to a thermal gradient of 52–60°C to denature the proteins. The lysates were then spun down to remove denatured protein aggregates and separated by SDS-PAGE. Western blotting was performed to detect residual C / EBPβ and β-tubulin in the cell lysates after heating.

[0115] Western blot analysis of thermally denatured cell lysates from untreated U251 cells showed that denatured C / EBPβ was lost from the solution at 58°C, as indicated by the loss of the C / EBPβ protein band on the Western blot (Figure 4, Panel A). Cells treated with 10 μM ST101 showed increased C / EBPβ stability at 58°C, as indicated by increased C / EBPβ detection (indicating an interaction between ST101 and C / EBPβ) (Figure 4, Panels A and B). In control experiments, ST101 did not increase the stability of ATF5 in the CETSA assay, demonstrating the specificity of its interaction with C / EBPβ, which is consistent with CD data.

[0116] Additional experiments using CD spectroscopy to evaluate dimer exchange showed that C / EBPβ exhibits higher affinity for ST101 than ATF5. In these experiments, when ST101 was pre-mixed with a peptide containing the bZIP domain of C / EBPβ, the addition of a peptide containing the bZIP domain of ATF5 failed to displace ST101, as indicated by the lack of signal increase (Figure 5, Panel A). In contrast, when ST101 was pre-mixed with the ATF5 bZIP peptide, the C / EBPβ bZIP peptide resulted in a signal increase, producing a peak identical to the peak of ST101 interacting with the C / EBPβ bZIP peptide (Figure 5, Panel B). These data indicate a preferential interaction between C / EBPβ and ST101 over ATF5, as indicated by the shift in the C / EBPβ+ATF5 spectrum upon ST101 addition.

[0117] To quantify the ST101 inhibition of the interaction between C / EBPβ and ATF5, a competitive enzyme-linked immunosorbent assay (ELISA) was used. C / EBPβ (3.6 ng / well) was immobilized in a 384-well plate (Nunc MaxiSorp, ThermoSci) by incubation overnight at 4°C. Unbound proteins were removed by washing three times with Tris-buffered saline (TBS) + 0.1% Tween (TBST), and then the wells were blocked with 5% bovine serum albumin in TBS at 4°C for 1 hour. After removing the blocking buffer, ST101 was diluted with TBS and added to the appropriate wells at 4°C for 1 hour, and the wells were washed three times with TBST.

[0118] Next, 1 ng of recombinant ATF5 was added to each well, incubated at 4°C for 18 hours, and washed three times with TBST to remove unbound proteins. The ATF5 bound to the plate was incubated at 4°C for 1 hour in the presence of a 1:1000 dilution rabbit anti-ATF5 antibody (ab60126, Abcam), followed by a 1:1000 dilution goat anti-rabbit IgG-HRP antibody (ab6721, Abcam). After incubation with each antibody, the plates were washed three times with TBST and detected using 50 μL of TMB substrate. The reaction was stopped by adding 25 μL of 2.5 M sulfuric acid, and absorbance was detected at 450 nm using a SpectraMax M3 plate reader (Molecular Devices). 50 This was calculated using GraphPad Prism 8.3.0 software via nonlinear regression with four parameters and a variable gradient.

[0119] In initial experiments, 3.6 ng of C / EBPβ was bound to the plate, and 1.0 nM of K was added via ATF5 binding. d The result was obtained (Figure 6, Panel A). In subsequent experiments, when gradually increasing concentrations of ST101 were added to a constant concentration of 1 nM ATF5, the detection of ATF5 decreased in a dose-dependent manner (Figure 6, Panel B). Nonlinear four-parameter analysis revealed that the IC5 of the inhibition of ATF5-C / EBPβ binding by ST101 was 50The concentration was determined to be 24.6 ± 0.9 nM. These data support the CD dimer exchange experiment.

[0120] Example 3. ST101 crosses the blood-brain barrier in naive C57BL / 6 mice. To determine whether systemic administration of ST101 can penetrate the brains of animals with an intact blood-brain barrier, immunohistochemical (IHC) analysis of ST101 was performed on naive C57BL / 6 brain sections after a single systemic administration. ST101 (25 mg / kg) or the vehicle was administered by intravenous injection into the lateral tail vein (n=3 / group). Two hours after injection, mice were euthanized. Brains were collected and stored in 4% paraformaldehyde. Tissue was processed for IHC staining of 2 μM sections using rabbit polyclonal anti-ST101 antibody detected with DAB-labeled anti-rabbit secondary reagent.

[0121] Brain sections from mice administered ST101 showed evidence of ST101 cell and microvascular staining (Figure 7, Panel B), but brain sections from mice administered the vehicle showed no evidence of ST101 staining (Figure 7, Panel A). These data indicate that ST101 crosses the blood-brain barrier in naive C57BL / 6 mice after systemic peptide administration.

[0122] Example 4. ST101 is safe. Safety pharmacology The potential pharmacological effects of ST101 on the central nervous system, cardiovascular system, and respiratory system were investigated in cynomolgus monkeys under 28-day toxicity, toxicological (TK), and good safety pharmacology (GLP) studies. No effects of ST101 were observed on any of these systems, as will be described in more detail below.

