Use of C / EBP-beta antagonists and immunomodulators

Abstract

Methods of administering a peptide antagonist of CCAAT / enhancer binding protein beta (C / EBP beta) in combination with an immunomodulator are provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 414,397, filed on October 7, 2022.

[0003] Reference Electronic Sequence Listing

[0004] The contents of the electronic sequence listing (file name: Sapience020WO1.xml; size: 4,534 bytes; creation date: October 6, 2023) are incorporated herein by reference in their entirety. Background Art

[0005] CCAAT / enhancer binding protein β (C / EBPβ) is a basic leucine zipper (bZIP) transcription factor that leads to aberrant gene activation in many cancers. Upregulated or overactivated C / EBPβ drives tumorigenesis by promoting tumor survival and proliferation and is a key regulator of the immunosuppressive environment (Homma 2006; Ruffell 2009). Specifically, C / EBPβ regulates macrophage differentiation, promoting the expression of M2 myeloid-derived suppressor cells (MDSCs), which contribute to the suppression of antitumor immunity and are associated with poor prognosis (Marigo 2010). Reprogramming tumor-associated macrophages (TAMs) from an M2 phenotype to an M1 phenotype represents a potential strategy to enhance antitumor immunity.

[0006] Immune checkpoint inhibitors (ICIs) have shown unprecedented success in immunogenic tumors such as melanoma, however many patients with advanced solid tumors respond poorly or not at all to ICI therapy. Refractory tumors are often classified as "cold" tumors, characterized by a lack of T cell infiltration into the tumor stroma or an immunosuppressive environment that precludes T cell infiltration and activity. Because the immunosuppressive tumor microenvironment (TME) is a major barrier to the success of ICI therapy in solid cancers, the ability to convert the TME to an immunoactive state holds great promise for improving responses.

[0007] ST101 is a novel peptide antagonist that prevents C / EBPβ dimerization and inhibits C / EBPβ-dependent gene expression. The confirmed responses in melanoma and other tumors prompted the evaluation of the effects of ST101 on macrophage differentiation. Summary of the invention

[0008] Some main aspects of the present invention are summarized below. Additional aspects are described in the detailed description, examples, drawings, and claims sections of the present disclosure. The description in each section of the present disclosure is intended to be read together with the other sections. In addition, the various embodiments described in each section of the present disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present invention.

[0009] We demonstrate that ST101 exposure reprograms M2-like macrophages to an immune-promoting M1-like phenotype in vitro and in vivo. Macrophage repolarization leads to restoration of cytotoxic T cell activity and synergizes with anti-PD-1 therapy to enhance antitumor responses in an in vivo triple-negative breast cancer model. These results identify ST101 as a novel approach to enhance macrophage antitumor activity and support its utility for combination strategies in cancers that respond poorly to ICl.

[0010] Therefore, the present disclosure provides a method for administering a combination of a peptide antagonist of CCAAT enhancer binding protein β (C / EBPβ) and an immunomodulator. In one embodiment, a method for inhibiting the growth of a solid tumor in a subject is provided, the method comprising performing a combined therapy with the following pharmaceutical composition: (i) a pharmaceutical composition comprising an effective amount of a peptide antagonist of C / EBPβ and (ii) a pharmaceutical composition comprising an effective amount of an immunomodulator; wherein the pharmaceutical composition comprising the antagonist of C / EBPβ and the pharmaceutical composition comprising the immunomodulator are administered to the subject together or separately.

[0011] Another embodiment is a method of reducing the volume of a solid tumor in a subject, the method comprising combination therapy with: (i) a pharmaceutical composition comprising an effective amount of a peptide antagonist of C / EBPβ and (ii) a pharmaceutical composition comprising an effective amount of an immunomodulator; wherein the pharmaceutical composition comprising the antagonist of C / EBPβ and the pharmaceutical composition comprising the immunomodulator are administered to the subject together or separately.

[0012] Another embodiment provides a method for treating a solid tumor in a subject, the method comprising performing a combination therapy with: (i) a pharmaceutical composition comprising an effective amount of a peptide antagonist of C / EBPβ and (ii) a pharmaceutical composition comprising an effective amount of an immunomodulator. The pharmaceutical composition comprising the antagonist of C / EBPβ and the pharmaceutical composition comprising the immunomodulator are administered to the subject together or separately.

[0013] Also provided are pharmaceutical compositions comprising an effective amount of a peptide antagonist of C / EBPβ and a pharmaceutical composition comprising an effective amount of an immunomodulator for use in a combined treatment method for inhibiting growth of a solid tumor in a subject, reducing the volume of a solid tumor in a subject and / or treating a solid tumor in a human subject.

[0014] In a preferred embodiment, the antagonist of C / EBPβ is ST101 and the immunomodulator is a PD-1 inhibitor.

[0015] In some embodiments, the solid tumor is a melanoma, carcinoma, or sarcoma. In certain embodiments, the subject is diagnosed with locally advanced or metastatic breast cancer (LA / MBC), melanoma, glioblastoma (GBM), or castration-resistant prostate cancer (CRPC).

[0016] In one embodiment, the peptide antagonist comprises the D-amino acid sequence VAEAREELERLEARLGQARGEL (SEQ ID NO: 1). In another embodiment, the peptide antagonist comprises the amino acid sequence LEGRAQGLRAELRELEERAEAV (SEQ ID NO: 3). In some embodiments, the peptide antagonist is a cell penetrating peptide. For example, the peptide antagonist may comprise a cell penetrating sequence. In a specific embodiment, the peptide antagonist is ST101 (SEQ ID NO: 2).

[0017] In some embodiments, the peptide antagonist is administered to a subject at a dose of about 0.5-16 mg / kg. In one embodiment, the dose of ST101 is about 500 mg.

