Treatment of beta-catenin related cancers

Abstract

The present invention provides an oligopeptide compound CyPep-1 for use in the treatment of cancers associated with abnormal activation of the Wnt / beta-catenin pathway. The invention also provides a method of treating a cancer associated with abnormal activation of the Wnt / beta-catenin pathway, the method comprising administering to a subject an oligopeptide compound, optionally in conjunction with a second therapeutic agent; and a method of identifying a subject suitable for treating cancer with an oligopeptide compound, the method comprising determining the presence of a biomarker indicative of an activated Wnt / beta-catenin pathway from a sample obtained from the subject. It has been found that CyPep-1, which was previously shown to preferentially target and disrupt the plasma membrane of cancer cells, also functions by inhibiting the Wnt / beta-catenin pathway. This dual therapeutic effect has a particular therapeutic significance because the use of CyPep-1 may have a particular effect on cancers showing abnormal activation of the Wnt / beta-catenin signaling pathway. The compound has a unique dual action mechanism of promoting immune activation and reversing immunological rejection.

Description

Technical Field

[0001] This disclosure and invention relates to the treatment of cancers associated with abnormal Wnt / β-catenin signaling pathways. Specifically, we have discovered that an oligopeptide compound (CyPep-1), previously shown to preferentially target and destroy the plasma membrane of cancer cells, also works by inhibiting the Wnt / β-catenin pathway. This dual therapeutic effect is of particular therapeutic significance because the use of CyPep-1 may have a special effect on cancers exhibiting abnormal activation of the Wnt / β-catenin signaling pathway. The compound possesses a unique dual mechanism of action, promoting immune activation and reversing immune rejection. Methods for identifying patients suitable for treatment of such cancers with this oligopeptide compound are also provided. Background Technology

[0002] Despite advances in cancer treatment in recent years, cancer remains a leading cause of morbidity and mortality worldwide. In 2020, approximately 19.3 million patients were diagnosed with cancer, and nearly 10 million died from it. Furthermore, it is projected that 28.4 million people will be diagnosed with cancer by 2040, a 47% increase from 2020; and with the aging population worldwide, cancer incidence is expected to rise further (Sung et al., CA Cancer J Clin 71(3): 209-249, 2021). Therefore, there is an urgent need for new and improved cancer treatments.

[0003] There is a growing recognition that different cancers may be associated with abnormalities in different mechanisms occurring within cells, opening up different treatment options for different groups of cancers. One such group of cancers is those associated with abnormal Wnt / β-catenin signaling.

[0004] β-catenin is a structural protein in intercellular adhesion. It is also a transcription factor in the Wnt signaling pathway. The Wnt / β-catenin pathway, also known as the classical Wnt pathway, is generally highly conserved and is activated via autocrine or paracrine mechanisms through the binding of extracellular Wnt ligands (including Wnt3a, Wnt1, and Wnt5a) to target membrane receptors. After activation, the Wnt pathway stabilizes β-catenin and translocates it to the nucleus, subsequently promoting the expression of genes involved in key cellular mechanisms, including cell proliferation, differentiation, migration, survival, renewal, and apoptosis (Liu et al., Sig Transduct Target Ther 7(3), 2022).

[0005] In the context of cancer, mutations in various genes involved in this pathway have been documented, and aberrant Wnt / β-catenin signaling has been reported to participate in cancer pathogenesis by promoting tumorigenesis, cancer cell renewal, proliferation, and immune response. Specifically, β-catenin activation is thought to drive immune cell rejection, promote cold tumor formation in the tumor microenvironment, and is considered a key factor in immune checkpoint inhibition resistance (Pai et al., J Hematol Oncol 10(1):101, 2017; Tissier et al., Cancer Res 65:(17), 2005).

[0006] Aberrant Wnt / β-catenin signaling is associated with many different types of cancer (i.e., in many different tissues). These include specific forms of colorectal cancer (CRC), melanoma, desmoidoma, lung cancer, kidney cancer, liver cancer, breast cancer, ovarian cancer, and adrenocortical carcinoma (ACC) (Pai et al., 2017; Tissier et al., 2005; and Koury et al., Stem Cells Int:2925869, 2017).

[0007] Adrenal choroidal cancer (ACC) is a cancer of the outer layer of the adrenal gland. It is a rare cancer with an incidence of 0.7–2 cases per million people per year, and patients diagnosed with ACC have a very poor prognosis; median survival after progression with standard treatment is less than one year, while progression-free survival for patients with advanced or metastatic disease is <2 months. Due to the rarity and malignancy of ACC, treating the disease is extremely challenging, and surgical resection is the only curative option for patients with early-stage ACC. Patients with ACC also have a high risk of recurrence after complete resection, whether local or distant. Unfortunately, ACC responds only moderately to standard cytotoxic chemotherapy (such as radiotherapy and ablation), and the approved adjuvant therapy mitotane for ACC has a low treatment index (see, for example, Tissier et al., 2005, ibid.). Therefore, ACC presents a unique unmet need for cancer treatment. However, more generally, there is a persistent need for new therapies for β-catenin-driven cancers; although the contribution of Wnt signaling to tumorigenesis has been recognized and Wnt signaling inhibition has shown promising effects in some preclinical models, no Wnt signaling-targeting drugs have proven clinically successful in cancer or other diseases.

[0008] β-catenin has been an elusive drug target to date. Despite initial promise, trial results for therapeutics designed to target the aberrantly activated Wnt / β-catenin signaling pathway for cancer treatment have been mixed (Zhang & Wang, J Hematol Oncol 13(1): 165, 2020). Various inhibitors targeting the Wnt / β-catenin pathway are currently under development in preclinical and clinical trials. Different classes of targets exist, including Wnt antagonists, porcupine (PORCN) inhibitors, β-catenin / T cell-specific (TCF) inhibitors, and monoclonal antibodies against the receptor protein Frizzled (FZD). While some candidates appear promising, issues remain regarding safety, efficacy, and drug delivery, and success is often seen only in a relatively small percentage of patients or in patients with only very specific types of cancer. Even where some efficacy has been shown, adverse reactions are currently common, and it remains unclear how the activity of some candidates can translate into sustained clinical outcomes in multiple cancers associated with the aberrantly activated Wnt / β-catenin pathway.

[0009] Therefore, given the extensive involvement of the Wnt / β-catenin pathway in many different cellular processes across various cell types and its interactions with other signaling pathways, the Wnt / β-catenin pathway presents a challenging therapeutic target in achieving desired therapeutic benefits while avoiding undesirable side effects.

[0010] WO 2011 / 092347 discloses oligopeptide compounds with selective cytotoxicity against tumor cells and proposes their use in the treatment of a range of cancers. These oligopeptide compounds comprise a peptide (named CyPep-1) consisting of the amino acid sequence shown in SEQ ID NO:1. These peptides are cationic and exhibit a high ability to bind to negatively charged membranes (e.g., those of many tumor cells, particularly cancer cells), thereby producing strong selective cytolytic activity against tumor / cancer cells (non-tumor / non-cancerous mammalian cells tend to have more neutrally charged membranes and are not targeted by CyPep-1). This peptide has not only been shown to exhibit selective cytotoxicity against a variety of cancer cell lines in vitro, leading to neoantigen release, but also strong antitumor activity against various types of cancer, and indeed against non-malignant tumor lesions such as warts, and is well-tolerated in animal disease models. CyPep-1's cytolytic activity is also evident in its effects on bacterial cells (possibly related to the negative charge on the cell membranes of many bacteria), and it has been shown to be effective against medically relevant Gram-positive and Gram-negative bacteria. Its use as an antimicrobial agent (including antibacterial and antifungal agents) has been proposed (see WO 2011 / 092347). Summary of the Invention

[0011] In the process of studying the tumor therapeutic potential of CyPep-1, the inventors were surprised to find that CyPep-1 can induce beneficial therapeutic effects and can be used to treat specific groups (i.e., types or categories) of cancers, namely cancers associated with abnormal activation of the Wnt / β-catenin pathway, particularly ACC.

[0012] As illustrated in the examples below, CyPep-1 demonstrated exceptional efficacy in treating ACC patients, with responses far exceeding expectations. This prompted the inventors to further investigate and consider the properties of CyPep-1, suggesting its role as an inhibitor of the Wnt / β-catenin pathway to reverse the immune response in the tumor microenvironment, i.e., initiating “immune reprogramming.” Specifically, as shown in the examples, CyPep-1 reduced the nuclear accumulation of β-catenin and downregulated Wnt-induced secretory protein-1 (WISP-1 / CCN). This gene is transcribed from β-catenin, and the downregulation was caused by CyPep-1's inhibition of the β-catenin pathway. Thus, the inventors have discovered an alternative mode of action for CyPep-1. Furthermore, nuclear localization of β-catenin was reduced after treatment with CyPep-1, and evidence of CyPep-1 activation of Axin2 was also observed. The results in ACC and other cancers provide proof of concept for its activity and validate the CyPep-1 platform with its dual mechanism of action as a novel treatment option for many cancer patients suffering from a previously difficult-to-treat group of cancers.

[0013] Therefore, the current advances provide a new and very promising therapy for treating specific types or subgroups of cancers (i.e., cancers associated with the Wnt / β-catenin pathway) using CyPep-1 peptides.

[0014] Therefore, in a first aspect, this article provides an oligopeptide compound comprising a D-amino acid sequence as shown in SEQ ID NO:1 or a D-amino acid sequence having at least 85% sequence identity with it, for treating a subject with cancer, wherein the cancer is associated with aberrant activation of the Wnt / β-catenin pathway.

[0015] Therefore, the oligopeptide compounds provided herein for medical use and methods are compounds containing inverted amino acid sequences.

[0016] In one embodiment, each amino acid of the oligopeptide compound is a D-amino acid. In this embodiment, the oligopeptide compound is correspondingly a reverse oligopeptide compound.

[0017] In one embodiment, the oligopeptide compound is a D-amino acid peptide having or consisting of the sequence shown in SEQ ID NO: 1.

[0018] This oligopeptide compound can be advantageously used in combination therapy with one or more other cancer therapeutic agents.

[0019] Therefore, in another respect, this article provides an oligopeptide compound comprising a D-amino acid sequence as shown in SEQ ID NO:1 or a D-amino acid sequence having at least 85% sequence identity with it, for treating cancers associated with aberrant activation of the Wnt / β-catenin pathway, optionally wherein each amino acid of the compound is a D-amino acid, and wherein the compound is used in combination with a second therapeutic agent effective in treating said cancer.

[0020] One related aspect provides a pharmaceutical product comprising: (i) an oligopeptide compound comprising a D-amino acid sequence as shown in SEQ ID NO:1 or having at least 85% sequence identity with it, optionally wherein each amino acid of the compound is a D-amino acid; and (ii) a second oncology therapeutic agent, as a combination formulation for use alone, simultaneously or sequentially in the treatment of a subject’s cancer, wherein the cancer is associated with aberrant activation of the Wnt / β-catenin pathway.

[0021] Another related aspect provides a method for treating cancer associated with aberrant activation of the Wnt / β-catenin pathway, comprising administering to a subject in need an oligopeptide compound comprising a D-amino acid sequence as shown in SEQ ID NO:1 or a D-amino acid sequence having at least 85% sequence identity therewith, optionally wherein each amino acid of the compound is a D-amino acid, optionally administered together with a second therapeutic agent.

[0022] Specifically, the oligopeptide compound and optionally (i.e., in the case of use) a second therapeutic agent are administered to the subject in an effective amount. More specifically, in the case of combination therapy, this effective amount is effective for treating cancer when administered in combination.

[0023] On the other hand, this document provides the use of an oligopeptide compound comprising a D-amino acid sequence as shown in SEQ ID NO:1 or a D-amino acid sequence having at least 85% sequence identity with it in the preparation of a medicament for treating cancers associated with aberrant activation of the Wnt / β-catenin pathway, optionally in combination with a second therapeutic agent, and optionally wherein each amino acid of the compound is a D-amino acid.

[0024] Therefore, more specifically, this aspect provides the use of an oligopeptide compound comprising a D-amino acid sequence as shown in SEQ ID NO:1 or a D-amino acid sequence having at least 85% sequence identity with it in the preparation of a medicament for treating cancers associated with aberrant activation of the Wnt / β-catenin pathway, optionally wherein each amino acid of the compound is a D-amino acid, and wherein the oligopeptide compound is used in combination with a second therapeutic agent.

[0025] In other words, it can also be seen that this aspect provides (i) the use of an oligopeptide compound and (ii) a second therapeutic agent in the preparation of a medicament for treating cancers associated with aberrant activation of the Wnt / β-catenin pathway, the oligopeptide compound comprising a D-amino acid sequence as shown in SEQ ID NO:1 or a D-amino acid sequence having at least 85% sequence identity with it, optionally wherein each amino acid of the compound is a D-amino acid.

[0026] In this context, the drug can be considered a kit or pharmaceutical product comprising a first therapeutic agent and a second therapeutic agent as defined above (i) and (ii), provided for the stated use. More broadly, in the aforementioned pharmaceutical uses, when the oligopeptide is used in combination therapy with another therapeutic agent, the oligopeptide can be considered the first therapeutic agent.

[0027] In the combination therapy described herein, the second therapeutic agent can be any tumor therapeutic agent, particularly any second therapeutic agent that is effective in treating cancer. In one embodiment, the second therapeutic agent is an immunotherapeutic agent. In a more specific embodiment, it is a checkpoint inhibitor.