[0123] Electrocardiograms (ECGs) were performed before administration and near the final dose. To minimize handling-related stress, monkeys were sedated by intramuscular injection of 100 mg / mL ketamine hydrochloride at a dose of approximately 10 mg / kg. Electrocardiograms were qualitatively and quantitatively evaluated (e.g., QT&HR;QTc) by a committee-certified veterinary cardiologist. Electrocardiograms were recorded using leads I, II, III, aVR, aVL, and aVF. No ST101-related changes were observed in the electrocardiogram evaluation.

[0124] For supplementary ECG and respiratory assessment, qualitative electrocardiogram and respiratory changes induced in animals administered either control (0 mg / kg) or 30 mg / kg of ST101 via an IV bolus were compared using ECG waveforms and waveforms derived from thoracic and abdominal distension during ventilation. Overall, ECG traces from a total of 16 monkeys (8 males, 8 females; n=4 / group / sex) were evaluated. Qualitative assessment involved visual inspection of traces from each animal for rhythm and / or morphological abnormalities in the electrocardiogram, as well as thoracic and abdominal respiratory leads. Qualitative analysis assessed trace interpretation quality, presence or absence of muscle tremor or 60 Hz artifacts, general voltage changes over time, ST-T composition, and rhythm, among other factors. Data were compared between control and high-dose animals, and between the control and high-dose groups, both post-administration and baseline.

[0125] All monkeys had a short recording period during which they could subjectively analyze their electrocardiograms and lung images (in most cases). No subjective changes were observed in either the electrocardiogram or electropulmonary electrocardiogram attributable to ST101.

[0126] Indirect fundus examinations were performed by committee-certified veterinary ophthalmologists in sedated monkeys prior to assignment to the study and towards the end of the treatment period. No ST101-related changes were observed in the group-average ophthalmic assessment.

[0127] During the acclimatization period (baseline before treatment) and near the end of the treatment and recovery period, a staff veterinarian or a trained designated person recorded the respiration of the monkeys. Respiratory rate was recorded for 30 seconds and multiplied by 2 to obtain respiratory rate / minute. No ST101-related changes were evident in the assessment of the group's mean respiration.

[0128] Blood pressure (systolic, diastolic, and mean arterial pressure) was collected in monkeys prior to assignment to the study and near the end of the treatment and recovery period. Blood pressure was measured concurrently with respiratory examinations. Monkeys were sedated to minimize handling-related stress. No ST101-related changes were evident in the assessment of the group's mean blood pressure.

[0129] Evaluation of false allergic reactions Clinical signs of a pseudo-allergic reaction may include decreased body temperature, decreased activity, and piloerection. No clinical signs of a pseudo-allergic reaction were observed after intravenous (IV) bolus administration of 10 mg / kg ST101 over 2-3 minutes to cynomolgus monkeys. However, administration of 30 mg / kg resulted in moderate decreased activity, and the animals recovered within 24 hours. Extending the infusion rate from 2-3 minutes to 1-2 hours resulted in good tolerability of the 30 mg / kg ST101 dose.

[0130] Intravenous infusions of 50 mg / kg over 1 hour or 30 mg / kg over 30 minutes resulted in decreased activity and flushing, with animals recovering within a few hours. Subsequent studies showed no signs of pseudo-allergy in monkeys after four 1-hour infusions at dose levels of 7.5, 15, and 30 mg / kg over 11 days (n=2 / sex / group).

[0131] In a 28-day GLP toxicity study in monkeys (see Example 5), one of six female monkeys showed signs of a pseudo-allergic reaction after initial exposure to ST101 at 30 mg / kg, while the males did not. The animal appeared to be recovering and began eating and moving around within a few hours, so no intervention was performed. Approximately 48 hours after ST101 exposure, the animal's condition deteriorated, becoming lethargic and having a slow heart rate, and it died shortly thereafter. No other signs of pseudo-allergic reactions were observed in this study (n=4 cores + 2 recovered monkeys / sex / group, total of 36 ST101-treated animals). The tolerable levels of ST101 exposure in these studies were significantly higher than those tested in clinical settings. Therefore, pseudo-allergies are not expected to occur in human subjects.

[0132] Example 5. ST101 is highly tolerable. Test 1 The tolerability of ST101 after repeated 1-hour intravenous infusions at doses of 7.5 mg / kg, 15 mg / kg, and 30 mg / kg was evaluated in cynomolgus monkeys in a 12-day study. The study design shown in Table 1 consists of four groups (one for each dose of ST101 and one for the vehicle control), each comprising four monkeys (two of each sex), which were administered either ST101 or the vehicle control four times over 11 days. [Table 2]

[0133] Blood samples were collected at multiple points in time, before and after administration, on days 1 and 11. Tissue samples were collected from various organs, including the brain, bladder, heart, intestines, kidneys, liver, and lungs, evaluated, and processed for evaluation.

[0134] The results showed no significant changes in clinical observation, body weight, clinical chemistry, or hematology. Clear fluid was observed at one or more injection sites in both males administered 7.5 mg / kg, one male administered 15 mg / kg, one male administered 30 mg / kg, and all treated females. No other macroscopic findings associated with ST101 administration were observed.

[0135] Mild to severe thrombosis was observed at the last injection site in one male treated with ST101 at a dose of 7.5 mg / kg, one male treated with ST101 at a dose of 15 mg / kg, both males treated with ST101 at a dose of 30 mg / kg, and all treated females. No other microscopic findings related to ST101 were observed.