[0018] In a certain aspect of the invention, the immunomodulator is an antibody or an antibody-drug conjugate, for example, selected from the group consisting of amivantamab, belantamab mafodotin-blmf, bevacizumab, cetuximab, denosumab, dinutuximab, enfortumab vedotin-ejfv, margetuximab, naxitamab-gqgk, necitumumab, panitumumab, pertuzumab, ramucirumab, sacituzumab govitecan-hzi y, tebentafusp-tebn, tisotumab vedotin-ejfv, tisotumab vedotin-gqgk, tisotumum ... In certain aspects, the antibody is an anti-PD-1 antibody.

[0019] In one aspect of the invention, the immunomodulator is selected from the group consisting of a checkpoint inhibitor, a cytokine, and an immune adjuvant. The checkpoint inhibitor can target, for example, at least one of PD-1, PD-L1, CTLA-4, or LAG-3. In a specific embodiment, the checkpoint inhibitor is atezolizumab, avelumab, cemiplimab, dostalimab, durvalumab, ipilimumab, nivolumab, pembrolizumab, relatimab, or a combination thereof.

[0020] In one embodiment, the immunomodulator is a cytokine that targets at least one of, for example, the IL-2 pathway, the IL-2R pathway, the IFNAR1 pathway, or the IFNAR2 pathway. Examples of cytokines used in the methods of the invention include aldesleukin, granulocyte-macrophage colony stimulating factor, interferon alpha-2a, interferon alpha-2b, and pegylated interferon alpha-2b.

[0021] The immunomodulator may be an immunoadjuvant. In one embodiment, the immunoadjuvant targets the Toll-like receptor 7 pathway or the Toll-like receptor 3 pathway. Specific examples of immunoadjuvants include imiquimod and polyICLC.

[0022] In one embodiment, the pharmaceutical composition comprising the antagonist of C / EBPβ and / or the pharmaceutical composition comprising the immunomodulator is administered parenterally, such as intravenously.

[0023] In some embodiments, the pharmaceutical composition comprising an antagonist of C / EBPβ and the pharmaceutical composition comprising an immunomodulator are administered to the subject on different days.

[0024] In certain embodiments, the pharmaceutical composition comprising an antagonist of C / EBPβ is administered once a week for at least three weeks, or once every two weeks for at least four weeks. In certain embodiments, the pharmaceutical composition comprising an immunomodulator is administered once a week for at least three weeks, or once every two weeks for at least four weeks, or once every three weeks for at least six weeks. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A schematic diagram showing the protocol of the human peripheral blood mononuclear cell (hPBMC) M1 / M2 polarization model described in Example 1 is shown.

[0026] Figure 2A-2B It is shown that in the case of Figure 2A ) and donor 2 ( Figure 2B ) in cultured hPBMCs, ST101 converts the M2 program to an M1-like phenotype. Cells were treated with the indicated concentrations of ST101 and the expression of CD163 and CD68 was measured by flow cytometry.

[0027] Figures 3A-3D It was shown that ST101 significantly reduced the expression of Figure 3A-3B ) and donor 2 ( Figure 3C-3D ) in culture of hPBMC-derived M2 cells. The cells were treated with the indicated concentrations of ST101, and the expression of CD163 and CD68 was measured by flow cytometry ( Figure 3A , 3C ). Figure 3B and 3D Shown are the ratios of M2 cells to M1 cells after treatment with the indicated concentrations of ST101.

[0028] Figure 4A-4B Shown that ST101 inhibits M2 macrophage polarization in hPBMCs derived from Donor 1. Cells were treated with the indicated concentrations of ST101 and CD163 ( Figure 4A ) and CD68( Figure 4B ) expression.

[0029] Figures 5A-5C ST101 is shown to affect the tumor microenvironment. Nanostring analysis of tumor samples from human patients treated with ST101 showed a reduction in IL6 signaling ( Figure 5A ), increased tumor-infiltrating macrophages ( Figure 5B ) and decreased regulatory T cells ( Figure 5C ).

[0030] Figure 6 It is shown that ST101 restores the activation of cultured human CD8+ T cells co-incubated with immunosuppressive M2 macrophages.

[0031] Figure 7 Enhanced tumor growth inhibition (TGI) by combined treatment with an antagonist of C / EBPβ (ST101) and an immune checkpoint inhibitor (anti-PD-1 antibody) was shown. DETAILED DESCRIPTION

[0032] In order to more easily understand the present invention, some terms are first defined. Additional definitions are set forth throughout this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention relates.

[0033] Any headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be obtained by reference to the specification as a whole. Accordingly, the terms defined immediately below are defined in more detail by reference to the specification as a whole.

[0034] All references cited in this disclosure are hereby incorporated by reference in their entirety. In addition, any manufacturer's instructions or catalogs of any products cited or mentioned herein are incorporated by reference. The documents incorporated by reference herein or any teaching therein can be used in the practice of the present invention. The documents incorporated by reference herein are not recognized as prior art.

[0035] I. Definitions

[0036] The phraseology or terminology in the present disclosure is for the purpose of description rather than limitation, so that the phraseology or terminology of the present specification will be interpreted by skilled artisans based on teachings and guidance.

[0037] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The term "a" (or "an") and the terms "one or more" and "at least one" are used interchangeably.

[0038] Furthermore, “and / or” should be considered as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” used in phrases such as “A and / or B” is intended to include: “A and B”; “A or B”; “A” (alone); and “B” (alone). Similarly, the term “and / or” used in 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).

[0039] Whenever an embodiment is described using the wording "comprising," other similar embodiments described in the terms "consisting of" and / or "consisting essentially of are included.