[0028] In one implementation, cancer is ACC.

[0029] In another aspect, the present invention provides a method for identifying subjects suitable for cancer treatment with an oligopeptide compound comprising a D-amino acid sequence as shown in SEQ ID NO:1 or having at least 85% sequence identity with it, optionally wherein each amino acid of the compound is a D-amino acid, the method comprising determining the presence of a biomarker indicating activation of the Wnt / β-catenin pathway in a sample obtained from the subject.

[0030] In a specific implementation, the present invention provides a method for identifying and treating cancers in a subject associated with abnormal activation of the Wnt / β-catenin pathway, the method comprising: (i) Identifying the subject suitable for cancer treatment by determining the presence of biomarkers indicating activation of the Wnt / β-catenin pathway in samples obtained from the subject; and (ii) administering to the subject an oligopeptide compound comprising a D-amino acid sequence as shown in SEQ ID NO: 1 or having at least 85% sequence identity with it, optionally wherein each amino acid of the compound is a D-amino acid, optionally administered together with a second therapeutic agent.

[0031] Therefore, subjects with cancer are tested to determine whether the cancer is associated with abnormal activation of the Wnt / β-catenin pathway by determining the presence of a biomarker indicating activation of the Wnt / β-catenin pathway, and if the biomarker is present, the oligopeptide compound is administered to treat the cancer.

[0032] Biomarkers can be mutations in genes involved in the pathway, and / or changes in gene expression (e.g., increases or decreases), and / or changes in the expression of proteins encoded by the gene (e.g., increases or decreases) (i.e., increases or decreases in the level of the gene product).

[0033] This can include any gene in the pathway, including genes encoding proteins such as β-catenin or Axin2, or genes encoding regulatory factors in the pathway. In a representative embodiment, the gene may encode a protein involved in the Axin2 regulatory network. For example, a biomarker may be one or more mutations in a gene encoding Axin2 or a regulatory factor of Axin2, the gene containing one or more mutations. However, biomarkers are not limited to Axin2 / Axin2 regulatory genes and can be any gene in the pathway that can show association with clinical response or disease control.

[0034] Detailed description This invention and disclosure relate to novel uses of previously identified oligopeptide compounds, particularly the peptide CyPep-1 (SEQ ID NO: 1) as described in WO 2011 / 092347, which have been identified as tumor therapeutics due to their selective cytolytic activity against cancer cells. We have now discovered that CyPep-1 possesses additional activity in inducing tumor-specific immune activation and reversing immune rejection. The compounds presented herein are intended for the treatment of a new group of patients with cancers associated with the aberrant Wnt / β-catenin pathway.

[0035] SEQ ID NO:1 is a 27-amino acid peptide composed of a fragment of the tumor suppressor protein Conductin / Axin2 (particularly the RGS domain aa 126-140) coupled to the C-terminus of the HIV-TAT cell-penetrating peptide: YGRKKRRQRRRGKTLRVAKAIYKRYIE (SEQ ID NO:1) The above fragment of Conductin / Axin2 has the amino acid sequence KTLRVAKAIYKRYIE (SEQ ID NO:2; corresponding to amino acid numbers 13-27 of SEQ ID NO:1), and the HIV-TAT cell-penetrating peptide has the amino acid sequence YGRKKRRQRRRG (SEQ ID NO:3; corresponding to amino acid numbers 1-12 of SEQ ID NO:1).

[0036] Conductin / Axin2 is a central protein in the Wnt / β-catenin pathway in tumorigenesis. It is a member of the β-catenin disruption complex, which targets β-catenin for proteasomal degradation. Therefore, Axin2 is an inhibitor of β-catenin—it inhibits β-catenin activity and induces its degradation. The Axin protein family can also directly promote the export of β-catenin from the nucleus.

[0037] The Wnt / β-catenin pathway is highly complex and regulated by several components, including proteins, genes, etc. As previously discussed, Axin2 is a target gene of β-catenin. High levels of β-catenin expression lead to high levels of Axin2 expression, and this expression pattern is observed in β-catenin-driven cancers, such as colorectal cancer. Axin2 is a negative regulator of the Wnt / β-catenin pathway because it promotes the phosphorylation and degradation of β-catenin, resulting in a decrease in the amount / level of β-catenin in cells. Cancer cells can “escape” this negative feedback, i.e., overcome the degradation / inhibition / reduction of β-catenin caused by high levels of Axin2 expression through mutations that inactivate Axin2 or genes that stabilize Axin2 (e.g., GNAI2 / 3). This mechanism is called mutational inactivation. This mechanism leads to cancer cells exhibiting high levels of inactivated Axin2 expression, where the RGS domain of Axin2 is aggregated. Therefore, a large amount of Axin2, or a “reservoir” of Axin2, exists in cancer cells. The compounds described in this article utilize this characteristic of β-catenin-driven cancer.

[0038] Therefore, the peptide CyPep-1 of SEQ ID NO: 1 has two domains: a first domain (Conductin / Axin2 domain) and a second domain. The first domain is derived from proteins involved in the Wnt / β-catenin pathway, and the second domain contains a cationic membrane-binding domain that specifically binds to negatively charged phospholipids such as phosphatidylserine (PS). The polar head of PS is normally only present inside the cell, but is present outside the cell in cancer cells. Therefore, PS represents a cancer-specific target. The binding of CyPep-1 to PS leads to aggregation and the formation of pores across the membrane, resulting in the release of cancer antigens and lytic cell death. Furthermore, CyPep-1 is believed to activate Axin2 by stabilizing the RGS domain, thereby leading to aggregation and target binding. In this respect, the RGS domain of Axin2 can aggregate, and in this aggregated form, Axin2 is inactive. By binding to the RGS domain, CyPep-1 can reverse this aggregation and activate Axin2, or promote its activity in a non-aggregated form. More specifically, in its non-aggregated form, Axin2 can aggregate or condense, and in this aggregated or condensed form, Axin2 is active. Data presented in the following examples demonstrate that CyPep-1 promotes the aggregation of Axin2 into microscopically visible "aggregates" or spots. CyPep-1 activation of Axin2 inhibits nuclear translocation and activation of β-catenin.

[0039] To avoid proteolytic degradation by CyPep-1, we replaced the naturally occurring L-amino acids with their D-amino acid enantiomers, as these enantiomers are not recognized by serum proteases. This yielded a stable peptide of 27 D-amino acids with a theoretical isoelectric point of 11.81 and a molecular weight of 3492.16 U. Genes & Cancer, Volume 5 (5-6), May 2014 ).

[0040] Therefore, the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 85% sequence identity with it, contained in the oligopeptide compound for the purposes proposed herein, is a D-amino acid sequence. In other words, the amino acid sequence consists entirely of D-amino acids (D-aa). It may also be referred to as a reverse amino acid sequence. In one embodiment, the oligopeptide compound as a whole consists entirely of D-amino acids, or in other words, it is a "D-oligopeptide compound". It may also be referred to as a reverse oligopeptide compound. A peptide consisting entirely of L-amino acids is referred to in the art as an L-peptide, while a peptide consisting entirely of D-amino acids is referred to in the art as a D-peptide. The term "reverse peptide" is used to refer to a peptide having the same amino acid sequence as an L-peptide but consisting entirely of D-amino acids (i.e., a D-peptide having the same sequence as the corresponding L-peptide). Reverse peptides / oligopeptide compounds have a structure that is a mirror image of their corresponding L-peptide / oligopeptide compounds (e.g., L-peptides with the same amino acid sequence). Reverse peptides / compounds can be advantageously used in clinical settings (as opposed to L-peptides / compounds) because they are generally not easily degraded by serum proteases (reverse peptides may not be recognized by proteases due to their non-native conformation). Oligopeptide compounds may in particular contain or consist of D-peptides, which are composed of the amino acid sequence shown in SEQ ID NO: 1.

[0041] In this regard, although a portion of the peptide is derived from Axin2, given its inverted (D-aa) structure, it cannot be assumed that the peptide will retain any functional effects associated with the biological activity of its parental protein, particularly any signaling effects (i.e., any functional / signaling effects of Axin2). Therefore, surprisingly and unexpectedly, this oligopeptide compound will be able to affect the expression of proteins involved in signaling (e.g., WISP-1), as shown in the following examples, and will be able to downregulate (i.e., inhibit) Wnt / β-catenin signaling.

[0042] As used herein, the term "oligopeptide compound" refers to a compound composed of amino acids or equivalent subunits linked together by peptide bonds or equivalent bonds. Therefore, the term "oligopeptide compound" includes both peptides and peptide mimics.

[0043] "Equivalent subunit" refers to a subunit that is structurally and functionally similar to an amino acid. The main chain portion of the subunit may differ from that of a standard amino acid; for example, it may incorporate one or more nitrogen atoms instead of one or more carbon atoms.

[0044] A "peptide mimic" is a compound that is functionally equivalent to or similar to a peptide and can have a three-dimensional structure similar to its peptide counterpart, but is not solely composed of amino acids linked by peptide bonds. The preferred category of peptide mimics is peptide-like compounds, i.e. N-Substituted glycine. Peptides are closely related to their natural peptide counterparts, but they differ in chemical properties; their side chains are attached to nitrogen atoms along the molecular backbone, rather than to α-carbons as they are in amino acids.

[0045] Peptides typically have a long half-life in the body, making them preferred in embodiments where a longer, sustained effect is required. This helps reduce the frequency of re-administration of the composition. However, for biosafety reasons, a shorter half-life may be preferred in other embodiments; in those embodiments, peptides are preferred.

[0046] Preferably, the oligopeptide compound is an oligopeptide (more specifically, a D-oligopeptide). The oligopeptide compound may contain di-amino acids and / or β-amino acids. Most preferably, the oligopeptide compound consists of α-amino acids.

[0047] Oligopeptides are polymers formed from amino acids linked together by peptide bonds. As defined herein, an oligopeptide contains at least three amino acids, although obviously, oligopeptide compounds used herein contain more than three amino acids. There is no specific maximum length for an oligopeptide compound or oligopeptide as defined herein; for example, it may contain up to 30, 40, 50, or 100 amino acids or more, but the prefix "oligopeptide" is generally used to indicate a relatively small number of subunits, such as amino acids, i.e., less than 200, preferably less than 100, 90, 80, 70, 60, or 50 subunits. Therefore, the oligopeptide compound of the present invention may contain at least 23 and no more than 200 subunits. In embodiments, it contains at least 24, 25, 26, or 27 subunits. Another definition is that it contains no more than 50, 45, 40, 35, 30, 29, 28, or 27 subunits. Therefore, an oligopeptide compound may contain a plurality of subunits within a range consisting of any integers listed above for the minimum or maximum number of subunits. Therefore, the representative subunit ranges include 23-150, 23-100, 23-80, 23-50, 23-40, 23-30, 25-150, 25-100, 25-80, 25-50, 25-40, 25-30, 26-150, 26-100, 26-80, 26-50, 26-40, 26-30, 27-150, 27-100, 27-80, 27-50, 27-40, 27-30, 27-29, and 27-28.

[0048] As defined herein, oligopeptide compounds can simply be oligopeptides, that is, polymers composed of amino acids linked by peptide bonds. Alternatively, oligopeptide compounds may contain additional functional groups, conjugates, etc.

[0049] The oligopeptide compounds used herein comprise the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity with it. For convenience, such sequences having at least 85% sequence identity may be referred to as substantially identical or equivalent sequences. In a specific embodiment, the oligopeptide compound comprises the amino acid sequence shown in SEQ ID NO:1. In another embodiment, the oligopeptide compound consists of the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity with it. In yet another embodiment, the oligopeptide compound consists of the amino acid sequence shown in SEQ ID NO:1.

[0050] The level of sequence identity between two sequences (e.g., an oligopeptide sequence and the sequence shown in SEQ ID NO:1) can be determined by sequence alignment. Sequence alignment can be performed using any suitable method, such as computer programs like the EMBOSS Needle or EMBOSS stretcher (both from Rice, P. et al.). Trends Genet .16(6): 276-277,2000) can be used for paired sequence alignment, while Clustal Omega (Sievers, F. et al.) Mol. Syst. Biol. 7:539, 2011) or MUSCLE (Edgar, RC, Nucleic Acids Res. (32(5):1792-1797, 2004) can be used for multiple sequence alignment. Such computer programs can be used with standard input parameters, such as the standard Clustal Omega parameters: matrix Gonnet, gap opening penalty 6, gap extension penalty 1; or the standard EMBOSS Needle parameters: matrix BLOSUM62, gap opening penalty 10, gap extension penalty 0.5. Any other suitable parameters can be used alternatively.

[0051] In addition to the D-amino acid of SEQ ID NO: 1 (or its equivalent sequence), the oligopeptide compound may contain one or more other amino acids, including L-amino acids, or engineered amino acids or naturally occurring non-proteinogenic amino acids, such as those formed through metabolic processes. Examples of non-proteinogenic amino acids that may be used include ornithine (a product of the urea cycle) and artificially modified amino acids, such as 9... H Amino acids protected by -fluorene-9-ylmethoxycarbonyl (Fmoc)-, tert-butoxycarbonyl (Boc)- and 2,2,5,7,8-pentamethylchromane-6-sulfonyl (Pmc), as well as amino acids with carboxybenzyl (Z) groups.