[0136] Administration of 7.5 mg / kg, 15 mg / kg, and 30 mg / kg doses to cynomolgus monkeys caused thrombosis, acute inflammation, red blood cell accumulation, fibrosis, and edema at the injection site. These findings were consistent with local irritation caused by ST101 administration. No other ST101-related macroscopic or microscopic findings were observed.

[0137] Based on the results of this study, a dose of 30 mg / kg was considered to be the maximum dose (HNSTD) and no-observed-adverse-effect level (NOAEL) of ST101 that does not cause serious toxicity.

[0138] Test 2 In this study, male and female cynomolgus monkeys were administered the test substance ST101 at doses of 7.5, 15, and 30 mg / kg (corresponding to groups 2, 3, and 4, respectively), or a vehicle control (group 1), as a 1-hour intravenous (IV) infusion twice a week for a total of 8 doses. The final dose was administered on day 24 for females and day 25 for males. The study design shown in Table 2 indicates that each of the four groups (each dose of ST101 and the vehicle control) consisted of 8 monkeys (4 of each sex) and 4 monkeys (2 of each sex) from the core study of the recovery group. After the final exposure to ST101, the recovery group was subjected to a 2-week treatment-free period. [Table 3]

[0139] Blood samples were collected on days 1 and 24 / 25 to analyze the plasma concentration of ST101 using a validated LC-MS / MS analysis method with a quantitative range of 20.0–2000 ng / mL. For groups 2 (7.5 mg / kg), 3 (15 mg / kg), and 4 (30 mg / kg), the toxicological (TK) parameters of ST101 were measured on days 1 and 24 / 25. Autopsies with macroscopic observation and organ weighing were performed on all animals, and histopathological diagnosis was performed on all 30 mg / kg dose and control core test animals.

[0140] Treatment-related findings included swelling / edema, scabbing, scaly skin, and erosion / ulceration at the injection site. The severity and incidence of these observations increased proportionally with the dose level. No ST101-related changes were evident in the assessment of mean body weight and weight gain in the group.

[0141] Clinical and chemical changes associated with ST101 occurred only in the 30 mg / kg dose group and were not observed in the 7.5 mg / kg or 15 mg / kg dose groups. Similarly, hematological changes such as increased platelet count and increased fibrinogen were limited to the 30 mg / kg dose group. Increased platelet and fibrinogen levels, as well as increased creatinine phosphokinase, correlate with vascular stimulation at the injection site.

[0142] Evaluation of conventional electrocardiogram and respiratory data collected using a DSI Jacketed (non-invasive) external telemetry system with a chest band showed no treatment-related changes.

[0143] Changes in organ weight parameters associated with ST101 included increased liver and kidney weight (absolute weight and relative weight to brain weight and body weight) at the final necropsy in males at a 30 mg / kg dose, and increased liver and kidney weight (absolute weight and relative weight to body weight) at the final necropsy in females at a 30 mg / kg dose. Absolute weight of the adrenal glands and kidneys increased in females at a 15 mg / kg dose. Increases in liver and kidney weight (absolute weight and relative weight to brain weight and body weight) and decreases in thymus weight (absolute weight and relative weight to brain weight and body weight) were still evident in animals at a 30 mg / kg dose at recovery necropsy.

[0144] Microscopic examination revealed minimal to moderate renal tubular necrosis in two males and three females administered 30 mg / kg of ST101, and mild protein cast formation in the renal tubules in one female administered 30 mg / kg of ST101. Minimal to severe renal tubular necrosis was observed in two males and one female administered 15 mg / kg. In recovered animals, minimal to mild renal tubular necrosis was observed in the kidneys of one male at 7.5 mg / kg, two males at 15 mg / kg, one male at 30 mg / kg, and one female at 15 mg / kg. The tubular changes were partially reversible during the period allocated to this study. The frequency and severity of renal lesions did not show a clear relationship with dose and occurred sporadically with low severity in core and recovered animals.

[0145] At end-stage necropsy, macroscopic lesions included clear fluid at one or more injection sites in two of the four males and two of the four females administered 30 mg / kg. One or more crusts / lesions / red pigmentation were observed at one or more injection sites in one of the four males and one of the four females administered 7.5 mg / kg, three of the four males and one of the four females administered 15 mg / kg, and three of the four males and all of the females administered 30 mg / kg. Mild to severe thrombosis at the last injection site was also observed in some animals in each dose group, occurring more frequently in the 30 mg / kg dose group. At recovery necropsy, one or more crusts / lesions / red pigmentation were observed at one or more injection sites in one of the two females administered 15 mg / kg and in both males administered 30 mg / kg.

[0146] In light of these results, the HNSTD dose was considered to be 15 mg / kg, and the NOAEL dose was considered to be 7.5 mg / kg.

[0147] Example 6: Administration of ST101 to patients with solid tumors An open-label Phase 1-2 dose-finding study will be conducted to determine the safety, tolerability, pharmacokinetics, pharmacodynamics (PD), and efficacy of ST101 administered intravenously to patients with advanced, unresectable, and metastatic solid tumors. This study will consist of two phases: (1) a dose escalation phase and (2) a dose expansion phase.

[0148] Dose escalation phase Test design The dose escalation phase includes intravenous administration of ST101 to patients diagnosed with locally advanced or metastatic melanoma, carcinoma, or sarcoma of any tumor type that is refractory or intolerant to all available therapies that may affect survival. Dose cohorts are administered once weekly (QW) at doses of 0.5, 1, 2, 4, 6, 8, and 16 mg / kg; the highest dose level may be administered every other week (Q2W). Administration is performed by IV infusion with a total infusion time of at least 90 minutes. ST101 is provided in a pharmaceutical composition containing trehalose and lactate, diluted in 0.9% saline.