[0040] Units, prefixes and symbols are expressed in a form acceptable to the International System of Units (SI). Numerical ranges include numbers that limit the range, and any single value provided herein can be used as the endpoints of the range including other single values ​​provided herein. For example, a set of values ​​(such as 1, 2, 3, 8, 9 and 10) is also disclosed in numerical ranges such as 1-10, 1-8, 3-9. Similarly, the disclosed range is the disclosure of each single value (i.e., intermediate value) contained in the range, including integers and fractions. For example, the range of 5-10 is also disclosed in 5, 6, 7, 8, 9 and 10, and 5.2, 7.5, 8.7, etc.

[0041] Unless otherwise indicated, the term "at least" or "approximately" preceding a series of elements should be understood to refer to each element in the series. The term "about" preceding a numerical value includes ±10% of the value. For example, a concentration of about 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of about 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v).

[0042] The terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to amino acid polymers of any length and their salts. The polymer may be straight or branched, may contain modified amino acids, and may be interspersed with non-amino acids. Unless otherwise indicated, for example, for the abbreviations of unusual or unnatural amino acids described herein, the three-letter and single-letter abbreviations used in the art are used herein to represent amino acid residues. Unless preceded by a "D" or a lowercase letter, an amino acid is an L-amino acid. Groups or strings of amino acid abbreviations are used to represent peptides. Unless otherwise indicated, peptides are indicated with the N-terminus on the left, and the sequence is written from the N-terminus to the C-terminus.

[0043] "Retro-inverso" peptides have an inverted amino acid sequence relative to a reference L-amino acid sequence and are composed of D-amino acids (reversing the chirality of the α-center of the amino acid subunit) to help maintain a side chain topology similar to the original L-amino acid peptide.

[0044] An "isolated" molecule is a molecule in a form not found in nature, including those that have been purified.

[0045] An "active agent" is an ingredient intended to provide biological activity. An active agent may be associated with one or more other ingredients. An active agent that is a peptide may also be referred to as an "active peptide."

[0046] An "effective amount" of an active agent is an amount sufficient to achieve a particular purpose.

[0047] The term "pharmaceutical composition" refers to a formulation that is in a form that allows the biological activity of the active ingredient to be effective and does not contain additional components that are unacceptably toxic to the subject to whom the composition is administered. Such compositions can be sterile and can contain a pharmaceutically acceptable carrier, such as normal saline. Suitable pharmaceutical compositions can contain one or more buffers (e.g., acetate, phosphate or citrate buffers), surfactants (e.g., polysorbates), stabilizers (e.g., polyols or amino acids), preservatives (e.g., sodium benzoate), and / or other conventional solubilizing agents or dispersants.

[0048] A "subject" or "individual" or "animal" or "patient" or "mammal" is any subject, particularly a mammalian subject, for whom diagnosis, prognosis or treatment is desired. Mammalian subjects include humans, livestock, farm animals, sports animals, and laboratory animals, including, for example, humans, non-human primates, dogs, cats, pigs, cattle, horses, rodents, including rats and mice, rabbits, and the like.

[0049] A "control patient" is a subject who has not received treatment of the present invention. A "control population" or "control patient population" is a group of subjects who have not received treatment of the present invention. The subjects in the control patient or control population have the same disease or condition as the subjects compared to the control patient or control population. For example, the clinical results of cancer patients who receive the composition or method of the present invention are compared with the average (median) results of subjects with the same type of cancer who have not received the pharmaceutical composition or method of the present invention. In some embodiments, the control patient or the patient in the control population has received a treatment different from the treatment of the present invention, such as standard of care treatment.

[0050] Terms such as "treating" or "treatment" or "alleviating" or "to alleviate" refer to therapeutic measures that cure, alleviate, relieve symptoms, and / or arrest the progression of a diagnosed pathological condition or disorder. In certain embodiments, a subject's disease or disorder is successfully "treated" if the patient exhibits complete, partial, or temporary relief or elimination of at least one symptom or measurable physical parameter associated with the disease or disorder.

[0051] An "antagonist" is a substance that prevents, blocks, inhibits, neutralizes or reduces the biological activity or effect of another molecule (eg, a receptor or ligand).

[0052] The terms "inhibit," "block," and "suppress" are used interchangeably and refer to any statistically significant reduction in the incidence or activity, including a complete blockade of the incidence or activity. For example, "inhibit" may refer to a reduction in activity or incidence of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. An "inhibitor" is a molecule, factor, or substance that produces a statistically significant reduction in the incidence or activity of a process, pathway, or molecule.

[0053] A "tumor" or "solid tumor" is a mass of neoplastic cells, such as cancer cells. The terms "advanced," "metastatic," and "advanced / metastatic" are used interchangeably to describe cancers in which malignant cells have migrated from the original tumor to another location in the patient's body, such as another organ.

[0054] "Tumor cells" or "tumors" have typically undergone some form of mutation / transformation compared to normal cells or tissues of the same type, resulting in abnormal growth. Tumors include morphological irregularities as well as pathological proliferations. Tumor cells can be benign or malignant. Malignant tumors (i.e., cancers) are distinguished from benign tumors in that they exhibit loss of cell differentiation and orientation and have the properties of invasion and metastasis.

[0055] II. Peptides and Compositions

[0056] C / EBPβ

[0057] Since C / EBPβ depends on the basic leucine zipper (bZIP) for its interaction with cofactors, it represents a prime target for the development of peptide antagonists. To associate with DNA and transcriptionally activate gene expression, C / EBPβ dimerizes with binding partners through interactions between their bZIP domains. In addition to homodimerization, C / EBPβ also forms heterodimers with bZIP-containing transcription factors such as Jun / Fos, C / EBPγ (Huggins 2013), delta-interacting protein A (Bezy 2005), and the CREB / ATF family (Zhao 2014).