[0052] The in vitro and / or in vivo stability of oligopeptide compounds can be improved or enhanced by using stabilizing or protecting methods known in the art, such as adding protecting or stabilizing groups, incorporating amino acid derivatives or analogs, or chemically modifying the amino acids. Such protecting or stabilizing groups can be added, for example, at the N-terminus and / or C-terminus. One example of such groups is an acetyl group, and other protecting groups or groups that may stabilize the peptide are known in the art.

[0053] As reported in WO 2011 / 092347, oligopeptide compounds as defined herein are active in inhibiting the growth and / or viability of cancer cells. For cells, “inhibition of growth” means a reduction, more particularly a measurable reduction, in any aspect of cell growth, whether it be an increase in cell size or an increase in the amount and / or volume of its components (more particularly, an increase in cell number). Thus, the term “growth” explicitly includes cell replication or proliferation. The rate of cell growth (e.g., in terms of the rate of increase in cell number) may be reduced. As representative examples, growth (e.g., cell number or growth rate) may be reduced by at least 50%, 60%, 70%, 80%, 90%, or 95%. In some cases, growth may be reduced by 100%, i.e., growth may be completely inhibited and stopped. Therefore, cell replication or proliferation can be reduced or inhibited. As described, the term “inhibition” includes any degree of reduction in growth.

[0054] Inhibition of cell growth can be identified by comparing the growth rate of control cells or cell populations cultured under standard laboratory conditions and in the absence of the oligopeptide of interest with the growth rate of the same or corresponding cells or cell populations cultured under conditions in the presence of the oligopeptide of interest but otherwise identical to the control cells or cell populations. The rate of cell replication or proliferation can be assessed, in particular, by determining the cell number at selected time points. A reduction in the cell number in a population cultured in the presence of the oligopeptide relative to the cell number in a control population indicates that the oligopeptide has inhibitory activity against cell growth. Cell number (and the resulting growth or other conditions) can be determined by cell counting, for example using a hematology counter.

[0055] Inhibition of cell viability includes any action that reduces cell viability or makes it less likely to survive or viable. Cell viability can be considered the ability of a cell to survive under given conditions. Inhibition of cell viability specifically includes killing or destroying cells, i.e., causing them to die. Cell death can be assessed using any standard laboratory technique. For example, the inability of cells or cell populations to grow (including replicate) or to utilize or absorb nutrients can be considered an indication of cell death (i.e., lack of viability). Cell viability can also be assessed by monitoring morphological changes in cells or tissues containing cells (e.g., tumors). Morphological changes can be analyzed using a microscope; for example, necrosis or cell lysis may be obvious during visual analysis of cells or tissues, indicating a lack of viability. Generally, if the cell membrane integrity is lost, the cell can be considered dead.

[0056] Inhibition of cell viability can be identified, for example, by comparing the viability of control cells or cell populations incubated under standard laboratory conditions and in the absence of the oligopeptide compound of interest with the viability of the same or corresponding cells or cell populations incubated under conditions in the presence of the oligopeptide compound of interest but otherwise identical to the control cells or cell populations. Cell viability is typically assessed using a crystal violet assay, as is known to those skilled in the art. In such an assay, a cell monolayer adhered to a surface (e.g., a culture plate) is brought into contact with (or not into contact with) the compound of interest. Cell death causes cells to detach from the surface. After contact with the compound of interest, the monolayer is washed to remove the detached cells and then stained with crystal violet, which binds to proteins and DNA, thereby staining the cells. The staining level can be used to determine viability; that is, if the cell population in contact with the compound of interest is stained less than the control population, the compound of interest can be considered to inhibit cell viability. The crystal violet staining level of a cell population can be visually assessed (simply by eye) or quantified by using a methanol-extracted dye followed by spectroscopic determination of the optical density of the methanol-extracted dye at 570 nm.

[0057] Many other methods for determining the viability or growth of cancer cells are well known in the art, and many routine assays can be used to determine whether cells are alive (viable) or dead. One option is to visually assess the morphological characteristics of cell death in cells of interest, such as necrotic or apoptotic bodies, membrane vesicles, nuclear condensation and DNA cleavage into regularly sized fragments, cell membrane rupture, and leakage of cell contents into the extracellular environment. Other methods utilize the characteristic loss of cell membrane integrity in dead cells. Membrane-impermeable dyes (e.g., trypan blue and propidium iodide) are commonly used to assess membrane integrity. These dyes are excluded from intact cells and therefore do not stain in such cells. If cell membrane integrity is impaired, these dyes can enter the cell and stain intracellular components. Alternatively or additionally, dyes that stain only cells with intact membranes can be used to give an indication of cell viability. The LIVE / DEAD cell viability assay, available from Thermo Fisher Scientific, is an assay that uses two different colored dyes, one to stain dead cells and the other to stain live cells, thus allowing both types of cells to be identified. Examples of suitable live-cell-specific dyes include calcein AM (green) and C12-rezazon (red); examples of suitable dead-cell-specific dyes include etidium homodimer-1 (red), propidium iodide (red), and SYTOX Green. Another method for assessing membrane integrity is to detect the release of cellular components (e.g., lactate dehydrogenase) into the culture medium.

[0058] Another option is to measure cellular metabolism. This can be routinely done in several ways, such as measuring ATP levels. Only living cells with intact membranes can synthesize ATP, and since ATP is not stored in cells, ATP levels decline rapidly after cell death. Therefore, monitoring ATP levels provides an indication of cellular state. Yet another option is to measure a cell's reducing potential. Living cells that metabolize nutrients produce reducing agents (e.g., NADH and NADPH), and thus a cell's reducing potential can be assessed by applying markers that give different outputs in reducing or oxidizing forms (e.g., fluorescent dyes). Cells lacking the ability to produce reducing markers can be considered dead. MTT and MTS assays are convenient examples of such assays.

[0059] Cancer cells divide unchecked and can be "immortal," meaning they express telomerase, allowing them to continue dividing indefinitely instead of dying or aging after reaching their Hayflick limit like healthy cells. Technicians can determine whether a particular cell is cancerous or healthy. Cancer cells typically exhibit distinctive histological features that allow them to be identified, such as a large, irregular nucleus and abnormalities within the cytoplasm. Genetic testing can also be used to determine whether a cell is cancerous.

[0060] The oligopeptide compounds used herein have activities that inhibit the growth and / or viability of both in vivo and in vitro cancer cells. This activity can be readily determined in vitro using suitable cell lines. Many laboratory cell lines are cancerous and convenient for research use due to their “immortality.” Any such cancer cell line can be used to determine the activity of the compound of interest, such as cell lines A172 (human glioblastoma), GAMG (human glioblastoma), U87 (human glioblastoma), 4T1 (mouse breast cancer), HOS (human osteosarcoma), and MC38 (mouse colon cancer). Many other cancer cell lines are also known to those skilled in the art. Such cells can be obtained from any suitable source, such as cell banks like ATCC (USA). The activity of the compound of interest is preferably determined using mammalian cancer cells. Human cancer cells can be used. Specifically, cell lines can be derived from cancers associated with aberrant activation of the Wnt / β-catenin pathway. Such cancers are listed below, and cell lines can be derived from any of these cancers.

[0061] Cancer cells used for testing can also be obtained from subjects (e.g., human cancer patients). Cancer cells can be surgically removed from a cancer patient and the activity of oligopeptide compounds of interest can be tested on them. Therefore, cancer cells can originate from cancer cell lines or from clinical or veterinary samples. Cancer cells can originate from tumors. Cancer cells can originate from any cancer, but particularly from cancers associated with abnormal activation of the Wnt / β-catenin pathway.

[0062] As described above, oligopeptide compounds exhibit selective cytotoxicity against cancer cells. As used herein, the term "cytotoxicity" has essentially the same meaning as "inhibition of the activity of..." as described above. In other words, oligopeptide compounds selectively inhibit or kill cancer cells (or more preferably, generally inhibit the activity of tumor cells).

[0063] If a compound exhibits greater cytotoxicity against cancer cells than against non-cancer cells, and particularly if it exhibits greater cytotoxicity against cancer cells than against healthy cells, then the compound can be said to have selective cytotoxicity against cancer cells. Specifically, oligopeptide compounds have little or no effect on healthy non-cancer cells but are cytotoxic to cancer cells.

[0064] The above describes a method for analyzing the effects of compounds of interest on cell growth and viability. The same method can be used to determine whether a compound of interest exhibits selective cytotoxicity against cancer cells. The viability of a cancer cell population exposed to the compound of interest is compared to the viability of a healthy cell population exposed to the compound of interest. If, under the same conditions, the viability of the cancer cell population decreases more significantly than that of the healthy cell population after exposure to the compound of interest, then the compound of interest can be said to exhibit selective cytotoxicity against cancer cells.

[0065] As described above, the pharmaceutical uses and methods of the present invention are based on the discovery of an alternative mode of action of oligopeptide compounds, namely their selective activity in inhibiting the Wnt / β-catenin pathway. This activity can be assessed by evaluating the compound's effect on signal transduction via this pathway (e.g., in in vitro assays in cell lines), according to methods known in the art. Downstream effects of inhibiting this pathway can also be assessed. For example, as described in the examples below, the activity of the compound in inhibiting the protein level and nuclear localization of β-catenin or in inhibiting the expression of downstream target genes (such as WISP-1, Axin2, and Myc mRNA) can be determined. Methods for assessing these effects include immunohistochemistry (IHC), Western blotting (WB), ELISA, polymerase chain reaction (PCR), and sequencing.

[0066] Furthermore, as described in the examples below, the effect of the compound on Axin2 polymerization can be detected by microscopy in in vitro cell studies, for example, using cancer cell lines, and using antibodies to visualize Axin2.

[0067] The oligopeptide compounds described herein can be synthesized by those skilled in the art using standard techniques. Chemical synthesis methods for oligopeptide compounds containing D-amino acids or other non-proteogenic amino acids are known. Liquid-phase or solid-phase protein synthesis can be used to generate polypeptides that can form or be contained within the oligopeptide compounds used in this invention. Such methods are well known to those skilled in the art, who can readily prepare oligopeptide compounds using appropriate methods common in the art.

[0068] Subjects receiving the oligopeptide compound are those suffering from cancer associated with aberrant activation of the Wnt / β-catenin pathway. Subjects are animals, which can be human or any non-human animal, but particularly mammals. This can include laboratory animals, livestock, animals, zoo animals, or sports animals. Subjects can be rodents, such as mice, rats, rabbits, or guinea pigs. Subjects can be pet animals, such as cats or dogs, or farm animals, such as horses, cattle, sheep, pigs, or goats. Subjects can be wild animals, such as animals in zoos or wildlife parks. In a specific embodiment, the subject is a primate, such as a monkey or ape. Most specifically, the subject is a human. Therefore, the therapy disclosed herein can be used for veterinary or clinical purposes, but is preferably used for clinical purposes, i.e., for treating human subjects suffering from cancer (i.e., cancer patients suffering from cancer caused by aberrant activation of the Wnt / β-catenin pathway).

[0069] Oligopeptide compounds were administered to subjects to treat cancers associated with abnormal activation of the Wnt / β-catenin pathway.

[0070] As used herein, the term “treatment” broadly refers to any effect or step (or intervention) that is beneficial to the management of a clinical condition. Treatment can include reducing, alleviating, improving, slowing the progression of an ongoing condition or one or more symptoms thereof, or eliminating it, or in any way improving the clinical condition of a subject, relative to the condition or symptoms prior to treatment. Treatment can include any clinical step or intervention that contributes to or is part of a treatment plan or protocol. Therefore, as used herein, “treatment” encompasses curative treatment (or treatment aimed at curative purposes) as well as treatment that is solely for prolonging life or palliative purposes (i.e., designed only to limit, alleviate, or improve the symptoms of the condition).

[0071] “Aberrant activation of the Wnt / β-catenin pathway” refers to increased or abnormal activation of this pathway compared to the pathway in healthy subjects, healthy tissues, or healthy (i.e., non-cancerous) cells, or in other words, disruption of this pathway. Consequently, signaling via this pathway is increased in cancer cells, such as in tumors or in the local microenvironment of tumors. Specifically, as determined by mRNA expression, aberrant or increased activation of this pathway can be characterized by increased protein levels of β-catenin or increased levels of downstream target genes such as WISP-1, Axin2, and Myc. Methods used to assess these effects include immunohistochemistry (IHC), Western blotting (WB), ELISA, and polymerase chain reaction (PCR). Furthermore, mutations in genes such as β-catenin, APC, ZNRF3, and MIP1 drive aberrant activation of this pathway and can be detected by sequencing.

[0072] Interference in this pathway may stem from one or more mutations that may occur in one or more proteins involved in the pathway, such as mutations in genes encoding proteins selected from: β-catenin, Axin1, Axin2, colonic adenomatous polyposis (APC), ZNRF3, MEN1, and GNAI2. This can lead to interference or abnormality in protein expression and / or function. For example, the expression of certain proteins may be reduced or increased, or functional proteins may be inactivated (e.g., inhibitory proteins or enzymes). For example, the formation of the degradation complex (DC), which is characteristic of this pathway, may be reduced. Therefore, as will be discussed in more detail below, this cancer can be identified by screening cancer samples or samples from patients suspected of having this cancer for one or more biomarkers indicating aberration or activation only of the Wnt / β-catenin pathway.

