Cancer treatment related to β-catenin

Abstract

The present invention provides the oligopeptide compound CyPep-1 for use in the treatment of cancers associated with abnormal activation of the Wnt / β-catenin pathway. The present invention also provides a method for treating cancers associated with abnormal activation of the Wnt / β-catenin pathway, comprising administering the oligopeptide compound to a target, or optionally administering it to a target together with a second therapeutic agent, and a method for identifying a target for cancer treatment with the oligopeptide compound, comprising determining the presence of a biomarker indicating an activated Wnt / β-catenin pathway in a sample obtained from the target. CyPep-1, which has been previously shown to preferentially target and destroy the cell membrane of cancer cells, has been found to also act by inhibiting the Wnt / β-catenin pathway. This dual therapeutic effect is particularly therapeutically interesting because the use of CyPep-1 may particularly affect cancers exhibiting abnormal activation of the Wnt / β-catenin signaling pathway. The compound has a distinct dual mechanism of action, promoting immune activation while simultaneously reversing immune elimination.

Description

TECHNICAL FIELD

[0001] field The present disclosure and the present invention relate to the treatment of cancers associated with aberrant Wnt / β-catenin signaling pathways. In particular, we have discovered that the oligopeptide compound (CyPep-1), which has previously been shown to selectively target and disrupt the cell membrane of cancer cells, also acts by inhibiting the Wnt / β-catenin pathway. This dual therapeutic effect is of particular therapeutic interest, as the use of CyPep-1 can particularly affect cancers that exhibit aberrant activation of the Wnt / β-catenin signaling pathway. This compound has a distinct dual mechanism of action that promotes immune activation and reverses immune exclusion. Also provided are methods of identifying patients suitable for the treatment of such cancers with the oligopeptide compound.

[0002] background Despite recent advances in cancer treatment, 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 cancer. Furthermore, 28.4 million people are expected to be diagnosed with cancer in 2040, representing a 47% increase from 2020, and the incidence of cancer is projected to rise as the global population ages (Sung et al., CA Cancer J Clin 71 (3): 209-249, 2021). Accordingly, there is an urgent need for new and improved therapies for cancer.

[0003] As is increasingly recognized, different cancers may be associated with different mechanistic abnormalities occurring within cells, allowing different therapeutic approaches for different groups of cancers. One such group of cancers is those associated with aberrant Wnt / β-catenin signaling. β-catenin is a protein that plays a structural role in cell-cell 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 usually highly conserved and is activated via the binding of extracellular Wnt ligands (including Wnt3a, Wnt1, and Wnt5a) to target membrane receptors via autocrine or paracrine mechanisms. After activation, the Wnt pathway stabilizes β-catenin, translocates it to the nucleus, and subsequently promotes the expression of genes involved in major cellular mechanisms such as cell proliferation, differentiation, migration, survival, regeneration, and apoptosis (Liu et al., Sig Transduct Target Ther 7 (3), 2022). In relation to cancer, mutations have been reported in various genes along the pathway, and Wnt / β-catenin signaling has been reported to be involved in cancer pathogenesis by promoting tumorigenesis, cancer cell regeneration, proliferation, and immune responses. Specifically, β-catenin activation is thought to promote the elimination of immune cells and promote cryotumorogenesis in the tumor microenvironment, and is considered an important factor in immune checkpoint inhibitor resistance (Pai et al., J Hematol Oncol 10(1): 101, 2017; Tissier et al., Cancer Res 65:(17), 2005). Abnormal Wnt / β-catenin signaling is associated with many different types of cancer, i.e., cancers in many different tissues. These include certain forms of colorectal cancer (CRC), melanoma, desmoid tumors, lung cancer, kidney cancer, liver cancer, breast cancer, ovarian cancer, and adrenocortical carcinoma (ACC) (Pai et al., 2017 (see above); Tissier et al., 2005 (see above); and Koury et al., Stem Cells Int:2925869, 2017)).

[0004] Adrenal cortex cancer (ACC) is a cancer of the outer layer of the adrenal gland. ACC is a rare cancer with an incidence of 0.7–2 cases per million people per year. Patients diagnosed with ACC have a very poor prognosis; if the disease progresses with standard treatment, the median survival time is less than one year, and progression-free survival is less than two months in patients with advanced or metastatic lesions. Treating ACC is challenging due to the rarity and aggressiveness of the disease, and surgical resection is the only curative option for patients with early-stage ACC. Furthermore, patients with ACC have a high risk of local or distant recurrence after complete resection. Unfortunately, ACC responds only moderately to standard cytotoxic chemotherapy such as radiotherapy and ablation, and mitotane, an approved adjuvant therapy for ACC, has poor therapeutic indicators (see, e.g., Tissier et al., 2005, above). Therefore, ACC presents a particularly unmet need for cancer treatment. However, more generally, there is a continuing need for new therapies for β-catenin-induced cancers, and despite the recognition of the contribution of Wnt signaling to tumorigenesis and Wnt signaling inhibition showing promising effects in some preclinical models, Wnt signaling-targeted drugs have not proven clinically successful in cancer or other diseases. Beta-catenin remains an elusive drug target. Despite its initial potential, the results of cancer therapies designed to target the abnormally activated Wnt / β-catenin signaling pathway have been mixed (Zhang & Wang, J Hematol Oncol 13(1): 165, 2020). A variety of inhibitors targeting the Wnt / β-catenin pathway are currently under development in both preclinical and clinical trials. A diverse category of targeted drugs exists, 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, concerns about safety, efficacy, and drug delivery remain, and success is typically seen only in relatively small percentages of patients or in patients with very specific types of cancer. Despite some demonstrated efficacy, side effects are currently common, and it remains unclear how the activity of several drug candidates translates into sustained clinical outcomes in multiple cancers associated with activated abnormal Wnt / β-catenin pathways.

[0005] Therefore, considering the extensive involvement of the Wnt / β-catenin pathway in many diverse cellular processes across different cell types, and its interactions with other signaling pathways, the Wnt / β-catenin pathway presents a challenging therapeutic target in terms of achieving desired therapeutic benefits while avoiding undesirable side effects.

[0006] WO2011 / 092347 discloses oligopeptide compounds that are selectively cytotoxic to neonatal cells and proposes their use for the treatment of a range of cancers. These oligopeptide compounds include a peptide (named CyPep-1) consisting of the amino acid sequence described in SEQ ID NO: 1. The peptide is cationic and exhibits a high ability to bind to negatively charged membranes, specifically those typical of many neonatal cells, including cancer cells, resulting in potent and selective cytolytic activity against neonatal / cancer cells (non-neonatal / 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 be selectively cytotoxic to various cancer cell lines in vitro, but also to have potent antitumor effects against various types of cancer, as well as potent antitumor effects against non-malignant neonatal lesions such as warts, and has also been shown to be well-tolerated in animal models of the disease. The cytolytic activity of CyPep-1 is evident even against bacterial cells (likely due to the negative charge maintained by many bacterial cell membranes), and it has been shown to have potent bactericidal effects against medically relevant species of Gram-positive and Gram-negative bacteria. Its use as an antimicrobial agent, including antibacterial and antifungal properties, has been proposed (see WO2011 / 092347).

[0007] Summary of the Invention In the process of investigating the potential of CyPep-1 for tumor treatment, the inventors surprisingly found that CyPep-1 can induce beneficial therapeutic effects and can be used to treat a specific group (i.e., type or class) of cancers, in other words, cancers associated with abnormal activation of the Wnt / β-catenin pathway, particularly ACC. As shown in the following examples, CyPep-1 proved remarkably effective in treating patients with ACC, demonstrating a much greater response than anticipated. This led the inventors to further investigate and examine the properties of CyPep-1, suggesting that it functions as an inhibitor of the Wnt / β-catenin pathway, reversing the immune response in the tumor microenvironment, i.e., initiating "immune reprogramming." Specifically, as shown in the examples, CyPep-1 can reduce nuclear accumulation of β-catenin and downregulate Wnt-induced secretory protein-1 (WISP-1 / CCN). This gene is transcribed by β-catenin, and the downregulation is caused by inhibition of the β-catenin pathway by CyPep-1. Thus, the inventors discovered a further mechanism of action of CyPep-1. Furthermore, nuclear localization of β-catenin decreased after treatment with CyPep-1, and evidence of CyPep-1-mediated activation of Axin2 was also confirmed. These results in ACC and other cancers demonstrate the concept and validity of CyPep-1 activity, and suggest that the CyPep-1 platform with its dual mechanism of action offers a new treatment option for many cancer patients with cancers that have previously been difficult to treat.

[0008] Therefore, this development provides a novel and highly advantageous therapeutic approach using the CyPep-1 peptide for treating specific types or subgroups of cancer, namely cancers associated with the Wnt / β-catenin pathway.

[0009] Accordingly, in a first embodiment, an oligopeptide compound comprising the D-amino acid sequence described in SEQ ID NO: 1, or a D-amino acid sequence having at least 85% sequence identity thereto, for use in the treatment of cancer in a subject, wherein the cancer is associated with abnormal activation of the Wnt / β-catenin pathway, is provided herein.

[0010] Therefore, the oligopeptide compounds provided herein for medical use and methods are compounds comprising an inverso amino acid sequence.

[0011] In one embodiment, all amino acids in the oligopeptide compound are D-amino acids. Therefore, in this embodiment, the oligopeptide compound is an inverso-oligopeptide compound.

[0012] In one embodiment, the oligopeptide compound has the sequence shown in SEQ ID NO: 1, or is a D-amino acid sequence consisting thereof.

[0013] Accordingly, in another embodiment, the following oligopeptide compounds are provided herein for the treatment of cancer associated with abnormal activation of the Wnt / β-catenin pathway, comprising a D-amino acid sequence described in SEQ ID NO: 1, or a D-amino acid sequence having at least 85% sequence identity thereto, wherein all amino acids of the compound are D-amino acids, and the compound is used in conjunction with a second therapeutic agent effective in treating the cancer.

[0014] Related embodiments provide a pharmaceutical composition comprising (i) an oligopeptide compound comprising the D-amino acid sequence described in Sequence ID No. 1, or a D-amino acid sequence having at least 85% sequence identity thereto, an oligopeptide compound in which all amino acids of the compound are optionally D-amino acids, and (ii) a second oncological therapeutic agent as a combination formulation for use separately, simultaneously, or sequentially in the treatment of cancer in a subject, wherein the cancer is related to abnormal activation of the Wnt / β-catenin pathway.

[0015] Further related embodiments provide a method for treating cancer associated with abnormal activation of the Wnt / β-catenin pathway, comprising administering to a subject requiring treatment an oligopeptide compound containing the D-amino acid sequence described in SEQ ID NO: 1, or a D-amino acid sequence having at least 85% sequence identity thereto, wherein all amino acids of the compound are D-amino acids, and optionally administered together with a second therapeutic agent.

[0016] In particular, oligopeptide compounds, and optionally (i.e., if used) a second therapeutic agent, are administered to the target in an effective dose. More specifically, in the case of combination therapy, the effective dose is the dose that is effective in treating cancer when administered in combination.

[0017] In another embodiment, the use of an oligopeptide compound comprising the D-amino acid sequence described in SEQ ID NO: 1, or a D-amino acid sequence having at least 85% sequence identity thereto, in the manufacture of a pharmacopoeia for the treatment of cancer associated with abnormal activation of the Wnt / β-catenin pathway is provided herein, optionally used in combination with a second therapeutic agent, and optionally all amino acids of the compound are D-amino acids.

