Complexes comprising alpha-lactalbumin and fatty acids or lipids for use in the treatment or prevention of cancer - Patent Application 20070122999

Abstract

The present invention provides a conjugate for use in various therapeutic applications, as well as a therapeutic method using the conjugate or a pharmaceutical composition containing the conjugate. The conjugate is particularly useful for treating cancer, especially when such transformation occurs at a site distant from the site of administration of the conjugate, and for treating metabolic-related conditions. The conjugate comprises a polypeptide having the sequence of naturally occurring alpha-lactalbumin or a functional variant thereof, or a peptide of up to 50 amino acids containing the alpha-helical domain of the polypeptide, and a fatty acid or a lipid or salt thereof.

Description

[Technical Field]

[0001] The present invention relates to a complex for the prevention or treatment of cancer and other conditions, including conditions related to metabolism, whether secondary or unrelated to cancer, comprising a polypeptide having the sequence of naturally occurring alpha-lactalbumin or a functional variant thereof, or a peptide of up to 50 amino acids comprising the alpha-helical domain of said polypeptide, and a fatty acid or a lipid or salt thereof for therapeutic use in tumor surveillance. [Background technology]

[0002] HAMLET (human α-lactalbumin rendered lethal to tumor cells) is the first member of a family of tumoricidal, unfolded protein-lipid complexes consisting of partially unfolded α-lactalbumin and oleic acid. Initially isolated in the form of a fraction obtained by passing a casein-containing fraction of human milk through an ion-exchange column under high-salt conditions ( WO 96 / 004929 ), it was found to be biologically active, particularly possessing antibacterial activity. Subsequently, other methods for preparing active complexes have been derived, including those in which α-lactalbumin from various sources and oleic acid are heated together in solution. In addition, however, HAMLET and related complexes, such as BAMLET derived from bovine α-lactalbumin, have also been found to kill transformed cells, such as tumor or papilloma cells, and to possess antiviral activity. HAMLET kills many types of tumor cells in vitro, as shown in human glioblastoma xenografts and animal models of bladder cancer, and this tumoricidal activity is maintained in vivo. Topical application of HAMLET eliminated or reduced skin papillomas, and local instillation of HAMLET killed bladder cancer cells but not healthy cells in the surrounding tissue, resulting in a reduction in tumor size. Tumor cell sensitivity to HAMLET reflects oncogenic transformation and is modified by the cellular glycolytic state (Storm P et al. (2011) Oncogene). ShRNA silencing of c-Myc or Ras pathway members conferred resistance to HAMLET, and the level of c-Myc expression was comparable to HALET sensitivity. Furthermore, glucose deprivation sensitized tumor cells to HAMLET, and HAMLET sensitivity was modified by shRNA targeting glycolytic enzymes. Additionally, HAMLET was shown to have a profound effect on overall metabolism, with rapid metabolic paralysis within tumor cells and a potential shift of glycolytic flux toward the pentose phosphate pathway.

[0003] Tumor surveillance is essential to prevent tumor cells from developing into tumor masses. However, the protective forces that eliminate emerging tumor cells or reprogram them to healthy tissue are not fully understood. While the tissue environment is expected to contain molecules that implement antitumor defenses, even the role of immune surveillance remains unclear, as immune deficiency itself does not appear to cause cancer. Tissue development in newborns presents similar challenges, as immature or virus-infected cells must be removed and replaced with cells that perform essential physiological functions in mature tissues. Molecules provided in milk have evolved to provide solutions locally within the respiratory and gastrointestinal tracts. Therefore, molecular solutions provided in milk that eliminate immature cells and drive tissue differentiation may be highly relevant for therapeutically achieving tumor surveillance.

[0004] Alpha-lactalbumin is the most abundant protein in human milk and is important for survival and progeny. Native alpha-lactalbumin acts as a substrate specifier in the lactose synthase complex; without lactose, milk cannot be produced due to its high viscosity. When partially unfolded, human alpha-lactalbumin acquires the ability to kill tumor and immature cells by forming an oleic acid conjugate. HAMLET (human alpha-lactalbumin rendered lethal for tumor cells) effectively kills a wide range of tumor cells and has demonstrated therapeutic efficacy in colon cancer and several other cancer models and clinical studies. A second HAMLET family member, alpha-oleic acid, formed by the N-terminal alpha-helical peptide of alpha-lactalbumin, has shown therapeutic efficacy in patients with bladder cancer.

[0005] While conjugates such as HAMLET have been demonstrated to promote cure in the treatment of a range of existing cancers ( WO 2005 / 082406 ) and for the preventative treatment of colon cancer ( WO 2014 / 023976 ), such conjugates have not previously been shown to be useful in the treatment of cancers where the conjugate cannot be administered directly (e.g., orally for cancers outside the gastrointestinal tract). Surprisingly, the inventors have demonstrated that the conjugate is useful in the treatment of cancers distant from the site of administration, as well as secondary cancers or tumor metastases. The properties of the conjugate are such that uptake from the site of administration would not be expected. Thus, the ability of the conjugate to function at sites distant from its original administration is highly surprising. Furthermore, the inventors have demonstrated that the conjugate has long-term effects that persist beyond the period of administration, thereby enabling the inventors to demonstrate the usefulness of the conjugate in the prevention or treatment of secondary or new cancers, which is also significant and surprising.

[0006] The present inventors have investigated the role of multiple adenomatous polyposis coli (APC), a disease that is prone to intestinal cancer. Min / +We investigated how the bovine α-lactalbumin conjugate BAMLET affects intestinal tumor development and tissue homeostasis in mice with BAMLET compared with C57BL / 6 controls. Results indicate that supplementing drinking water with BAMLET is sufficient to delay tumor growth and increase survival in tumor-prone mice. In-depth analysis revealed significant effects on transcriptional mechanisms affecting the tumor microenvironment. These effects included inhibiting the programmed cell death-1 (PD-1) signaling pathway. Unexpected effects on Wnt / β-catenin signaling in the lung, liver, and kidney were also observed, suggesting a protective effect of BAMLET beyond the intestine. In contrast, healthy mice exhibited a weak intestinal response affecting metabolic functions such as lipid and glucose metabolism and insulin resistance, without evidence of systemic effects. Results demonstrate how tumor surveillance meets the demand for milk components that preferentially target tumor cells in a proximal host without adverse effects on a healthy host background. The response to BAMLET in extraintestinal tissues further suggested a more global role for α-lactalbumin for tissue development in the neonate and tumor surveillance.

[0007] Based on the collected evidence, it is clear that the complex has a systemic effect and alters the overall tumor environment. As described below, the inventors have generated evidence of both a shift in gene expression and physiological effects in response to administration of the complex. Furthermore, while most cancer treatments are directed at a single point attack, for example, by targeting one specific gene, the complex of the present invention has a very broad effect on cancer through multiple routes. This allows the complex to be useful in targeting many different cancers, including metastasis.

[0008] The present inventors have also confirmed that administration of the conjugate provides long-term protection from cancer.

[0009] The inventors have further identified other systemic effects, particularly metabolic effects. Such effects have been found in otherwise healthy animals. The effects may affect cancer development and / or the overall progression of cancer patients by reducing conditions secondary to cancer that significantly impact health. The effects also demonstrate the usefulness of the conjugate in treating such conditions, even when they are unrelated to cancer. Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention provides conjugates for use in a variety of therapeutic applications, as well as methods of treatment using the conjugates or pharmaceutical compositions comprising the conjugates. The conjugates are particularly useful for treating malignant transformation, particularly cancer, especially when such transformation is found at sites distant from the site of administration of the conjugate. [Means for solving the problem]

[0011] The complex comprises a polypeptide having the sequence of naturally occurring alpha-lactalbumin or a functional variant thereof, or a peptide of up to 50 amino acids comprising the alpha helical domain of said polypeptide, and a fatty acid or a lipid or salt thereof.

[0012] The defined conjugates are provided for use in tumor surveillance. Further, the defined conjugates are provided for use in modifying the tumor environment. Also provided are methods for treating or preventing cancer, comprising the steps of tumor surveillance and / or modifying the tumor environment.

[0013] Tumor surveillance refers to the identification of cancerous or precancerous cells or other indicators of a cancer or precancerous condition. It can also include initiating a response to the presence of such cells or indicators, for example, by modifying the tumor environment.

[0014] Modifying the tumor environment means modifying the conditions that affect tumor development or growth, such as, but not limited to, immune cells, signaling molecules, extracellular matrix, blood supply, etc. It can refer to the tumor microenvironment, i.e., the environment surrounding the tumor, or the broader environment of the body. In particular, modifying the tumor environment means modifying the tumor environment so that tumor development, growth, or metastasis is reduced or prevented, or the likelihood of tumor development, growth, or metastasis is reduced or prevented.

[0015] There is provided a conjugate as defined for use in the prevention or treatment, particularly the treatment, of cancer, wherein the conjugate is for administration at a first site and the cancer is at a second site.

[0016] Also provided is a method for preventing or treating, particularly a method for treating, cancer, comprising administering to a subject having, or prone to, or at high risk of developing cancer, an effective amount of the conjugate or a pharmaceutical composition comprising the conjugate, wherein the conjugate or composition is for administration at a first site and the cancer is at a second site.

[0017] The conjugate or composition comprising the conjugate is for administration to a first site on or within the body. By this, we contemplate that the conjugate is formulated for administration to a specific site, for example, it is formulated for oral administration, intravesical administration, intracerebral administration or local administration.

[0018] The cancer is found at a second site, for example, the cancer is found in one or more of the nasal passages, digestive tract (e.g., one or more of the oral cavity, stomach, colon, intestine), brain, lung, kidney, vagina, bladder, liver, skin, breast, prostate, and / or ovary.

[0019] In particular, the cancer may be found in the lung, kidney, or liver. Preferably, it is found in the lung. Alternatively, it may be found in the kidney. Alternatively, it may be found in the liver.