[0073] The abnormal Wnt / β-catenin pathway is known to be associated with many types of cancer; however, it should be understood that not every type of cancer (e.g., in a specific organ and / or tissue) will be associated with this abnormal pathway. In some cancer cases, clinicians can use existing techniques to determine whether a particular cancer is associated with abnormal activation of this pathway without testing it. In some cases, this can be inferred. In other cases, subjects can be screened or tested to investigate or determine whether the cancer is associated with the abnormal pathway. This is discussed further below.

[0074] Cancers associated with abnormal activation of the Wnt / β-catenin pathway may alternatively be termed β-catenin-driven cancers.

[0075] These types of cancers can include eye cancer, vulvar cancer, endocrine cancers (including, for example, adrenocortical tumors, particularly ACC, and parathyroid or thyroid cancer), anal cancer, pancreatic cancer, colorectal cancer, stomach cancer, bile duct cancer, liver cancer (particularly hepatocellular carcinoma (HCC)), kidney cancer (e.g., renal cell carcinoma), gallbladder cancer, bladder cancer, skin cancer (including melanoma, although melanoma can occur anywhere in the body, such as uveal melanoma), prostate cancer, penile cancer, breast cancer, head and neck cancers (e.g., head and neck squamous cell carcinoma (HNSCC), parotid gland cancer), cervical cancer, esophageal cancer, endometrial cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer (e.g., lung adenocarcinoma)), glioma, medulloblastoma, ovarian cancer, nephroblastoma, bile duct cancer, neuroendocrine cancers (carcinoids), HPV-positive cancers, squamous cell carcinomas, and sarcomas or desmoidomas.

[0076] Squamous cell carcinoma (SCC) can include, in particular, HNSCC (as described above), as well as squamous cell carcinoma of the cervix, lung, esophagus, vulva, anus, etc.

[0077] The specific cancers treated according to the uses and methods described in this article include: ACC, undifferentiated thyroid carcinoma, lung adenocarcinoma, parathyroid carcinoma, head and neck squamous cell carcinoma, melanoma (e.g., uveal melanoma), neuroendocrine carcinoma (carcinoid), vulvar squamous cell carcinoma, chondrosarcoma, anal cancer, small cell lung cancer, and non-small cell lung cancer.

[0078] In some embodiments, the cancer is selected from: ACC, HNSCC, parotid gland cancer, parathyroid cancer, thyroid cancer, and uveal melanoma. In other embodiments, the cancer is selected from: ACC, parotid gland cancer, thyroid cancer, or parathyroid cancer.

[0079] In some implementations, the cancer is not colorectal cancer, or not colon or rectal cancer. In a specific implementation, the cancer to be treated is ACC.

[0080] Therefore, cancers, especially those associated with solid tumors, are those that clinically present as solid tumors. However, the use of oligopeptide compounds is not limited to this type of cancer and also includes other cancers, including hematopoietic system cancers such as acute myeloid leukemia (AML).

[0081] Therefore, cancer can be any cancer associated with the abnormal Wnt / β-catenin pathway. This includes both primary and secondary cancers. As will be discussed in more detail below, oligopeptide compounds are particularly effective in treating primary and secondary tumors or metastases in subjects.

[0082] As described above, oligopeptide compounds can be used in combination with a second therapeutic agent, particularly a second agent effective in treating cancer. The second therapeutic agent can be a second anticancer agent, but in other embodiments it can have different activities; for example, it can be an anti-inflammatory agent or any other agent that can be used to treat a patient.

[0083] In a specific implementation plan, the second therapeutic agent may be selected from chemotherapeutic agents, immunotherapeutic agents, hormone therapy, radiotherapy, or photodynamic therapy.

[0084] As mentioned in this article, chemotherapy agents are administered drugs that target and work against cancer, such as drugs that destroy malignant cells and tissues. Chemotherapy drugs are typically small molecule agents. Typical chemotherapy agents are cytotoxic or cell-inhibiting and work by killing or inhibiting cancer growth. Any class of chemotherapy agents can be used, such as taxanes (e.g., paclitaxel and docetaxel), topoisomerase inhibitors (e.g., topotecan), anthracyclines (e.g., doxorubicin and epirubicin), nucleoside analogs (e.g., gemcitabine), platinum-based agents (e.g., cisplatin and carboplatin), alkylating agents (e.g., cyclophosphamide), and kinase inhibitors (e.g., imatinib), or other chemotherapy agents or drugs as described above.

[0085] As used herein, an immunotherapeutic agent is any agent that affects a subject's immune system or immune response. For example, an immunotherapeutic agent can be administered to induce, enhance, or suppress an immune response. Such immunotherapeutic agents may include, for example, antibodies (especially monoclonal antibodies), checkpoint inhibitors, cytokines, cells used in adoptive cell transfer therapy (ACT) (e.g., natural or modified immune cells, which may be autologous or allogeneic (donor cells), such as chimeric antigen receptor (CAR) T cells, tumor-infiltrating lymphocytes (TILs) obtained from or derived from a patient, genetically modified immune cells, NK cells, etc.), and vaccines.

[0086] Hormone therapy (also known as hormone therapy, anti-hormone therapy, hormone treatment, or endocrine therapy), as referred to in this article, is used to treat hormone-dependent cancers by removing, blocking, or adding specific hormones into the body to alter the activity or production of those hormones. Examples of hormone therapy agents include, for example, aromatase inhibitors, luteinizing hormone-releasing hormone (LHRH) agonists or blockers or LH blockers, fulvestrant, anti-androgens, gonadotropin-releasing hormone (GnRH) blockers, enzalutamide, abiraterone, dalotamide, medroxyprogesterone acetate, and megestrol acetate.

[0087] As used in this article, radiation therapy (also known as radiotherapy) refers to the use of high doses of ionizing radiation to control or kill cancer cells. Such radiation therapy can include, for example, external beam radiation therapy and internal beam radiation therapy (e.g., brachytherapy), including whole-body radiation therapy (e.g., radiopharmaceuticals, radioactive iodine, etc.).

[0088] As used herein, photodynamic therapy refers to the combined use of photosensitizing agents (e.g., photosensitizers (e.g., sodium porphyrin) or agents with photosensitizing effects) and light that may originate from, for example, a laser or other light source (e.g., a light-emitting diode (LED)). Such photodynamic therapies may include, for example, extracorporeal photodissociation (ECP) or photoimmunotherapy (PIT).

[0089] In a specific implementation, the second therapeutic agent is a cytotoxic agent, cells used for adoptive cell transfer therapy, an antibody, a hormone, or a checkpoint inhibitor.

[0090] Checkpoint inhibitors are agents that bind to and inhibit the function of immune checkpoints. Immune checkpoints are regulators of the immune system that function to promote antigen-specific activation of immune cells and achieve self-tolerance, thereby supporting immune activity against antigen targets and preventing autoimmune diseases and abnormal immune system activity against host tissues. Immune checkpoints can be stimulatory or inhibitory. Stimulatory immune checkpoints modulate the activity of immune cells against antigen targets by stimulating proliferation and effector responses after binding to their homologous ligands or agonists. Examples of stimulatory immune checkpoints include CD28, which acts as a co-stimulatory factor for T-cell activity and initiates T-cell proliferation upon binding to its ligands CD80 and CD86.

[0091] Suppressive immune checkpoints, upon binding to their homologous ligands or agonists, downregulate or inhibit immune cell function, thereby promoting self-tolerance and preventing autoimmune activity or excessive and aberrant immune responses that could lead to host damage, such as cytokine storms. However, activation of suppressive immune checkpoints can prevent the immune system from targeting cancer cells. Examples of such suppressive immune checkpoints include PD-1 and CTLA-4. Checkpoint inhibitors, as defined herein (and generally in the art), are agents that inhibit the activity of suppressive immune checkpoints. Except in the paragraphs for which their meaning is clearly defined above, throughout this disclosure, the term "immune checkpoint" means suppressive immune checkpoint.

[0092] As defined herein, a checkpoint inhibitor is any agent that binds to an immune checkpoint or its ligand and is used directly to prevent immune checkpoint activation. Therefore, a checkpoint inhibitor can be an antagonist of an immune checkpoint. All currently clinically available checkpoint inhibitors work by blocking their target immune checkpoints, i.e., binding to the checkpoint or its ligand and thus preventing the interaction between the checkpoint and the ligand (a mechanism known as immune checkpoint blocking). However, the checkpoint inhibitors used in combination with oligopeptide compounds in this paper can work through any mechanism, including immune checkpoint blocking, non-competitive inhibition of immune checkpoints, covalent or structural alterations of immune checkpoints (or their ligands), etc. Ideally, a checkpoint inhibitor should expose cancer cells to the immune system without causing the same system to attack healthy tissue.

[0093] Therefore, a checkpoint inhibitor can be any agent that binds to an immune checkpoint or immune checkpoint ligand and inhibits the activity of the immune checkpoint. Checkpoint inhibitors can be, for example, small molecules, ligand antagonists, affimers, or antibodies. An antibody, as referred to herein, can be a natural or synthetic antibody, or a fragment or derivative thereof. The term "antibody" is used broadly herein to include any type of antibody or antibody-based molecule. This includes not only natural antibody molecules but also modified, synthetic, or recombinant antibodies, and their derivatives or fragments. Therefore, an antibody can be any molecule or entity or construct having an antibody-based binding region derived from an antibody-derived binding domain.

[0094] Therefore, an antibody can alternatively be defined as a binding molecule containing an antigen-binding domain obtained from or derived from an antibody. An antibody can be or may be derived from / based on any convenient or desired type, class, or subtype of antibody. As mentioned above, an antibody can be natural, derived, or synthetic. It can be monoclonal or polyclonal. Therefore, an antibody can bind a single epitope, or it can be a mixture of antibodies (or antibody molecules) that bind different epitopes.

[0095] Therefore, a checkpoint inhibitor can be a binding molecule comprising an antigen-binding domain from an antibody that is specific to (or targets) an immune checkpoint or its ligand. Examples of such “antibodies” (i.e., antibody-based binding molecules) include monoclonal and polyclonal antibodies, antibody fragments (including Fab, Fab', F(ab')2, and Fv fragments, and any fragment lacking an Fc region), chimeric (e.g., humanized or CDR transplanted) antibodies, single-chain antibodies (e.g., scFv antibodies), antibodies identified or obtained from phage display, etc. In a specific embodiment, the checkpoint inhibitor is a monoclonal antibody.

[0096] Affinities are engineered non-antibody proteins that mimic the binding of antibodies to their targets. Affinities originate from the cystatin family and share a common structure: an α-helix atop an antiparallel β-sheet. Affinities and their generation methods are described in WO2009 / 136182.

[0097] In specific implementations, checkpoint inhibitors inhibit PD-1 activity. Checkpoint inhibitors, in particular, can block the interaction between PD-1 and PD-L1 (or the interaction between PD-1 and PD-L2), thereby preventing PD-1 activation (as mentioned above, PD-1 activation inhibits T-cell effector function). Checkpoint inhibitors that block the interaction between PD-1 and PD-L1 / PD-L2 bind to one of these proteins and prevent the interaction between the two proteins from occurring. Therefore, checkpoint inhibitors that block the interaction between PD-1 and PD-L1 can bind to PD-1, or they can bind to either PD-L1 or PD-L2. In specific implementations, checkpoint inhibitors bind to either PD-1 or PD-L1. Specifically, such checkpoint inhibitors can bind to the PD-L1 binding site of PD-1, or the PD-1 binding site of PD-L1. To block the interaction between PD-1 and both PD-L1 and PD-L2, it may be advantageous to use a PD-1-binding checkpoint inhibitor to block the interaction between PD-1 and its ligand.

[0098] In specific embodiments, the checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1 / PD-L2 is an antibody that binds to PD-1 (particularly a monoclonal antibody, or a derivative or fragment thereof). In other embodiments, the checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1 is an antibody that binds to PD-L1 (particularly a monoclonal antibody, or a derivative or fragment thereof). Many such antibodies are known in the art, such as nivolumab (Bristol-Myers Squibb), a human monoclonal anti-PD1 IgG4 antibody; pembrolizumab, a humanized IgG4 anti-PD-1 antibody (Merck); atezolizumab, a fully humanized anti-PD-L1 antibody (Genentech); and durvalumab, a humanized anti-PD-L1 antibody (Medimmune / AstraZeneca), all of which have been regulatory approved and may be used herein. Many other such antibodies are currently under development / experimentation, such as tislelizumab, a humanized anti-PD-1 antibody (BeiGene); and avelumab, a fully human anti-PD-L1 antibody (Pfizer / Merck), and can also be used according to the present invention. Similarly, antibodies that bind to PD-L2 (preferably monoclonal antibodies, or derivatives or fragments thereof) can be used to block the interaction between PD-1 and PD-L2.

[0099] As described above, another immune checkpoint that can be targeted by checkpoint inhibitors is CTLA-4. Therefore, in another embodiment, the checkpoint inhibitor blocks the interaction between CTLA-4 and its ligands CD80 and CD86. As detailed above regarding PD-1 / PD-L1 interaction, an agent that blocks the interaction between CTLA-4 and CD80 / CD86 binds to one of these proteins and prevents CTLA-4 from interacting with CD80 and / or CD86. Such an agent can bind to CTLA-4, CD80, or CD86. However, CD80 and CD86 also act as co-stimulatory molecules for T cells by binding to CD28. Therefore, any checkpoint inhibitor that blocks the interaction between CTLA-4 and CD80 / CD86 cannot block the interaction between CD28 and CD80 / CD86. Therefore, a checkpoint inhibitor that blocks the interaction between CTLA-4 and CD80 / CD86 preferably binds to CTLA-4 rather than CD80 and / or CD86. Specifically, checkpoint inhibitors can bind CTLA-4 at the binding sites where CTLA-4 interacts with CD80 or CD86.