[0018] Therefore, more specifically, this embodiment provides the use of an oligopeptide compound comprising the D-amino acid sequence described in SEQ ID NO: 1, or a D-amino acid sequence having at least 85% sequence identity thereto, in the manufacture of a pharmacopoeia for treating cancer associated with abnormal activation of the Wnt / β-catenin pathway, wherein all amino acids of the compound are D-amino acids, and the oligopeptide compound is used in combination with a second therapeutic agent. This embodiment also provides, in other words, the use of (i) an oligopeptide compound comprising the D-amino acid sequence described in Sequence ID No. 1, or a D-amino acid sequence having at least 85% sequence identity thereto, wherein all amino acids of the compound are optionally D-amino acids, and (ii) a second therapeutic agent, in the manufacture of a pharmaceutical product for treating cancer associated with abnormal activation of the Wnt / β-catenin pathway. In this regard, the pharmaceutical product can be considered a kit and pharmaceutical product, comprising the first and second therapeutic agents as defined in (i) and (ii) above, provided for the aforementioned use. More broadly, in the medical use described above, if an oligopeptide is used in combination therapy with another therapeutic agent, it can be considered the first therapeutic agent.

[0019] In the combination therapies described herein, the second therapeutic agent may be any anti-cancer agent, in particular any second therapeutic agent effective in treating cancer. In one embodiment, the second therapeutic agent is an immunotherapy agent; more specifically, a checkpoint inhibitor.

[0020] In one embodiment, the cancer is ACC.

[0021] In another embodiment, the present invention provides a method for identifying targets for cancer treatment using an oligopeptide compound comprising the D-amino acid sequence described in SEQ ID NO: 1, or a D-amino acid sequence having at least 85% sequence identity thereto, wherein all amino acids of the compound are optionally D-amino acids, and the method comprises determining the presence of a biomarker indicating an activated Wnt / β-catenin pathway in a sample obtained from the target.

[0022] In a particular embodiment, a method for identifying and treating cancer associated with abnormal activation of the Wnt / β-catenin pathway in a subject, (i) identifying a subject for cancer treatment by determining the presence of a biomarker indicative of an activated Wnt / β-catenin pathway in a sample obtained from the subject; and (ii) administering to the said subject an oligopeptide compound comprising the D-amino acid sequence set forth in SEQ ID NO: 1, or a D-amino acid sequence having at least 85% sequence identity thereto, wherein optionally all amino acids of the said compound are D-amino acids, and optionally the administration is carried out together with a second therapeutic agent, A method comprising the same is provided. Accordingly, a subject having cancer is tested for whether the cancer is associated with aberrant activation of the Wnt / β-catenin pathway by determining whether a biomarker indicative of the activated Wnt / β-catenin pathway is present, and if present, the said oligopeptide compound is administered to treat the cancer.

[0023] The biomarker can be a mutation of a gene in the pathway, and / or a change (e.g., increase or decrease) in the expression of the gene, and / or a change (e.g., increase or decrease) in the expression of a protein encoded by the gene (i.e., an increase or decrease in the level of the gene product). This may include any gene in the pathway, including genes encoding proteins such as β-catenin or Axin2, or any gene in a gene encoding a regulatory factor in the pathway. In a representative embodiment, the gene may encode a protein involved in the Axin2 regulatory network. For example, the biomarker may be one or more mutations in the gene encoding Axin2 or the gene encoding a regulator of Axin2. However, the biomarker is not limited to Axin2 / Axin2 regulators, and may be any gene in the pathway that can be shown to be associated with clinical response or disease control.

[0024] Detailed explanation This invention and disclosure relate to novel applications of the previously identified WO2011 / 092347 oligopeptide compound, specifically the peptide CyPep-1 (SEQ ID NO: 1), which was identified as an oncological therapeutic based on its selective cytolytic activity against cancer cells. We have now found that CyPep-1 has further activity to induce tumor immune-specific activation and reverse immune elimination. This compound is proposed herein for the treatment of a new group of patients, namely patients with cancers associated with an abnormal Wnt / β-catenin pathway.

[0025] Sequence ID 1 is a fragment of the tumor suppressor protein conductin / axin 2, specifically the RGS domain, a 27-amino acid peptide consisting of amino acids 126-140, bound to the C-terminus of an HIV-TAT cell membrane permeable peptide. YGRKKRRQRRRGKTLRVAKAIYKRYIE(Sequence ID 1)

[0026] The above conductin / axin 2 fragment 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 membrane permeable peptide has the amino acid sequence YGRKKRRQRRRG (SEQ ID NO: 3; corresponding to amino acid numbers 1-12 of SEQ ID NO: 1).

[0027] Conductin / Axin 2 is a central protein in the Wnt / β-catenin pathway of tumorigenesis. It is a member of the β-catenin degradation complex and targets β-catenin for proteasomal degradation. Therefore, Axin 2 is a β-catenin inhibitor, inhibiting β-catenin activity and inducing its degradation. The Axin protein family can also directly promote the transport of β-catenin from the nucleus.

[0028] The Wnt / β-catenin pathway is highly complex and regulated by several components (i.e., proteins, genes, etc.), and as mentioned earlier, Axin2 is a target gene for β-catenin. High levels of β-catenin expression lead to high levels of Axin2 expression, and this expression pattern is observed in β-catenin-dependent 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 loop by inactivating either Axin2 or genes that stabilize Axin2 (e.g., b, CNAI2 / 3), thus avoiding β-catenin degradation / inhibition / degradation through high levels of Axin2 expression (this mechanism is known as mutagenesis). This mechanism leads to high levels of inactivated Axin2 expression in cancer cells, causing the RGS domain of Axin2 to aggregate. As a result, large amounts of Axins are present in cancer cells, essentially acting as a "storage." The compounds described herein exploit or take advantage of this β-catenin-dependent characteristic of cancer.

[0029] Therefore, the peptide of Sequence ID No. 1, CyPep-1, has two domains: a first domain, conductin / axin 2, derived from proteins involved in the Wnt / β-catenin pathway, and a second domain having a cationic plasma membrane-binding domain that specifically binds to negatively charged phospholipids such as phosphatidylserine (PS). The polar head of PS is normally found only inside cells, but is found outside cancer cells. Thus, PS exhibits cancer-specific targeting. Binding to PS by CyPep-1 leads to membrane-mediated aggregation and pore formation, resulting in cancer antigen and lytic cell death. Furthermore, it is thought that CyPep-1 activates Axins 2 by stabilizing the RGS domain, leading to polymerization and target binding. In this regard, the RGS domain of Axin 2 can aggregate, and in this aggregated form, Axin 2 is inactive. By binding to the RGS domain, CyPep-1 can reverse this aggregation and activate Axin 2, or promote its active non-aggregated form. More specifically, in its non-aggregated form, Axin2 can polymerize or condense, and in this polymerized or condensed form, Axin2 is active. The data shown in the following examples demonstrate that CyPep-1 can promote the polymerization of Axins into microscopically visible "condensates" or punctate particles. Activation of Axin2 by CyPep-1 inhibits nuclear translocation and β-catenin activation.

[0030] To avoid proteolytic degradation of CyPep-1, naturally occurring L-amino acids were substituted with their D-amino acid enantiomers, which are not recognized by serum proteases. This resulted in 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, Vol. 5(5-6), May 2014).

[0031] Therefore, the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 85% sequence identity thereto, contained in the oligopeptide compounds proposed for use herein, is a D-amino acid sequence. In other words, the amino acid sequence consists entirely of D-amino acids (D-aa). This may also be referred to as an inverso-amino acid sequence. In one embodiment, the oligopeptide compound consists entirely of a D-amino acid sequence, or in other words, is a "D-oligopeptide compound." This may also be referred to as an inverso-oligopeptide compound. Peptides consisting entirely of L-amino acids are known in the art as L-peptides, and peptides consisting entirely of D-amino acids are known in the art as D-peptides. The term "inversopeptide" is used to refer to a peptide that has the same amino acid sequence as an L-peptide but consists entirely of D-amino acids. An inversopeptide / oligopeptide compound has a mirror image structure with respect to its corresponding L-peptide / oligopeptide compound (e.g., an L-peptide with the same amino acid sequence). Inversopeptides / compounds are generally not sensitive to degradation by serum proteases, which may be advantageous for use in clinical settings (due to their unnatural conformations, inversopeptides cannot be recognized by protease enzymes). Oligopeptide compounds, in particular, may contain or consist of a D-peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0032] In this regard, although a portion of the peptide is derived from Axin2, given that it has an inverso structure (D-aa), it cannot be assumed that the peptide retains any functional effects related to the biological activity of its parent protein, particularly any signaling effects (i.e., any functional / signaling effects of Axin2). Therefore, it was surprising and unpredictable that the oligopeptide compound could affect the expression of signaling-related proteins, such as WISP-1, and downregulate (i.e. inhibit) Wnt / β-catenin signaling, as shown in the following examples.

[0033] As used herein, the term “oligopeptide compound” means a compound composed of amino acids or equivalent subunits linked to one another by peptide bonds or equivalent bonds. Therefore, the term “oligopeptide compound” includes peptides and peptide mimes.

[0034] An "equivalent subunit" refers to a subunit that is structurally and functionally similar to an amino acid. The backbone 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.

[0035] A "peptide mime" refers to a compound that is functionally equivalent to or similar to a peptide and can adopt a similar three-dimensional structure to its peptide counterpart, but is not composed solely of amino acids linked by peptide bonds. A preferred class of peptide mimes is peptoids, i.e., N-substituted glycines. Peptoids are closely related to their natural peptide counterparts, but chemically differ in that their side chains are attached to nitrogen along the molecular backbone rather than to the α-carbon in the amino acid. Peptide mimes are typically preferred in embodiments where a longer-lasting effect is desired, as they have a longer half-life in the patient's body. This can help reduce the frequency with which the composition needs to be re-administered. However, for biological safety reasons, shorter half-lives may be preferred in other embodiments, and peptides are preferred in those embodiments.

[0036] Preferably, the oligopeptide compound is an oligopeptide (more specifically, a D-oligopeptide). The oligopeptide compound may incorporate di-amino acids and / or β-amino acids. More preferably, the oligopeptide compound consists of α-amino acids.

[0037] Oligopeptides are polymers formed from amino acids linked to each other by peptide bonds. As defined herein, an oligopeptide contains at least three amino acids, but the oligopeptide compounds used herein clearly contain three or more amino acids. As defined herein, an oligopeptide compound or oligopeptide does not have a specific maximum length and may contain, for example, 30, 40, 50 or 100 or more amino acids, but typically the prefix "oligo" is used to indicate a relatively small number of subunits, such as amino acids, i.e., fewer than 200, preferably 100, 90, 80, 70, 60 or fewer subunits. Thus, the oligopeptide compounds of the present invention may contain at least 23 and no more than 200 subunits. In embodiments, they may contain at least 24, 25, 26 or 27 subunits. Alternatively, they are defined as containing 50, 40, 35, 30, 29, 28 or 27 or fewer subunits. Therefore, oligopeptide compounds may contain a number of subunits within the range of the minimum or maximum number of subunits, which is composed of one of the integers shown above. Typical 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.

[0038] The oligopeptide compounds defined herein may simply be oligopeptides, i.e., polymers consisting of amino acids linked by peptide bonds. Alternatively, the oligopeptide compounds may contain additional functional groups, conjugates, etc.

[0039] The oligopeptide compounds used herein comprise the amino acid sequence described in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto. Such sequences having at least 85% sequence identity may, for convenience, be referred to as substantially identical or equivalent sequences. In certain embodiments, the oligopeptide compound comprises the amino acid sequence described in SEQ ID NO: 1. In other embodiments, the oligopeptide compound comprises the amino acid sequence described in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto. In yet another embodiment, the oligopeptide compound comprises the amino acid sequence described in SEQ ID NO: 1.