[0020] In some embodiments, the second site is not one or more of the nasal passages, the digestive tract (one or more of the oral cavity, stomach, colon, and intestines), the brain, the lungs, the kidneys, the vagina, the bladder, the liver, the skin, the breast, the prostate, and / or the ovaries. In particular, in some embodiments, it is not the nasal passages. In some embodiments, it is not the digestive tract. In some embodiments, it is not the brain. In some embodiments, it is not the lungs. In some embodiments, it is not the kidneys. In some embodiments, it is not the vagina. In some embodiments, it is not the bladder. In some embodiments, it is not the liver. In some embodiments, it is not the skin. In some embodiments, it is not the breast. In some embodiments, it is not the prostate. In some embodiments, it is not the ovaries.

[0021] The first and second sites are preferably different, and more preferably separate from each other, i.e., found in different parts of the body or different organs. For example, if the conjugate is for oral administration, the cancer is not a cancer of the digestive tract. Table 1 provides further examples of first and second sites. [Table 1]

[0022] The treatment or prevention of cancer may include steps of tumor surveillance and / or modification of the tumor environment.

[0023] There is also provided a conjugate as described above for use as a checkpoint inhibitor, particularly an inhibitor of PD-1. The invention provides the conjugate for use in the prevention or treatment, particularly the treatment, of PD-L1 positive cancers or other cancers amenable to PD-1 targeting.

[0024] The present invention further provides a method for preventing or treating a cancer that is PD-L1 positive or otherwise susceptible to PD-1 targeting, the method comprising administering to a subject in need thereof a therapeutically effective amount of the conjugate, or a composition comprising the conjugate.

[0025] Cancer can be found at any site in the body, for example, any of the sites listed in connection with other aspects of the invention.

[0026] The conjugate may be for administration via any suitable route, such as those described in connection with other aspects of the invention. It may be for administration directly to the site of the cancer or for administration at a different site. For example, the conjugate may be for oral administration for the treatment of PD-L1 positive cancer in the gastrointestinal tract or elsewhere in the body, such as the liver, lungs, or kidneys.

[0027] The cancer may be a primary cancer or a metastasis.

[0028] Also provided by the present invention is a conjugate as described above for use in preventing, reducing or treating metastasis.

[0029] The present invention further provides a method for preventing or treating metastatic cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the conjugate, or a composition comprising the conjugate.

[0030] The primary tumor from which the metastasis arises, or the metastasis itself, may be found at any site in the body, for example, at any of the sites listed in connection with other aspects of the invention.

[0031] The complex may be for administration via any suitable route, such as those described in connection with other aspects of the invention. It may be for administration directly to the site of the primary cancer or metastasis, or for administration at a different site. For example, the complex may be for oral administration for the prevention, reduction, or treatment of metastatic cancer in the digestive tract, or metastatic cancer arising from cancer in the digestive tract. Alternatively, it may be for the prevention, reduction, or treatment of metastatic cancer in other locations in the body, such as the liver, lungs, or kidneys, or metastatic cancer arising from cancer in those locations.

[0032] Preventing, reducing, or treating metastasis may include steps of tumor surveillance and / or modification of the tumor environment.

[0033] Also provided is a conjugate as described above for use in the treatment or prevention of a metabolic-related condition such as insulin resistance, type II diabetes, metabolic syndrome, non-alcoholic fatty acid liver disease, cirrhosis, hypertension, etc. The conjugate may be used to regulate insulin resistance or sensitivity, lipid metabolism and / or glucose metabolism, and is therefore useful in treating conditions resulting from challenges with such processes.

[0034] The present invention further provides methods of treating such metabolic-related conditions comprising administering the conjugate to a subject as directed.

[0035] Such metabolic conditions may be associated with the presence of cancer, e.g., they may be secondary to cancer, or may be unrelated to cancer. The complex is particularly useful for improving the health of subjects with cancer by treating the cancer, treating conditions secondary to cancer, or both. Thus, the present invention provides a complex as described above for use in treating or preventing metabolic conditions, such as insulin resistance, type II diabetes, metabolic syndrome, non-alcoholic fatty acid liver disease, cirrhosis, and hypertension, in subjects with or previously had cancer. Also provided are complexes as described above for use in treating or preventing metabolic conditions, such as insulin resistance, type II diabetes, metabolic syndrome, non-alcoholic fatty acid liver disease, cirrhosis, and hypertension, in subjects who do not or have never had cancer.

[0036] Complex The complex comprises a polypeptide having the sequence of naturally occurring alpha-lactalbumin or a functional variant thereof, or a peptide of up to 50 amino acids comprising the alpha helical domain of said polypeptide, and a fatty acid or a lipid or salt thereof.

[0037] In one embodiment the polypeptide has the sequence of naturally occurring alpha-lactalbumin, preferably human or bovine alpha-lactalbumin, more preferably bovine alpha-lactalbumin.

[0038] In one embodiment, the alpha helix domain is the α1 (residues 1-39) or α2 (residues 81-123) domain of human α-lactalbumin, SEQ ID NO:3 or SEQ ID NO:4. KQFTK XELSQLLKDIDGYGGIALPELI XTMFHTSGYDTQ (SEQ ID NO: 3) LDDDITDDIM XAKKILDIKGIDYWLAHKALXTEKLEQWL XEKL (SEQ ID NO: 4) Here, X is an amino acid residue other than cysteine.

[0039] In one embodiment, the conjugate comprises a peptide of about 45, 42 or 40 amino acids, in particular 39 amino acids, preferably corresponding to the α1 domain of human α-lactalbumin.

[0040] In one embodiment, the functional variant consists of a sequence that lacks disulfide bonds, hi one embodiment, the functional variant consists of a sequence in which the cysteine ​​residues in native alpha-lactalbumin are changed to other amino acid residues, preferably alanine residues.

[0041] In one embodiment, the fatty acid or lipid thereof or salt thereof is a fatty acid or a salt thereof. In one embodiment, the fatty acid or a salt thereof is oleic acid or an oleate.

[0042] In one embodiment, the polypeptide has the sequence of bovine alpha-lactalbumin and the fatty acid or salt thereof is oleic acid or oleate.

[0043] The polypeptide present in the complex may have the sequence of alpha-lactalbumin or a variant thereof as described above.

[0044] The complex may be referred to as a biologically active complex. As used herein, the term "biologically active" means that the complex has a biological activity that is different from or stronger than that of the individual components. In particular, the complex may selectively induce cell death, particularly in tumor cells, and / or have bactericidal or antiviral effects not found in natural proteins, including, for example, monomeric α-lactalbumin forms, although other therapeutic effects may also be available.

[0045] The term "variant" refers to a protein or polypeptide that has a similar biological function but differs from the base sequence from which it is derived in that one or more amino acids in the sequence have been replaced with other amino acids. Amino acid substitutions can be considered "conservative," in which an amino acid is replaced with a different amino acid that has broadly similar properties. Non-conservative substitutions are when an amino acid is replaced with an amino acid of a different type.

[0046] A "conservative substitution" refers to the substitution of an amino acid with another amino acid of the same class, where classes are defined as follows: [Table 2]

[0047] As is well known to those skilled in the art, altering the primary structure of a peptide by conservative substitutions may not significantly alter the activity of the peptide, since the side chain of the amino acid inserted into the sequence may be able to form similar bonds and contacts as the side chain of the substituted amino acid, even if the substitution is in a region that is important in determining the conformation of the peptide.

[0048] Non-conservative substitutions are possible as long as they do not disrupt the function of the DNA-binding domain polypeptide. Generally speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptide.

[0049] Determining the effect of any substitution (and indeed any amino acid deletion or insertion) is well within the routine capabilities of one of ordinary skill in the art, who can readily determine whether a variant polypeptide retains the essential properties and activity of the basic protein. For example, in determining whether a variant of a polypeptide is within the scope of the present invention, one of ordinary skill in the art would determine whether a complex containing the variant retains the biological activity (e.g., tumor cell killing) of a complex formed with the unfolded form of the native protein, and whether the polypeptide has at least 60%, preferably at least 70%, more preferably at least 80%, and even more preferably 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the native protein.

[0050] A variant of a polypeptide may comprise or consist essentially of an amino acid sequence having at least 70% identity to a naturally occurring protein sequence, such as an alpha-lactalbumin or lysozyme sequence, for example at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity.

[0051] The level of sequence identity is suitably determined using the BLASTP computer program, which uses the native protein sequence as the base sequence. This means that the native protein sequence forms the sequence to which the percentage identity is determined. BLAST software is publicly available at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi (accessed March 12, 2009).

[0052] In certain embodiments, the polypeptide is α-lactalbumin, such as human, bovine or ovine α-lactalbumin.As mentioned above, these variants can be useful in the present invention, especially in dietary supplement applications, but it may be preferable to use natural protein in the product.In certain embodiments, human α-lactalbumin is used.In another embodiment, α-lactalbumin is bovine α-lactalbumin.A wide range of α-lactalbumin sequences are known in the literature, for example, as shown in Watanabe et al., J.Vet Med Sci, (2000) 62(11), 1217-1219.

[0053] In another embodiment, the polypeptide comprises a recombinant protein having the sequence of α-lactalbumin or a fragment thereof, but lacking intramolecular disulfide bonds or bridges. By ensuring that the recombinant protein lacks intramolecular disulfide bridges, the molecule becomes three-dimensionally non-natural and completely inactive with respect to its original intrinsic biological activity. This is achieved by changing the cysteine ​​residues in natural α-lactalbumin to other residues, particularly alanine residues. Preferably, all cysteine ​​residues are changed to other residues, such as alanine residues. In particular, the recombinant protein is based on the sequence of human α-lactalbumin, although α-lactalbumin from other sources, including bovine or ovine α-lactalbumin, can be used to derive the recombinant protein.