[0058] Activating transcription factor 5 (ATF5) is a CREB / ATF factor that has been identified to associate with and activate C / EBPβ in HEK293 and HCT116 cancer cells, leading to transactivation of a pro-survival phenotype (Zhang 2015). ATF5 is highly expressed in many cancers, including gliomas, where it contributes to an oncogenic phenotype by driving overexpression of Bcl-2 family proteins and survivin, but is rarely found in differentiated cell types (Sheng 2010). Overexpression of a truncated bZIP domain of ATF5 lacking the DNA binding domain leads to cancer cell toxicity in glioma and other tumor cells (Angelastro 2006); administration of a peptide containing the truncated bZIP domain also produces similar results (Cates 2016; Karpel-Massler 2016).

[0059] C / EBPβ antagonist peptide

[0060] In some embodiments, the methods of the present invention include treating a patient with a solid tumor with a combination therapy comprising an effective amount of a peptide antagonist of C / EBPβ and an immunomodulator. In one embodiment, the peptide antagonist of C / EBPβ comprises the D-amino acid sequence VAEAREELER LEARLGQARGEL (SEQ ID NO: 1), which is a retro-inverse variant of the wild-type ATF5 bZIP domain. The peptide antagonist of C / EBPβ can be designed, for example, as described in Example 1 of WO 2021 / 262604.

[0061] The peptide antagonist of C / EBPβ can be a cell penetrating peptide. In one embodiment, the peptide comprises a cell penetrating domain. Many cell penetrating peptide sequences have been described and characterized in the literature (see WO 2019 / 136125). In one embodiment, the peptide is a cyclic peptide. For example, using hydrocarbon nails (Bernal 2007; Bird 2016) or other cyclization methods known in the art, cyclized peptides can enter cells by passive diffusion, endocytosis / endosome escape or other mechanisms (Dougherty 2019). The peptide can also be delivered to cells by mechanisms utilizing cell receptors (e.g., integrin targeting, RGD-like sequences). Alternatively, the peptide can be encapsulated in a vesicle (such as an exosome or liposome) or in a micelle and delivered to the cell.

[0062] The ability of peptides based on the native ATF5 bZIP domain to antagonize C / EBPβ activity can be measured by the methods described herein, for example, in Example 2 of WO2021 / 262604. The cytotoxic activity of peptide antagonists of C / EBPβ can be measured in vitro by known assays and / or in vivo using known tumor models; for example, WO 2019 / 136125 describes such assays and models.

[0063] ST101 is an all-D-amino acid peptide that exhibits potent antitumor activity and resistance to proteolytic degradation in vitro and in vivo. Specifically, we have previously demonstrated the cytotoxicity of ST101 in HL60 (promyelocytic leukemia), AMLl4 (acute myeloid leukemia), SET2 (megakaryocytic leukemia), A375 (melanoma), MCF7 (breast cancer), U87 (glioblastoma), U251 (glioblastoma), DU145 (prostate cancer), A549 (lung cancer), peripheral blood mononuclear cells (PBMC) and bone marrow mononuclear cells (BMMC) (see WO 2019 / 136125). In addition, subcutaneous administration of ST101 in a xenograft mouse model using A375, HL60, MCF7 and U251 cells resulted in a significant reduction in tumor volume (see WO2019 / 136125).

[0064] ST101 consists of a modified domain based on the ATF5 bZIP domain and the antennapedia penetratin domain to allow cell penetration. The D-amino acid sequence of ST101 is VAEAR EELERLEARLGQARGELKKWKMRRNQFWLKLQR (SEQ ID NO: 2), where the cell penetration region is in italics. ST101 promotes cytotoxic activity in tumor cells by disrupting the association of C / EBPβ with anti-apoptotic transcription factors (see WO 2021 / 262604).

[0065] We have demonstrated for the first time that ST101 alters the tumor microenvironment and reprograms macrophage differentiation to an M1 phenotype. The potential effects of ST101 on anti-tumor immunity prompted us to test whether the combination of C / EBPβ antagonists (such as ST101) and immunomodulators (such as immune checkpoint inhibitors) would enhance anti-tumor activity. The combination of ST101 and PD-1 unexpectedly reduced tumor volume in a triple-negative breast cancer mouse model, demonstrating an approximately 1.8 to 2.0-fold increase in activity compared to either agent alone. (See Example 3; Figure 7 ).

[0066] IV. Methods for preparing C / EBPβ antagonists

[0067] Peptide antagonists of C / EBPβ may be chemically synthesized, for example using solid phase peptide synthesis or liquid phase peptide synthesis, or a combination of both. The synthesis may optionally be performed as peptide fragments that are subsequently joined chemically or enzymatically.

[0068] Alternatively, a peptide antagonist of C / EBPβ can be expressed using a recombinant method. 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 those codons that are selected to be favorable in the host cell that will produce recombinant ST101. Standard methods can be used to synthesize nucleic acid molecules encoding a peptide antagonist of C / EBPβ, such as ST101.

[0069] Once prepared, the nucleic acid encoding the peptide can be inserted into an expression vector and operably linked to an expression control sequence suitable for expressing the peptide in the desired host. In order to obtain high expression levels of the peptide, the nucleic acid can be operably linked or associated with transcriptional and translational expression control sequences that are functional in the selected expression host.

[0070] Any known person skilled in the art can use a variety of expression host / vector combinations. Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli (E. coli), including pCR1, pBR322, pMB9 and derivatives thereof, plasmids with a wider host range, such as M13, and filamentous single-stranded DNA phage.

[0071] Suitable host cells include prokaryotes, yeast, insects or higher eukaryotic cells under the control of suitable promoters. Prokaryotes include gram-negative or gram-positive organisms, such as Escherichia coli or bacillus. Cell lines of higher eukaryotic cells or mammalian sources can be established, examples of which include Pichia pastoris, 293 cells, COS-7 cells, L cells, C127 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, HeLa cells and BHK cells. Cell-free translation systems can also be used.

[0072] The peptides may be purified using methods including, for example, reverse phase high performance liquid chromatography (RP-HPLC), multi-column countercurrent solvent gradient purification (MCSGP), and ion exchange chromatography.