[0149] Infusion-related reactions (IRRs) may occur during and / or after administration of ST101. Such reactions may include fever, chills / rigidity, tachycardia, tachypnea, hypotension, bronchospasm, and other observed symptoms. Unless the associated symptoms persist for more than two days, an IRR is not considered a DLT. IRRs are used by the DRC to assess the feasibility of the administration route and duration.

[0150] Supportive therapies may be implemented to maintain patient comfort through standard medical management. Such measures include discontinuing or delaying infusions, diphenhydramine, acetaminophen, and NSAIDs, crystalloid solutions, oxygen, bronchodilators, and other clinically necessary drug therapies, for example: a. Acetaminophen / paracetamol 650-1000 mg administered intravenously or orally (PO); b. Diphenhydramine 25-50 mg administered intravenously or by pre-ovenous injection; c. Chlorphenamine 5-10 mg administered intravenously or by prophylaxis; d. Ranitidine 50 mg administered intravenously or by pre-oral administration; e. Ibuprofen 200-400 mg administered as polyoxygenated solution; f. Naproxen 500 mg administered via polyoxygenated solution; g. IV (warmed) administration of 0.9% physiological saline, 1 L or PRN; h. Prochlorperazine 5-10 mg administered intravenously or by pre-ovenous injection; i. Ondansetron 4-16 mg administered intravenously or by proprioception; j. Dexamethasone 5-10 mg administered intravenously; k. Hydrocortisone 100 mg administered intravenously once; 1. Hydrocortisone 50 mg administered intravenously for 6 hours.

[0151] Patients who experience an associated IRR during the initial infusion will receive prophylaxis with minimal doses of diphenhydramine and acetaminophen in all subsequent infusions, which should be administered before ST101 administration in anticipation of IRR occurring during the infusion.

[0152] result The dose escalation phase is underway. All patients in cohorts 1-4 (dose levels 0.5, 1, 2, and 4 mg / kg, respectively; n=3 for each of cohorts 1-3; n=6 for cohort 4) completed the 21-day dose-limiting therapy (DLT) period, however, one patient in cohort 4 did not receive all doses due to disease progression. In cohort 5 (6 mg / kg), one patient completed the DLT period, and two patients are currently in the DLT period. Patients in cohort 5 received 10 mg of montelukast orally two days prior to administration and on the day of administration, 20 mg of famotidine orally or intravenously on the day of administration, and either 10 mg of chlorfenamine orally or 50 mg of diphenhydramine intravenously on the day of administration. Patients with other conditions, such as allergies, may receive secondary medications such as montelukast daily.

[0153] One patient in Cohort 1 and two patients in Cohort 3 showed no change in tumor size, as assessed by radiological evaluation (30% decrease to 20% increase), for at least 18 weeks. In particular, the patient in Cohort 1 showed no change for 46 weeks. The disease status of the patients is shown in Figure 8.

[0154] A 62-year-old woman with metastatic cutaneous melanoma refractory to all available therapies achieved a partial response after 9 weeks of treatment with 4 mg / kg ST101. The patient had previously experienced four local recurrences, which were resected and treated with isolated limb perfusion of melphalan. Lung metastases were treated with combination therapy of ipilimumab and nivolumab, followed by nivolumab monotherapy, but were discontinued due to hypophysitis. Subsequent frontal lobe brain metastases were treated with complete resection, radiotherapy, and nivolumab until progressive disease (PD). Subsequent miliary lung metastases were treated with temozolomide for approximately 16 months until PD, following resection of a large abdominal mass. The patient was diagnosed with piriformis muscle metastasis immediately prior to the initiation of this study. Table 3 shows the evaluation of the patient's response to ST101 therapy as measured by contrast-enhanced CT. [Table 4]

[0155] All but one of the patient's lesions shrank, with the largest lesion showing substantial reduction. Furthermore, several non-targeted lung lesions decreased in size, and no new lesions were detected.

[0156] Most adverse events (AEs) experienced by patients were internal reflex reactions (IRRs) occurring within the first six hours, which were controlled with antihistamines / anti-inflammatory drugs. To manage IRRs, the following measures were used in patients: antihistamines (58% of 12 patients), NSAIDs (17%, none in patients in cohorts 1, 3, or 4), acetaminophen / paracetamol (50%), corticosteroids (42%, none in patients in cohorts 1 or 2), famotidine (8%, none in patients in cohorts 1, 2, or 4), interrupted infusion (42%, none in patients in cohorts 1 or 2), and slowed infusion (42%, none in patients in cohorts 1 or 2).