[0054] In certain embodiments, the polypeptide is a recombinant protein having the sequence of native mature alpha-lactalbumin but with all of the cysteines found at positions 6, 28, 61, 73, 77, 91, 111 and 120 in the full-length sequence of mature human alpha-lactalbumin mutated to other amino acids that do not form disulfide bridges, such as alanine. Thus, characteristics of proteins that may be utilized in accordance with the present invention include the protein of SEQ ID NO: 1. JPEG2025534430000003.jpg19158 where the bold type indicates the position of the cysteine ​​mutation in native human alpha-lactalbumin.

[0055] As reported in WO2010079362, additional amino acid residues, for example up to 20 amino acids, can be attached to the N- and / or C-terminus of the protein if convenient, for example for expression purposes. Thus, in particular, the recombinant protein shown in SEQ ID NO: 1 but with an additional methionine at the N-terminus has been used in the conjugates of the invention (SEQ ID NO: 2 shown below). JPEG2025534430000004.jpg19158

[0056] The polypeptide used in the complex is suitably in pure form and is suitably prepared by conventional peptide synthesis or by recombinant expression.In particular, the DNA encoding the required recombinant alpha-lactalbumin can be inserted into a suitable expression vector such as a plasmid, and then be used to transform host cells, for example, prokaryotic cells such as E. coli or eukaryotic cells such as certain insect cells, using conventional methods.

[0057] Suitable fatty acids or lipids include those known to produce biologically active complexes. These include, for example, the fatty acids described in WO2008058547. When salts are used, they are suitably water-soluble salts. Specific examples of suitable salts can include alkali or alkaline earth metal salts. In certain embodiments, the salt is an alkali metal salt, such as sodium salt or potassium salt. When used in pharmaceuticals, the salt will be pharmaceutically acceptable.

[0058] Particular examples of fatty acids or lipids for use in the present invention are those having 4 to 30 carbon atoms, such as 6 to 28, e.g., 8 to 26. In particular embodiments, the fatty acids or lipids have 10 to 24 carbon atoms, such as 12 to 22, e.g., 14 to 20, carbon atoms. In particular, the fatty acids or lipids will have 16, 17, 18, or 20 carbon atoms. The fatty acids may be saturated or unsaturated.

[0059] In particular, however, the conjugates of the present invention utilize fatty acids or fatty acid salts having 18 carbon atoms. In one embodiment, the conjugates of the present invention utilize fatty acids or fatty acid salts having 18 carbon atoms, wherein the fatty acid chain is unsaturated. In one embodiment, the fatty acid or fatty acid salt is a C18:1 fatty acid or a salt thereof. Specific examples include C18:1 fatty acids or salts thereof represented by the following formulas: CH3(CH2)7CH=CH(CH2)7COOH or CH3(CH2)7CH=CH(CH2)7COO. - In one embodiment, the fatty acid or salt thereof is oleic acid or oleate.

[0060] The complexes can be prepared using methods similar to those described in, for example, WO99 / 26979, WO2008 / 138348, WO2010 / 131237, WO2014 / 023976, WO2018 / 210759, and WO2022 / 073982, the contents of which are incorporated herein by reference. The complexes can be prepared by contacting the unfolded α-lactalbumin or a derivative thereof with a particular oleic acid or salt cofactor under ion exchange conditions, such as those found on an ion exchange column, or by incubating a solution of α-lactalbumin or a derivative thereof with a cofactor at elevated temperatures, for example, 50-80°C, for example, 50-70°C, particularly 55-60°C, to produce complexes suitable for use in the present invention.

[0061] However, these methods have generally focused on attempting to recreate the conditions under which proteins are unfolded and complexed with oleate ions. Such work has focused on using pure proteins, including recombinant mutant versions of the base protein, to facilitate the production of active complexes. However, such starting materials can also increase production costs.

[0062] It is known that complexes obtained with α-lactalbumin from sources other than human milk, in particular BAMLET obtained with bovine α-lactalbumin, have qualitatively similar effects on cells, in particular tumor cells, as HAMLET (see, for example, Rammer et al. (2010) Mol. Cancer Ther. 9 (1) 24-32). Therefore, if HAMLET or a composition based on HAMLET is used instead of BAMLET, the effects shown below using BAMLET will also be observed.

[0063] Dosage The amount of conjugate administered to an individual will depend on various factors, including the nature of the composition and risk factors. However, as a general rule, when administered orally, a single dose of 1 mg to 20 g of biologically active conjugate is used, with this amount being appropriately administered daily. A daily dosage can be, for example, at least about 1 mg, 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 750 mg, 1 g, 2 g, 3 g, 4 g, 5 g, 7.5 g, 10 g, 12.5 g, 15 g, or 17.5 g. Alternatively, or in addition, the daily dosage can be less than 25 g, 22.5 g, 20 g, 17.5 g, 15 g, 10 g, 7.5 g, 5 g, 4 g, 3 g, 2 g, 1 g, 750 mg, 500 mg, 400 mg, 300 mg, 200 mg, 100 mg, 75 mg, 50 mg, 25 mg, 20 mg, 15 mg, 10 mg, or 5 mg. Alternatively, the daily dosage of the complex can be between 0.1 g and 1 g per kg of body weight. The daily dosage can be at least or about 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.8 g, 0.9 g, or 1 g per kg of body weight. Alternatively, or in addition, the daily dosage may be less than 1.5g, 1g, 0.9g, 0.8g, 0.7g, 0.6g 0.5g, 0.4g, 0.3g, 0.2g, 0.1g per kg of body weight.

[0064] Food and drink composition The complex or pharmaceutical composition may be in the form of an additive or ingredient for a beverage or food, such as a beverage, especially drinking water, or a food, such as baby food, or a powder for mixing into a beverage, e.g., a protein shake. Such food and beverage compositions may be prepared using standard techniques. Similarly, the complex or pharmaceutical composition may be in the form of a composition for parenteral, or preferably, intravenous, nutritional provision.

[0065] The present invention also provides a food, beverage, food additive, or other nutritional composition comprising the complex as defined in the preceding aspects of the invention, in particular for use in the treatment of cancer or a metabolic-related condition.

[0066] Examples of such compositions include aqueous or dairy drinks, especially drinking water, baby food, nutritional compositions for parenteral or intravenous administration, food additives, such as powders, nutritional capsules, gels or tablets for mixing into drinks or foods.

[0067] overview Throughout the description and claims of this specification, the terms "comprises" and "includes" and variations of these terms, such as "comprising" and "comprises," mean "including but not limited to" and do not exclude other elements, wholes or steps. Furthermore, unless the context requires otherwise, the singular encompasses the plural, and in particular where the indefinite article is used, the specification should be understood as contemplating both the plural and the singular unless the context requires otherwise.

[0068] Preferred features of each aspect of the invention may be those described in relation to any of the other aspects. Within the scope of this application, it is expressly intended that the various aspects, embodiments, examples, and alternatives described in the preceding paragraphs, claims, and / or the following description and drawings, and particularly their individual features, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment may be combined in any way and / or combination, unless such features are inconsistent. [Brief explanation of the drawings]