[0040] The level of sequence identity between two sequences (for example, the target oligopeptide sequence and the sequence described in Sequence ID No. 1) can be determined by sequence alignment. Sequence alignment can be performed using appropriate methods; for example, computer programs such as EMBOSS Needle or EMBOSS stretcher (both from Rice, P. et al., Trends Genet. 16(6): 276-277, 2000) are used for pairwise 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) are used for multiple sequence alignment. Such computer programs may also be used with standard input parameters. Standard input parameters include, for example, 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. Other suitable parameters may be used instead.

[0041] In addition to the D-amino acid of Sequence ID No. 1 (or an equivalent sequence), the oligopeptide compound may contain one or more other amino acids, including an L-amino acid, or an artificially modified amino acid or a naturally occurring non-proteinogenic amino acid (e.g., an amino acid formed during metabolic processes). Examples of non-proteinogenic amino acids that can be used include ornithine (a product of the urea cycle), and artificially modified amino acids such as 9H-fluoren-9-ylmethoxycarbonyl (Fmoc)-, tert-butyloxycarbonyl (Boc)-, and 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc)-protected amino acids, as well as amino acids having a carboxybenzyl (Z) group.

[0042] The in vitro and / or in vivo stability of oligopeptide compounds can be improved or enhanced by stabilizing or protecting means known in the art, such as the addition of protecting or stabilizing groups, the incorporation of amino acid derivatives or amino acid analogs, or the chemical modification of amino acids. Such protecting or stabilizing groups can be added, for example, to the N-terminus and / or C-terminus. Examples of such groups include acetyl groups, and other protecting groups and groups that can stabilize peptides are known in the art.

[0043] As reported in WO2011 / 092347, the oligopeptide compounds as defined herein have activity that inhibits the proliferation and / or viability of cancer cells. “Inhibiting cell proliferation” means that any aspect of cell proliferation is reduced, more specifically, measurably reduced, in terms of the increase in cell size or the amount and / or volume of its components, more specifically, the increase in cell number. Thus, the term “proliferation” explicitly includes cell replication or regeneration. The rate of cell proliferation may decrease, for example, with respect to the rate of increase in cell number. Typical examples include proliferation (e.g., cell number, or growth rate) which may be reduced by at least 50%, 60%, 70%, 80%, 90%, or 95%. In some cases, proliferation may be reduced by 100%, i.e., proliferation may be completely inhibited and stopped. Thus, cell replication or regeneration may be reduced or inhibited. As stated, the term “inhibit” includes any degree of reduction in proliferation.

[0044] Inhibition of cell proliferation can be identified by comparing the proliferation rate of control cells or cell populations cultured in the absence of the oligopeptide compound of interest under standard laboratory conditions with the proliferation rate of the same or corresponding cells or cell populations cultured in the presence of the oligopeptide compound of interest, but otherwise under the same conditions as the control cells or cell population. The rate of cell replication or regeneration can be assessed, in particular, by determining the cell number at a selected time point. A decrease in the cell number in the population cultured in the presence of the oligopeptide compound compared to the cell number in the control population indicates that the oligopeptide compound has activity that inhibits cell proliferation. The cell number (and therefore proliferation or other) can be determined by the cell count, for example, using a hemocytometer.

[0045] To “inhibit” a cell’s viability includes any effect that reduces the cell’s ability to survive, makes it difficult for the cell to survive, or renders it unviable. Cell viability can be considered the ability of a cell to survive under given conditions. Inhibition of cell viability includes, in particular, killing or destroying cells, i.e., cell death. Cell death can be assessed by any standard experimental technique. For example, failure of proliferation of a cell or cell population, including replication or utilization or assimilation of nutrients, may be considered to indicate cell death (i.e., lack of viability). Cell viability can also be assessed by monitoring morphological changes in cells or tissues containing cells, such as tumors. Morphological changes can be analyzed by microscopy, and for example, necrosis or cytolysis are evident by visual analysis of cells or tissues and indicate a lack of viability. Typically, if the integrity of the cell membrane is lost, a cell can be considered dead. Inhibition of cell viability can be identified, for example, by comparing the viability of control cells or cell populations cultured in the absence of the target oligopeptide compound under standard laboratory conditions with the viability of the same or corresponding cells or cell populations cultured in the presence of the target oligopeptide compound, but otherwise under the same conditions as the control cells or cell populations. Cell viability is generally evaluated using the crystal violet assay 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) the target compound. Cell death results in the detachment of cells from the surface. After contact with the target compound, the monolayer is washed to remove the detached cells, and then stained with crystal violet, which stains cells by binding to proteins and DNA. The level of staining can be used to determine viability; i.e., if the cell population in contact with the target compound is stained less than the control population, the target compound can be considered to inhibit cell viability. The level of crystal violet staining in a cell population can be determined visually (simply by eye) or quantitatively by dye extraction using methanol, followed by spectroscopy determination of the optical density of the methanol-extracted dye at 570 nm.

[0046] Many other methods for determining the viability or proliferation of cancer cells are well known in the art, and many routine assays are available to determine whether cells are alive (viable) or dead. One option is to visually assess the cells of interest for morphological features characteristic of cell death, such as necrotic or apoptotic bodies, membrane blebs, nuclear condensation and DNA breaks into regularly sized fragments, cell membrane disruption, and leakage of cellular contents into the extracellular environment. Another method takes advantage of the characteristic loss of cell membrane integrity in dead cells. Membrane-impermeable dyes (e.g., trypan blue and propidium iodide) are routinely used to assess membrane integrity. These dyes are excluded from intact cells, so no staining occurs in such cells. If cell membrane integrity is compromised, these dyes can access the cell and stain intracellular components. Alternatively, or additionally, indicators of cell viability can be obtained using dyes that stain only cells with intact membranes. The LIVE / DEAD cell viability assay available from Thermo Fisher Scientific is an assay that uses two dyes of different colors, one to stain dead cells and the other to stain live cells, thus allowing for their identification. Examples of suitable live cell-specific dyes include calcein AM (green) and C-12 resazurin (red), while examples of suitable dead cell-specific dyes include ethidium homodimer-1 (red), propidium iodide (red), and SYTOX Green. Another approach to assessing membrane integrity is to detect the release of cellular components in the culture medium, such as lactate dehydrogenase. Another option is to measure cellular metabolism. This is done routinely in many ways, for example, by measuring ATP levels. Only living cells with intact membranes can synthesize ATP, and since ATP is not stored in cells, ATP levels rapidly decrease with cell death. Therefore, monitoring ATP levels provides an indicator of the cell's state. Yet another option is to measure the reduction potential of cells. Living cells that metabolize nutrients produce reducing agents (e.g., NADH and NADPH), and therefore, the reduction potential of cells can be assessed by applying markers (e.g., fluorescent dyes) to the cells, which give different outputs, whether reduced or oxidized. A nuclear cell that cannot reduce the marker can be considered dead. MTT and MTS assays are convenient examples of this type of assay.

[0047] Cancer cells can divide in an unrestricted manner and be "immortal." That is, they express telomerase and can therefore continue to divide indefinitely instead of dying or aging like healthy cells after reaching the Hayflick limit. Those skilled in the art can determine whether certain cells are cancerous or healthy. Cancer cells often exhibit prominent histological features that allow for their identification, such as large, irregular nuclei and intracellular abnormalities. The determination of whether cells are cancerous can also be made by genetic testing.

[0048] The oligopeptide compounds used herein have activity that inhibits the proliferation and / or viability of cancer cells both in vivo and in vitro. This activity can be easily determined in vitro using a suitable cell line. Many laboratory cell lines are cancerous and are convenient for research purposes due to their "immortality." The activity of the compound of interest can be determined using any such cancer cell line, e.g., cell lines A172 (human glioblastoma), GAMG (human glioblastoma), U87 (human glioblastoma), 4T1 (mouse mammary cancer), HOS (human osteosarcoma), and MC38 (mouse colon cancer). Many others are also known to those skilled in the art. Such cells can be obtained from any suitable source, e.g., cell depository such as ATCC (USA). The activity of the compound of interest is preferably determined using mammalian cancer cells. Human cancer cells can be used. In particular, the cell line may be derived from cancer associated with abnormal activation of the Wnt / β-catenin pathway. Such cancers are listed below, and the cell line may originate from any of these.

[0049] Cancer cells for testing may be obtained from subjects, such as human cancer patients. Cancer cells may be surgically removed from cancer patients, and the activity of the target oligopeptide compound may then be tested. Therefore, cancer cells may be derived from cancer cell lines, or from clinical trials or veterinary samples. Cancer cells may be derived from tumors. Cancer cells may be derived from any cancer, but in particular from cancers associated with abnormal activation of the Wnt / β-catenin pathway.

[0050] As described above, oligopeptide compounds are selectively cytotoxic to cancer cells. The term "cytotoxic" as used herein is essentially synonymous with "inhibits viability." In other words, oligopeptide compounds selectively inhibit or kill the viability of cancer cells (or, more preferably, generally inhibit the viability of newly formed cells).

[0051] A compound can be said to be selectively cytotoxic to cancer cells if it has a greater cytotoxic effect on cancer cells than on non-cancerous cells, and especially if it has a greater cytotoxic effect on cancer cells than on healthy cells. In particular, oligopeptide compounds may have no effect or minimal effect on healthy non-cancerous cells, but are cytotoxic to cancer cells.

[0052] The method for analyzing the effects of a target compound on cell proliferation and survival is described above. Whether a target compound is selectively cytotoxic to cancer cells can be determined by the same method. The survival rate of a population of cancer cells exposed to the target compound is compared to the survival rate of a population of healthy cells exposed to the target compound. If, after exposure to the target compound under identical conditions, the survival rate of the cancer cell population decreases compared to that of the healthy cell population, then the target compound can be said to be selectively cytotoxic to cancer cells.

[0053] As described above, the medical use and methods described herein are based on the discovery of further mechanisms of action of oligopeptide compounds, namely their selective activity in inhibiting the Wnt / β-catenin pathway. This activity can be evaluated by assessing the effect of the compound on signal transduction via the pathway, for example, in in vitro studies in cell lines, according to methods known in the art. Downstream effects of inhibiting the pathway can also be evaluated. For example, the activity of the compound in inhibiting β-catenin protein levels and nuclear localization, or in inhibiting downstream target genes such as WISP-1, Axin2, and Myc mRNA expression, can be determined, as described in the following examples. Methods for evaluating these effects include immunohistochemistry (IHC), Western blotting (WB), ELISA, polymerase chain reaction (PCR), and sequencing. Furthermore, as described in the following examples, the effect of the compound on Axin2 polymerization can be detected microscopically in in vitro cell studies, for example, using cancer cell lines and antibodies that visualize Axin2.

[0054] The oligopeptide compounds described herein can be synthesized by those skilled in the art using standard techniques. Chemical synthesis methods are known for oligopeptide compounds containing D-amino acids or other non-protein constituent amino acids. Liquid-phase or solid-phase protein synthesis can be used to produce the polypeptides constituting the oligopeptide compounds used in the present invention, or the polypeptides contained therein. Such methods are well known to those skilled in the art, and they can readily produce the oligopeptide compounds using common and appropriate techniques in the art.

[0055] The subjects to whom the oligopeptide compounds are administered are those suffering from cancer associated with abnormal activation of the Wnt / β-catenin pathway. The subjects are animals, which may be humans or any non-human animals, but are particularly mammals. These may include laboratory animals, domesticated animals, livestock, and animals in zoos or amusement parks. The subjects may be rodents such as mice, rats, rabbits, or guinea pigs. The subjects may be companion animals such as cats or dogs, or livestock such as horses, cattle, sheep, pigs, or goats. The subjects may be wild animals, such as animals in zoos or amusement parks. In certain embodiments, the subjects are primates such as monkeys or apes. Most specifically, the subjects are humans. Therefore, the treatments disclosed herein may be for veterinary or clinical purposes, but are preferably for clinical purposes, i.e., for the treatment of human subjects with cancer (i.e., cancer patients with cancer caused by abnormal activation of the Wnt / β-catenin pathway).