[0157] Blood samples were collected at various points before administration and after the entire infusion period during the first cycle, and at less frequent thereafter. For patients belonging to the QW dosing cohort and therefore following a 21-day treatment cycle, blood samples were collected at the following points as shown in Table 4. [Table 5]

[0158] For patients belonging to the Q2W medication cohort and therefore following a 28-day treatment cycle, blood samples are collected at the following time points as shown in Table 5. [Table 6]

[0159] The pharmacokinetics (PK) of ST101 in plasma were evaluated by liquid chromatography-tandem mass spectrometry (LC-MS). The PK results for cohorts 1-4 are shown below. max and AUC t The saturation rate was proportional to the dose (Figure 9; Figure 10, Panels A and B), and each cohort showed minimal accumulation of ST101 4 hours after infusion (Figure 11). Furthermore, although the mean excretion half-life differed among the cohorts, the T saturation rate of each cohort was max This occurred at or near the end of the infusion. The PK results are summarized in Table 6. [Table 7]

[0160] Tumor uptake of ST101 was measured by immunohistochemical analysis. Biopsy samples taken from patients during screening, i.e., before ST101 treatment and during cycle 2 of treatment, were processed and immunoassayed for ST101 using rabbit polyclonal anti-ST101 antibody. Sections were analyzed by committee-certified pathologists and scored based on the incidence of cells showing ST101 uptake. The data show that tumor uptake of ST101 increases with increasing ST101 dose (Figure 12). All biopsies taken from patients during screening prior to ST101 exposure were negative for ST101 immunostaining.

[0161] The effect of ST101 on tumor cell proliferation was determined by immunohistochemical analysis. Biopsy samples taken from patients during screening and during cycle 2 of treatment were processed and immunoassayed for the presence of Ki67. Sections were analyzed by committee-certified pathologists and scored based on the intensity of the Ki67 signal. The data showed a decrease in Ki67 signal after ST101 exposure, as evidenced by the decrease in Ki67 staining (Figure 13).

[0162] Pharmacodynamic evaluation includes circulating cell-free deoxynucleic acid (DNA) analysis of mechanistically related genetic abnormalities and their temporal changes, as well as baseline and on-treatment tumor analysis using quantitative reverse transcriptase (qRT)-polymerase chain reaction (PCR), nanostrings, and immunohistochemistry (IHC).

[0163] Expansion phase The trial will proceed to the expansion phase once the highest escalation cohort is reached, or once significant efficacy is observed at a given dose level and a recommended phase 2 dose (RP2D) is selected. The selection of the RP2D will be based on safety, PK, PD, and efficacy data. The RP2D may be the maximum tolerated dose (MTD), the "active dose" (a dose of ST101 considered to have antitumor activity, selected based on preclinical, safety, PK, PD, and efficacy data), or another dose between the MTD and the active dose. The expansion phase will consist of cohorts of four specific tumor types, as described below.

[0164] At least 10 patients in the expanded cohort underwent a mandatory core biopsy or excisional biopsy at screening within 28 days prior to enrollment. Where possible, and in patients who underwent a screening biopsy and still have residual tumor, a post-treatment biopsy of the same lesion will be performed. Additional optional biopsies may be performed for other lesions.

[0165] Administration of ST101 to breast cancer patients Hormone receptor-positive (HR) hormonal receptors that have progressed after prior treatment with 1-2 types of hormone-based therapies. posPatients diagnosed with locally advanced / metastatic breast cancer (LA / MBC) were administered ST101 at doses of 0.5, 1, 2, 4, 6, 8, 12, or 16 mg / kg once weekly, or 16 mg / kg of ST101 every other week. Prior treatment with cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitors, mammalian target of rapamycin (mTOR) inhibitors, or chemotherapy was permitted as monotherapy or in combination.

[0166] RECIST 1.1 (Eisenhauer 2009) is used to evaluate tumor reduction, disease control rate (DCR), duration of response (DOR), and / or progression-free survival (PFS). Either calculated CT or MRI is used to evaluate tumor reduction, but CT is the preferred imaging technique. For radiological evaluation of CR or PR, a confirmatory image is required at least 4 weeks after the initial assessment of the response. For radiological evaluation of SD, a confirmatory image is required at least 6 weeks after the initial assessment of the response. PFS is determined from the time of the first administration of treatment until the first recorded disease progression or death. DOR is determined from the time the initial response was observed until the first recorded disease progression or death.

[0167] Administration of ST101 to melanoma patients Patients diagnosed with advanced / metastatic melanoma that has progressed after or during treatment with immune checkpoint inhibitors (CPIs) and have received one or two prior therapies should be administered ST101 at a dose of 0.5, 1, 2, 4, 6, 8, 12, or 16 mg / kg once weekly, or 16 mg / kg every other week. Patients with BRAF mutations must also be receiving one of the appropriate targeted therapies.

[0168] RECIST 1.1 (Eisenhauer 2009) is used to evaluate tumor reduction, disease control rate (DCR), duration of response (DOR), and / or progression-free survival (PFS). Either calculated CT or MRI is used to evaluate tumor reduction, but CT is the preferred imaging technique. For radiological evaluation of CR or PR, a confirmatory image is required at least 4 weeks after the initial assessment of the response. For radiological evaluation of SD, a confirmatory image is required at least 6 weeks after the initial assessment of the response. PFS is determined from the time of the first administration of treatment until the first recorded disease progression or death. DOR is determined from the time the initial response was observed until the first recorded disease progression or death.

[0169] Administration of ST101 to glioblastoma patients Patients diagnosed with primary (new-onset) GBM that has relapsed or progressed (according to modified RANO criteria) after one standard treatment regimen should receive ST101 at a dose of 0.5, 1, 2, 4, 6, 8, 12, or 16 mg / kg once weekly, or 16 mg / kg of ST101 every other week. "Standard treatment" is defined as maximum surgical resection, radiotherapy, and temozolomide or temozolomide adjuvant chemotherapy in combination with radiotherapy. Patients receiving tumor field therapy as an adjuvant to first-line treatment are eligible.