[0069] One or more embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Figure 1]BAMLET treatment delays tumor progression in ApcMin / + mice. (a) Schematic of the treatment model. Ten-week-old female ApcMin / + mice were gavaged twice daily with 20 mg of BAMLET or PBS (sham) for 10 days. Mice were sacrificed 2 weeks (PBS: n = 4 + 5, BAMLET: n = 5 + 5) or 5 weeks (PBS: n = 5 + 5, BAMLET: n = 5 + 5) after the end of treatment (2 weeks or 5 weeks post-treatment, pt). (b) Ten-week-old female mice were administered 20 mg of BAMLET or PBS daily in their drinking water (dw) for 8 weeks (PBS: n = 5 + 5 + 5, BAMLET: n = 5 + 5 + 9) or for extended periods (8 w dw). (c) H&E-stained intestinal sections showing the progression of colon cancer from microadenomas (2 w pt) to polyps (5 w pt) and larger tumors (8 w dw) in the sham group. Representative sections are from n = 4-5 mice per group. (d) BAMLET prevented or delayed colon cancer progression at each time point. (e) The total number of polyps was significantly reduced in ApcMin / + mice treated with BAMLET. (f) The tumor area was smaller compared to the sham group. Data are presented as the mean ± SEM of two to three experiments (n = 9–15 mice in the sham group, n = 10–19 mice in the BAMLET-treated group). [Figure 2]The robust effect of BAMLET on intestinal gene expression is shown in (a) a heatmap comparing the intestinal gene expression profiles of BAMLET-treated ApcMin / + mice and sham-treated ApcMin / + mice. A time-dependent increase in the number of regulated genes was observed in mice treated with BAMLET via gavage (2 or 5 weeks after treatment, 2w or 5w pt), and the effect was confirmed in mice receiving BAMLET in their drinking water (8 weeks, 8w dw). (Red: upregulated genes; blue: downregulated genes; black: unregulated genes; cutoff fold change ≥ 2.0 compared to sham). (b) Venn diagram of significantly regulated genes in BAMLET-treated ApcMin / + mice confirming regulated genes at all time points. (c) Network analysis of these shared genes (n = 2339) revealed a major treatment effect. (d) Wnt / β-catenin signaling was inhibited in ApcMin / + mice treated with BAMLET. (e) Genes defining the tumor microenvironment were broadly inhibited, predicting reduced proliferation, angiogenesis, metastasis, and the PD-1 pathway. (e) Biological functions such as tumor growth, cell migration, invasion, and metastasis were inhibited. (f) Tumor microenvironment genes were also significantly modulated. [Figure 3]The long-term effects of BAMLET administration in drinking water are shown. (a) Schematic of the long-term treatment model. ApcMin / + mice received drinking water (dw) supplemented with BAMLET (20 mg / day) (n = 6 + 9) or PBS (sham) (n = 5 + 8) from the age of 10 weeks. (b) and (c) BAMLET treatment increased survival compared with sham-treated mice. (d) Reduction in polyp number and (e) weight loss in BAMLET-treated ApcMin / + mice compared with sham-treated ApcMin / + mice. (f) Gene expression analysis confirmed that the Wnt / β-catenin signaling pathway was activated in sham-treated ApcMin / + mice (drinking water supplemented with PBS for 15 weeks, 15w dw) but inhibited or unregulated in BAMLET-treated ApcMin / + mice (drinking water supplemented with BAMLET for 27 weeks, 27w dw). (g) β-catenin staining in intestinal sections of BAMLET-treated mice was reduced compared with sham-treated ApcMin / + mice. (h) Quantification of β-catenin in intestinal, lung, liver, and kidney tissue sections. Data are presented as the mean ± SEM of two experiments (n = 5 + 8 mice in the sham group, n = 6 + 9 mice in the BAMLET-treated group). [Figure 4]Figure 1 shows inhibition of PD-1 signaling by BAMLET supplementation in drinking water. (a) Gene expression analysis of intestinal RNA confirmed that the PD-1 pathway was strongly upregulated in sham-treated APCMin / + mice compared with healthy C57BL / 6 mice over a long-term follow-up period. (b) Genes in the PD-1 pathway were not modulated in BAMLET-treated APCMin / + mice compared with healthy C57BL / 6 mice (cutoff FC2, p<0.05). (c) Genes in the PD-1 pathway were inhibited in BAMLET-treated APCMin / + mice compared with sham-treated APCMin / + mice. (d, e) PD-1 staining of intestinal sections from sham-treated or BAMLET-treated APCMin / + mice was performed with an anti-PD-1 antibody (red = PD-1, blue = DAPI). (d) Swiss-roll preparation of intestinal sections from sham- or BAMLET-treated mice. The arrow indicates the location of the magnified tumor area in the representative image in (e) (n = 3 mice per group). (f) Quantification of PD-1 staining in Swiss-roll preparations comparing tumor and healthy areas in sham- and BAMLET-treated APCMin / + mice. Data are presented as mean ± SEM for n = 3 mice per group. (g, h) Quantification of PD-1 staining in intestinal sections from individual mice comparing tumor and healthy areas. [Figure 5]Figure 1 shows the inhibition of lung cancer by long-term BAMLET treatment. (a) Intestinal sections from BAMLET-treated or sham-treated APCMin / + mice were stained with β-catenin or TTF-1-specific antibodies, followed by H&E counterstaining and immunohistochemistry. (a, b, c) β-catenin staining and (d, e, f) TTF-1 staining were performed on healthy C57 / BL6 mice and sham- or BAMLET-treated APCMin / + mice. (a) C57 / BL6 mice showed a normal pattern of weak β-catenin staining in bronchial and bronchiolar epithelial cells. (b) A significant increase in overall β-catenin staining in sham-treated APCMin / + mice, including foci adjacent to bronchiolar epithelial cells with increased staining. (c) A decrease in β-catenin in BAMLET-treated APCMin / + mice compared with sham-treated APCMin / + mice. (d) Quantification of β-catenin staining in lung tissue for (a-c) n = 3 mice per group. (e) C57 / BL6 mice showed weak TTF-1 staining in bronchial and bronchiolar epithelial cells. (f) Significant increase in overall TTF-1 staining in sham-treated APCMin / + mice, including areas of proliferating cells with increased staining from bronchiolar epithelial cells. (g) Decreased TTF-1 staining in BAMLET-treated APCMin / + mice. (h) Quantification of TTF-1-positive areas in overall lung sections (see inset). Data are presented as mean ± SEM for n = 3 mice per group. [Figure 6]The effects of BAMLET in healthy C57BL / 6 mice were shown. BAMLET was administered to healthy C57BL / 6 mice by gavage (for 2 and 5 weeks) (n = 5) or in their drinking water (for 8 weeks) (n = 5). Gene expression analysis of intestinal tissue was performed. (a) Heatmap comparing gene expression profiles of mice that received BAMLET by gavage and were followed for 2 or 5 weeks (2w or 5w pt) or supplemented their drinking water with BAMLET for 8 weeks (8w dw) (red: up-regulated genes, blue: down-regulated genes, black: unregulated genes; cutoff fold change ≥ 1.5 compared to placebo, P < 0.05). (b) The total number of regulated genes was low in healthy C57BL / 6 mice (approximately 150 genes) without evidence of a toxic response to BAMLET. (c) Venn diagram of significantly regulated genes in BAMLET-treated C57BL / 6 mice. (d) Biofunction analysis of common genes confirmed in (c) with predicted effects on lipid metabolism, glucose metabolism, insulin resistance, and inflammation. (e) P-values ​​and Z-scores of regulated biological functions in BAMLET-treated C57BL / 6 mice after 5 weeks. (f) The highest regulated common genes were mostly enzymes related to carbohydrate, lipid, and protein degradation. (g) Schematic of the BAMLET effects discussed in this study. [Figure 7]Supplementary data for Figure 1 are shown, demonstrating that BAMLET treatment delays tumor progression in ApcMin / + mice. (a)–(e) Data from ApcMin / + mice treated by oral gavage and followed for 2 weeks after treatment (2w pt) (n = 5 + 5 for BAMLET, n = 4 + 5 for sham). (f)–(j) Data from ApcMin / + mice treated by oral gavage and followed for 5 weeks (5w pt) (n = 5 + 10 for BAMLET, n = 5 + 5 for sham). (a) and (f) Dissected micrographs of small pieces of intestine showing tumors (arrowheads) in BAMLET- or sham-treated ApcMin / + mice. (b) and (g) Whole histology specimens (arrowheads, tumors) of dissected intestinal sections stained with methylene blue (n = 4 mice per group). (c, h) The total number of polyps was significantly reduced in BAMLET-treated ApcMin / + mice compared with sham-treated ApcMin / + mice over the course of 2 and 5 weeks. (d, i) The number and size of detected polyps were reduced by BAMLET treatment (<0.5 mm, 0.5-2 mm, >2 mm). (e, j) Swiss roll sections of H&E-stained intestines show that BAMLET-treated ApcMin / + mice had smaller and fewer polyps than sham-treated mice (n = 4 mice per group). Data are presented as the mean ± SEM of 2-3 experiments (n = 9-15 mice for the sham group, n = 10-19 mice for the BAMLET-treated group). [Figure 8]ApcMin / + mice were administered BAMLET-supplemented drinking water or PBS for 8 weeks (8 weeks dw). (a) Micrographs of small sections of intestine showing tumors (arrowheads) in BAMLET- or sham-treated ApcMin / + mice after 8 weeks. (b) The total number of polyps was significantly reduced in BAMLET-treated intestines compared with sham. (c) The reduction in polyp number reflected all three sizes analyzed (<0.5 mm, 0.5–2 mm, >2 mm). Data are presented as the mean ± SEM of two experiments (n = 5 + 8 mice for the sham-treated ApcMin / + mice group and n = 6 + 9 mice for the BAMLET-treated ApcMin / + mice group). (d) Whole-tissue preparations of intestinal sections stained with methylene blue (arrowheads, tumors) (n = 4 mice per group). (e) Swiss-roll sections of H&E-stained intestines showing smaller and fewer polyps in BAMLET-treated ApcMin / + mice 8 weeks after treatment compared with sham (n = 4 mice per group). (f) Heatmap comparing gene expression profiles between sham-treated APCMin / + mice and mice administered BAMLET in drinking water (red: up-regulated genes, blue: down-regulated genes, black: unregulated genes, cutoff fold change ≥ 2.0 compared with healthy intestinal tissue). (g) Histogram showing the number of regulated genes. (h) Biological function analysis of regulated genes showed a significant reduction in cancer function in the BAMLET-treated group compared with sham. [Figure 9]Figure 1 shows the effect of BAMLET treatment on intestinal gene expression. (a) and (d) are heat maps comparing gene expression profiles between sham-treated ApcMin / + mice and mice treated with BAMLET via gavage. Mice were sacrificed 2 weeks (upper panel) or 5 weeks (lower panel) after the end of treatment (2w or 5w pt). (Red: upregulated genes, blue: downregulated genes, black: unregulated genes; cutoff fold change ≥ 2.0 compared to healthy intestinal tissue). (b) and (e) are histograms showing the number of regulated genes. (Top panel: 2w pt, bottom panel: 5w pt). Gene expression increased in sham-treated mice after 2 weeks, with a further increase observed after 5 weeks. In contrast, the number of regulated genes significantly decreased in BAMLET-treated ApcMin / + mice after 2 weeks, with a further decrease observed after 5 weeks. (c, f) Biofunctional analysis revealed a significant decrease in cancer-related functions in all treatment groups compared with the sham group. (g) Highly regulated genes in sham- and BAMLET-treated ApcMin / + mice. Cancer-related biofunctional functions were predominant in the sham group but inhibited in the BAMLET-treated ApcMin / + group. (h) Principal component analysis of mRNA profiles in whole intestinal tissue. BAMLET-treated mice clustered near healthy mice and away from the sham group. (i) A time-dependent decrease in the molecular mechanisms of cancer pathway genes was observed. [Figure 10]Figure 1 shows the inhibition of colon cancer-related gene expression by BAMLET treatment. (a) Heatmap showing the reduction in the number of colon cancer-related genes in ApcMin / + mice treated with BAMLET by gavage (2 or 5 weeks after treatment, 2w or 5w pt) and in drinking water (8 weeks, 8w dw) compared with sham-treated ApcMin / + mice (red: up-regulated genes, blue: down-regulated genes, black: unregulated genes, cutoff fold change ≥ 2.0 compared to healthy intestinal tissue). (b) Histogram showing the number of colon cancer-related genes. (c) Highly regulated colon cancer genes confirmed by biofunctional analysis. [Figure 11] The effect of BAMLET treatment on tumor markers by immunohistochemistry of intestinal sections is shown. The levels of tumor markers VEGF, Ki67, cyclin D1, and β-catenin were reduced in BAMLET-treated ApcMin / + mice compared to sham-treated ApcMin / + mice. (a) Quantification of (b) 5 weeks after oral gavage administration. (c) Quantification of (d) 8 weeks after BAMLET-supplemented drinking water administration. Data are presented as mean ± SEM, n = 5 mice per group. [Figure 12]Figure 1 shows the effect of BAMLET on colon cancer cells. (a-b) Live-cell confocal images showing the uptake of BAMLET labeled with AleXa Fluor-568 (21 μM, magenta) by DLD1 colon cancer cells. Nuclei are counterstained with DAPI (blue). (b) Quantification of uptake in (a). (c) Membrane response to BAMLET in giant unilamellar vesicles (GUVs, magenta). (d) Quantification of the response in (c). (e) Dose-dependent increase in TUNEL staining in BAMLET-treated DLD1 colon adenocarcinoma cells (n = 50 cells per group). Scale bar = 20 μm. (f) Quantification of TUNEL staining. (g) Tumor cell death quantified by PrestoBlue and ATPlite™. BAMLET (orange) was compared to α1-oleic acid (blue) in DLD1 cells. (h) Tumor cell survival determined by colony assay of human colon cancer DLD1 and HT29 cells. (i) Quantification of the colony assay in (h) compares the effects of BAMLET (orange) with those of BAMLET and α1-oleic acid (blue). (j) In vivo imaging of tumor-bearing APCMin / + or C57BL / 6 mice (both 18 weeks old) treated with VivoTag 680-labeled BAMLET via gavage. Compared to C57BL / 6 mice (n = 4 + 4), BAMLET retention was higher at 24 hours (n = 3) and 48 hours (n = 3). (k) Quantification of fluorescence intensity in intestinal sections 24 hours (top) and 48 hours (bottom) after BAMLET administration. (l) BAMLET staining in the tumor area of ​​intestinal sections from (k) using immunohistochemistry. m shows quantification of BAMLET staining from (l). Data are presented as mean ± SEM from three independent experiments for all cell culture experiments, or n = 3–4 mice per group. [Figure 13]Supplementary data for Figure 3 are shown, showing gene expression analysis of intestinal tissue from BAMLET-treated ApcMin / + mice compared with sham-treated ApcMin / + mice. (a)–(d) show that the molecular mechanisms of cancer, colon cancer metastasis, tumor microenvironment, and Wnt / β-catenin signaling pathway were downregulated in the BAMLET-treated group. [Figure 14] Supplementary data for Figure 3 are shown, illustrating the effects of long-term treatment on major extraintestinal tissues. (a) Macroscopic appearance of the lungs, liver, kidneys, and spleen from sham-treated (15 weeks, dw) and BAMLET-treated (27 weeks, dw) APCMin / + mice compared with healthy C57BL / 6 mice sacrificed after long-term follow-up. Changes in tissue morphology indicated systemic complications in the sham group. These effects were reduced in the group receiving BAMLET-supplemented drinking water. (b) Histological analysis of H&E-stained sections. n = 4 per group. (c) Gene expression analysis of lungs, liver, kidneys, and spleen from BAMLET-treated and sham-treated APCMin / + mice. Heatmap comparing gene expression profiles (red: up-regulated genes; blue: down-regulated genes; cutoff fold change ≥ 2.0 compared to sham). (d) The total number of regulated genes in lung, liver, kidney, and spleen tissues of BAMLET-treated mice compared with those of sham (cutoff fold change ≧2.0 compared with sham). (e) The molecular mechanisms of cancer pathways were strongly regulated by BAMLET treatment, as well as colon cancer metastasis, tumor microenvironment, and Wnt signaling pathway. [Figure 15]Figure 1 shows the effect of BAMLET on systemic β-catenin staining. β-catenin staining was quantified in tissue sections from liver and kidney tissues of sham-treated and BAMLET-treated APCMin / + mice and compared with healthy C57BL / 6 controls. Representative sections, n = 3 mice per group. (a-b) Decreased levels of β-catenin staining in liver and kidney tissues from APCMin / + mice treated chronically with drinking water supplemented with BAMLET are consistent with inhibition of Wnt / β-catenin signaling outside the intestinal compartment. [Figure 16] Supplementary data are shown in Figure 3, showing gene expression analysis of lungs, liver, and kidneys from BAMLET-treated mice compared with sham-treated ApcMin / + mice. The Wnt / β-catenin signaling pathway was strongly upregulated in lungs, liver, and kidneys from sham-treated ApcMin / + mice, but downregulated in BAMLET-treated ApcMin / + mice. DETAILED DESCRIPTION OF THE INVENTION