[0100] In specific implementations, the checkpoint inhibitor that blocks the interaction between CTLA-4 and CD80 / CD86 is an antibody (preferably a monoclonal antibody, or a derivative or fragment thereof) that binds to CTLA-4. Many such antibodies are known in the art, such as ipilimumab, a human IgG1 monoclonal antibody (Bristol-Myers Squibb), which has been approved by regulatory authorities. Other such antibodies are under development / experimentation, such as trimemumab, a human IgG2 monoclonal antibody (Medimmune / AstraZeneca).

[0101] PD-1 and CTLA-4 are expressed on T cells. PD-1 and CTLA-4 inhibition aims to promote T cell activity; therefore, when antibodies targeting PD-1 or CTLA-4 are used as checkpoint inhibitors, it is preferable that the binding of the antibody to its target does not induce antibody-dependent cytotoxicity (ADCC), which can lead to the death of target T cells. ADCC is primarily mediated by natural killer (NK) cells expressing Fc receptors (e.g., CD16), which recognize and bind to the Fc (i.e., constant) domain of antibodies that bind to target antigens. The binding of NK cell Fc receptors to the Fc domain of antigen-binding antibodies leads to NK cell activation, thereby releasing cytotoxic agents that kill antibody-bound cells.

[0102] Antibodies capable of binding to target cells without inducing ADCC can be specific IgG subclasses unrelated to ADCC activity, or can be rationally designed by introducing point mutations to inhibit Fc receptor binding. Such rational design will be apparent to those skilled in the art. For example, a mutation at position 228 in the constant region of human IgG4 can prevent antibody binding to the Fc receptor. Therefore, nivolumab and pembrolizumab (both human IgG4 antibodies, as described above) both contain the S228P mutation in their constant regions that prevents Fc receptor binding, meaning that neither antibody mediates ADCC. Any PD-1 antibody used as a checkpoint inhibitor according to the present invention can contain the same or equivalent mutations. An equivalent mutation means a mutation at a different residue (or a corresponding residue in the constant region of a different antibody isotype) having the same effect (i.e., inhibiting Fc receptor binding).

[0103] However, in other cases, it may be preferred that checkpoint inhibitors mediate ADCC. The anti-CTLA-4 antibody ipilimumab has been shown to mediate ADCC against Treg cells through monocytes expressing atypical CD16, thus providing a second mechanism to prevent the downregulation of immune effector cells (Romano et al., PNAS 112(19) 6140-6145, 2015).

[0104] While not as prominent as PD-1 and CTLA-4, other immune checkpoints besides these two are also known and can be targeted by checkpoint inhibitors. These other checkpoints include, for example, LAG-3 (also known as CD223). Specifically, such agents can be antibodies that bind to LAG-3, many of which are under development, such as BMS-986016 (Bristol-Myers Squibb).

[0105] In another alternative approach, inhibitors of killer cell immunoglobulin-like receptors (KIRs) can be used as checkpoint inhibitors. For example, lirelurumab (Bristol-Myers Squibb), a fully human monoclonal antibody targeting KIRs, can be used as a checkpoint inhibitor.

[0106] Other immune checkpoints that can be targeted by checkpoint inhibitors to prevent their activation (e.g., by blocking their interaction with homologous ligands) include B7-H3 (also known as CD276), BTLA (also known as CD272), VISTA, and TIM-3 (also known as HAVCR2). Where appropriate, the ligands of these checkpoints can also be targeted by checkpoint inhibitors to block the interaction between the ligand and its immune checkpoint receptor. For example, the TIM-3 ligand phosphatidylserine (PS) can be targeted by checkpoint inhibitors to block its interaction with TIM-3, such as using anti-PS antibodies. An example of such an antibody is bavituximab (Oncologie Inc.), which is currently under development.

[0107] Any checkpoint inhibitor can be used. As detailed above, many checkpoint inhibitors are known to those skilled in the art or can be developed, for example, by rational design or by generating antibodies targeting appropriate targets. In specific implementations, more than one checkpoint inhibitor can be used in combination with an oligopeptide compound. For example, two or more different checkpoint inhibitors can be used, each inhibiting the activation of different immune checkpoints. For example, a checkpoint inhibitor that blocks PD-1 activation can be used in combination with a checkpoint inhibitor that blocks CTLA-4 activation. It has been previously shown that the combination of multiple checkpoint inhibitors produces improved therapeutic outcomes in some cancers compared to the use of any single checkpoint inhibitor.

[0108] As stated above, oligopeptide compounds, and any second agent actually used in conjunction with them, can be administered via any convenient or desired route, which may depend on the subject, condition, and nature of the agent, etc. Choosing an appropriate route of administration is entirely within the scope of the routine skills of clinicians in this field.

[0109] Possible routes of administration include oral, rectal, nasal, topical, vaginal, and parenteral administration. Oral administration, as used herein, includes buccal and sublingual administration. Topical administration, as used herein, includes transdermal administration. Parenteral administration, as defined herein, includes subcutaneous, intramuscular, intravenous, intraperitoneal, and intradermal administration. Oligopeptide compounds can be administered to subjects, in particular, for systemic delivery, such as via oral or parenteral administration, or topically to the site of cancer to be treated, such as local or direct application to the tumor. Possible routes of local administration include local application, delivery to the cancer (e.g., tumor) site by direct application (e.g., via injection or infusion), and inhalation, depending on the location of the cancer (tumor).

[0110] In a specific implementation, the oligopeptide compound is administered to the subject via intratumoral administration, such as by direct injection or infusion into the tumor. This has been found to be particularly advantageous. In the case of oligopeptide compounds, intratumoral administration into a tumor (e.g., a primary tumor) has been shown to cause regression not only in the injected tumor but also in tumors at other sites (e.g., secondary tumors). This has indeed been confirmed by injecting secondary tumors and observing tumor regression at other sites. This is further evidenced by... Figure 9 This has been confirmed, as shown in the following examples. Therefore, a strong remote effect of the oligopeptide compound can be observed. Thus, the most convenient or easily accessible tumor, or perhaps the largest or most mature tumor, can be selected as the application site. However, other forms of administration, including systemic administration, are not excluded.

[0111] The generally accepted principle is that metastatic lesions of solid tumors require systemic administration of anticancer drugs; therefore, the idea of ​​treating metastatic disease through intratumoral drug administration seems counterintuitive. Indeed, for the millions of cancer patients treated each year, systemic anticancer drugs rarely achieve a cure. Liver metastases are associated with poor prognosis compared to metastases in other organs, and liver resection and other liver-directed procedures (such as cryotherapy, thermal ablation, or chemoembolization) are generally accepted procedures for prolonging the survival of ACC patients. Intratumoral administration of drugs has several significant advantages compared to systemic administration, including achieving intratumoral concentrations of the drug by several orders of magnitude and potentially reducing systemic toxicity. Preliminary efficacy data for CyPep-1 in ACC, as reported in the following examples, suggest that intratumoral administration (including liver metastases) represents a promising advance in the management of metastatic ACC patients.

[0112] When an oligopeptide compound is used in combination with another therapeutic agent, the two agents may be administered alone, simultaneously, or sequentially. As used herein, "alone" administration means administering the oligopeptide compound and the second agent to the subject simultaneously or at least substantially simultaneously, but via different routes of administration. As used herein, "simultaneous" administration means administering the oligopeptide compound and the second agent to the subject simultaneously or at least substantially simultaneously via the same route of administration. As used herein, "sequential" administration means administering the oligopeptide compound and the second agent to the subject at different times. Specifically, the administration of the first therapeutic agent is completed before the administration of the second therapeutic agent begins. When administered sequentially to the subject, the first and second therapeutic agents may be administered via the same route of administration or via different routes of administration.

[0113] The administration of the oligopeptide compound and / or the second agent may be repeated (i.e., twice or more) during the subject's treatment. For example, the subject may receive multiple treatment cycles in which both the oligopeptide compound and the second agent are administered. Alternatively, the subject may receive a single dose of one therapeutic agent and repeated doses of the other.

[0114] If multiple additional therapeutic agents are administered to a subject in combination with the oligopeptide compound, the two or more additional agents may be administered individually, simultaneously, or sequentially.

[0115] In specific implementations, the second agent (e.g., a checkpoint inhibitor) is administered parenterally to the subject. For example, the second agent may be administered intravenously to the subject. In one implementation, the oligopeptide compound is administered intratumorally, and the second agent (e.g., a checkpoint inhibitor) is administered parenterally, such as intravenously.

[0116] Oligopeptide compounds, and when used with a second or additional therapeutic agent, are formulated for administration according to principles known in the art. Therefore, they are provided in the form of pharmaceutical compositions comprising the compound and / or a second or additional therapeutic agent, and one or more pharmaceutically acceptable carriers or excipients.

[0117] As described above, oligopeptide compounds and / or checkpoint inhibitors (or pharmaceutical compositions comprising them) may be administered to subjects in a manner suitable for the cancer to be treated. The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, but may also be determined through clinical trials to determine the appropriate dosage. Conveniently, the oligopeptide compound and / or second agent may be provided to subjects at daily, weekly, or monthly doses or at moderate frequencies, for example, every 2, 3, 4, 5, or 6 days, every 2, 3, 4, 5, or 6 weeks, every 2, 3, 4, 5, or 6 months, annually, or every two years. As described above, the same dosing regimen or different dosing regimens may be used to administer the oligopeptide compound and second agent or additional agents to subjects.

[0118] The dosage may be determined based on the subject's body size. The oligopeptide compound may be administered at doses ranging from 10 μg / kg to 100 mg / kg body weight, such as 10 μg / kg to 50 mg / kg body weight, 10 μg / kg to 20 mg / kg body weight, 10 μg / kg to 10 mg / kg body weight, 10 μg / kg to 5 mg / kg body weight, 10 μg / kg to 2.5 mg / kg body weight, 100 μg / kg to 2.5 mg / kg body weight, 500 μg / kg to 5 mg / kg body weight, or 1 mg / kg to 5 mg / kg body weight. In a specific embodiment, the oligopeptide compound may be administered at a dose of approximately 2 mg / kg body weight, such as 1 mg / kg to 2.5 mg / kg body weight, 1.5 mg / kg to 2.5 mg / kg body weight, or 1.8 mg / kg to 2.2 mg / kg body weight. A skilled clinician will be able to calculate the appropriate dosage for a patient based on all relevant factors, such as age, height, weight, the condition to be treated, and its severity.

[0119] Similar considerations apply to the second or additional therapeutic agent. The dosage may be determined based on the subject's body size. The second therapeutic agent may be administered at doses ranging from 10 μg / kg to 100 mg / kg body weight, such as 10 μg / kg to 50 mg / kg body weight, 10 μg / kg to 10 mg / kg body weight, 10 μg / kg to 5 mg / kg body weight, 10 μg / kg to 2.5 mg / kg body weight, 100 μg / kg to 5 mg / kg body weight, 100 μg / kg to 2.5 mg / kg body weight, 500 μg / kg to 5 mg / kg body weight, or 1 mg / kg to 5 mg / kg body weight. In a specific embodiment, the second therapeutic agent may be administered at a dose of approximately 2 mg / kg body weight, such as 1 mg / kg to 2.5 mg / kg body weight, 1.5 mg / kg to 2.5 mg / kg body weight, or 1.8 mg / kg to 2.2 mg / kg body weight. A skilled clinician will be able to calculate the appropriate dosage for a patient based on all relevant factors, such as age, height, weight, the condition to be treated, and its severity.

[0120] As described above, the second agent (e.g., a checkpoint inhibitor) can be administered at the same dose as the oligopeptide compound, or at a higher or particularly lower dose. When the compound and agent are used in combination, the dose may be lower than when the compound or agent is used alone (e.g., in monotherapy). For example, checkpoint inhibitors can be administered at doses from 100 μg / kg to 100 mg / kg body weight, such as 500 μg / kg to 50 mg / kg body weight, or 1 mg / kg to 10 mg / kg body weight. Exemplary doses include 1 mg / kg body weight, 2 mg / kg body weight, 3 mg / kg body weight, 4 mg / kg body weight, 5 mg / kg body weight, 6 mg / kg body weight, 7 mg / kg body weight, 8 mg / kg body weight, 9 mg / kg body weight, and 10 mg / kg body weight. Checkpoint inhibitors can be administered at a fixed dose, such as 100 mg to 1.5 g. Exemplary doses of checkpoint inhibitors include 100 mg, 200 mg, 240 mg, 250 mg, 300 mg, 400 mg, 480 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, and 1500 mg.

[0121] Appropriate dosing regimens for many checkpoint inhibitors are known. For example, nivolumab, when used alone, is administered at a dose of 240 mg intravenously every 2 weeks or 480 mg intravenously every 4 weeks; ipilimumab, when used alone in melanoma therapy, is administered at a dose of 3 mg / kg intravenously every 3 weeks. These dosing regimens, and many other such checkpoint inhibitor dosing regimens, are known to those skilled in the art and can be found in licensing approvals issued by regulatory agencies such as the FDA and EMA.