[0170] The modified RANO (Ellingson 2017) is used to evaluate tumor reduction, disease control rate (DCR), duration of response (DOR), and / or progression-free survival (PFS). Gadolinium-enhanced MRI is used to evaluate tumor reduction. For radiological evaluation of CR or PR, a confirmatory image is required at least 4 weeks after the initial assessment of the response. For radiological evaluation of SD, a confirmatory image is required at least 6 weeks after the initial assessment of the response. PFS is determined from the time of the first administration of treatment until the first recorded disease progression or death. DOR is determined from the time the initial response was observed until the first recorded disease progression or death.

[0171] Administration of ST101 to prostate cancer patients In patients diagnosed with castration-resistant prostate cancer (CRPC) that has progressed after prior treatment with taxanes, abiraterone, and enzalutamide / apalutamide (unless contraindicated or the patient is intolerant to these drugs), ST101 should be administered once weekly at a dose of 0.5, 1, 2, 4, 6, 8, 12, or 16 mg / kg, or every other week at a dose of 16 mg / kg.

[0172] The response will be scored using the PCWG3 guidelines (Scher 2016). Tumor reduction will be assessed using either CT or MRI. Imaging assessments will be performed until disease progression (PD) occurs. For radiological evaluations of CR or PR, a confirmatory image is required at least 4 weeks after the initial assessment of the response. For radiological evaluations of stable disease (SD), a confirmatory image is required at least 6 weeks after the initial assessment of the response. DCR, DOR, and PFS will be assessed based on PCWG3 measurements. The time from the first administration of the study treatment to the first recorded disease progression or death determines PFS. The time from the first observed response to the first recorded disease progression or death determines DOR. References Aguilar-Morante D, et al. Decreased CCAAT / enhancer binding protein beta expression inhibits the growth of glioblastoma cells. Neuroscience 176:110-119 (2011). Angelastro JM, et al. Selective destruction of glioblastoma cells by interference with the activity or expression of ATF5. Oncogene 2006;25: 907-916. Asada R, et al. The signalling from endoplasmic reticulum-resident bZIP transcription factors involved in diverse cellular physiology. J Biochem 2011;149: 507-518. Bernal F, et al. Reactivation of the p53 Tumor Suppressor Pathway by a Stapled p53 Peptide. J. Am. Chem. Soc. 2007;129:2456-2457. Bezy O, et al. Delta-interacting protein A, a new inhibitory partner of CCAAT / enhancer-binding protein beta, implicated in adipocyte differentiation. J Biol Chem 2005;280: 11432-11438. Bird GH, et al. Biophysical Determinants for Cellular Uptake of Hydrocarbon-Stapled Peptide Helices. Nat. Chem. Biol. 2017;12:845-852. Bruzzoni-Giovanelli H, et al. Interfering peptides targeting protein-protein interactions: the next generation of drugs? Drug Discov Today 2018;23: 272-285. Bushweller JH. Targeting transcription factors in cancer - from undruggable to reality. Nat Rev Cancer 2019;19: 611-624.Cates CC, et al. Regression / eradication of gliomas in mice by a systemically-deliverable ATF5 dominant-negative peptide. Oncotarget 2016;7: 12718-12730. Demma MJ, et al. Omomyc Reveals New Mechanisms To Inhibit the MYC Oncogene. Mol Cell Biol 2019;39. 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. Ellingson B, et al. Modified Criteria for Radiographic Response Assessment in Glioblastoma Clinical Trials. Neurotherapeutics. 2017;14:307-320. Homma J, et al. Increased expression of CCAAT / enhancer binding protein beta correlates with prognosis in glioma patients. Oncol Rep 2006;15: 595-601. Huggins CJ, et al. C / EBPgamma suppresses senescence and inflammatory gene expression by heterodimerizing with C / EBPbeta. Mol Cell Biol 2013;33: 3242-3258. Karpel-Massler G, et al. A Synthetic Cell-Penetrating Dominant-Negative ATF5 Peptide Exerts Anticancer Activity against a Broad Spectrum of Treatment-Resistant Cancers. Clin Cancer Res 2016;22: 4698-4711. Kim MH, et al. Translationally regulated C / EBP beta isoform expression upregulates metastatic genes in hormone-independent prostate cancer cells.Prostate 68:1362-1371 (2008). Lamb J, et al. A mechanism of cyclin D1 action encoded in the patterns of gene expression in human cancer. Cell 2003;114: 323-334. Lambert M, et al. Targeting Transcription Factors for Cancer Treatment. Molecules 2018;23. Lathbridge A, et al. Computational Competitive and Negative Design To Derive a Specific cJun Antagonist. Biochemistry 2018;57: 6108-6118. Lathbridge A, et al. Coupling Computational and Intracellular Screening and Selection Toward Co-compatible cJun and cFos Antagonists. Biochemistry 2019. Lekstrom-Himes J, et al. Biological role of the CCAAT / enhancer-binding protein family of transcription factors. J Biol Chem 1998;273: 28545-28548. Oya M, et al. Increased activation of CCAAT / enhancer binding protein-beta correlates with the invasiveness of renal cell carcinoma. Clin Cancer Res 2003;9: 1021-1027. Pal R, et al. C / EBPbeta regulates transcription factors critical for proliferation and survival of multiple myeloma cells. Blood 114:3890-3898 (2009). Potapov V, et al. Data-driven prediction and design of bZIP coiled-coil interactions. PLoS Comput Biol 2015;11: e1004046. Scher HI, et al. End Points and Outcomes in Castration-Resistant Prostate Cancer: From Clinical Trials to Clinical Practice. J. Clin. Oncology. 2011:29(27):3695-3704. Scher HI, et al. Trial Design and Objectives for Castration-Resistant Prostate Cancer: Updated Recommendations from the Prostate Cancer Clinical Trials Working Group 3. J Clin Oncology. 