[0070] Early, localized targeting of growing tumors can reduce the risk of tumor progression and metastatic disease. While surgery is highly effective and can permanently remove tumors, chemotherapy is often required depending on the tumor's classification, predicted risk, and lymph node spread prior to surgery. Chemotherapeutic agents also target healthy tissue, but the benefits of treatment must be weighed against the risks of toxicity and associated morbidity.

[0071] Colorectal cancer is the leading cause of death, with over 180,000 cases diagnosed annually in the United States (1). Genetic predisposition is a risk factor and mutations affecting the APC gene can cause both typical and attenuated familial adenomatous polyposis (FAPC). 2(Ref. 1). The APC gene and the Wnt / β-catenin signaling network regulate intestinal cell proliferation and physiology, and loss-of-function mutations can lead to cellular overgrowth and polyp formation (Ref. 3). Patients with APC mutations can develop multiple tumors in the colon during the first decades of life (Ref. 4), and intestinal tumors from Apc mutant mice show similar kinetics (Ref. 5). In addition, several lifestyle-related factors have been linked to colorectal cancer, including diet, lack of exercise, smoking, and alcoholism (Refs. 6–9).

[0072] As disclosed herein, BAMLET complexes (bovine α-lactalbumin made lethal to tumor cells) were administered to APCs of intestinal cancer. Min / + BAMLET was investigated as an oral therapeutic agent in a model. BAMLET is a complex formed by partially unfolded bovine α-lactalbumin and oleic acid, belonging to a novel class of oncogenic molecules with documented cancer specificity (see 10-13). In this study, intestinal polyp formation was inhibited by 10 days of BAMLET gavage, and long-term protection was achieved by administering BAMLET in drinking water. Surprisingly, however, health benefits of oral BAMLET treatment, including effects on lipid metabolism, glycolysis, and insulin resistance, were detected in C57BL / 6 mice, whose systemic disease development was prevented by BAMLET treatment and who did not develop tumors. These beneficial effects and the apparent lack of toxicity open up the possibility of local administration of BAMLET to prevent or treat intestinal cancers and their systemic consequences.

[0073] This study investigated the potential of BAMLET as an oral therapeutic tool for intestinal cancer. BAMLET was retained in tumor tissue for at least 48 hours after the first oral dose. A 10-day gavage treatment was sufficient to suppress tumor growth, and supplementation of drinking water with BAMLET for 8 weeks prevented tumor growth, suppressed oncogene expression, and resulted in a nearly healthy phenotype. Long-term treatment resulted in prolonged tumor growth delay and APC suppression. Min / +Remarkably, long-term BAMLET treatment inhibited the PD-1 signaling pathway and prevented the progression of systemic disease affecting the lungs, liver, kidneys, and spleen. Min / + These compelling therapeutic effects in mice suggest that the therapeutic and preventive potential of BAMLET should be further investigated.

[0074] The molecular basis of these therapeutic effects was analyzed by gene expression analysis. Genes defining the tumor microenvironment were significantly increased in placenta-treated APCs. Min / + These findings suggest that BAMLET actively protects the tissue environment by inhibiting or preventing key cancer-related gene networks from being expressed. Several genes were strongly upregulated in the treated mice but absent or weakly regulated at some early time points in the BAMLET group. This included genes driving metastasis, tumor growth, angiogenesis, and the Wnt / β-catenin signaling pathway. While these effects may reflect delayed tumor growth in the treated mice, the observations suggest that BAMLET actively protects the tissue environment by inhibiting or preventing key cancer-related gene networks from being expressed. Some of these effects were still detectable after long-term follow-up, suggesting that BAMLET administration in drinking water maintains antitumor pressure by eliminating emerging cancer cells and reprogramming gene expression in intestinal tissue.

[0075] Unexpectedly, the PD-1 signaling pathway was strongly inhibited by BAMLET for a long period of time. PD-1 and its ligand, programmed cell death ligand 1 (PD-L1), are immunotherapy targets with promising results in several clinical trials for colorectal cancer, lung cancer, renal cell carcinoma, and breast cancer (see 16-23). ​​Immune checkpoint therapy blocks the PD-1 / PD-L1 interaction by directly targeting tumor cells or by indirectly enhancing or restoring T cell function and thus antitumor activity (see 24, 25). BAMLET-treated APCs Min / +Mice showed that the intestinal PD-1 signaling pathway was upregulated and PD-1 pathway activation was reduced compared to the sham group, which showed enhanced PD-1 staining. Therefore, BAMLET-treated APC Min / + Although mice were observed to have residual polyps over time, there was no evidence of an ongoing response in their tumors, suggesting a protective and largely healthy PD-1 phenotype. This observation, and the lack of PD-1 signaling, suggests that in addition to killing tumor cells, BAMLET treatment reprograms local tumor tissue to a more passive state.