[0056] Oligopeptide compounds are administered to patients to treat cancers associated with abnormal activation of the Wnt / β-catenin pathway.

[0057] As used herein, the terms “to treat” or “treatment” broadly mean any effect or stage (or intervention) that is beneficial in the management of a clinical condition. Treatment may include reducing, alleviating, improving, slowing the progression or eliminating the condition or one or more symptoms being treated, or improving the clinical condition in question in any way, compared to the condition or symptoms before treatment. Treatment may include any clinical stage or intervention that contributes to or is part of a treatment program or regimen. Thus, as used herein, “treatment” includes curative treatment (i.e., treatment intended to be curative) and treatment that is merely life-sustaining or palliative (i.e., treatment designed solely to limit, reduce or improve the symptoms of a condition).

[0058] "Abnormal activation of the Wnt / β-catenin pathway" refers to increased, abnormal, or in other words, confused activation of the pathway compared to the pathway in healthy subjects, healthy tissues, or healthy (i.e., non-cancerous) cells. Therefore, signaling through this pathway is increased in cancer cells, e.g., in tumors or the local microenvironment of tumors. In particular, abnormal or increased activation of the pathway can be characterized by increased levels of β-catenin protein or increased levels of downstream target genes such as WISP-1, Axin2, and Myc, as determined by mRNA expression. Methods for evaluating these effects include immunohistochemistry (IHC), Western blotting (WB), ELISA, and polymerase chain reaction (PCR). Furthermore, gene mutations in β-catenin, APC, ZNRF3, and MIP1 that cause abnormal activation of the pathway can be detected by sequencing.

[0059] Disruption in the pathway can result from one or more mutations in one or more proteins involved in the pathway, such as mutations in genes encoding proteins selected from β-catenin, Axin1, Axin2, adenomatous polyposis (APC), ZNRF3, MEN1, and GNAI2. This can lead to disruption or abnormality of protein expression and / or function. For example, the expression of certain proteins may be decreased or increased, or functional proteins (e.g., suppressor proteins or enzymes) may be inactivated. For example, the formation of degradation complexes (DCs) characteristic of the pathway may be reduced. Thus, as will be discussed in more detail below, such cancers can be identified by screening cancer samples, or samples from patients suspected of having such cancers, for one or more biomarkers indicating an abnormal or simply activated Wnt / β-catenin pathway.

[0060] While abnormal Wnt / β-catenin pathways are known to be associated with many types of cancer, it will be understood that not all cancers of a particular type (e.g., those affecting a specific organ and / or tissue) are associated with abnormal pathways. In some cases, clinicians can know from their level of expertise that a particular cancer is associated with abnormal activation of the pathway, without needing to test it. In some cases, this can be inferred. In other cases, the patient can investigate or determine whether the cancer is associated with an abnormal pathway. This will be discussed further below.

[0061] Cancers associated with abnormal activation of the Wnt / β-catenin pathway may alternatively be referred to as β-catenin-induced cancers. Such cancers may include eye cancer, vulvar cancer, endocrine cancer (e.g., including adrenocortical tumors, especially ACC), parathyroid or thyroid cancer, anal cancer, pancreatic cancer, colorectal cancer, stomach cancer, bile duct cancer, liver cancer, especially hepatocellular carcinoma (HCC), kidney cancer (e.g., renal cell carcinoma), gallbladder cancer, bladder cancer, and 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 cancer (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), such as lung adenocarcinoma, glioma, medulloblastoma, ovarian cancer, Wilms' tumor, bile duct cancer, neuroendocrine (carcinoid) cancer, HPV-positive cancer, squamous cell carcinoma and sarcoma, or desmoid tumors. Squamous cell carcinoma (SCC) may include, in particular, HNSCC (described above), and squamous cell carcinomas of the cervix, lungs, esophagus, vulva, anus, etc.

[0062] Specific cancers for treatment by the use and methods described herein include ACC, anaplastic thyroid cancer, lung adenocarcinoma, parathyroid cancer, head and neck squamous cell carcinoma, melanoma, such as uveal melanoma, neuroendocrine (carcinoid) carcinoma, vulvar squamous cell carcinoma, chondrosarcoma, anal cancer, small cell lung cancer, and non-small cell lung cancer. In certain 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. In some embodiments, the cancer is not colorectal cancer, or is neither colorectal nor rectal cancer. In certain embodiments, the cancer being treated is ACC. Therefore, cancer is defined as cancer that is particularly associated with solid tumors, i.e., cancer that clinically manifests as solid tumors. However, the use of oligopeptide compounds is not limited to such cases, and also includes other cancers, such as hematopoietic cancers, including acute myeloid leukemia (AML).

[0063] Therefore, cancer can be any cancer associated with an abnormal Wnt / β-catenin pathway. This includes primary and secondary cancers. As will be discussed in detail below, oligopeptide compounds are particularly effective in treating both primary and secondary or metastatic tumors in the subject.

[0064] As described above, the oligopeptide compound may be used in combination with a second therapeutic agent, particularly a second therapeutic agent useful for treating cancer. The second therapeutic agent may be a second anticancer agent, but in other embodiments it may have different activity, for example, an anti-inflammatory agent, or any other agent useful for treating the patient. In certain embodiments, the second therapeutic agent may be selected from chemotherapeutic agents, immunotherapeutic agents, hormone therapy, radiotherapy, or photodynamic therapy. As referred to herein, chemotherapeutic agents are drugs that are administered and act against cancer, such as drugs that destroy malignant cells and tissues. Chemotherapy agents are generally small molecule drugs. Typical chemotherapeutic agents are cytotoxic or inhibitory, acting to kill cancer cells or inhibit their growth. Any chemotherapeutic agent of any class may be used, for example, taxanes (such as paclitaxel and docetaxel), topoisomerase inhibitors (such as topotecan), anthracyclines (such as doxorubicin and epirubicin), nucleoside analogs (such as gemcitabine), platinum-based drugs (such as cisplatin and carboplatin), alkylating agents (such as cyclophosphamide), and kinase inhibitors (such as imatinib), or any other chemotherapeutic agent or drug mentioned above. As referred to herein, an immunotherapeutic agent is any agent that affects the immune system or immune response of a target. For example, it may be administered to induce, proliferate, or suppress an immune response. Such immunotherapeutic agents include, for example, antibodies, especially monoclonal antibodies, checkpoint inhibitors, cytokines, cells for adoptive cell transfer therapy (ACT), such as native or modified immune cells that 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 such as NK cells, and vaccines. As referred to herein, hormone therapy, also known as hormonal therapy, anti-hormonal therapy, hormone treatment, or endocrine therapy, is used to treat cancers that utilize hormones to grow by altering the activity or production of specific hormones by removing, blocking, or adding those hormones to the body. Examples of hormonal 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, darolutamide, medroxyprogesterone acetate, and megestrol. As referred to herein, radiotherapy, also known as radiotherapy, refers to the use of high doses of ionizing radiation to control or kill cancer cells. Such radiotherapy may include, for example, whole-body radiotherapy (e.g., radiopharmaceuticals, radioactive iodine, etc.), external beam radiotherapy, and internal radiotherapy (e.g., close-range radiotherapy, etc.). As referred to herein, photodynamic therapy refers to the use of a combination of a photosensitizer, such as a photosensitizer (e.g., porfimer sodium), and light, which may originate from another light source, such as a laser or light-emitting diode (LED). Such photodynamic therapies may include, for example, extracorporeal photodynamic therapy (ECP) or photoimmunotherapy (PIT).

[0065] In certain embodiments, the second therapeutic agent is a cytotoxic agent, cells for adoptive cell transfer therapy, antibodies, hormones, or checkpoint inhibitors.

[0066] Checkpoint inhibitors are drugs that bind to immune checkpoints and inhibit their function. Immune checkpoints are regulators of the immune system that promote antigen-specific activation of immune cells and enable self-tolerance, thus supporting immune activity against antigen targets and preventing autoimmune diseases and abnormal immune system activity against host tissues. Immune checkpoints can be stimulant or inhibitory. Stimulant immune checkpoints act to modulate immune cell activity against antigen targets by stimulating proliferation and effector responses through the binding of their homologous ligands or agonists. An example of a stimulant immune checkpoint is CD28, which acts as a co-stimulator of T cell activity and initiates T cell proliferation by binding to its ligands, CD80 and CD86.

[0067] Inhibitory immune checkpoints, by binding to their homologous ligands or agonists, downregulate or inhibit immune cell function, promote self-tolerance, and prevent excessive and abnormal immune responses that could cause host damage, such as autoimmune activity or 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. Throughout this disclosure, except for the paragraphs above where its meaning is explicitly defined, the term “immune checkpoint” means suppressive immune checkpoint.

[0068] As defined herein, a checkpoint inhibitor refers to any agent that binds to an immune checkpoint or immune checkpoint ligand and acts directly to prevent the activation of the immune checkpoint. Therefore, a checkpoint inhibitor may be an antagonist of an immune checkpoint. All checkpoint inhibitors currently available for clinical use act by blocking their target immune checkpoint, i.e., by binding to it or its ligand and thus preventing the interaction between the checkpoint and the ligand (a mechanism known as immune checkpoint blockade). However, checkpoint inhibitors used herein in combination with oligopeptide compounds may act by any mechanism including immune checkpoint blockade, non-competitive inhibition of immune checkpoints, covalent binding or structural alteration of immune checkpoints (or their ligands). Ideally, checkpoint inhibitors should expose cancer cells to the immune system without attacking healthy tissue in the same system.

[0069] 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, affirmers, or antibodies. Antibodies as referred to herein may be natural or synthetic antibodies, or fragments or derivatives thereof. The term “antibody” is used herein in a broad sense to include any type of antibody or antibody-based molecule. This includes not only natural antibody molecules, but also modified antibodies, synthetic antibodies, or recombinant antibodies, as well as their derivatives or fragments. Therefore, an antibody can be any molecule, entity, or construct that has a binding region derived from an antibody, i.e., an antibody domain derived from an antibody.

[0070] 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 may be any convenient or desired species, class, or subtype of antibody, or may be derived from / based on one. As described above, an antibody may be native, derivatized, or synthetic. It may be monoclonal or polyclonal. Therefore, an antibody may bind to a single epitope, or it may be a mixture of antibodies (or antibody molecules) that bind to different epitopes.

[0071] Therefore, checkpoint inhibitors can be binding molecules containing an antigen-binding domain derived from an antibody specific to (or against) an immune checkpoint or its ligand. Examples of such “antibodies” (i.e., antibody-based binding molecules) include monoclonal and polyclonal antibodies, Fab, Fab', F(ab')2 and Fv fragments, as well as any fragment lacking an Fc region, chimeric (e.g., humanized or CDR transplanted) antibodies, single-chain antibodies (e.g., scFv antibodies), and antibodies identified or obtained from phage display. In certain embodiments, the checkpoint inhibitor is a monoclonal antibody.

[0072] Affimers are modified non-antibody proteins that mimic antibody binding to a target. Affimers are derived from the cystatin protein family and share a common structure of an α-helix on an antiparallel β-sheet. Affimers and methods for their production are described in WO2009 / 136182.

[0073] In certain embodiments, checkpoint inhibitors inhibit the activity of PD-1. In particular, checkpoint inhibitors can block the interaction between PD-1 and PD-L1 (or between PD-1 and PD-L2), and thus prevent PD-1 activation (as described above, PD-1 activation inhibits T cell effector function). A checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1 / PD-L2 binds to one of these proteins, preventing the interaction between the two proteins from occurring. Therefore, a checkpoint inhibitor that blocks the interaction between PD-1 and PD-L1 may bind to PD-1, or to PD-L1 or PD-L2. In certain embodiments, checkpoint inhibitors bind to PD-1 or PD-L1. In particular, such checkpoint inhibitors may bind to the PD-L1 binding site of PD-1, or to the PD-1 binding site of PD-L1. To block the interaction between PD-1 and both PD-L1 and PD-L2, and to block the interaction between PD-1 and its ligand, it may be advantageous to use checkpoint inhibitors that bind to PD-1.