[0073] Immunomodulators

[0074] The methods of the present invention involve combination therapy, comprising administering a C / EBPβ antagonist and an immunomodulator. The terms "immunomodulator" and "immunotherapeutic agent" are used interchangeably and refer to an agent that stimulates or suppresses a subject's immune system to fight a disease or infection. In the context of the present invention, the disease is a tumor or cancer.

[0075] Immunomodulators include, for example, immune checkpoint inhibitors; cytokines; targeted antibodies and drug-antibody conjugates; adjuvants, such as imiquimod and polyinosinic-polycytidylic acid (polyICLC); oncolytic viruses, such as T-VEC; and small molecule drugs, such as thalidomide, lenalidomide, or pomalidomide.

[0076] Immune checkpoint inhibitors target immune checkpoint proteins or their ligands. Immune checkpoint proteins include but are not limited to cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), also known as CD152, programmed cell death protein 1 (PD-1), also known as CD279, lymphocyte activation gene 3 (LAG-3), also known as CD223, T cell immunoglobulin mucin (TIM-3), also known as HAVcr2, and T cell immune receptor with Ig and ITIM domains (TIG IT). Examples of immune checkpoint inhibitors include atezolizumab, avelumab, cemiplimab, dostalimab, durvalumab, ipilimumab, nivolumab, pembrolizumab and relatlimab.

[0077] Some cytokines, such as interferons, can disrupt cancer cell division and slow tumor growth. Other cytokines, such as interleukins (IL), stimulate immune cell growth and proliferation. Examples of immunomodulatory cytokines include aldesleukin, granulocyte macrophage colony stimulating factor, interferon α-2a, interferon α-2b, and pegylated interferon α-2b.

[0078] Targeted antibodies can be customized to target antigens on the surface of cancer cells, thereby destroying cancer activity, particularly uninhibited growth. Some targeted antibodies are conjugated with anticancer drugs. Other targeted antibodies have bispecificity, such as binding cancer cells and T cells to enhance anticancer immune responses. Examples of targeted antibodies and drug-antibody conjugates include ervantumab, mabelantuzumab-blmf, bevacizumab, cetuximab, denosumab, dinutuximab, enrokumab-ejfv, magetuximab, nacitumomab-gqgk, nexitumomab, panitumumab, pertuzumab, ramucirumab, gosartumomab-hziy, tebenfos-tebn, vetisoltuzumab, trastuzumab, detrastuzumab and emmet trastuzumab.

[0079] Composition and administration

[0080] In certain aspects, the present invention provides a combination method comprising administering a composition, such as a pharmaceutical composition comprising an effective amount of a peptide antagonist of C / EBPβ (such as ST101), and administering a composition, such as a pharmaceutical composition comprising an effective amount of an immunomodulator (such as a checkpoint inhibitor). For example, methods of administering a C / EBPβ peptide antagonist are described in WO 2021 / 262604.

[0081] Because the combination therapy of the present invention demonstrates synergy between C / EBPβ antagonists and immunomodulators, in some embodiments, the effective amount of each active agent may be lower than the effective amount when each active agent is administered alone. For example, the effective amount of each of the peptide antagonists of C / EBPβ and the immunomodulator may be a subtherapeutic dose.

[0082] The composition is preferably administered parenterally. Parenteral administration routes include intravenous (IV), intramuscular, intraperitoneal, intrathecal, and subcutaneous. In certain embodiments, the composition comprising a peptide antagonist of C / EBPβ and / or the composition comprising an immunomodulator is administered to a subject by intravenous infusion.

[0083] The composition containing the peptide antagonist of C / EBPβ and the composition containing the immunomodulator can be the same composition or different compositions. If the peptide antagonist and the immunomodulator are contained in two separate compositions, the two compositions can be administered to the subject at the same time or at different times (including different days).

[0084] Each of the compositions comprising peptide antagonists and compositions comprising immunomodulators is administered to a subject more than once. In one embodiment, the administration of the peptide antagonist of C / EBPβ can be once a week for at least three weeks (i.e., three administrations), six weeks (i.e., six administrations), nine weeks, twelve weeks, three months, six months, nine months, or twelve months. In another embodiment, administration can be performed once every two weeks for at least four weeks (i.e., two administrations), eight weeks (i.e., four administrations), twelve weeks, three months, six months, nine months, or twelve months. In some embodiments, the peptide antagonist of C / EBPβ can be administered to a patient once a week for at least three weeks, six weeks, nine weeks, twelve weeks, three months, six months, nine months, or twelve months, and then administered once every two weeks for at least four weeks, eight weeks, twelve weeks, three months, six months, nine months, or twelve months.

[0085] In one embodiment, the administration of immunomodulators can be carried out once a week, for at least three weeks (i.e., three administrations), six weeks (i.e., six administrations), nine weeks, twelve weeks, three months, six months, nine months or twelve months. In another embodiment, the administration can be carried out once every two weeks, for at least four weeks (i.e., two administrations), eight weeks (i.e., four administrations), twelve weeks, three months, six months, nine months or twelve months. In another embodiment, the administration of immunomodulators can be carried out once every three weeks, for at least six weeks (i.e., two administrations), nine weeks (i.e., three administrations), twelve weeks, three months, six months, nine months or twelve months. In another embodiment, the administration of immunomodulators can be carried out once every four weeks, for at least eight weeks (i.e., two administrations), twelve weeks (i.e., three administrations), three months, six months, nine months or twelve months. Similarly, the administration of immunomodulators can be carried out once a month, for at least two months (i.e., two administrations), three months (i.e., three administrations), six months, nine months or twelve months.

[0086] For the purposes of this disclosure, "combination therapy" means that a treatment period including administration of a peptide antagonist of C / EBPβ overlaps with a treatment period including administration of an immunomodulator.