[0076] Furthermore, BAMLET treatment reduced Wnt / β-catenin signaling and β-catenin protein levels in the intestine. Notably, BAMLET treatment also affected major organs outside the intestine, reducing Wnt / β-catenin signaling and β-catenin staining in the lungs, liver, and kidneys, potentially increasing the risk of cancer progression. In parallel, significant effects on these organs were detected, including fibrotic changes in the lungs, hyperlipidemia in the liver, and changes in the renal cortex and papilla. In the lungs, these changes were accompanied by the formation of proliferating foci of proliferation that protruded from the bronchial lining into the parenchyma. These foci stained for the lung adenocarcinoma marker TTF-1 (see reference 15), suggesting a pulmonary origin rather than metastasis from an intestinal tumor. BAMLET-treated APCs Min / + The frequency and size of these tumor-like areas were reduced in mice. The Wnt / β-catenin pathway functions in adult lung epithelium, and increased Wnt / β-catenin signaling can be associated with epithelial cell damage and hyperplasia, impairing epithelial-mesenchymal crosstalk in idiopathic pulmonary fibrosis (IPF) (26). Furthermore, patients with familial colonic polyposis have been reported to develop lung cancer (27), suggesting that aberrant Wnt / β-catenin signaling may drive tumor development, a process that appears to be affected by BAMLET administration in drinking water.

[0077] The effects of BAMLET on health parameters in tumor-free mice are remarkable. The beneficial effects on lipid metabolism, glucose metabolism, and reduced insulin resistance suggest that BAMLET may accelerate lipid breakdown in intestinal tissue, reduce glucose levels, and increase insulin sensitivity in pancreatic tissue. This study suggests that the complex formed by α-lactalbumin and oleic acid may provide fundamental health benefits in the intestinal tract and other organs in addition to beneficial therapeutic properties in cancer models. Collectively, these results suggest that oral treatments may have widespread systemic effects, a potential paradigm shift for cancer prevention and therapeutic intervention.

[0078] method Preparation of BAMLET and α1-oleic acid The BAMLET conjugate was made by mixing bovine α-lactalbumin (Sigma, Cat# L5385) with oleic acid (Sigma, Cat# O1008). α1 was synthesized using Fmoc solid-phase chemistry (Mimotope). The α1 sequence is aa 1-39 Ac-KQFTKAELSQLLKDIDGYGGIA-LPELIATMFHTSGYDTQ-OH.

[0079] Apc Min / + Intestinal cancer model in mice Apc Min / + Mice were obtained from Jackson Laboratories at approximately 8 weeks of age. Genotyping was performed by PCR analysis of genomic DNA obtained from blood drawn from the retro-orbital sinus. Multiple tumors appeared in the small intestine between 8 and 10 weeks of age. To reduce stress from transportation, mice were acclimated for approximately 2 weeks in the local animal facility at BMC, Lund University.

[0080] In the BAMLET treatment protocol, 10-week-old female mice (n = 10 per group) were orally gavaged with 20 mg of BAMLET in 400 μl of PBS twice daily for 10 days. Mice were deprived of water or food for 5 hours prior to BAMLET administration. Food and water were provided 30 minutes after oral BAMLET administration. Sham-treated mice were gavaged with 400 μl of PBS. After the end of treatment, mice were sacrificed at 2 and 5 weeks, and intestinal tissue samples were collected for further analysis.

[0081] In the BAMBLET prevention protocol, 10-week-old female Apc Min / + Mice (n = 10 mice per group) were given BAMLET (20 mg / day in 5 ml PBS) daily in their drinking water for 8 weeks and sacrificed at 18 weeks of age. Similar treatments were used in the survival study group, where mice were observed up to 37 weeks of age.

[0082] Tumor enumeration and sample collection Mice were sacrificed by isoflurane inhalation. Tumor enumeration and sample collection were as previously described (28). Tumor number and size were determined using a dissecting microscope (Olympus) and assessed by three anonymous investigators.

[0083] Methylene blue staining The opened intestinal segments were spread flat between sheets of filter paper and fixed overnight in 10% neutral buffered formalin. Formalin-fixed sections were transferred to 70% ethanol and stained with 0.2% methylene blue (Sigma, #M9140). Stained sections were rinsed with deionized water and imaged under a dissecting microscope.

[0084] Histology and immunohistochemistry Swiss rolls of longitudinally opened intestinal segments were fixed overnight in 10% neutral-buffered formalin. Samples were embedded in paraffin, and 5-μm-thick sections were further processed for histology and immunohistochemistry using antibodies against bovine α-lactalbumin (ThermoFisher Scientific, Cat#A10-128A), β-catenin (CellSignaling, Cat#9562), cyclin D1 (ThermoFisher Scientific, Cat#SC8396), Ki-67 (BD Biosciences, Cat#556003), VEGF (Abcam, Cat#ab46154), PD-1 (Abcam, Cat#ab214421), and TTF-1 (Abcam, Cat#ab227652) as previously described (28) with minor modifications. Citrate buffer (Dako Target Retrieval Solution, Agilent, Cat. #S1699) was used for antigen retrieval of cyclin D1, Ki-67, VEGF, bovine α-lactalbumin, and β-catenin staining. EDTA buffer (Abcam, Cat. #ab64216) was used for antigen retrieval of TTF-1 staining. Immunohistochemistry was quantified using ImageJ. Hematoxylin and eosin (H&E) staining was performed using hematoxylin (ThermoFisher Scientific, Cat. #7211) followed by counterstaining with Eosin-Y (ThermoFisher Scientific, Cat. #7111). Images were captured using an AX10 microscope or a Hamamatzu Nanozoomer scanner (Carl Zeiss), and the DAB-positive β-catenin antibody areas (4–10) of each organ cross-section were quantified using ImageJ.

[0085] For PD-1 immunofluorescence staining, paraffin sections were deparaffinized in xylene, rehydrated in reduced ethanol, and then washed with deionized water. Slides were then immersed in target retrieval solution (Dako, S1699) and boiled for 20 minutes, followed by permeabilization with 0.25% Triton in PBS at room temperature for 30 minutes. Sections were then incubated overnight at 4°C in a blocking solution consisting of 5% goat serum in PBS for 1 hour at room temperature before being treated with rabbit monoclonal anti-mouse PD-1 antibody (Abeam-ab214421, 1:150) in 1% goat serum. Slides were then washed with 0.025% PBS-T and stained with goat anti-rabbit AleXa Fluor-568 secondary antibody (1:200, 1 hour at room temperature, Invitrogen cat#A11034). Nuclei were counterstained with DAPI for 15 min, washed with PBS, and then mounted with Fluoromount aqueous mounting medium (Sigma, F4680). Images were captured with a Hamamatzu Nanozoomer scanner, and fluorescence intensity was quantified using ImageJ.

[0086] BAMLET real-time in vivo fluorescence imaging BAMLET was labeled using the VivoTag 680XL Protein Labeling Kit (Perkin Elmer). Min / + Mice were orally gavaged with 10 mg of VivoTag 680-labeled BAMLET in 200 μl of PBS. Intestinal tissue samples were collected 24 or 48 hours later and imaged using an IVIS Spectral Imaging System (Perkin Elmer). BAMLET signals were acquired using a fluorescence setting of 680 nm excitation.

[0087] Transcriptome analysis Approximately 5 mg of tissue was homogenized using a Tissuelyser (Qiagen), total RNA was extracted using the RNeasy kit (Qiagen), amplified using the GeneChip 3'IVT Express kit, hybridized to mouse genome 430 PM array strips, and scanned using the GeneAtlas system (Affymetrix). Data were normalized using Robust Multi Average implemented in Transcriptome Analysis Console software (v.4.0.1.36, Applied Biosystems, ThermoFisher Scientific). Relative expression was analyzed by ANOVA using empirical Bayes methods, and genes with absolute fold changes greater than 1.5 or 2.0 were considered differentially expressed. Heatmaps were constructed using Graphpad Prism 9, and differentially expressed genes were analyzed using Ingenuity Pathway Analysis software (IPA, Qiagen).

[0088] cell culture Colorectal adenocarcinoma cells (DLD1) and colorectal adenocarcinoma cells (HT29) were purchased from the American Type Culture Collection (ATCC, VA, USA). A549 and DLD1 cells were cultured in RPMI-1640 supplemented with 1% non-essential amino acids, 1 mM sodium pyruvate, 50 μg / ml gentamicin, and 5–10% fetal calf serum (FCS) at 37°C and 5% CO2. All cell culture reagents were purchased from ThermoFisher Scientific. Cells were subcultured every 3 days.

[0089] Cell death assay Two assays were used as indirect measures of cell death: the luminescence-based ATPlite™ kit (Perkin Elmer) and the Prestoblue assay (ThermoFisher Scientific). 4Cells (1000 cells / well) were seeded in serum-free RPMI-1640 on 96-well plates and treated with BAMLET and α-1-oleic acid at different concentrations (7, 21, and 35 μM) and cultured for 1 h. Then, FCS was added at a concentration of 5%, and the cells were continuously cultured at 37°C for 2 h, at the end of which, two kits were used according to the manufacturer's instructions. Brightness and fluorescence were measured using a microplate reader (Infinite F200, Tecan). Experiments were performed in triplicate and in duplicate.

[0090] Colony assay Cells were plated in 12-well plates (1 × 10 3 Cells were seeded at 1000 x g (7, 21, and 35 μM) and cultured overnight. Cells were treated with different complexes of BAMLET (7, 21, and 35 μM) or αl-oleic acid (7, 21, and 35 μM) in serum-free medium and cultured at 37°C and 5% CO2 for 1 hour. Culture was continued after adding FCS to the medium. Ten days after treatment, cells were washed once and fixed with cold methanol (300 μl) on ice for 15 minutes. Finally, cell colonies were stained with hematoxylin (ThermoFisher Scientific, Cat#7211) for 5 minutes, and images were captured under a dissecting microscope (Carl Zeiss). Experiments were repeated twice for each cell line.