[0074] In certain 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, and for example, nivolumab (Bristol-Myers Squibb), a human monoclonal anti-PD1 IgG4 antibody; pembrolizumab (Merck), a humanized IgG4 anti-PD-1 antibody; atezolizumab (Genentech), a fully humanized anti-PD-L1 antibody; and durvalumab (Medimmune / AstraZeneca), a human anti-PD-L1 antibody, are all regulatory approved and can be used herein. Many other such antibodies, such as the humanized anti-PD-1 antibody tislerizumab (BeiGene) and the fully human anti-PD-L1 antibody avelumab (Pfizer / Merck), are currently under development / testing and can be used in accordance with the present invention. Similarly, the interaction between PD-1 and PD-L2 can be blocked using antibodies that bind to PD-L2 (preferably monoclonal antibodies, or their derivatives or fragments).

[0075] As mentioned 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 described above with respect to PD-1 / PD-L1 interaction, a drug that blocks the interaction between CTLA-4 and CD80 / CD86 binds to one of these proteins, preventing CTLA-4 from interacting with CD80 and / or CD86. Such a drug may bind to CTLA-4, CD80, or CD86. However, CD80 and CD86 also function as co-stimulatory molecules for T cells through binding to CD28. Therefore, any checkpoint inhibitor that blocks the interaction between CTLA-4 and CD80 / CD86 should not block the interaction between CD28 and CD80 / CD86. Thus, a checkpoint inhibitor that blocks the interaction between CTLA-4 and CD80 / CD86 preferably binds to CTLA-4 rather than CD80 and / or CD86. In particular, checkpoint inhibitors can bind to CTLA-4 at binding sites that interact with CD80 or CD86.

[0076] In certain embodiments, 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, for example, ipilimumab (Bristol-Myers Squibb), a human IgG1 monoclonal antibody that has received regulatory approval. Other such antibodies include, for example, tremelimumab (Medimmune / AstraZeneca), a human IgG2 monoclonal antibody, which is currently under development / investigation.

[0077] PD-1 and CTLA-4 are expressed on T cells. PD-1 and CTLA-4 inhibition is designed to promote T cell activity, and therefore, when antibodies targeting PD-1 or CTLA-4 are used as checkpoint inhibitors, it may be preferable that the binding of the antibody to its target does not initiate antibody-dependent cytotoxicity (ADCC), which can lead to the death of the target T cells. ADCC is primarily mediated by natural killer (NK) cells that express Fc receptors (such as CD16) that recognize and bind to the Fc (i.e., constant) domain of antibodies bound to the target antigen. When the Fc receptor on an NK cell binds to the Fc domain of an antigen-binding antibody, the NK cell is activated and releases a cytotoxic agent that kills the cell to which the antibody is bound.

[0078] Antibodies capable of binding to target cells without inducing ADCC may be from a specific IgG subclass unrelated to ADCC activity, or may be rationally designed by introducing point mutations to inhibit Fc receptor binding. Such rational design is readily apparent to those skilled in the art. For example, a mutation at position 228 in the human IgG4 constant region can prevent the antibody from binding to the Fc receptor. Thus, both nivolumab and pembrolizumab (both human IgG4 antibodies, as described above) contain the S228P mutation in the constant region that prevents Fc receptor binding, meaning that neither antibody mediates ADCC. Any antibody against PD-1 for use as a checkpoint inhibitor according to the present invention may contain identical or equivalent mutations. Equivalent mutations mean mutations at different residues (or corresponding residues in the constant regions of different antibody isotypes) that have the same effect, i.e., inhibition of Fc receptor binding.

[0079] However, in other situations, it may be preferable for checkpoint inhibitors to mediate ADCC. The anti-CTLA-4 antibody ipilimumab has been shown to mediate ADCC against Treg cells mediated by non-classical CD16-expressing monocytes, thus providing a second mechanism to prevent downregulation of immune effector cells (Romano et al., PNAS 112 (19) 6140-6145, 2015).

[0080] Although less prominent, other immune checkpoints besides PD-1 and CTLA-4 are also known and can be targeted by checkpoint inhibitors. One such checkpoint is LAG-3 (also known as CD223). In particular, such drugs are antibodies that bind to LAG-3, and several are currently under development, such as BMS-986016 (Bristol-Myers Squibb).

[0081] In a further alternative approach, inhibitors of killer cell immunoglobulin-like receptors (KIRs) can be used as checkpoint inhibitors. For example, lirirumab (Bristol-Myers Squibb) is a fully human monoclonal antibody against KIRs that can be used as a checkpoint inhibitor.

[0082] For example, other immune checkpoints that can be targeted by checkpoint inhibitors to prevent their activation by blocking interactions with their congener 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 be targeted by checkpoint inhibitors to block interactions between the ligand and its immune checkpoint receptor. For example, phosphatidylserine (PS), a TIM-3 ligand, can be targeted by checkpoint inhibitors to block interactions with TIM-3, for example, using an anti-PS antibody. An example of such an antibody is bavituximab (Oncologie Inc.), which is currently under development.

[0083] 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 the production of antibodies against suitable targets. In certain embodiments, one or more checkpoint inhibitors may be used in combination with an oligopeptide compound. For example, two or more different checkpoint inhibitors, each inhibiting the activation of a different immune checkpoint, can be used. 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 combined use of multiple checkpoint inhibitors results in improved treatment outcomes in some cancers compared to the use of any single checkpoint inhibitor.

[0084] As described above, oligopeptide compounds, and any second drug actually used in combination with them, can be administered by any convenient or desired route, which may depend on the subject, condition, and properties of the drug. Selecting an appropriate mode of administration is within the scope of routine practice for clinicians in this field.

[0085] 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. In particular, oligopeptide compounds may be administered to a subject for systemic delivery, for example, via oral or parenteral administration routes, or locally to the site of cancer being treated, for example, locally or directly to the tumor. Possible routes of topical administration, depending on the site of the cancer (tumor), include topical administration, direct delivery, for example, injection or infusion into the site of cancer (e.g., tumor), and inhalation.

[0086] In certain embodiments, oligopeptide compounds are administered to the target by intratumoral administration, e.g., direct administration by 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., primary tumor) has been found to induce regression not only in the injected tumor but also in tumors at other sites (e.g., secondary tumors). In fact, this has been observed when injected into secondary tumors, as shown in Figure 9, as shown in the following examples. Thus, a strong abscopal effect of the oligopeptide compound may be observed. Therefore, the most convenient or easily accessible, or perhaps largest or most developed, tumor may be selected as the administration site. However, other forms of administration, including systemic administration, are not excluded.

[0087] The generally accepted principle that metastases from solid tumors require systemic administration of anticancer drugs makes the concept of treating metastatic disease with intratumoral drug administration counterintuitive. In reality, systemically administered anticancer drugs are little curative for the millions of cancer patients treated each year. Liver metastases are associated with a poorer prognosis compared to metastases in other organs, and hepatic resection and other liver-targeted procedures, such as cryotherapy, thermal ablation, or chemoembolization, are well-accepted procedures to extend the survival of ACC patients. Intratumoral drug administration has several clear advantages over systemic administration, including achieving several orders of magnitude higher intratumoral drug concentrations and a potential reduction in systemic toxicity. As reported in the following examples, preliminary efficacy data of CyPep-1 in ACC suggest that its intratumoral administration, including liver metastases, represents a promising advance in the management of metastatic ACC patients.

[0088] When an oligopeptide compound is used in combination with another therapeutic agent, the two agents may be administered separately, simultaneously, or sequentially. As used herein, “separate” administration means that the oligopeptide compound and the second agent are administered to the subject simultaneously, or at least substantially simultaneously, via different routes of administration. As used herein, “simultaneous” administration means that the oligopeptide compound and the second agent are administered to the subject simultaneously, or at least substantially simultaneously, via the same route of administration. As used herein, “sequential” administration means that the oligopeptide compound and the second agent are administered to the subject at different times. In particular, the administration of the first therapeutic agent is completed before the administration of the second therapeutic agent is initiated. 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.

[0089] The administration of the oligopeptide compound and / or the second drug may be repeated (i.e., two or more times) during the course of treatment for the subject. For example, the subject may receive multiple cycles of treatment in which both the oligopeptide compound and the second drug are administered. Alternatively, the subject may receive a single dose of one of the therapeutic agents and repeated doses of the other therapeutic agent.

[0090] When multiple additional therapeutic agents are administered to a target in combination with an oligopeptide compound, two or more of these additional therapeutic agents may be administered separately, simultaneously, or sequentially to each other.

[0091] In certain embodiments, a second agent, such as a checkpoint inhibitor, is administered parenterally to the subject. For example, the second agent may be administered intravenously to the subject. In one embodiment, the oligopeptide compound is administered intratumorally, and the second agent, such as a checkpoint inhibitor, is administered parenterally, for example, intravenously.

[0092] When oligopeptide compounds and / or second or further therapeutic agents are used, they are formulated for administration in accordance with principles well known in the art. Therefore, they are provided in the form of pharmaceutical compositions comprising the compound and / or second or further therapeutic agent together with one or more pharmaceutically acceptable carriers or excipients.

[0093] As described above, oligopeptide compounds and / or checkpoint inhibitors (or pharmaceutical compositions containing them) may be administered to subjects in a manner appropriate to the cancer being treated. The dose and frequency of administration are determined by factors such as the patient's condition, as well as the type and severity of the patient's disease, although the appropriate dose may be determined by clinical trials. Preferably, the oligopeptide compounds and / or the second agent may be provided to subjects in daily, weekly, or monthly doses, or in doses of intermediate frequency. For example, doses may be provided 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 biennially. As described above, the same or different dosing plans may be used for the administration of the oligopeptide compounds and the second agent or further agents to subjects.

[0094] The dosage can be administered in an amount that depends on the size of the target. The oligopeptide compound can be administered in doses of 10 μg / kg to 100 mg / kg body weight, for example, 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 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 certain embodiments, the oligopeptide compound is administered in doses of about 2 mg / kg body weight, for example, 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 can 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.

[0095] Similar considerations apply to a second or further therapeutic agent. The dose may be administered in an amount dependent on the size of the target. The second therapeutic agent may be administered in doses of 10 μg / kg to 100 mg / kg body weight, for example, 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 certain embodiments, the second therapeutic agent is administered in doses of about 2 mg / kg body weight, for example, 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 can 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.

[0096] As described above, a second drug, such as a checkpoint inhibitor, can be administered at the same dose as the oligopeptide compound, or at a higher dose or especially a lower dose than the oligopeptide compound. When the compound and drug are used in combination, the dose may be lower than the dose when the compound or drug is used individually (e.g., monotherapy). For example, a checkpoint inhibitor may be administered at a dose of 100 μg / kg to 100 mg / kg body weight, for example, 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. A checkpoint inhibitor may also be administered at a fixed dose, for example, 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.

[0097] Appropriate dosing regimens are known for many checkpoint inhibitors. For example, nivolumab, when used alone, is administered according to a dosing regimen of 240 mg IV every two weeks or 480 mg IV every four weeks. Ipilimumab, when used alone in the treatment of melanoma, is administered according to a dosing regimen of 3 mg / kg IV every three weeks. Dosing regimens for these and many other such checkpoint inhibitors are known to those skilled in the art and can be found within the approvals issued by regulatory bodies such as the FDA and EMA.