[0087] III. How to use

[0088] The subject in need of the method of the present invention is a patient diagnosed with a solid tumor. For example, the subject may suffer from a locally advanced solid tumor or a metastatic inoperable tumor. In some embodiments, the subject suffers from basal cell carcinoma, bladder cancer, cervical cancer, bile duct cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, head and neck cancer, Merkel cell carcinoma, melanoma, renal cell carcinoma, squamous cell carcinoma, triple negative breast cancer or urothelial cell carcinoma.

[0089] In a specific embodiment, the subject suffers from melanoma, cancer or sarcoma. In one embodiment, the melanoma is a skin melanoma or a mucosal melanoma. In one embodiment, the cancer is an adenocarcinoma, such as a bladder adenocarcinoma, a colorectal adenocarcinoma, a pancreatic adenocarcinoma, a stomach / signet ring adenocarcinoma or a small intestine adenocarcinoma. In one embodiment, the sarcoma is an abdominal sarcoma or a myofibroblastic sarcoma. In a specific embodiment of the invention, a combination therapy of a peptide antagonist of C / EBPβ (e.g., ST101) and an immunomodulator can inhibit tumor growth, reduce tumor volume, or a combination thereof.

[0090] The effect of treatment can be evaluated by one or more known methods. For example, compared with the same results in patients (i.e., control patients) who have not received the methods of the present invention, patients who receive the methods of the present invention may experience results including prolonged survival, improved progression-free survival, improved response duration, prolonged remission, reduced risk of recurrence, and / or improved tumor responses to the combined therapy of the present invention. For example, the results of patients treated with the methods of the present invention can be compared with the median results in the control patient population. The control patient population can be administered with a regimen such as a group selected from a placebo, surgery, radiotherapy, chemotherapy, immunotherapy, hormone-based therapy, or targeted therapy. In another embodiment, patients receiving the combined therapy of the present invention can be compared with a control patient population treated with only one of the peptide antagonists or immunomodulators of C / EBPβ. Comparisons can be statistically analyzed using, for example, the Wilcoxon signed rank test or the Kaplan-Meier method.

[0091] The treatment response compares one or more efficacy measurements following the treatment regimen to a baseline (e.g., before treatment with the combination therapy). The baseline assessment is preferably performed within 24, 48, or 72 hours, or within 1, 2, 3, or 4 weeks prior to the first treatment. In a preferred embodiment, the baseline assessment is performed within 24 hours prior to the first treatment.

[0092] “Tumor burden” is the total mass or size of cancerous tissue in a patient. Tumor response can be assessed using metrics including objective response rate, disease control rate, and duration of response. Depending on the type of tumor, these parameters can be determined using the revised Response Evaluation Criteria in Solid Tumors (RE CIST 1.1) (Eisenhauer 2009), modified Response Assessment in Neuro-Oncology (mRANO) (Ellingson 2017), or PCWG3 guidelines (Scher 2016).

[0093] The objective response rate assesses a reduction in tumor size (e.g., tumor diameter), which can be determined by clinical examination and / or imaging. When a patient has multiple tumors, tumor size can optionally be expressed as the average diameter of all tumors or the sum of all tumor diameters. Superficial tumors can be measured clinically, for example using a caliper or by photography and a ruler. Imaging methods include computed tomography (CT), typically with a contrast agent; X-ray; magnetic resonance imaging (MRI); and positron emission tomography (PET), such as (18)F-fluorodeoxyglucose PET. In a preferred embodiment, CT is used to assess tumor response, for example in LA / MBC patients or melanoma patients. In another preferred embodiment, MRI, such as gadolinium-enhanced MRI, is used to assess tumor response, for example in GBM patients. Therefore, in one aspect, the present invention provides a method for reducing a patient's tumor burden, i.e., tumor mass and / or tumor size, comprising administering to the patient a combination therapy comprising a peptide antagonist of C / EBPβ (such as ST101) and an immunomodulator. The reduction in tumor burden is measured relative to baseline.

[0094] In certain embodiments, particularly those assessed by RECIST 1.1, the disease control rate defines the level of tumor response as a complete response (CR), i.e., disappearance of the tumor; a partial response (PR), i.e., a reduction in tumor size of at least 30%; stable disease, i.e., no change in tumor size; or progressive disease, i.e., an increase in tumor size and / or new lesions of at least 20%.

[0095] The duration of response is the length of time from when a response is achieved until disease progression, i.e., the period during which the tumor does not grow or spread or dies. The duration of response of a patient receiving the combination therapy of the present invention can be, for example, at least 4, 6, 8, 10 or 12 weeks, at least 4, 6, 8, 10, 12, 16, 18 or 24 months, or at least 3, 4 or 5 years. Therefore, in one aspect, the present invention provides a method for increasing the duration of response in a patient, the method comprising administering to the patient a combination therapy comprising a peptide antagonist of C / EBPβ and an immunomodulator. The increase in the duration of response is measured relative to the median duration of response of a control population.

[0096] Survival can be assessed as overall survival, i.e., the length of time a patient survives, or as progression-free survival, i.e., the length of time a patient receives treatment and the disease does not progress or worsen. Survival can be calculated 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 based on the response to treatment by, for example, Kaplan-Meier analysis. Therefore, in one aspect, the present invention provides a method for increasing the overall survival of a patient, the method comprising administering to the patient a combination therapy comprising a peptide antagonist of C / EBPβ and an immunomodulator. The increase in overall survival is measured relative to the median overall survival of a control population. On the other hand, the present invention provides a method for increasing the progression-free survival of a patient, the method comprising administering to the patient a combination therapy comprising a peptide antagonist of C / EBPβ and an immunomodulator. The increase in progression-free survival is measured relative to the median progression-free survival of a control population.