[0091] Live cell imaging assay To visualize the cellular uptake of BAMLET, cells were cultured in a 6-well ibidi chamber (3.5 x 10 4 Cells / well) were seeded overnight and then treated with Janelia Fluor-549-labeled (TOCRIS, Cat#6147) BAMLET mixed with unlabeled BAMLET (21 μM) for 1 hour at 37°C. Nuclei were counterstained with DAPI (Abcam Cat#ab228549, 1:1,000) for 5 minutes, and uptake of labeled BAMLET was captured with an LSM900 laser scanning confocal microscope with an oil-immersion ×63 objective (Carl Zeiss).

[0092] Giant unilamellar vesicle experiments Giant unilamellar vesicles (GUVs) were formed by hydrogel-assisted swelling according to established protocols (see 29, 30) with the modifications described above (see 31). Briefly, cover glasses were sonicated in 1 M NaOH solution (30 min), rinsed three times in Milli-Q water, and sonicated again (30 min). Cover glasses were plasma-etched (1 min) using a BD-20 laboratory corona treatment device (Electro Technic Products Inc.) to make the surface clean and hydrophilic. A thin film of a 1% (w / v) solution of molten ultra-low gelling temperature type IX-A agarose (Sigma) was deposited on the cover glasses to prepare the reaction bed for GUV formation. Cover glasses were placed in an AttoFluor® cell chamber (ThermoFisher Scientific). Subsequently, 25 μl of egg phosphatidylcholine (#840051P - Avanti Polar Lipids) in chloroform (25 mg / ml) doped with 4% v / v rhodamine C (1 mg / ml, Sigma) was deposited onto the gelled agarose surface, and the solvent was evaporated with nitrogen gas. The lipid hydrogel film was rehydrated with 200 mM sucrose in PBS, pH 7.2, for 1 hour and then transferred to 200 mM glucose in PBS, pH 7.2, for sedimentation. GUVs were treated with 21 μM BAMLET for 1 hour and then allowed to sediment overnight before being seeded onto glass coverslips for visualization.

[0093] TUNEL assay DNA fragmentation was detected using a terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay (Click-iT TUNEL Alexa Fluor 488 Imaging Assay Kit, ThermoFisher Scientific, #C10245). DLD1 cells were cultured in 8-well chamber slides (2 × 10 4Cells were seeded in 1000 x g (1000 x g) of 1000 x g (1000 x g) in 1000 x g of ... Fluorescence intensity was quantified by ImageJ.

[0094] statistical analysis method All in vitro experiments were repeated at least twice. Data are expressed as mean ± SEM. Gaussian distribution was determined by the D'Agostino-Pearson normality test. For data that followed a Gaussian distribution, a Student's t-test was used. Other data sets were analyzed by the Mann-Whitney U test. Differences between control and treatment groups were determined by ANOVA followed by Tukey's multiple comparison test. Significance was designated *P<0.05, **P<0.01, and ***P<0.001. Differences in survival rates were assessed by Kaplan-Meier analysis using the log-rank (Mantel-Cox) test.

[0095] research approval The experiments were approved by the Malmo / Lund Animal Experimentation Ethics Committee of the District Court of Lund, Sweden (#01302-20). Animal care and procedures followed institutional, national, and European Union guidelines, including Directive 2016 / 63 of the European Parliament and of the Council, the Swedish Animal Welfare Act (Djurskyddslagen 1988:534), the Swedish Welfare Act (Djurskydssforordningen 1988:539), and Institutional Animal Care and Use Committee (IACUC) guidelines. [Example]

[0096] Example 1: Oral BAMLET treatment reduces intestinal tumor growth Local targeting of growing tumors is essential to reduce tumor growth and the risk of metastatic disease. This study demonstrates that Apc, a mutation associated with hereditary and sporadic human colorectal cancer, causes intestinal polyps to grow to form large tumors. Min / + We focused on tumor surveillance by the BAMLET complex in mice.

[0097] Apc starting at 10 weeks of age, when tumor formation begins Min / + Two treatment protocols were used for the administration of BAMLET to the mouse intestinal tract (Figure 1a, b). According to protocol 1, mice were administered BAMLET by oral gavage twice daily for 10 days (Figure 1a) and sacrificed 2 or 5 weeks after treatment. According to protocol 2, mice were continuously administered BAMLET in their drinking water and sacrificed after 8 weeks or were followed for a longer period until their health deteriorated. Control mice were administered PBS (sham group) (Figure 1b).

[0098] In this study, all placebo-treated APCs Min / +Mice developed intestinal tumors. Tumor growth was quantified as an increase in tumor number and size. Rapid tumor growth in sham-treated mice was visualized by high-resolution imaging of H&E-stained tissue sections, showing an increase in tumor size from focal lesions (2 weeks) and fully formed polyps (5 weeks) to confluent tumors occupying most of the intestinal wall (Figure 1c). The progressive increase in polyp number and area was further visualized in H&E-stained "Swiss-roll" preparations of whole intestinal sections (Figure 1c and Figure 7). Polyps were counted by visual inspection, and counts were confirmed after methylene blue staining (Figure 7).

[0099] BAMLET treatment reduced the number of small tumors, fully formed polyps, and confluent tumors along the intestinal wall (Figure 1d). Normal villous structure was detected in the majority of sections after short-term BAMLET treatment (Figure 1d). The total number of polyps decreased 2 and 5 weeks after oral gavage administration, suggesting a rapid and sustained therapeutic effect (Figures 1e, 1f, and 7). Administration of BAMLET in drinking water reproduced the protective effect of oral gavage administration (Figure 1d). After 8 weeks, polyp number and polyp size were significantly reduced compared to the placebo group (Figures 1e, 1f, and 8). These results suggest a potent antitumor effect of BAMLET, which affects established tumors and prevents tumor growth.

[0100] Example 2: Inhibition of cancer-related gene expression by BAMLET To characterize tumor response to BAMLET, whole-genome transcriptome profiling of total intestinal RNA was performed. Gene expression profiles were compared between tumor-growing sham and BAMLET-treated APCs. Min / + Compared to healthy C57BL / 6 mice without intestinal tumors, a large number of genes were up-regulated in the sham-treated mice, and the most highly up-regulated biological functions were all cancer-related (Figures 8-10). In contrast, gene expression was significantly higher in the BAMLET-treated APCs, including cancer-related biological functions that were predominant in the sham group. Min / +Mice were significantly less affected. Principal component analysis (PCA) showed that BAMLET-treated mice clustered near healthy mice and away from the sham group, consistent with the lack of tumor growth. The inhibitory effects involved the molecular mechanisms of cancer gene networks (Figures 8 and 9) and colon cancer-related genes (Figure 10).

[0101] Example 3: Primary effect of BAMLET on the tumor environment To further analyze the mechanism of protection, gene expression profiles were analyzed in BAMLET-treated APCs. Min / + Direct comparisons were made between mice treated with BAMLET and the placebo group (Figure 2a, b). A strong therapeutic effect was noted across gene expression in mice receiving BAMLET via oral gavage or drinking water. Modulated genes included the Wnt / β-catenin signaling pathway (Figure 2c), which is known to regulate angiogenesis, tumor cell mobility, tumor growth, metastasis, and specifically colorectal cancer metastasis, as well as genes defining the tumor microenvironment (Figure 2d). Cancer-related biological functions were inhibited (Figure 2e), and molecular mechanisms of cancer pathway genes, including genes in the Ras signaling pathway, were weakly expressed. CD44, which binds SPP1 (osteopontin) and its ligand, was the most strongly inhibited gene in the tumor microenvironment pathway (Figure 2f).

[0102] The protective effect was confirmed by staining of intestinal tissue sections for the tumor markers vascular endothelial growth factor (VEGF), Ki67, cyclin D1, and β-catenin (Figure 11). All markers were significantly higher in BAMLET-treated APCs. Min / + It was less strongly expressed in the mouse group, consistent with the lack of tumor growth.

[0103] Example 4: BAMLET is internalized by cancer cells and retained in cancer tissues The efficacy of BAMLET treatment is consistent with a direct effect on emerging tumor cells and reprogramming of the active tissue environment. Cell studies demonstrated a direct, dose-dependent effect of BAMLET on colorectal adenocarcinoma cell lines (Figure 12). BAMLET was rapidly internalized into the cytoplasm and nucleus of DLD1 cells (Figure 12a, b). A rapid membrane response to the complex was recorded within giant unilamellar vesicles (GUVs) composed of phosphatidylcholine, where BAMLET induced rapid blebbing, tubulation, and eventual vesicle division (Figure 12c, d). An apoptosis-like response, evidenced by DNA strand breaks, was detected in the nuclei of treated cells (Figure 12e, f). BAMLET induced a rapid, dose-dependent decrease in cell viability (Figure 12g), and a sustained effect was recorded in colony assays, quantifying colony-forming cells after 10 days of proliferation (Figure 12h, i). The tumoricidal effect of BAMLET was similar to that of the α1-oleate conjugate currently being used in clinical trials. Cell death was accompanied by DNA strand breaks detected by TUNEL staining, suggesting the effect of BAMLET on chromatin structure, which was also observed for HAMLET and α1-oleate (Figure 6g, h). Cell studies demonstrated a rapid, sustained, and dose-dependent effect of BAMLET on the viability of colorectal adenocarcinoma cells.

[0104] To investigate whether BAMLET is retained in the intestine of tumor-bearing mice, VivoTag 680-labeled BAMLET was administered by oral gavage to 18-week-old APCs. Min / + BAMLET-treated healthy C57BL / 6 mice were used as controls and observed by whole-body imaging. Significant retention of BAMLET was observed after 24 and 48 hours. Min / + The BAMLET-treated APCs were detected in mice but not in C57BL / 6 mice treated with BAMLET, suggesting that BAMLET is retained in intestinal tumor tissues in vivo (Fig. 12k, l). Min / +Intestinal tissue sections from the mice were further examined immunohistochemically using a BAMLET-specific antibody. BAMLET staining was detected in intestinal tissue sections from BAMLET-treated mice. Peripheral exfoliation of tumor fragments stained for BAMLET was detected in some sections (Figure 12).