2016:34(12):1402-1418. Sheng Z, et al. An activating transcription factor 5-mediated survival pathway as a target for cancer therapy? Oncotarget 2010;1: 457-460. Takada K, et al. Targeted disruption of the BCL9 / beta-catenin complex inhibits oncogenic Wnt signaling. Sci Transl Med 2012;4: 148ra117. Walensky LD, et al. Hydrocarbon-stapled peptides: principles, practice, and progress. J Med Chem 2014;57: 6275-6288. Yan C, et al. Drugging the undruggable: transcription therapy for cancer. Biochim Biophys Acta 2013;1835: 76-85. Zahnow CA. CCAAT / enhancer-binding protein beta: its role in breast cancer and associations with receptor tyrosine kinases. Expert Rev Mol Med 2009;11: e12. Zhang ZY, et al. Stabilization of ATF5 by TAK1-Nemo-like kinase critically regulates the interleukin-1beta-stimulated C / EBP signaling pathway. Mol Cell Biol 2015;35: 778-788. Zhao Y, et al. p300-dependent acetylation of activating transcription factor 5 enhances C / EBPbeta transactivation of C / EBPalpha during 3T3-L1 differentiation. Mol Cell Biol 2014;34: 315-324. *** The present invention is further described by the following claims. In some embodiments, the present invention may be described as follows. [Aspect 1] A method for treating a solid tumor in a human patient, the method comprising parenterally administering to the patient a pharmaceutical composition containing an effective amount of a CCAAT enhancer-binding protein β (C / EBPβ) peptide antagonist. [Aspect 2] The method according to aspect 1, wherein the solid tumor is a melanoma, carcinoma, or sarcoma. [Aspect 3] The method according to aspect 1, wherein the patient has been diagnosed with locally advanced / metastatic breast cancer (LA / MBC), melanoma, glioblastoma (GBM), or castration-resistant prostate cancer (CRPC). [Aspect 4] The method according to aspect 1, wherein the patient has received prior treatment selected from the group consisting of chemotherapy, hormone therapy, immunotherapy, targeted therapy, and combinations thereof. [Aspect 5] The peptide antagonist is a D-amino acid sequence VAEAREELERLEARLGQARGEL (Sequence ID) 1 The method according to embodiment 1, including ) [Aspect 6] The peptide antagonist has the amino acid sequence LEGRAQGLRAELRELEERAEAV (Sequence ID)4 The method according to embodiment 1, including ) [Aspect 7] The method according to aspect 1, wherein the peptide antagonist is a cell-permeable peptide. [Aspect 8] The method according to aspect 1, wherein the peptide antagonist is ST101. [Aspect 9] The method according to aspect 8, wherein the peptide antagonist is administered to the patient at a dose of approximately 0.5 to 16 mg / kg. [Aspect 10] The method according to aspect 1, wherein the pharmaceutical composition is administered intravenously. [Aspect 11] The method according to aspect 10, wherein the pharmaceutical composition is administered by injection. [Aspect 12] The method according to aspect 11, wherein the pharmaceutical composition is administered by intravenous infusion with a total infusion time of approximately 30 to approximately 360 minutes. [Aspect 13] The method or composition according to aspect 12, wherein the total injection time is approximately 60 to approximately 180 minutes. [Aspect 14] The method according to aspect 1, wherein the pharmaceutical composition is administered once a week. [Aspect 15] The method according to aspect 1, wherein the pharmaceutical composition is administered once every two weeks. [Aspect 16] The method according to aspect 1, wherein the pharmaceutical composition is administered for at least 4 weeks. [Aspect 17] The method according to aspect 1, wherein one or more secondary agents selected from the group consisting of antihistamines, leukotriene inhibitors, nonsteroidal anti-inflammatory drugs, acetaminophen, corticosteroids, antiemetics, intravenous saline, and electrolytes are administered simultaneously with, before, or after the administration of the pharmaceutical composition. [Aspect 18] The method according to aspect 17, wherein an antihistamine and / or a leukotriene inhibitor is administered to the subject within approximately 48 hours prior to administration of the pharmaceutical composition. [Aspect 19] The method according to aspect 1, wherein the pharmaceutical composition comprises a buffer and a bulking agent. [Aspect 20] The method according to aspect 1, wherein the pharmaceutical composition comprises lactic acid and trehalose. [Aspect 21] The method according to aspect 1, wherein the pharmaceutical composition has a pH of about 3.0 to 8.0. [Aspect 22] HR in human patientspos A method for treating LA / MBC, the method comprising administering a pharmaceutical composition containing an effective amount of ST101 to the patient by intravenous infusion. [Aspect 23] A method for treating melanoma in a human patient, the method comprising administering to the patient by intravenous infusion a pharmaceutical composition containing an effective amount of ST101, wherein the patient has received at least one treatment prior to the administration of ST101. [Aspect 24] The method according to aspect 23, wherein the patient has melanoma that is progressing after or during treatment with an immune checkpoint inhibitor, and the patient has progressive / metastatic melanoma. [Aspect 25] The method according to aspect 23, wherein the patient has melanoma containing a BRAF mutation and the patient is receiving at least one targeted therapy. [Aspect 26] A method for treating primary GBM in a human patient, the method comprising administering a pharmaceutical composition containing an effective amount of ST101 to the patient by intravenous infusion. [Aspect 27] The method according to aspect 26, wherein the GBM has relapsed or progressed after prior treatment with maximum surgical resection, radiotherapy, and temozolomide or adjuvant chemotherapy with temozolomide in combination with radiotherapy. [Aspect 28] A method for treating CRPC in a human patient, the method comprising administering a pharmaceutical composition containing an effective amount of ST101 to the patient by intravenous infusion. [Aspect 29] The method according to aspect 28, wherein the patient has CRPC that has progressed after prior treatment with taxane, abiraterone, dalalutamide, and / or enzalutamide / apalutamide. [Aspect 30] The method according to any one of aspects 22 to 29, wherein ST101 is administered to the patient at a dose of approximately 0.5 to 16 mg / kg. [Aspect 31] The method according to aspect 30, wherein the pharmaceutical composition is administered once a week for at least three weeks. [Aspect 32] The method according to aspect 30, wherein the pharmaceutical composition is administered once every two weeks for at least four weeks. [Aspect 33] The method according to any one of aspects 22 to 32, wherein the pharmaceutical composition comprises trehalose and lactic acid. [Aspect 34] The method according to any one of aspects 22 to 33, wherein the pharmaceutical composition is administered by intravenous infusion with a total infusion time of approximately 60 to approximately 360 minutes. [Aspect 35] The method according to any one of aspects 22 to 34, wherein the clearance of ST101 is 0.75 to 3.5 liters per hour. [Aspect 36] Half-life of ST101 (t 1 / 2 The method according to any one of embodiments 22 to 35, wherein the duration is 10 to 70 hours. [Aspect 37] A pharmaceutical composition for parenteral administration comprising an effective amount of a C / EBPβ peptide antagonist for use in the treatment of solid tumors in human patients. [Aspect 38] Melanoma in human patients, HR pos A pharmaceutical composition for intravenous infusion containing an effective amount of ST101 for use in the treatment of LA / MBC, primary GBM, or CRPC.