[0097] A patient is successfully treated according to the methods of the present invention if the patient experiences or exhibits at least one of the following results following administration of the combination therapy:

[0098] - Undetectable tumor (or at least one tumor if multiple tumors were present at baseline);

[0099] - A decrease in tumor size of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to baseline;

[0100] - No significant increase in tumor size compared to baseline (e.g., less than 20%);

[0101] - a significant increase in duration of response, optionally compared to the median duration of response in a control patient population;

[0102] - A significant increase in progression-free survival, optionally compared to the median progression-free survival in a control patient population;

[0103] - A significant increase in overall survival, optionally compared to the median overall survival of a control patient population.

[0104] Example

[0105] Embodiments of the present disclosure may be further defined by reference to the following non-limiting examples. It will be apparent to those skilled in the art that many modifications to the materials and methods may be made without departing from the scope of the present disclosure.

[0106] Example 1. Antagonists of C / EBPβ reprogram myeloid-derived suppressor cell polarization and reduce tumor-associated Treg

[0107] Myeloid-derived suppressor cells (MDSCs) play a role in cancer progression and other related diseases by suppressing innate and adaptive immune responses. For example, depletion of MDSCs by conditional depletion of C / EBPβ in hematopoietic lineage cells inhibits the immunosuppressive activity of MDSCs and leads to significantly enhanced anti-tumor immunity.

[0108] Primary human macrophages were cultured from peripheral blood mononuclear cells (hPBMCs) and activated to M1 or M2 phenotypes by lipopolysaccharide (LPS) and TNFα (M1) or IL-4 (M2) in the presence of the C / EBPβ antagonist ST101, respectively. Figure 1 The expression of macrophage M1 (CD80, CD86) and M2 (CD163, CD206) was analyzed by flow cytometry and rtPCR ( Figure 1 Paired biopsies from the ST101 Phase 1-2 clinical study in patients with advanced unresectable and metastatic solid tumors were collected during screening (before ST101 exposure) and within 24 hours of ST101 administration during treatment cycle 2. Nanostring gene expression analysis was performed to determine differential gene expression and the effects of ST101 on the tumor microenvironment.

[0109] Treatment with pharmacologically relevant concentrations of ST101 (2.5, 5, or 10 μM) resulted in a dose-dependent reduction in M2+ macrophages and a corresponding induction of M1+ macrophages ( Figure 2A , 2B ; Figure 3A , 3C ; Figure 4A-4B At the highest ST101 concentration, a 12-fold decrease in the M2 to M1 ratio was observed, but there was no significant effect on cell viability ( Figure 3B , 3D ).

[0110] Paired patient biopsies in the ST1011-2 clinical study demonstrated reduced C / EBPβ target gene IL-6 signaling, an important driver of the M2 macrophage phenotype. Reduced IL-6 signaling resulted in an increased ratio of tumor-infiltrating macrophages to tumor-infiltrating lymphocytes (TILs) and a decreased ratio of regulatory T cells (Tregs) to TILs in tumor samples from treated patients. Figures 5A-5C ).

[0111] Overall, these results validate the potential of ST101 in reprogramming M2 macrophages to proinflammatory M1 macrophages, support a novel, macrophage-driven mechanism of action for ST101 as an anticancer agent, and support further exploration of ST101 in immuno-oncology therapeutic strategies. Importantly, ST101's effects on macrophage polarization may work in concert with direct cytotoxicity in C / EBPβ-driven cancers and raise the possibility that ST101's target population may extend beyond C / EBPβ-driven cancer types.

[0112] Example 2. Antagonists of C / EBPβ effectively activate CD8+ T cells

[0113] Cultured human T cells (matched donors) were co-incubated with cultures of M1 or M2 macrophages. T cell activation in CD8+ cells was measured by intracellular interferon-γ staining. In the presence of M2 macrophages, T cell activation was inhibited, as shown by a decrease in the percentage of interferon-γ positive cells. Adding ST101 to matched donor human T cell cultures with M2 macrophages restored T cell activity. Treatment of matched donor human T cell cultures with M1 macrophages with ST101 increased activity relative to untreated matched donor human T cell cultures with M1 macrophages. The results are shown in Figure 6 middle.

[0114] Example 3. C / EBPβ antagonists and checkpoint inhibitors show anti-tumor activity

[0115] Using a triple-negative breast cancer (TNBC) mouse model, we show that inhibition of C / EBPβ and PD-1 results in enhanced antitumor activity compared to either treatment alone. Briefly, 4T1-1uc TNBC tumor cells were orthotopically implanted into the mammary fat pad of immunocompetent Balb / c mice. When treatment was initiated, tumors reached approximately 100 mm at day 10 post-implantation. 3 Animals received 10 mg / kg ST101 subcutaneously on days 10, 12, 14, 16, 18, and 22 after tumor inoculation and / or 12.5 mg / kg ant-PD-1 antibody (BioXCell, Lebanon, NH) intraperitoneally on days 10, 14, and 22 after tumor inoculation. Results are shown in Figure 7 middle.

[0116] References

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[0134] ***

[0135] The invention is further described by the accompanying claims.

Claims

1. A method for inhibiting the growth of a solid tumor in a subject, the method comprising performing a combination therapy with the following pharmaceutical compositions: (i) a pharmaceutical composition comprising an effective amount of a peptide antagonist of CCAAT enhancer binding protein β (C / EBPβ) and (ii) a pharmaceutical composition comprising an effective amount of an immunomodulator; wherein the pharmaceutical composition comprising the antagonist of C / EBPβ and the pharmaceutical composition comprising the immunomodulator are administered to the subject together or separately.

2. A method for reducing the volume of a solid tumor in a subject, the method comprising combining therapy with the following pharmaceutical compositions: (i) a pharmaceutical composition comprising an effective amount of a peptide antagonist of CCAAT enhancer binding protein β (C / EBPβ) and (ii) a pharmaceutical composition comprising an effective amount of an immunomodulator; wherein the pharmaceutical composition comprising the antagonist of C / EBPβ and the pharmaceutical composition comprising the immunomodulator are administered to the subject together or separately.