[0122] For combination therapies, the oligopeptide compound and the second or additional therapeutic agent may be provided in the form of a kit containing two or more components. For example, the oligopeptide compound and the second therapeutic agent may be provided in separate containers (i.e., separate compositions) or in a single composition within a single container. Each therapeutic agent may be provided in any suitable form, such as in an aqueous solution or as a lyophilized product.

[0123] As described above, and in accordance with the pharmaceutical uses of this article, companion diagnostics are also provided, i.e., methods for identifying or determining which subjects are suitable or appropriate for treatment with oligopeptide compounds, based on the pharmaceutical uses and treatment methods described herein. Performing such screening is not necessary or even important, but may be desirable or useful in some cases.

[0124] Therefore, in some implementations, it is possible to determine whether a subject has cancer associated with abnormal activation of the Wnt / β-catenin pathway. As described above, this can be done by identifying the presence of biomarkers indicating abnormal or activated Wnt / β-catenin pathways in samples obtained from the subject.

[0125] Therefore, subjects (e.g., patients with signs of tumors or cancer who visit a hospital) can be screened or tested as part of their evaluation to determine the presence of such a biomarker. This can be done at the initial visit, or subsequently during the subject's evaluation or treatment (e.g., when further treatment is considered, or in case of recurrence, etc.), or during treatment.

[0126] The sample can be any sample in which such a biomarker is present, and this can depend on the biomarker and / or cancer in question. For example, the sample can be a sample of cells or tissue from a cancer site, such as a tumor biopsy. It can be a solid or liquid biopsy of the cancer. In other embodiments, it can be a sample of body tissue or fluid in which the cancer biomarker may be present, such as a blood-derived sample, such as serum or plasma containing circulating cancer DNA (e.g., cell-free DNA).

[0127] As mentioned above, mutations in various genes along the Wnt / β-catenin pathway have been identified and reported, and these mutations can have various effects, such as on protein expression levels (e.g., protein expression may be upregulated or downregulated) and / or protein function (e.g., protein may be inactivated). Therefore, a biomarker can be a mutation, or it can be an increase or decrease in the level (i.e., amount) of a specific protein or protein complex in a specific tissue or cell or subcellular compartment. For example, the amount of β-catenin in the cell nucleus can be increased. Increased expression can be measured at the protein or mRNA level. Furthermore, a biomarker can be an alteration in protein (e.g., enzyme) activity, thus allowing for functional assays of protein (e.g., enzyme) activity. Therefore, a biomarker can be an increase or decrease in the level of protein (e.g., enzyme) activity.

[0128] In one implementation, the biomarker is: (i) Increased expression of one or more of the following proteins in tumor samples: β-catenin, WISP-1, FZD3, FZD6, FZD7, Wnt3, Wnt4, Wnt5A, RSPO1, RSPO2, RSPO3, RSPO4, c-MYC, cyclin-D, TCF21; (ii) Mutations in one or more of the following genes: β-catenin (CTNNB1), AXIN1, AXIN2, APC, ZNRF3, MEN1, GNAI2, RNF43; (iii) The expression of one or more of the following genes is upregulated: WISP-1, c-MYC, cyclin-D, TCF21, FZD3, FZD6, FZD7, Wnt3, Wnt4, Wnt5A, RSPO2, CLDN1, LGR5.

[0129] In another embodiment, the biomarker is selected from any one or more of (i), (ii), and (iii) above or below: (i) Increased expression of one or more of the following proteins in tumor samples: β-catenin, WISP-1, Axin2, FZD3, FZD6, FZD7, Wnt3, Wnt4, Wnt5A, RSPO1, RSPO2, RSPO3, RSPO4, c-MYC, cyclin-D, TCF21; (ii) Mutations in one or more of the following genes: β-catenin (CTNNB1), AXIN1, AXIN2, APC, APC2, ZNRF3, MEN1, GNAI2, GNAI3, RNF43, DVL1, LPR5, LPR6, MED12, and BCL9L; (iii) The expression of one or more of the following genes is upregulated: WISP-1, Axin2, c-MYC, cyclin-D, TCF21, FZD3, FZD6, FZD7, Wnt3, Wnt4, Wnt5A, RSPO2, CLDN1, LGR5.

[0130] In a specific representative embodiment, the biomarker may be an increase in the expression of the proteins β-catenin, Wisp1, or Axin2. In another embodiment, the biomarker may be an increase in the expression of β-catenin or Wisp1.

[0131] Clinical studies can identify which genes / proteins in this pathway can serve as useful biomarkers. Therefore, suitable biomarkers can be identified by studying gene / protein expression, the presence of mutations in specific genes, or, in fact, the identification of specific mutations in those genes, in subjects who responded to treatment with compounds such as CyPep-1 compared to non-responders. Suitable biomarker genes identified in this way are described in the examples below.

[0132] Respondents in this regard can be subjects who have demonstrated any positive clinical response; this can include complete or partial remission, such as in tumor regression (e.g., tumor size reduction and / or number reduction), or in other clinical signs, or in the slowing or suppression of disease progression (i.e., disease stabilization). Therefore, for example, suitable biomarkers can be identified in subjects demonstrating partial remission or disease stabilization. This is described in the examples below.

[0133] As representative examples, the presence of one or more mutations in one or more genes in the following gene groups can be used as biomarkers according to the methods described herein: (i) Axin2, DVL1, APC2, LRP5, MEN1, GNAI3, MED12, CTNNB1, LPR6; (ii) Axin2, DVL1, APC2, LRP5, GNAI3, MED12, CTNNB1, LPR6; (iii) Axin2, DVL1, APC2, LRP5, MEN1, GNAI3, MED12, CTNNB1; (iv) Axin2, DVL1, APC2, LRP5, GNAI3, MED12, CTNNB1; (v) Axin2, DVL1, APC2, LRP5, MEN1, GNAI3, MED12; (vi) Axin2, DVL1, APC2, LRP5, GNAI3, MED12; (vii) Axin2, DVL1, APC2, LRP5, MEN1; (viii) Axin2, DVL1, APC2, LRP5.

[0134] As previously mentioned, Conductin / Axin2 is a central protein in the Wnt / β-catenin pathway in tumorigenesis, and CyPep-1 is believed to activate Axin2, thereby preventing the nuclear translocation and activation of β-catenin. Due to this mechanism of action, patients carrying one or more mutations in genes that reduce Axin2 activity or increase inactive Axin2 may particularly benefit from CyPep-1 treatment. Therefore, the presence of one or more mutations in one or more genes encoding proteins involved in the Axin2 regulatory network can represent a useful biomarker for such patients, especially mutations that inactivate or reduce Axin2 activity.

[0135] Therefore, a representative subgroup of biomarkers representing the biomarkers in the Axin2 regulatory network may include mutations in the genes listed in (v) through (viii) above. Representative mutations in these genes are shown in Table 1.

[0136] Table 1

[0137] Methods for detecting mutations or determining gene expression levels are widely described in the art, as are methods for determining the amount of a given protein in a sample. The determination of the activity of various proteins involved in the Wnt / β-catenin pathway is described in this art. Attached Figure Description

[0138] The invention can be more fully understood from the following non-limiting embodiments and with reference to the accompanying drawings, wherein: Figure 1 The results presented indicate that CyPep-1 inhibits Wnt / β-catenin signaling. A) Quantification of nuclear levels of β-catenin in adrenocortical cell line Y1, colorectal cell line CT26, and melanoma cell line B16-F10. B) Quantification of Wnt-induced secretory protein-1 (WISP-1 / CCN4), a target gene of β-catenin, by PCR in CT26 cells incubated with different concentrations of CyPep-1. C) Quantification of WISP-1 in colorectal cell lines CT26, melanoma cell lines B16-F10 and Yumm1.7, and glioma cell line GL261. D) Schematic diagram illustrating the mechanism of action (MOA) of CyPep-1 on the Wnt / β-catenin pathway.

[0139] Figure 2 The results presented demonstrate that CyPep-1 induces a pro-inflammatory response in vivo. A) In vivo tumor growth is shown on day 17 after implantation of the colorectal cell line CT26 and the melanoma cell line B16-F10 treated with either control (saline) or CyPep-1 monotherapy. B) CyPep-1 treatment in B16-F10 and CT26 tumor-bearing mice induced profound changes in the tumor immune microenvironment. Flow cytometry quantified the percentages of CD45+ leukocytes (gated in live cells) and CD8+ lymphoid cells after treatment with the solvent (CT) and CyPep-1. C) CyPep-1 increased inflammatory cytokines in B16-F10 and CT26 tumors. IL2, TNFα, and IFNγ secreted in the microenvironment of B16-F10 melanoma (top) and CT26 colorectal tumors (bottom) treated with either the control solvent (CT) or CyPep-1 (CyPep-1) were quantified by ELISA. Data are reported in pg / ml, standardized according to the weight of the excised tumor (g), and expressed as the mean of 5 tumors per group (each point represents one mouse). All results are expressed as mean ± SEM (error bars). Unpaired two-tailed Student's t-test was used (ns = not significant, ...). =p<0.005 and =p<0.0005) is used to calculate statistically significant differences compared to the control condition (indicated by an asterisk).

[0140] Figure 3 This paper presents the results of CyPep-1 monotherapy in a patient with ACC previously treated with mitotane (standard of care). The patient had metastatic disease in the liver, lungs, and bones. A) CT images before treatment and CT images 6 months after CyPep-1 treatment. According to the Recognition of Efficacy in Solid Tumors (RECIST) criteria, the sum of longest diameters (SOD) showed a 47% reduction in the sum of the diameters of the target lesions (partial response). B) Liver parameters measured before and during CyPep-1 treatment.

[0141] Figure 4 The results of CyPep-1 monotherapy in a second patient with ACC previously treated with mitotane are shown. This patient had metastatic disease that had spread to the liver, lungs, and bones.

[0142] A) Histopathological examination of biopsies obtained after 6 weeks of CyPep-1 treatment, showing necrosis and immune infiltration. B) RECIST measurements of the target lesions during treatment. C) Liver parameters measured before and during CyPep-1 treatment.

[0143] Figure 5 The results presented indicate that CyPep-1 treatment in melanoma cells reduced the expression of target genes Axin2 and Ccn4 / Wisp1, and increased the levels of two phosphorylated forms of β-catenin. A) Quantification of cytoplasmic and nuclear levels of β-catenin in the B16-F10 melanoma cell line after incubation with increased CyPep-1 concentration for 24 hours. B) Quantification of β-catenin target genes Axin2 and Wnt-induced secretory protein-1 (Ccn4 / Wisp1) by RT-qPCR in the B16-F10 melanoma cell line after incubation with 7.5 μM CyPep-1 for 24 hours. C) Quantification of phosphorylated β-catenin levels (Ser33 / 37-Thr41 and Ser45-Thr41) in the B16-F10 melanoma cell line after incubation with increased CyPep-1 concentration for 24 hours. D) Quantification of phosphorylated β-catenin levels (Ser33 / 37-Thr41 and Ser45-Thr41) in melanoma cell line B16-F10 after incubation with 7.5 μM CyPep-1 for 24 hours.

[0144] Figure 6 The results presented indicate that CyPep-1 treatment increased the levels of two phosphorylated forms of β-catenin in adrenocortical carcinoma cells. A) Quantification of phosphorylated β-catenin levels (Ser33 / 37-Thr41 and Ser45-Thr41) in the adrenocortical carcinoma cell line H295R after incubation with increased concentrations of CyPep-1 for 24 hours. B) Quantification of phosphorylated β-catenin levels (Ser33 / 37-Thr41 and Ser45-Thr41) in the adrenocortical carcinoma cell line H295R after incubation with 9.375 μM CyPep-1 for 24 hours.

[0145] Figure 7 The results of subcutaneous injection of CyPep-1 monotherapy into immunodeficient NSG mice with H295R human adrenocortical carcinoma cells are shown. When the tumor reached 250 mm... 3 Mice were treated with 25 mg / kg CyPep-1 intratumorally once a week. A) Tumor volume (mm) from day 0 to day 31 after CyPep-1 or solvent control injection. 3 The number of days mice were treated with CyPep-1 or a solvent control is indicated by dashed lines. On day 8, five mice from each group were euthanized to harvest tumors for RNA analysis (presented in D). B) Tumor volumes (mm) on days 0 and 31 after injection with CyPep-1 (CY-101) or a solvent control. 3C) Quantification of the survival probability within 31 days after treatment with CyPep-1 or solvent control. D) Quantification of β-catenin target genes Axin2 and Wnt-induced secretory protein-1 (Ccn4 / Wisp1) by RT-qPCR in tumors harvested from euthanized mice 8 days after initial treatment with CyPep-1 or solvent control.

[0146] Figure 8 Imaging of B16-F10 mouse melanoma cells treated with 20 μg / ml CyPep-1 for 24 hours is shown. Cells were stained with anti-Axin2 antibody and DAPI (for staining the cell nucleus) and imaged using confocal microscopy. A) Images of treated and untreated cells stained with Axin2 and DAPI, scale bar 20 μm. B) Individual portions of the image in section A (represented by white dashed rectangles in A), scale bar 5 μm. C) Schematic illustration of different cytoplasmic distributions of Axin2 (conductin) in inactive (diffuse) or active (aggregate) states. D) Schematic illustration of how CyPep-1 can bind to the RGS domain of Axin2, reverse their aggregation, and activate Axin2, leading to Axin2 polymerization.