Claims

1. A pharmaceutical composition for the treatment of solid tumors in human patients, for parenteral administration to the patient, wherein the pharmaceutical composition comprises a peptide antagonist of CCAAT enhancer-binding protein β (C / EBPβ) containing the D-amino acid sequence VAEAREEELLEARLGQARGELKKWKMRRNQFWLKLQR (SEQ ID NO: 2), wherein the peptide antagonist is a cell-permeable peptide, and the peptide antagonist is administered to the patient at a dose of 0.5 to 16 mg / kg.

2. The pharmaceutical composition according to claim 1, wherein the solid tumor is a melanoma, carcinoma, or sarcoma.

3. The pharmaceutical composition according to claim 1, wherein the patient has been diagnosed with locally advanced / metastatic breast cancer (LA / MBC), melanoma, glioblastoma (GBM), or castration-resistant prostate cancer (CRPC).

4. The pharmaceutical composition according to claim 1, wherein the patient has received prior treatment selected from the group consisting of chemotherapy, hormone therapy, immunotherapy, targeted therapy, and combinations thereof.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the peptide antagonist is ST101.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is administered intravenously.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein one or more secondary agents selected from the group consisting of antihistamines, leukotriene inhibitors, nonsteroidal anti-inflammatory drugs, acetaminophen, corticosteroids, antiemetics, intravenous saline, and electrolytes are administered simultaneously with, before, or after the administration of the pharmaceutical composition.

8. The pharmaceutical composition according to claim 7, wherein an antihistamine and / or a leukotriene inhibitor is administered to the subject within approximately 48 hours prior to administration of the pharmaceutical composition.

9. HR in human patients ｐｏｓ The pharmaceutical composition according to claim 5, for the treatment of LA / MBC.

10. The pharmaceutical composition according to claim 5, for the treatment of melanoma in human patients.

11. The pharmaceutical composition according to claim 10, wherein the patient has melanoma that is progressing after or during treatment with an immune checkpoint inhibitor, and the patient has progressive / metastatic melanoma.

12. The pharmaceutical composition according to claim 10, wherein the patient has melanoma containing a BRAF mutation, and the patient is receiving at least one targeted therapy.

13. The pharmaceutical composition according to claim 5, for the treatment of primary GBM in human patients.

14. The pharmaceutical composition according to claim 13, wherein the GBM has relapsed or progressed after prior treatment with maximum surgical resection, radiotherapy, and temozolomide or adjuvant chemotherapy with temozolomide in combination with radiotherapy.

15. The pharmaceutical composition according to claim 5, for the treatment of CRPC in human patients.

16. The pharmaceutical composition according to claim 15, wherein the patient has CRPC that has progressed after prior treatment with taxane, abiraterone, dalalutamide, and / or enzalutamide / apalutamide.

17. The pharmaceutical composition according to any one of claims 9 to 16, wherein the clearance of ST101 is 0.75 to 3.5 liters per hour.

18. Half-life of ST101 (t １／２ The pharmaceutical composition according to any one of claims 9 to 17, wherein the duration is 10 to 70 hours.

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