[0098] For combination therapy, the oligopeptide compound and the second or further therapeutic agent may be provided in the form of a kit containing both or more of the components. For example, the oligopeptide compound and the second therapeutic agent may be provided in separate containers, i.e., in separate compositions, or in a single composition in a single container. Each therapeutic agent may be provided in any suitable form, for example, in an aqueous solution or as a lyophilized product.

[0099] As described above, as with the medical uses described herein, companion diagnostics are also provided, i.e., methods for identifying or determining which subjects are suitable or appropriate for treatment with oligopeptide compounds in accordance with the medical uses and treatment methods described herein. While performing such screening is not essential or even important, it may be desirable or useful in some cases.

[0100] Therefore, in certain embodiments, 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 determining the presence of biomarkers indicating an abnormal or activated Wnt / β-catenin pathway in a sample obtained from the subject.

[0101] Therefore, subjects, such as patients presenting with signs of a tumor or cancer and visiting a hospital, may be screened or tested as part of their evaluation to determine whether such biomarkers are present. This can be done at the initial visit, or thereafter during the evaluation or course of treatment of the subject, for example, when considering further treatment, or in the event of recurrence, or during the course of treatment.

[0102] The sample can be any sample in which such biomarkers are present, which may depend on the biomarker and / or cancer in question. For example, the sample could be a sample of cells or tissue from the site of cancer, e.g., a tumor biopsy. It could be a solid or liquid biopsy of the cancer. In other embodiments, it could be a sample of body tissue or fluid in which cancer markers may be present, e.g., a blood-derived sample containing circulating cancer DNA, e.g., cell-free DNA, e.g., serum or plasma.

[0103] As described above, mutations in various genes of the Wnt / β-catenin pathway have been identified and reported, and these can have various effects on, for example, the expression level of proteins. For example, proteins may be upregulated or downregulated, and / or their function may be altered, for example, proteins may be inactivated. Therefore, biomarkers may be mutations, or increases or decreases in the level (i.e., amount) of a particular protein or protein complex in a particular tissue, cell, or intracellular compartment. For example, the amount of β-catenin in the nucleus may increase. Increased expression can be determined at the level of the protein or mRNA. Furthermore, biomarkers may be modified activity of a protein, for example, an enzyme, and therefore, functional assays may be performed on the activity of a protein, for example, an enzyme. Therefore, biomarkers may be increases or decreases in the level of activity of a protein, for example, an enzyme.

[0104] In one embodiment, 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 genes selected from β-catenin (CTNNB1), AXIN1, AXIN2, APC, ZNRF3, MEN1, GNAI2, and RNF43; (iii) Upregulation of the expression of one or more of the following genes: WISP-1, c-MYC, cyclin-D, TCF21, FZD3, FZD6, FZD7, Wnt3, Wnt4, Wnt5A, RSPO2, CLDN1, LGR5.

[0105] In another embodiment, the biomarker is selected from one or more of (i), (ii), and (iii) above, or from the following: (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 genes selected from β-catenin (CTNNB1), AXIN1, AXIN2, APC, APC2, ZNRF3, MEN1, GNAI2, GNAI3, RNF43, DVL1, LPR5, LPR6, MED12, and BCL9L; (iii) Upregulation of the expression of one or more of the following genes: WISP-1, Axin2, c-MYC, cyclin-D, TCF21, FZD3, FZD6, FZD7, Wnt3, Wnt4, Wnt5A, RSPO2, CLDN1, LGR5.

[0106] In a particularly representative embodiment, the biomarker may be an increase in the expression of the protein β-catenin, Wisp1, or Axin2. In another embodiment, the biomarker may be an increase in the expression of β-catenin or Wisp1.

[0107] Clinical studies can identify which genes / proteins in the pathway may serve as useful biomarkers. Therefore, appropriate biomarkers can be identified by studying gene / protein expression, the presence of mutations in specific genes, or the identification of specific mutations in those genes, in subjects who responded to treatment with the compounds described herein (e.g., CyPep-1) compared to non-responders. The identification of appropriate biomarker genes in this method is described in the following examples.

[0108] Responders in this regard may be any subject that exhibits a positive clinical response, which may include, for example, tumor regression (such as a decrease in tumor size and / or number), other clinical signs, or a reduction or inhibition of disease progression, i.e., a complete or partial response to stable disease. Therefore, for example, appropriate biomarkers may be identified in subjects exhibiting a partial response or stable disease. This is described in the following examples.

[0109] As a typical example, the presence of one or more mutations in one or more genes in the following gene group can function as a biomarker 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.

[0110] As previously mentioned, conductin / Axin2 is a central protein in the Wnt / β-catenin pathway of tumorigenesis, and CyPep-1 is thought to prevent β-catenin nuclear translocation and activation by activating Axin2. Due to this mechanism of action, patients with one or more mutations in genes that result in decreased Axin2 activity or increased inactive Axin2 can particularly benefit from treatment with CyPep-1. Therefore, the presence of one or more mutations in one or more genes encoding proteins involved in the Axin2 regulatory network can be a useful biomarker for such patients, especially those with mutations that inactivate Axin2 or result in decreased Axin2 activity.

[0111] Therefore, one representative subgroup of biomarkers that represent biomarkers in the Axin2 regulatory network may include mutations in the genes listed in (v) to (viii) above. Representative mutations in these genes are shown in Table 1. [Table 1]

[0112] Methods for detecting mutations or determining gene expression levels are widely described in the art, as are methods for determining the actual amount of a particular protein in a sample. Assays for the activity of various proteins involved in the Wnt / β-catenin pathway have also been described in the art.

[0113] Description of the drawing The present invention can be more fully understood by referring to the following non-limiting embodiments and drawings. [Brief explanation of the drawing]

[0114] [Figure 1-1]Figure 1 shows that CyPep-1 inhibits Wnt / β-catenin signaling. A) Quantification of nuclear levels of β-catenin in adrenal cortical cell line Y1, colorectal cell line CT26, and melanoma cell line B16-F10. [Figure 1-2] B) Quantification of β-catenin target gene Wnt-inducible secretory protein-1 (WISP-1 / CCN4) by PCR in CT26 cells incubated with different concentrations of CyPep-1. C) Quantification of WISP-1 in colorectal cell lines CT26, black oil cell lines B16-F10, Yumm1.7, and glioma cell line GL261. [Figure 1-3] D) Schematic diagram showing the mechanism of action (MOA) of CyPep-1 on the Wnt / β-catenin pathway. [Figure 2] Figure 2 shows results demonstrating that CyPep-1 induces a pro-inflammatory response in vivo. A) shows in vivo tumor growth 17 days post-transplant in colorectal cell line CT26 and melanoma cell line B16-F10 treated with control (saline) or CyPep-1 monotherapy. B) CyPep-1 treatment of mice with B16-F10 and CT26 tumors induces significant changes in the tumor immune landscape. Flow cytometry quantification of the percentage of CD45+ leukocytes (gated in vistatic cells) and CD8+ lymphocytes after vehicle (CT) and CyPep-1. C) CyPep-1 increases inflammatory cytokines in B16-F10 and CT26 tumors. ELISA quantification of IL2, TNFα, and IFNγ secreted in the microenvironment of B16-F10 melanoma (upper panel) and CT26 colorectal (lower panel) tumors treated with a control vehicle (CT) or CyPep-1 (CyPep-1). Data are reported as standardized pg / ml relative to resected tumor weight (g) and expressed as the mean of 5 tumors per group (each dot represents 1 mouse). All results are shown as mean ± SEM (error bars). Statistical significance (indicated by asterisks) is shown against the control condition using unpaired two-sided Student's t-tests (ns = no significant difference, ** = p < 0.005, and *** = p < 0.0005). [Figure 3]Figure 3 shows the results of CyPep-1 monotherapy in one ACC patient previously treated with mitotane (standard treatment). The patient had metastatic disease in the liver, lungs, and bones. A) CT images before treatment with CyPep-1 and 6 months after treatment with CyPep-1. Response based on the sum of maximum diameters (SOD) of solid tumor evaluation criteria (RECIST) showed a 47% reduction in the sum of target lesion diameters (partial response). Liver parameters measured before and during treatment with CyPep-1. [Figure 4] Figure 4 shows the results of CyPep-1 monotherapy in a second patient with ACC previously treated with mitotane. The patient had metastatic spread to the liver, lungs, and bones. A) Histopathological examination of a biopsy obtained at 6 weeks after treatment with CyPep-1 shows necrosis and immune infiltration. B) RECIST measurements of the target lesion during treatment. C) Liver parameters measured before and during treatment with CyPep-1. [Figure 5-1] Figure 5 shows results indicating that CyPep-1 treatment in melanoma cells reduces the expression of target genes Axin2 and Ccn4 / Wisp1 and increases the levels of both phosphorylated forms of β-catenin. A) Quantification of cytoplasmic and individual levels of β-catenin in melanoma cell line B16-F10 incubated with gradually increasing CyPep-1 concentrations for 24 hours. B) Quantification of β-catenin target genes Axin2 and Wnt-inducible secretory protein-1 (Ccn4 / Wisp1) by RT-qPCR in melanoma cell line B16-F10 incubated with 7.5 μM CyPep-1 for 24 hours. [Figure 5-2] C) Determination of phosphorylated β-catenin levels (Ser33 / 37 / Thr41 and Ser45-Thr41) in melanoma cell line B16-F10 incubated with CyPep-1 at gradually increasing concentrations for 24 hours. D) Determination of phosphorylated β-catenin levels (Ser33 / 37-Thr41 and Ser45-Thr41) in melanoma cell line B16-F10 incubated with 7.5 μM CyPep-1 for 24 hours. [Figure 6]Figure 6 shows the results of CyPep-1 treatment increasing the levels of both phosphorylated forms of β-catenin in adrenocortical cancer cells. A) Quantification of phosphorylated β-catenin levels (Ser33 / 37-Thr41 and Ser45-Thr41) in adrenocortical cancer cell line H295R incubated with progressively increasing levels of CyPep-1 for 24 hours. B) Quantification of phosphorylated β-catenin levels (Ser33 / 37-Thr41 and Ser45-Thr41) in adrenocortical cancer cell line H295R incubated with a concentration of 9.375 μM CyPep-1 for 24 hours. [Figure 7-1] Figure 7 shows the results of CyPep-1 monotherapy in immunodeficient NSG mice subcutaneously injected with H295R human adrenocortical carcinoma cells. When the tumor reached 250 mm3, the mice were treated with 25 mg / kg CyPep-1 once a week. A) Tumor volume (mm3) 0-31 days after injection of CyPep-1 or vehicle control. The days on which mice were treated with CyPep-1 or vehicle control are indicated by dashed lines. On day 8, 5 mice per group were euthanized and tumors were collected for RNA analysis (shown in D). B) Quantification of tumor volume (mm3) at 0 and 31 days after injection of CyPep-1 (CY-101) or vehicle control. [Figure 7-2] C) Kaplan-Meier graph of survival probability 31 days after treatment with CyPep-1 or vehicle control. D) Quantification of β-catenin target gene Axin2 and Wnt-inducible secretory protein-1 (Ccn4 / Wisp1) by RT-qPCR in tumors isolated from mice euthanized 8 days after initial treatment with CyPep-1 or vehicle control. [Figure 8-1] Figure 8 shows imaging of B16-F10 mouse melanoma cells treated with 20 μg / ml CyPep-1 for 24 hours. Cells were stained with anti-Axin2 antibody and DAPI (to stain the nuclei) and imaged using a confocal microscope. A) Images of treated and untreated cells stained with Axin2 and DAPI are shown on a 20 μm scale. [Figure 8-2] B) Magnified view of a portion of A on a 5 μm scale (indicated by a dashed white rectangle in A). [Figure 8-3] C) Schematic diagram showing the different cytoplasmic distributions of Axin2 (conductin) in the inactive (diffused) or active (aggregated) states. D) Schematic diagram showing that CyPep-1 can bind to the RGS domain of Axin2, reverse their aggregation, activate Axin2, and lead to Axin2 polymerization. [Figure 9-1] Figure 9 shows the results of CyPep-1 treatment in combination with the anti-PD-1 antibody pembrolizumab in a patient with parathyroid cancer who had previously been treated with surgery and radiotherapy (standard treatment) and nivolumab (anti-PD-1 antibody). The patient had metastatic disease with multiple lesions in different areas of the body. CyPep-1 was injected into only one lesion. A1) CT image of the injected lesion (indicated by a dashed white circle) at baseline. A2) CT image of the injected lesion 3 months after treatment with CyPep-1 (indicated by a dashed white circle). A3) Biopsy image of the injected lesion at baseline, scaled to 50 μm. A4) Biopsy image of the injected lesion 3 months after treatment with CyPep-1, scaled to 50 μm. A5) CT image of the uninjected lesion (indicated by a white arrow) at baseline. A6) CT image of the non-injected lesion (indicated by the white arrow) 20 months after the patient started treatment with CyPep-1. [Figure 9-2] B) Graph showing the percentage change in tumor size of injected and non-injected lesions over 24 months. [Figure 10-1] Figure 10 shows a graph illustrating the pharmacokinetic profile of CyPep-1 as measured using liquid chromatography (LC)-tandem mass spectrometry (MS). [Figure 10-2] Continued [Figure 11] Figure 11 shows the percentage change in tumor size of non-injectable target lesions over 18 months in eight patients with seven different cancer types. Patients received treatment with CyPep-1 for more than 6 months as monotherapy, in combination with the anti-PD-1 antibody pembrolizumab (indicated by asterisks), or via intrahepatic injection. [Figure 12]Figure 12 shows the survival of six patients with adrenocortical carcinoma during and after CyPep-1 intrahepatic injection. [Examples]