3. A method for treating a solid tumor in a subject, the method comprising performing a combination therapy with the following pharmaceutical compositions: (i) a pharmaceutical composition comprising an effective amount of a peptide antagonist of CCAAT enhancer binding protein β (C / EBPβ) and (ii) a pharmaceutical composition comprising an effective amount of an immunomodulator; wherein the pharmaceutical composition comprising the antagonist of C / EBPβ and the pharmaceutical composition comprising the immunomodulator are administered to the subject together or separately.

4. The method of any one of the preceding claims, wherein the solid tumor is a melanoma, a carcinoma or a sarcoma.

5. The method of any one of the preceding claims, wherein the subject has been diagnosed with locally advanced or metastatic breast cancer (LA / MBC), melanoma, glioblastoma (GBM), or castration-resistant prostate cancer (CRPC).

6. The method of any one of claims 1 to 3, wherein the peptide antagonist comprises the D-amino acid sequence VAEAREELERLEARLGQARGEL (SEQ ID NO: 1).

7. The method of any one of claims 1 to 3, wherein the peptide antagonist comprises the amino acid sequence LEGRAQGLRAELRELEERAEAV (SEQ ID NO: 3).

8. The method of any one of the preceding claims, wherein the peptide antagonist is a cell penetrating peptide.

9. The method of any one of claims 1 to 3, wherein the peptide antagonist is ST101.

10. The method of claim 9, wherein the peptide antagonist is administered to the subject at a dose of about 0.5-16 mg / kg.

11. The method of claim 9, wherein the peptide antagonist is administered to the subject at a dose of about 500 mg.

12. The method of any one of claims 1 to 3, wherein the immunomodulator is selected from the group consisting of a checkpoint inhibitor, a cytokine, and an immune adjuvant.

13. The method of any one of claims 1 to 3, wherein the immunomodulator is an antibody or an antibody-drug conjugate.

14. The method of claim 13, wherein the antibody or antibody-drug conjugate is selected from the group consisting of ervantumab, mabelantuzumab-blmf, bevacizumab, cetuximab, denosumab, dinutuximab, enrofloxacin-ejfv, magetuximab, nasituximab-gqgk, necituzumab, panitumumab, pertuzumab, ramucirumab, gosartumumab-hziy, tebenfos-tebn, veltisotumumab, trastuzumab, detrastuzumab and emtansine.

15. The method of claim 13, wherein the antibody is an anti-PD-1 antibody.

16. The method of claim 12, wherein the checkpoint inhibitor targets at least one of PD-1, PD-L1, CTLA-4, or LAG-3.

17. The method of claim 12, wherein the checkpoint inhibitor is selected from the group consisting of atezolizumab, avelumab, cemiplizumab, dostalimumab, durvalumab, ipilimumab, nivolumab, pembrolizumab, reluzumab, or a combination thereof.

18. The method of claim 12, wherein the cytokine targets at least one of the IL-2 pathway, the IL-2R pathway, the IFNAR1 pathway, or the IFNAR2 pathway.

19. The method of claim 12, wherein the cytokine is selected from the group consisting of aldesleukin, granulocyte-macrophage colony stimulating factor, interferon alpha-2a, interferon alpha-2b, and pegylated interferon alpha-2b.

20. The method of claim 12, wherein the immune adjuvant targets the Toll-like receptor 7 pathway or the Toll-like receptor 3 pathway.

21. The method of claim 12, wherein the immunoadjuvant is selected from the group consisting of imiquimod and poly-ICLC.

22. The method of any one of the preceding claims, wherein the pharmaceutical composition comprising an antagonist of C / EBP[beta] and / or the pharmaceutical composition comprising an immunomodulator is administered intravenously.

23. The method of any one of the preceding claims, wherein the pharmaceutical composition comprising an antagonist of C / EBP[beta] and the pharmaceutical composition comprising an immunomodulator are administered to the subject on different days.

24. The method of any one of claims 1 to 23, wherein the pharmaceutical composition comprising an antagonist of C / EBP[beta] is administered once a week for at least three weeks.

25. The method of any one of claims 1 to 23, wherein the pharmaceutical composition comprising an antagonist of C / EBP[beta] is administered once every two weeks for at least four weeks.

26. The method of any one of claims 1 to 25, wherein the pharmaceutical composition comprising an immunomodulator is administered once a week for at least three weeks.

27. The method of any one of claims 1 to 25, wherein the pharmaceutical composition comprising an immunomodulator is administered once every two weeks for at least four weeks.

28. The method of any one of claims 1 to 25, wherein the pharmaceutical composition comprising an immunomodulator is administered once every three weeks for at least six weeks.

29. A pharmaceutical composition comprising an effective amount of a peptide antagonist of CCAAT enhancer binding protein beta (C / EBPβ) and a pharmaceutical composition comprising an effective amount of an immunomodulator for use in a combined treatment method for inhibiting the growth of a solid tumor in a subject.

30. A pharmaceutical composition comprising an effective amount of a peptide antagonist of CCAAT enhancer binding protein beta (C / EBPβ) and a pharmaceutical composition comprising an effective amount of an immunomodulatory agent for use in a combined therapeutic method for reducing the volume of a solid tumor in a subject.

31. A pharmaceutical composition comprising an effective amount of a peptide antagonist of CCAAT enhancer binding protein beta (C / EBPβ) and a pharmaceutical composition comprising an effective amount of an immunomodulatory agent for use in a combined therapeutic approach for treating a solid tumor in a human subject.

32. The pharmaceutical composition of any one of claims 29 to 31, wherein the antagonist of C / EBPβ is ST101, and wherein the immunomodulator is a PD-1 inhibitor.

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