[0147] Figure 9 This presentation shows the results of CyPep-1 treatment in combination with an anti-PD-1 antibody (pembrolizumab) in a patient with parathyroid carcinoma who had previously received surgery and radiation therapy (standard of care) and nivolumab (an anti-PD-1 antibody). The patient had metastatic disease with multiple lesions in different parts of the body. Only one lesion was injected with CyPep-1. A1) CT image of the injected lesion at baseline (indicated by a white dashed circle). A2) CT image of the injected lesion three months after CyPep-1 treatment (indicated by a white dashed circle). A3) Histological image of the injected lesion biopsy at baseline, scale bar 50 μm. A4) Histological image of the injected lesion biopsy three months after CyPep-1 treatment, scale bar 50 μm. A5) CT image of the uninjected lesion at baseline (indicated by a white arrow). A6) CT images of uninjected lesions 20 months after the patient started CyPep-1 treatment (indicated by white arrows). B) The figure shows the percentage change in tumor size between injected and uninjected lesions over 24 months.

[0148] Figure 10 A graph showing the pharmacokinetic characteristics of CyPep-1 as measured using liquid chromatography (LC)-tandem mass spectrometry (MS) is presented.

[0149] Figure 11 This study shows the percentage change in tumor size of untreated target lesions over 18 months in eight patients with seven different types of cancer. Patients were treated with CyPep-1 for >6 months as monotherapy, in combination with the anti-PD-1 antibody pembrolizumab (indicated by an asterisk), or via intrahepatic injection.

[0150] Figure 12 The study presented the survival of six patients with adrenocortical cancer during and after receiving intrahepatic CyPep-1 injections. Detailed Implementation

[0151] Example Example 1 Inhibit β-catenin and WISP-1 (CNN4) CyPep-1, the D-peptide of SEQ ID NO: 1, was prepared by Bachem AG (Switzerland). The role of CyPep-1 in inhibiting the Wnt / β-catenin pathway was evaluated by assessing the expression of WISP-1 (a well-described downstream target gene of β-catenin) in various cancer cell lines. Downregulation of WISP-1 indicated reduced transcriptional activity of β-catenin in CyPep-1-treated cells.

[0152] In short, cell lines were cultured for 24 hours with 0 μM to 5 μM CyPep-1 added to the culture medium. Control cells were cultured in CyPep-1-free medium (in PBS). Cells were washed with PBS and lysed using a suitable cell separation kit, and cytoplasmic and nuclear fractions were recovered according to the supplier's instructions. Cytoplasmic and nuclear fractions were separated on an SDS-PAGE gel, and proteins were transferred to nitrocellulose membranes incubated with anti-β-catenin and anti-α-tubulin antibodies (specific cytoplasmic proteins) or anti-histone H3 antibodies (specific nuclear proteins). Quantification of β-catenin bands in the cytoplasm and nucleus was performed using ImageJ. The β-catenin / α-tubulin ratio corresponds to the cytoplasmic fraction of β-catenin, and the β-catenin / histone H3 ratio (Y1) or the β-catenin / lamin A / C ratio (B16-F10 and CT26) corresponds to the nuclear fraction of β-catenin. Results are reported as the fold change in the cytoplasmic and nuclear fractions of β-catenin in cells treated with a specified concentration of CyPep-1 compared to untreated control cells. Figure 1A). To assess CyPep-1 mRNA expression, melanoma (B16-F10 and Yumm1.7), colorectal cancer (CT26), or glioma (GL261) were treated with CyPep-1 as described above. For CT26 cells, dose-dependent decreases in WISP-1 expression were assessed using different doses of CyPep-1.

[0153] Figure 1 A shows the nucleoprotein levels of β-catenin in the Y1 adrenocortical cell line, CT26 colorectal cell line, and B16-F10 melanoma cell line treated with CyPep-1 or as a control. Results are reported as fold changes compared to the control. Figure 1 B and Figure 1 C shows the mRNA expression levels of WISP-1 / CCN4 in melanoma (B16-F10), colorectal cancer (CT26), glioma (GL261), and melanoma (Yumm1.7) cell lines treated with CyPep-1. Results are reported as fold changes in WISP-1 levels in treated cells compared to controls. In all cell lines, CyPep-1 treatment significantly reduced WISP-1 / CCN4 mRNA expression.

[0154] Example 2 CyPep-1 induces pro-inflammatory responses in vivo B16-F10 and CT26 cell lines were obtained from ATCC. RPMI 1640, DMEM, FBS, and antibiotics were obtained from Life Technologies. B16-F10 and CT26 cells were cultured in DMEM and RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, respectively, at 37°C and 5% CO2. These cell lines were mycoplasma-free according to the Mycoalert kit (Lonza). C57BL / 6 and BALB / C (7 weeks old) were obtained from Janvier and fed under pathogen-free conditions for one week prior to the experiment. On day 0, mice were subcutaneously injected with 0.2 × 10⁻⁶ styrosine diluted in 100 μL PBS. 6 One B16-F10 cell or 10 6 CT26 cells. The median tumor volume reached 100 mm. 3 At that time, administer 2 mg / kg CyPep-1 or solvent (PBS) intratumorally.

[0155] Tumor weight results were reported as an average of 10 mice per group, which was derived from two independent experiments, with 5 mice per group in each experiment.

[0156] Figure 2A showed that CyPep-1 reduced tumor weight in B16-F10 and CT26 tumor-bearing mice compared to controls. Results are presented as mean ± SEM (error bars). Unpaired two-tailed Student's t-tests were used to calculate statistically significant differences (indicated by asterisks).

[0157] Figure 2 B indicates that CyPep-1 treatment in B16-F10 and CT26 tumor-bearing mice induced profound changes in the tumor immune microenvironment. Tumors were harvested and mechanically dissociated into fragments (<4 mm), which were then enzymatically digested at 37°C for 45 min using a mouse tumor dissociation kit (Miltenyi Biotec). Single-cell suspensions were prepared, and erythrocytes were lysed using ACK (10-548E, Lonza).

[0158] Cells were counted using a Countess automated cell counter (Invitrogen) and blocked on ice for 30 minutes with an Fc blocker (TruStain fcX™ (anti-mouse CD16 / 32) antibody 101320 BioLegend). The following antibodies (BioLegend): FITC anti-mouse CD45, APC anti-mouse CD8a, APC / Fire 750 anti-mouse CD4, and the LIVE / DEAD fixable blue-dead cell staining kit (Thermo Fisher Scientific) were used as viability dyes. For compensatory controls, single-dye staining was performed, and fluorescence diffusion was examined by performing an FMO control. The level of nonspecific binding was evaluated on isotype controls.

[0159] Figure 2 C indicates that CyPep-1 increases inflammatory cytokines in B16-F10 and CT26 tumors. IL2, TNFα, and IFNγ secreted in the microenvironment of B16-F10 melanoma (top) and CT26 colorectal tumors (bottom) treated with either the control solvent (CT) or CyPep-1 (CyPep-1) were quantified by ELISA. Data are reported in pg / ml, normalized to the weight of excised tumor (g), and expressed as the mean of 5 tumors per group (each point represents one mouse). IL2, TNFα, and IFNγ were quantified according to the manufacturer's protocol using the R&Dsystems Mouse IL2 (Catalog No. DY410-05), TNFα (Catalog No. DY410-05), and IFNγ (Catalog No. DY485-05) DuoSet ELISA Kit.

[0160] Example 3 Clinical research Material CyPep-1 peptide was synthesized by Bachem AG (Switzerland). CyPep-1 is a full-D-amino acid peptide consisting of the amino acid sequence shown in SEQ ID NO:1.

[0161] method Clinical trials evaluated CyPep-1 monotherapy. The patient cohort included all participants enrolled in a basket trial (which included all types of solid tumors) who had advanced disease (stage IV metastatic disease, with most patients exhibiting stage IVC disease) and had exhausted all other available treatment options.

[0162] Patients received 20 mg CyPep-1 injections every two weeks (weekly). Patient response was defined as an objective response according to RECIST 1.1 guidelines, or if the disease remained stable for >16 weeks. RECIST 1.1 endpoints used to assess target lesions included: • Complete remission (CR): All target lesions disappear; • Partial response (PR): The total target lesion LD is reduced by at least 30% compared to the baseline total LD; • Disease progression (PD): The minimum total LD ​​recorded after the start of treatment or the appearance of one or more new lesions is used as a reference, with the total LD ​​of the target lesions increasing by at least 20%; • Stable disease (SD): Based on the minimum total LD ​​after the start of treatment, the reduction in disease does not reach the PR level, and the increase in disease does not reach the PD level.

[0163] RECIST 1.1 indicators used to assess non-target lesions include: • Complete remission (CR): All non-target lesions disappear and tumor marker levels return to normal; • Stable disease (SD): Persistent presence of one or more non-target lesions and / or tumor marker levels remaining above the normal range; • Disease progression (PD): The appearance of one or more new lesions, and / or clear progression of existing non-target lesions.

[0164] result CyPep-1 monotherapy has shown promising results in advanced IVC cancers associated with aberrantly activated Wnt / β-catenin pathway: CyPep-1 monotherapy Figure 3The results of CyPep-1 monotherapy in an ACC patient are shown. A) CT scans of the patient's ACC at baseline and 6 months after CyPep-1 treatment are shown. Partial response (47%) was observed at 6 months according to the RECIST criteria for efficacy in solid tumors. B) Evaluation of liver enzymes during treatment showed that values ​​had returned to normal.

[0165] Figure 4 The results of CyPep-1 monotherapy in another ACC patient are shown. A) Histological H&E staining of biopsies following CyPep-1 monotherapy showed significant cell death. B) Target lesions in the lungs and liver during 6 months of treatment. Tumor shrinkage was observed not only in the injected tumors but also in other uninjected metastases in the liver (same organ as the injected lesion) and lungs (distal organs to the injected lesions). The shrinkage of uninjected metastases in distal organs is considered the strongest evidence of the immune-mediated remote effect of intratumoral drug administration. C) Evaluation of liver enzymes during treatment.

[0166] Example 4 CyPep-1 induces phosphorylation of β-catenin. Melanoma (B16-F10) and adrenocortical carcinoma (H295R) cells were plated. After 24 hours, B16-F10 cells were treated with increased concentrations of CyPep-1 (0 μM to 7.5 μM); H295R cells were treated with increased concentrations of CyPep-1 (0 μM to 9.375 μM) when 80% confluence was reached. Both cell lines were treated with CyPep-1 for 24 hours, and control cells were cultured in CyPep-1-free medium (in PBS). Cells were then harvested and washed with PBS. RIPA buffer (containing phosphatase and protease inhibitors) was used to lyse cells and extract proteins and mRNA. A suitable cell isolation kit was also used for B16-F10 cells to recover cytoplasmic and nuclear fractions according to the supplier's instructions.

[0167] The extracted proteins were isolated on an SDS-PAGE gel and transferred to a nitrocellulose membrane, where they were incubated with either anti-β-catenin antibody and anti-α-tubulin antibody (specific cytoplasmic proteins) or anti-histone H3 antibody (specific nucleoproteins). Figure 5 A); Anti-β-catenin (Ser33 / 37-Thr41) antibody or anti-β-catenin (Ser45-Thr41) antibody and anti-actin antibody (control) Figure 5 C and Figure 6A). Quantification of the phosphate-β-catenin band was performed using Image J. The ratio of phosphate-β-catenin / actin protein expression was reported as the fold change in phosphate-β-catenin and actin in cells treated with 7.5 μM (B16-F10) or 9.375 μM (H295R) CyPep-1 compared to untreated control cells. Figure 5 D and Figure 6 B).

[0168] Figure 5 A indicates that with increasing CyPep-1 concentration, the nuclear fraction of β-catenin decreases, while the cytoplasmic fraction increases.

[0169] Figure 5 D and Figure 6 B indicates that in melanoma and adrenocortical cancer cells, treatment with CyPep-1 increased both phosphorylated forms of phospho-β-catenin (approximately 2-fold). Phosphorylation of β-catenin at target residues of GSK3 (pS33, pS37, and pT41) and CKI (pS45) tagged β-catenin for ubiquitination and subsequent proteasome degradation. These results suggest that CyPep-1 increased phosphorylation of these residues and enhanced β-catenin degradation.

[0170] To assess CyPep-1 mRNA expression, melanoma (B16-F10) cells were treated with CyPep-1 as described above. RT-qPCR was performed to evaluate the mRNA expression levels of the Wnt / β-catenin downstream target genes Axin2 and Ccn4 / Wisp1. The ratio of target gene mRNA expression was reported as the fold change in target gene mRNA expression in cells treated with 7.5 μM CyPep-1 compared to untreated control cells. Figure 5 B).

[0171] Figure 5 B indicates that, compared with untreated control cells, CyPep-1 reduced the expression of Wnt / β-catenin downstream target genes Axin2 and Ccn4 / Wisp1 in treated cells.

[0172] Example 5 In vivo study of CyPep-1 in adrenocortical carcinoma Human adrenocortical carcinoma cells (H295R cell line) were subcutaneously injected into immunodeficient NGS mice. When the tumor reached 250 mm... 3Mice were treated weekly with 25 mg / kg CyPep-1 or a solvent control. Eight days after the first treatment, five mice from each group were euthanized to harvest tumors for mRNA analysis. Other animals received further treatment and were used for tumor growth (…). Figure 7 A and Figure 7 B) and survival ( Figure 7 C) Analysis. When the tumor volume reaches 1500 mm... 3 At that time, the mice were euthanized.