[0115] Examples Example 1 Inhibition of β-catenin and WISP-1 (CNN4) CyPep-1, the D-peptide of Sequence ID No. 1, was prepared by Bachem AG (Switzerland). The effect of CyPep-1 on inhibiting the Wnt / β-catenin pathway was evaluated by assessing the expression of WISP-1, a well-known downstream target gene of β-catenin, in various cancer cell lines. Decreased WISP-1 expression indicates a decrease in β-catenin transcriptional activity in CyPep-1 treated cells. In short, cell lines were cultured for 24 hours in the presence of CyPep-1 added to culture medium at a concentration of 0–5 μM. Control cells were cultured in medium (PBS) without CyPep-1. The cells were washed with PBS and lysed using an appropriate cell fractionation kit according to the supplier's instructions, and the cytoplasmic fraction and each fraction were collected. The cytoplasmic fraction and each fraction were separated on an SDS-PAGE gel, and the proteins were transferred onto a nitrocellulose membrane incubated with either anti-β-catenin antibody and anti-α-tubulin antibody (specific cytoplasmic protein) or anti-histone H3 antibody (specific nucleoprotein). The cytoplasmic β-catenin band and nuclear β-catenin band were quantified by ImageJ. The β-catenin / α-tubulin ratio corresponds to the cytoplasmic fraction of β-catenin, while the β-catenin / histone H3 (Y1) or β-katenin / laminin A / C (B16-F10 and CT26) ratios correspond to the nuclear fraction of β-catenin. Results are reported as a fold change in the expression levels of the cytoplasmic and nuclear fractions of β-catenin in cells treated with the indicated concentrations of CyPep-1 compared to untreated control cells (Figure 1A). To evaluate CyPep-1 mRNA expression, melanoma (B16-F10 and Yumm1.7), colorectal cancer (CT26), or glioma (GL261) cells were treated with CyPep-1 as described above. For CT26 cells, dose-dependent reduction in WISP-1 expression was evaluated using different doses of CyPep-1. Figure 1A shows the nucleoprotein levels of β-catenin in adrenocortical cell line Y1, colorectal cell line Ct26, and melanoma cell line B16-F10 treated with CyPep-1 or control. Results are reported as fold change compared to control. Figures 1B and C show 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 change of WISP-1 in treated cells compared to control. In all cell lines, treatment with CyPep-1 significantly reduced WISP-1 / CCN4 mRNA expression.

[0116] Example 2 CyPep-1 induces pro-inflammatory responses in vivo. The 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 at 37°C and 5% CO2 in DMEM and RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, respectively. The cell lines were mycoplasma-free based on testing using the Mycoalert kit (Lonza). C57BL / 6 and BALB / C (7 weeks old) mice were obtained from Janvier and reared under pathogen-free conditions one week prior to the experiment. On day 0, 0.2 × 10⁶ cells diluted in 100 μl of PBS were placed on the right flank of the mice. 6 individual B16-F10 cells or 10 6 Individual CT26 cells were subcutaneously injected. The median tumor volume was 100 mm³. 3 Upon reaching this point, CyPep-1 was administered intratumor at a dose of 2 mg / kg or via vehicle (PBS). Tumor weight results are reported as the average of 10 mice per group from two independent experiments conducted with 5 mice per group. Figure 2A shows that CyPep-1 reduced tumor weight compared to controls in both mice with B16-F10 tumors and mice with CT26 tumors. Results are shown as mean ± SEM (error bars). Statistical significance (indicated by asterisks) is calculated using an unpaired two-sided Student's t-test. Figure 2B shows that CyPep-1 treatment induces significant changes in the tumor immune landscape in mice with B16-F10 tumors and mice with CT26 tumors. Tumors were harvested, mechanically dissociated into fragments (<4 mm), and then enzymatically digested at 37°C for 45 minutes using a mouse tumor dissociation kit (Miltenyi Biotec). Single-cell suspensions were prepared, and erythrocytes were lysed with ACK (10-548E, Lonza). Cells were counted using a Countess Automated Cell Counter (Invitrogen), and Fc blocks (TruStain fcX) were used. TM Cells were blocked on ice for 30 minutes using the anti-mouse CD16 / 32 antibody (101320 BioLegend). The following antibodies were used (BioLegend): FITC anti-mouse CD45, APC anti-mouse CD8a, APC / Fire750 anti-mouse CD4. The LIVE / DEAD Fixable Blue Dead Cell Stain Kit (Thermo Fisher Scientific) was used as the survival stain. As a corrective control, single-dye staining was performed and fluorescence diffusion was confirmed by FMO control. The level of nonspecific binding was evaluated using isotype controls. Figure 2C shows that CyPep-1 increases inflammatory cytokines in B16-F10 and CT26 tumors. ELISA quantification of IL2, TNFα, and IFNγ secreted in the microenvironment of B16-F10 melanoma (upper panel) and CT26 colorectal (lower panel) tumors treated with a control vehicle (CT) or CyPep-1 (CyPep-1). Data are reported as standardized pg / ml relative to resected tumor weight (g) and expressed as the mean of 5 tumors per group (each dot represents 1 mouse). IL2, TNFα, and IFNγ were quantified using the mouse IL2 (ref DY410-05) and IFNγ (ref DY485-05) DuoSet ELISA kits according to the manufacturer's protocol.

[0117] Example 3 Clinical trials material The CyPep-1 peptide was synthesized by Bachem AG (Switzerland). CyPep-1 is a total D-amino acid peptide consisting of the amino acid sequence described in SEQ ID NO: 1. method The clinical trial evaluated CyPep-1 monotherapy. The patient population consisted of all patients enrolled in a basket trial (all solid tumor types were enrolled in the trial) with advanced cancer (stage IV metastatic disease, many patients showing stage IVC disease) and all other available treatment options had been exhausted. Patients received 20 mg CyPep-1 injections every two weeks. Patient response is defined as objective response according to the RECIST 1.1 guidelines, or stable disease condition for at least 16 weeks. The following are RECIST 1.1 evaluation criteria for assessing target lesions: Complete response (CR): Disappearance of all target lesions; • Partial response (PR): A reduction of at least 30% in the total LD ​​of the target lesion, relative to the total LD ​​at baseline; • Disease progression (PD): When the total LD ​​of the target lesion increases by at least 20% relative to the lowest total LD ​​recorded since the start of treatment, or when one or more new lesions appear; • Disease Stability (SD): Based on the lowest total LD ​​since the start of treatment, there is neither a sufficient reduction to be judged as a partial response (PR) nor a sufficient increase to be judged as a PR. The following are some of the RECIST 1.1 criteria for evaluating non-target lesions: Complete response (CR): Remission of all non-target lesions and normalization of tumor marker levels; • Disease stability (SD): Persistence of one or more non-target lesions and / or maintenance of tumor marker levels above the normal range; • Disease progression (PD): The appearance of one or more new lesions and / or clear progression of existing non-target lesions. result CyPep-1 monotherapy showed promising results in advanced IVC cancer associated with abnormally activated Wnt / β-catenin pathways.

[0118] CyPep-1 monotherapy Figure 3 shows the results of CyPep-1 monotherapy in patients with ACC. A) shows CT scans of ACC in patients before treatment with CyPep-1 and 6 months after treatment with CyPep-1. The Solid Tumor Evaluation Criteria (RECIST) showed a partial response (47%) at 6 months. B) Evaluation of liver enzymes during administration shows normalization of values. Figure 4 shows the results of CyPep-1 monotherapy in other patients with ACC. A) Biopsy histological H&E staining showing extensive cell death after treatment with CyPep-1 monotherapy. B) Target lesions in the lungs and liver during 6 months of treatment. Tumor reduction is observed not only in the injected tumor but also in other non-injectable metastases, the liver (same organ as the injected lesion) and lungs (organs distant from the injected lesion). Reduction of non-injectable metastases in distant organs is considered the strongest evidence of the immune-mediated abscopal effect of the intratumoral-administered drug. C) Evaluation of liver enzymes during treatment.

[0119] Example 4 CyPep-1 induces phosphorylation of β-catenin. Melanoma (B16-F10) and adrenocortical carcinoma (295R) cells were seeded. B16-F10 cells were treated with CyPep-1 (0–7.5 μM) at progressively increasing concentrations after 24 hours, and H295R cells were treated with CyPep-1 (0–9.375 μM) at progressively increasing concentrations when they reached 80% confluence. Both cell lines were treated with CyPep-1 for 24 hours, and control cells were cultured in CyPep-1-free medium (in PBS). The cells were then harvested and washed with PBS. The cells were lysed using RIPA buffer (containing phosphatase and protease inhibitors), and proteins and mRNA were extracted. Appropriate cell fractionation kits were also used for B16-F10 cells, and cell and nuclear fractions were collected according to the supplier's instructions. The extracted proteins were separated on an SDS-PAGE gel and transferred to a nitrocellulose membrane incubated with either an anti-β-catenin antibody and either an anti-α-tubulin antibody (cytoplasm-specific protein) or an anti-histone H3 antibody (nucleus-specific protein) (Figure 5A), or either an anti-phospho-β-catenin (Ser33 / 37-Thr41) antibody or an anti-phospho-β-catenin (Ser45-Thr41) antibody and an anti-actin antibody (control) (Figures 5C and 6A). Phospho-β-catenin bands were quantified using ImageJ. The phospho-β-catenin / actin protein expression ratio is reported as the fold change in phospho-β-catenin and actin in cells treated with 7.5 μM (B16-F10) or 9.375 μM (H295R), compared to untreated control cells (Figures 5D and 6B). Figure 5A shows that as the concentration of CyPep-1 increases, the nuclear fraction of β-catenin decreases and the cytoplasmic fraction increases. Figures 5D and 6B show that both phosphorylated forms of phospho-β-catenin are increased (by more than twofold) upon treatment with CyPep-1 in melanoma and adrenocortical carcinoma cells. Phosphorylation of β-catenin at GSK3 (pS33, pS37, and pT41) and CKI (pS45) target residues targets β-catenin for ubiquitination and subsequent proteasomal degradation. These results indicate that CyPep-1 increases phosphorylation of these residues and, consequently, increases β-catenin degradation. To evaluate CyPep-1 mRNA expression, melanoma (B16-F10) cells were treated with CyPep-1 as described above. RT-qPCR was performed to assess the mRNA expression levels of the Wnt / β-catenin downstream target genes Axin2 and Ccn4 / Wisp1. The ratio of target gene mRNA expression is reported as the fold change in target gene mRNA in cells treated with 7.5 μM CyPep-1 compared to untreated control cells (Figure 5B). Figure 5B shows that CyPep-1 reduces the expression of Wnt / β-catenin downstream target genes Axin2 and Ccn4 / Wisp1 in treated cells compared to untreated control cells.

[0120] Example 5 In vivo trials of CyPep-1 for adrenocortical cancer Human adrenocortical carcinoma cells (H295R cell line) were subcutaneously injected into immunodeficient NGS mice. The tumor size was 250 mm. 3 When the tumor volume reached 1500 mm³, mice were administered intratumorally once a week with 25 mg / kg of CyPep-1 or a vehicle control. Five mice per group were euthanized 8 days after the initial treatment, and tumors were collected for mRNA analysis. The remaining animals were further treated and used for tumor growth (Figures 7A and 7B) and survival analysis (Figure 7C). 3 When the condition was reached, the mice were euthanized. Figures 7A and 7B show that CyPep-1 significantly reduced tumor volume compared to the vehicle-treated mice after initial treatment until the end of the experiment. Mice treated with CyPep-1 appeared to have no tumors at the end of the experiment. Figure 7C shows that CyPep-1 significantly increased mouse survival (p=0.0062) compared to mice treated with the vehicle. mRNA was collected from tumors of mice euthanized on day 8. 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 is reported as a fold change in target gene mRNA in tumors treated with CyPep-1, compared to untreated control cells (Figure 7D). Figure 7D shows that CyPep-1 reduces the expression of Wnt / β-catenin downstream target genes Axin2 and Ccn4 / Wisp1. This reduction was significant for Ccn4 / Wisp1 (p=0.0083).

[0121] Example 6 Confocal microscopy observation of Axin2 Melanoma cells (B16-F10) were seeded on ibidi plates. After 24 hours, the cells were treated with CyPep-1 (0-20 μg / ml) at gradually increasing concentrations for 24 hours. Subsequently, the cells were fixed with 4% PFA and stained with rabbit anti-Axin2 antibody. An anti-rabbit secondary antibody conjugated with AlexaFluor633 was used. The nuclei were stained using DAPI. The samples were observed using a confocal microscope (Figures 8A and 8B). Figures 8A and 8B show the diffusive staining of Axin2 in untreated samples. This observation indicates inactive Axin2. When inactive, the RGS domain of Axin2 aggregates (Figure 8D), and Axin2 shows a diffuse distribution in the cytoplasm (Figure 8C). Figures 8A and 8B also show that condensates / punctate structures are formed in most cells treated with CyPep-1. This observation indicates activated Axin2. When activated, the RGS domain does not aggregate, and Axin2 polymerizes (condenses) (Figure 8C). This observation indicates that Axin2 polymerizes upon treatment with CyPep-1, suggesting that CyPep-1 activates Axin2. It is thought that CyPep-1 binds to the RGS domain of Axin2, reverses their condensation, and activates Axin2 (Figure 8D).

[0122] Example 7 Clinical trials material The CyPep-1 peptide was synthesized by Bachem AG (Switzerland). CyPep-1 is a total D-amino acid peptide consisting of the amino acid sequence described in SEQ ID NO: 1. method Following the clinical trials described in Example 3, a recommended dose for further development (RP2D = 20 mg) was investigated in the following three expanded cohorts: A) CyPep-1 monotherapy, patients received 20 mg of CyPep-1 every two weeks (every other week); B) CyPep-1 and anti-PD-1 antibody, patients received 20 mg of CyPep-1 every two weeks (every other week) and 400 mg of pembrolizumab every six weeks; C) Intrahepatic CyPep-1 monotherapy, patients received 20 mg of CyPep-1 every two weeks (every other week) to intrahepatic lesions. Response evaluations were performed every eight weeks. Patient response was defined as objective response according to the RECIST 1.1 guidelines, or stable disease status for 16 weeks or more. The RECIST 1.1 evaluation criteria for assessing target lesions are described in Example 3. result CyPep-1 monotherapy showed promising results in advanced IV cancer associated with abnormally activated Wnt / β-catenin pathways.

[0123] CyPep-1 and anti-PD-1 antibodies Figure 9 shows the results of combination therapy with CyPep-1 and an anti-PD-1 antibody (pembrolizumab) in patients with parathyroid cancer. A1-2) shows CT scans of the CyPep-1 injected lesions at baseline and 3 months post-treatment. A3-4) shows biopsy images from the injected lesions at baseline and 3 months post-treatment. The baseline tissue was malignant, but the tissue 3 months post-treatment was benign. A5-6) shows CT scans of the non-injected lesions at baseline and 20 months after initiation of CyPep-1 treatment in the injected lesions. The tumors have visibly decreased in size. B) Evaluation of the rate of change in tumor size in the injected and non-injected lesions shows a decrease in the size of both tumors over 24 months. These results not only demonstrate the efficacy of CyPep-1 when injected directly into the lesion, but also provide strong evidence of the immune-mediated abscopal effect of the intratumor-administered drug by reducing non-injectable metastases in distant organs.

[0124] Pharmacokinetics of CyPep-1 Figure 10 shows the favorable pharmacokinetic profile of CyPep-1. Serum concentrations peaked approximately 15 minutes after CyPep-1 administration. The limit of quantification (LLOQ) was 10 ng / mL.

[0125] Tumor growth dynamics of non-injectable target lesions Figure 11 shows the percentage change in tumor size of non-injectable target lesions over 24 months for eight patients with various types of cancer. Patients received CyPep-1 for at least 6 months. The patient details are as follows: sarcoma, CyPep-1 monotherapy, disease stable (SD); chondrosarcoma, CyPep-1 monotherapy, disease stable (SD); uveal melanoma, intrahepatic injection, disease progression (PD); melanoma, CyPep-1 monotherapy, disease stable (SD); adrenocortical carcinoma, intrahepatic injection, partial response (PR); head and neck squamous cell carcinoma, CyPep-1 and anti-PD-1 antibody, disease stable (SD); adrenocortical carcinoma, intrahepatic injection, disease stable (SD); parathyroid carcinoma, CyPep-1 and anti-PD-1 antibody, unconfirmed disease progression (UPD); melanoma, CyPep-1 and anti-PD-1 antibody, partial response (PR). These results also provide strong evidence for the immune-mediated abscopal effect of CyPep-1, due to a reduction in non-injectable metastases in distant organs. This effect is observed across multiple cancer types and multiple dosage forms.

[0126] intrahepatic injection Figure 12 shows the survival of six adrenocortical cancer patients who received CyPep-1 intrahepatic injections. The patient details are shown in Table 2 below. [Table 2]

[0127] These results indicate a positive correlation between better iRECIST response and longer survival in patients with mutations in the β-catenin pathway, particularly those whose genes encode proteins involved in the Axin2 network. This suggests that patients with β-catenin pathway mutations resulting in abnormal Wnt / β-catenin signaling respond best to CyPep-1 treatment. Therefore, mutations in the β-catenin pathway may serve as a biomarker for patients suitable for CyPep-1 treatment. In this regard, it should be noted that patient 10-022, who did not demonstrate disease control (demonstrated disease progression), had a very advanced stage of the disease and participated in the trial at a later stage. Therefore, a negative response from this one patient does not indicate that LRP6 cannot function as a biomarker or that CyPep-1 treatment is generally ineffective, but rather indicates that a response is not observed in every patient, in all patients, and at all stages of the disease. This patient can be considered an outlier.

Claims

1. An oligopeptide compound for use in the treatment of cancer in a subject, comprising the D-amino acid sequence described in Sequence ID No. 1, or a D-amino acid sequence having at least 85% sequence identity thereto, wherein the cancer is associated with abnormal activation of the Wnt / β-catenin pathway.

2. The oligopeptide compound for use according to claim 1, wherein the oligopeptide compound comprises the D-amino acid sequence described in SEQ ID NO: 1, or consists of the D-amino acid sequence described 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, nettle, dog, pig, horse, cattle, sheep, goat, monkey, or primate.

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

5. The oligopeptide compound for use according to claim 4, wherein the cancer is selected from eye 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, Wilms' tumor, neuroendocrine (carcinoid) cancer, HPV-positive cancer, squamous cell carcinoma and sarcoma, or desmoid tumor.

6. The oligopeptide compound for use according to claim 5, wherein the cancer is selected from melanoma, parathyroid cancer, thyroid cancer, squamous cell carcinoma of the head and neck (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 together with a second therapeutic agent effective for treating the cancer.

9. The oligopeptide compound for use according to claim 8, wherein the second therapeutic agent is selected from chemotherapy agents, immunotherapy agents, hormone therapy, radiotherapy, or photodynamic therapy.

10. The oligopeptide compound for use according to claim 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. The compound is formulated for use according to any one of claims 1 to 12, wherein the compound is an oligopeptide compound for use according to any one of claims 1 to 12, which is formulated for intratumoral injection or infusion.

14. The oligopeptide compound for use according to any one of claims 1 to 13, wherein the compound is a compound for local administration to a primary tumor or metastatic tumor.

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

16. A method for treating cancer associated with abnormal activation of the Wnt / β-catenin pathway, comprising administering an oligopeptide compound to a subject of interest, optionally co-administering it with a second therapeutic agent, wherein the oligopeptide compound, subject, cancer, treatment, and 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 pharmacopoeci for treating cancer associated with abnormal activation of the Wnt / β-catenin pathway in a subject, which may be optionally combined with a second therapeutic agent, wherein the oligopeptide compound, subject, cancer, treatment, and second therapeutic agent are as defined in any one of claims 1 to 15.

18. A product comprising, as a combination formulation for use separately, simultaneously, or sequentially in the treatment of cancer in a target, an oligopeptide compound according to claim 1 or 2 and a second oncological therapeutic agent, wherein the cancer is associated with abnormal activation of the Wnt / β-catenin pathway.

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

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

21. The method according to claim 20, wherein the biomarker is (i), (ii), and (iii) below. (i) Increased expression of one or more of the following proteins in the tumor sample: 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 genes selected from β-catenin (CTNNNB1), AXIN1, AXIN2, APC, APC2, ZnRF3, MEN1, GNAI2, RNF43, DVL1, LPR5, LPR6, MED12, and BCL9L; (iii) Upregulation of the expression of one or more of the following genes: WISP-1, Axin2, c-MYC, Cyclin-D, TCF21, FZD3, FZD6, FZD7, Wnt3, Wnt4, Wnt5A, RSPO2, CLDN1, LGR5.

22. The method according to claim 20 or 21, wherein the biomarker is a mutation in a gene encoding a protein involved in the Axin2 regulatory network, and the gene comprises 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, and LRP6.

24. A method for identifying and treating cancer associated with abnormal activation of the Wnt / β-catenin pathway in a subject, comprising (i) and (ii) below: (i) Identifying a target for cancer treatment by determining the presence of a biomarker indicating the activated Wnt / β-catenin pathway in a sample obtained from the target, wherein the identification is performed by the method according to any one of claims 20 to 23; (ii) Administer to the subject an oligopeptide compound containing the D-amino acid sequence described in Sequence ID No. 1, or a D-amino acid sequence having at least 85% sequence identity thereto, wherein all amino acids of the compound are D-amino acids, and the compound is administered together with a second therapeutic agent, optionally.

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