[0173] Figure 7 A and Figure 7 B indicates that, after the first treatment, CyPep-1 significantly reduced tumor volume compared to the solvent until the end of the experiment. No tumors were observed in mice treated with CyPep-1 at the end of the experiment. Figure 7 C indicates that CyPep-1 also significantly (p=0.0062) increased the survival rate of mice compared with solvent-treated mice.

[0174] mRNA was extracted from tumors harvested from mice euthanized on day 8. RT-qPCR was performed to evaluate the mRNA expression levels of the downstream target genes Axin2 and Ccn4 / Wisp1 of Wnt / β-catenin. The ratio of target gene mRNA expression was reported as the fold change in target gene mRNA expression in CyPep-1-treated tumors compared to untreated control tumors. Figure 7 D).

[0175] Figure 7 D indicates that, compared with untreated control tumors, CyPep-1 reduced the expression of Wnt / β-catenin downstream target genes Axin2 and Ccn4 / Wisp1 in treated mouse tumors. For Ccn4 / Wisp1, this reduction was significant (p=0.0083).

[0176] Example 6 Confocal microscopy observation of Axin2 Melanoma cells (B16-F10) were seeded in ibidi plates. After 24 hours, the cells were treated with increased concentrations of CyPep-1 (0 μg / ml to 20 μg / ml) for another 24 hours. The cells were then fixed with 4% PFA and stained with rabbit anti-Axin2 antibody. Anti-rabbit secondary antibody conjugated with AlexaFluor633 was used. DAPI was used for nuclear staining. Samples were observed using a confocal microscope. Figure 8 A and Figure 8 B).

[0177] Figure 8 A and Figure 8B shows diffuse staining of Axin2 in untreated samples; this observation indicates that Axin2 is inactive. When inactive, the RGS domains of Axin2 aggregate ( Figure 8 D), and Axin2 is diffusely distributed in the cytoplasm ( Figure 8 C). Figure 8 A and Figure 8 B also showed the formation of aggregates / spots in most cells treated with CyPep-1; this observation indicates that Axin2 is active. When active, the RGS domains do not aggregate, and Axin2 aggregates (aggregates). Figure 8 C). This observation indicates that Axin2 aggregates after treatment with CyPep-1, suggesting that CyPep-1 activates Axin2. It is believed that CyPep-1 binding to the RGS domains of Axin2 reverses their aggregation, thereby activating Axin2 (C). Figure 8 D).

[0178] Example 7 Clinical research Material CyPep-1 peptide was synthesized by Bachem AG (Switzerland). CyPep-1 is a full-D-amino acid peptide consisting of the amino acid sequence shown in SEQ ID NO:1.

[0179] method Following the clinical trials described in Example 3, the recommended dose (RP2D = 20 mg) for further development was explored in three expanded cohorts: A) CyPep-1 monotherapy, with patients receiving 20 mg CyPep-1 injections every two weeks; B) CyPep-1 and anti-PD-1 antibody, with patients receiving 20 mg CyPep-1 injections every two weeks and 400 mg pembrolizumab every 6 weeks; C) Intrahepatic CyPep-1 monotherapy, with patients receiving 20 mg CyPep-1 injections every two weeks at the intrahepatic lesion. Response was assessed every 8 weeks. Patient response was defined as an objective response according to RECIST 1.1 guidelines, or if disease stability persisted for >16 weeks. RECIST 1.1 measures used to assess target lesions were as described in Example 3.

[0180] result CyPep-1 monotherapy has shown promising results in advanced IV cancers associated with aberrantly activated Wnt / β-catenin pathway: CyPep-1 and anti-PD-1 antibody Figure 9 The efficacy of combination therapy with CyPep-1 and an anti-PD-1 antibody (pembrolizumab) in a patient with parathyroid carcinoma is shown. A1-2) CT scans of the lesion injected with CyPep-1 are shown at baseline and 3 months after treatment. A3-4) Histological images of the injected lesion biopsy are shown at baseline and 3 months after treatment. The tissue at baseline indicated malignancy, but the tissue at 3 months after treatment indicated benign tissue. A5-6) CT scans of the uninjected lesion at baseline and the injected lesion 20 months after starting CyPep-1 treatment are shown. The tumor size has significantly decreased. B) Evaluation of the percentage change in tumor size between the injected and uninjected lesions shows that both tumors shrank in size over 24 months.

[0181] These results demonstrate the efficacy of CyPep-1 when injected directly into the lesion, and also provide strong evidence for the immune-mediated remote effect of intratumorally administered drugs due to the shrinkage of uninjected metastases in distant organs.

[0182] Pharmacokinetics of CyPep-1 Figure 10 Favorable pharmacokinetic curves for CyPep-1 are shown. Peak serum concentrations were reached approximately 15 minutes after CyPep-1 administration. The limit of quantitation (LLOQ) was 10 ng / mL.

[0183] Tumor growth dynamics of uninjected target lesions Figure 11 The percentage change in tumor size of uninjected target lesions over 24 months is shown in eight patients with different types of cancer. Patients were treated with CyPep-1 for >6 months.

[0184] Patient details are as follows: Sarcoma, CyPep-1 monotherapy, stable disease (SD); Chondrosarcoma, CyPep-1 monotherapy, stable disease (SD); Uveal melanoma, intrahepatic injection, progressive disease (PD); Melanoma, CyPep-1 monotherapy, stable disease (SD); Adrenocortical carcinoma, intrahepatic injection, partial response (PR); Head and neck squamous cell carcinoma, CyPep-1 and anti-PD-1 antibody, stable disease (SD); Adrenocortical carcinoma, intrahepatic injection, stable disease (SD); Parathyroid carcinoma, CyPep-1 and anti-PD-1 antibody, unconfirmed progressive disease (UPD); Melanoma, CyPep-1 and anti-PD-1 antibody, partial response (PR).

[0185] These results provide strong evidence for the immune-mediated remote effect of CyPep-1 due to the shrinkage of uninjected metastases in distant organs. This effect was observed across multiple cancer types and administration modalities.

[0186] Intrahepatic injection Figure 12 The survival of six patients with adrenocortical carcinoma who received intrahepatic CyPep-1 injections is shown.

[0187] The patient's detailed information is shown in Table 2 below: Table 2

[0188] PR = Partial remission, SD = Stable disease, PD = Disease progression PR and SD stand for Disease Control (i.e., effective management of disease). These results indicate that patients carrying mutations in the β-catenin pathway, particularly those whose genes encode proteins involved in the Axin2 network, are positively correlated with better iRECIST remission and longer survival. This suggests that patients carrying mutations in the β-catenin pathway that lead to aberrant Wnt / β-catenin signaling respond best to CyPep-1 treatment. Therefore, mutations in the β-catenin pathway can be used as biomarkers for patients suitable for CyPep-1 treatment.

[0189] In this regard, it is worth noting that patient 10-022, who did not demonstrate disease control (disease progression), had very advanced disease and was only enrolled in the trial at a late stage; therefore, this patient's negative response does not indicate that LRP6 cannot be used as a biomarker, nor does it indicate that CyPep-1 therapy is generally ineffective, but rather that not every patient will experience remission at every stage of the disease. This patient can be considered an exception.

Claims

1. An oligopeptide compound comprising a D-amino acid sequence as shown in SEQ ID NO:1 or a D-amino acid sequence having at least 85% sequence identity therewith, for treating a subject with cancer, wherein the cancer is associated with aberrant activation of the Wnt / β-catenin pathway.

2. The oligopeptide compound for the use of claim 1, wherein the oligopeptide compound comprises or is composed of the D-amino acid sequence shown in SEQ ID NO:

1.

3. The oligopeptide compound for use according to claim 1 or 2, wherein the subject is a mammal, preferably a mouse, rat, guinea pig, cat, dog, pig, horse, cow, sheep, goat, monkey or ape.

4. The oligopeptide compound for the use of claim 3, wherein the subject is a human.

5. The oligopeptide compound for the use according to claim 4, wherein the cancer is selected from ocular cancer, vulvar cancer, endocrine cancer, anal cancer, pancreatic cancer, colorectal cancer, bile duct cancer, liver cancer, kidney cancer, gallbladder cancer, bladder cancer, skin cancer, prostate cancer, breast cancer, head and neck cell carcinoma, parotid gland cancer, cervical cancer, esophageal cell carcinoma, endometrial cancer, lung cancer, adrenocortical carcinoma, glioma, ovarian cancer, nephroblastoma, neuroendocrine carcinoma (carcinoid), HPV-positive carcinoma, squamous cell carcinoma, and sarcoma or desmoidoma.

6. The oligopeptide compound according to claim 5, wherein the cancer is selected from melanoma, parathyroid carcinoma, thyroid cancer, head and neck squamous cell carcinoma (HNSCC), squamous cell carcinoma of the esophagus, cervix, vulva or lung, renal cell carcinoma, small cell lung cancer, non-small cell lung cancer, colon cancer or rectal cancer.

7. The oligopeptide compound for use according to claim 6, wherein the cancer is adrenocortical carcinoma.

8. An oligopeptide compound for use according to any one of claims 1 to 7, wherein the compound is used in conjunction with a second therapeutic agent that is effective in treating the cancer.

9. The oligopeptide compound for the use of claim 8, wherein the second therapeutic agent is selected from chemotherapeutic agents, immunotherapeutic agents, hormone therapy, radiotherapy, or photodynamic therapy.

10. An oligopeptide compound for use according to any one of claims 8 or 9, wherein the second therapeutic agent is a cytotoxic agent, a cell for adoptive cell transfer therapy, an antibody, a hormone, or a checkpoint inhibitor.

11. The oligopeptide compound for use according to claim 10, wherein the checkpoint inhibitor blocks the interaction between PD-1 and PD-L1, or between CTLA-4 and CD80 or CD86.

12. The oligopeptide compound for use according to claim 11, wherein the checkpoint inhibitor is an antibody that binds to PD-1, PD-L1, or CTLA-4.

13. An oligopeptide compound for use according to any one of claims 1 to 12, wherein the compound is formulated for intratumoral injection or infusion.

14. An oligopeptide compound for use according to any one of claims 1 to 13, wherein the compound is for topical application to primary or secondary tumors.

15. The oligopeptide compound for the use of claim 14, wherein the compound causes regression of primary and secondary tumors in the subject.

16. A method of treating cancer associated with aberrant activation of the Wnt / β-catenin pathway, the method comprising administering an oligopeptide compound to a subject in need, optionally together with a second therapeutic agent, wherein the oligopeptide compound, the subject, the cancer, the treatment, and the second therapeutic agent are as defined in any one of claims 1 to 15.

17. Use of an oligopeptide compound in the manufacture of a medicament for treating a subject with cancer associated with aberrant activation of the Wnt / β-catenin pathway, optionally in combination with a second therapeutic agent, wherein the oligopeptide compound, the subject, the cancer, the treatment, and the second therapeutic agent are as defined in any one of claims 1 to 15.

18. A product comprising an oligopeptide compound as defined in claim 1 or 2 and a second oncology therapeutic agent as a combination formulation for use alone, simultaneously or sequentially in the treatment of cancer in a subject, wherein the cancer is associated with aberrant activation of the Wnt / β-catenin pathway.

19. The product of claim 18, wherein the oligopeptide compound, the subject, the cancer, and the second therapeutic agent are as defined in any one of claims 2 to 15.

20. A method for identifying a subject suitable for treatment of cancer with an oligopeptide compound according to any one of claims 1 to 15, the method comprising determining the presence of a biomarker indicating activation of the Wnt / β-catenin pathway in a sample obtained from the subject.

21. The method of claim 20, wherein the biomarker is: (i) Increased expression of one or more of the following proteins in tumor samples: β-catenin, WISP-1, Axin2, FZD3, FZD6, FZD7, Wnt3, Wnt4, Wnt5A, RSPO1, RSPO2, RSPO3, RSPO4, c-MYC, cyclin-D, TCF21; (ii) Mutations in one or more of the following genes: β-catenin (CTNNB1), AXIN1, AXIN2, APC, APC2, ZNRF3, MEN1, GNAI2, RNF43, DVL1, LPR5, LPR6, MED12, and BCL9L; (iii) The expression of one or more of the following genes is upregulated: WISP-1, Axin2, c-MYC, cyclin-D, TCF21, FZD3, FZD6, FZD7, Wnt3, Wnt4, Wnt5A, RSPO2, CLDN1, LGR5.

22. The method of claim 20 or claim 21, wherein the biomarker is a mutant gene encoding a protein involved in the Axin2 regulatory network, wherein the gene contains one or more mutations.

23. The method according to any one of claims 20 to 22, wherein the gene is selected from: APC2, AXIN2, DVL1, LRP5, MEN1, GNAI3, MED12, CTNNB1, LRP6.

24. A method for identifying and treating cancers in a subject associated with aberrant activation of the Wnt / β-catenin pathway, the method comprising: (i) Identifying said subjects suitable for cancer treatment by determining the presence of biomarkers indicating activation of the Wnt / β-catenin pathway in samples obtained from the subjects, said identification being performed by a method as defined in any one of claims 20 to 23; and (ii) administering to the subject an oligopeptide compound comprising a D-amino acid sequence as shown in SEQ ID NO: 1 or having at least 85% sequence identity with it, optionally wherein each amino acid of the compound is a D-amino acid, optionally administered together with a second therapeutic agent.

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