[0105] Example 5: Long-term effects of BAMLET on tumor growth and systemic disease Supplementation of drinking water with BAMLET had a sustained protective effect against tumor growth (Fig. 3a). Kaplan-Meier analysis showed that BAMLET-treated APCs Min / + Increased survival rates were detected in the treated mice compared to the sham group (Figure 3b, c). Long-term BAMLET treatment reduced total polyp number and polyp size and prevented weight loss compared to the sham group (Figure 3d, e).

[0106] Intestinal gene expression profiles were compared between sham-treated and APCs at long-term follow-up. Min / + Mouse groups and BAMLET-treated Apc Min / + The expression patterns of the oncogenes and the Wnt / β-catenin signaling pathway, which were upregulated in the sham group and inhibited in the BAMLET-treated group, were significantly different between the two groups. This included the molecular mechanisms of oncogenes and the Wnt / β-catenin signaling pathway, which were upregulated in the sham group and inhibited in the BAMLET-treated group (Fig. 3f, Fig. 13). Further support for this difference in gene expression was provided by β-catenin staining, which was significantly reduced in the BAMLET-treated mice (Fig. 3g), demonstrating the inhibition of the Wnt / β-catenin pathway by BAMLET at the protein level.

[0107] Example 6: Inhibition of PD-1 signaling pathway in BAMLET-treated mice Gene expression analysis further confirmed that the programmed death receptor 1 (PD-1) signaling pathway was upregulated in the sham group compared with healthy mice. Affected genes included the T cell-related genes Rasgrp1, Cblb, and Lcp2 (Figure 4a). In contrast, BAMLET-treated APCs Min / +Mice showed reduced activation of the PD-1 pathway in tumor-free and tumor areas (Fig. 4b).

[0108] BAMLET-treated Apc Min / + In a direct comparison between mice and the sham group, the PD-1 pathway was identified as the most strongly downregulated (Figure 4c). Genes related to HLA class II histocompatibility antigens (Hla-dmb, Hla-dqb1, Hla-dqa1, Hla-drb5, and Hla-dma), IL2 receptors (IL2rg, Il2rb), and growth factors (Tgfb1) were significantly downregulated in BAMLET-treated APCs. Min / + It was down-regulated in mice compared with sham (Fig. 4c).

[0109] PD-1 staining was clearly detected in the sham group by immunohistochemistry and was more prominent in the tumor area than in the adjacent healthy tissue (Figure 4d). In contrast, PD-1 staining was significantly higher in the BAMLET-treated APCs. Min / + In mice, it was significantly lower in tumor and healthy tissue areas, suggesting the effect of BAMLET on PD-1 at the protein level.

[0110] Example 7: Evidence of protection against systemic disease During follow-up, macroscopic changes were observed in the intestine, peritoneal cavity, and major organs. The intestine and spleen were enlarged, the liver and kidneys were discolored, and the lungs appeared pale and firm. These changes were consistent with those observed in BAMLET-treated Apc mice, except for a moderate enlargement of the spleen in individual mice. Min / + It was much less apparent in mice (Fig. 14a).

[0111] Disease response was further examined by histopathology. Min / +The lungs of the mice showed evidence of thickened alveolar septa and reduced alveolar spaces, suggesting cellular hyperplasia or focal collapse of the lung parenchyma. Liver tissue showed evidence of centrilobular micro- and macro-vacuolar steatosis, or "fatty liver," and binucleated stem cells were observed. Spleens in the sham group showed a loss of lymphoid foci and a more disorganized arrangement of lymphoid cells (Figure 14b).

[0112] The systemic disease response was accompanied by increased β-catenin staining in different organs (Figure 15). Min / + In mice, staining was intense in the multi-layered lining of the bronchial tree and in the thickened septa between alveoli. Focal cell aggregates also formed along the renal pelvis, with greater overall staining intensity within the renal papilla. Min / + Intense and diffuse β-catenin staining was also detected in the liver of mice. In contrast, BAMLET-treated Apc Min / + Mice showed an overall decrease in β-catenin staining in all tissues, suggesting a therapeutic effect outside the intestinal compartment. In contrast to the intestine, BAMLET staining was not detected in the lung, liver, or kidney (Figure 15).

[0113] β-catenin staining was observed in mock-treated APCs Min / + In the lungs of mice, we further detected intensely stained areas corresponding to cross sections of the bronchi. Patterns of cellular proliferation were detected in these areas generating multilayered bronchial walls and in cell clusters apparently extending from the bronchial walls, suggesting tumor formation (Fig. 5a-d). Further staining was performed using an antibody against thyroid transcription factor-1 (TTF-1), which is highly expressed in lung adenocarcinoma and is used as a diagnostic marker for lung cancer (see 15). TTF-1 is strongly expressed by proliferating cells, and TTF-1 staining was overlaid with β-catenin staining (Fig. 5). The number of areas with proliferating cells, as well as the level of TTF-1 staining in those areas, were significantly correlated with the progression of BAMLET-treated APCs. Min / + It was significantly reduced in mice (Fig. 5e).

[0114] The systemic therapeutic effect was compared between the sham group and the BAMLET-treated APC Min / + This was confirmed by gene expression analysis comparing BAMLET-treated APC mice (Figures 14 and 16). Min / + Further differences were observed in colon cancer metastasis genes and tumor microenvironment pathways in these organs, with up-regulated genes being upregulated in the lungs, liver, and kidneys of sham-treated mice compared to the mice group (Figure 14).

[0115] The results suggest that in addition to its antitumor effects in the intestine, BAMLET treatment may modulate other tissues to make them less susceptible to cancer development by inhibiting the molecular mechanisms of cancer pathways and metastasis-promoting genes within the tumor microenvironment network.

[0116] Example 8: Lack of toxicity and beneficial health effects in healthy mice To confirm the effect on healthy tissue, healthy C57BL / 6 mice were administered BAMLET via oral gavage or in drinking water (Figure 6). This effect was assessed by macroscopic examination of intestinal tissue and gene expression analysis. Compared to untreated controls, there was no evidence of inflammatory or necrotic changes in the intestine or peripheral organs in these mice. The number of regulated genes was low (approximately 150 genes), and there was no evidence of a toxic response (Figure 6a-c). Interestingly, genes involved in lipid metabolism, glucose metabolism, insulin resistance, and immune regulation were significantly affected in the BAMLET group (Figure 6d, e). The most highly upregulated genes included genes encoding amylase (Amy2b), important for carbohydrate digestion, lipase (Pnlip), for lipid digestion, and several proteases for protein digestion (Cpb1, Prss3, Cela3b, Cele2a) (Figure 6f). No changes in macroscopic appearance were detected in extraintestinal tissues, and there were no changes in organ weights in healthy mice treated with BAMLET.

[0117] Consideration We have demonstrated that tumor-prone ApcMin / + We investigated the potential of BAMLET as an oral tumor surveillance molecule by assessing its preventive and therapeutic effects on intestinal tumor growth and extraintestinal organs in mice. While a strong antitumor effect was demonstrated in these mice, healthy C57BL / 6 mice were virtually unresponsive to BAMLET, except for its effects on lipid and glucose metabolism. These findings demonstrate how a single protein complex can address multiple essential host needs, in this case, lactose synthesis in the mammary gland, purging cancer cells from intestinal and extraintestinal tissues, and metabolic functions in healthy mice. Without being bound by theory, we believe this mechanism may have evolved to clear the intestinal mucosa of virally transformed and immature cells that resemble cancer cells. These findings further highlight the potential of BAMLET as a novel preventive or therapeutic tool in cancer treatment.

[0118] The molecular basis of these therapeutic effects was investigated by gene expression analysis. Genes defining the tumor microenvironment were strongly upregulated in this model of spontaneous carcinogenesis but were absent or weakly expressed at some early time points in the BAMLET-treated group. There was a sustained effect of BAMLET treatment 5 weeks after oral gavage administration, and genes known to drive metastasis, tumor growth, and angiogenesis were strongly downregulated. In addition, the Wnt / β-catenin signaling pathway was inhibited, suggesting that BAMLET may protect the tissue environment by preventing the expression of key cancer-related gene networks. While these effects may reflect the delayed tumor growth observed in BAMLET-treated mice, the results further suggest that BAMLET restores tissue homeostasis by actively promoting cell differentiation and directing abnormally proliferating cells back to normal. Furthermore, some of these effects were still detectable after long-term follow-up, suggesting that BAMLET administration in drinking water maintains anti-tumor and differentiation-inducing pressure by eliminating emerging cancer cells and reprogramming gene expression in intestinal tissue.

[0119] Notably, BAMLET treatment also affected major organs outside the intestine, reducing β-catenin levels in the lungs, liver, and kidneys, and strongly affecting genes that define the tumor environment and cancer-related genes, potentially reducing the risk of cancer transformation and tumor growth. The extraintestinal effects of BAMLET treatment were also observed in Apc Min / + While this model is typically used as a model of colorectal cancer metastasis, this was unexpected because most mice died of anemia or intussusception before progressing to the expected lifespan of approximately 100 days (24). BAMLET-treated mice showed significantly increased survival compared with placebo-treated mice, which survived to 180 days. TTF-1 staining in the lungs (23) suggests proliferating foci of pulmonary origin rather than metastasis from intestinal tumors. The Wnt / β-catenin pathway operates in adult lung epithelium (25), and patients with familial adenomatous polyposis have been reported to develop lung cancer (26), suggesting that aberrant Wnt / β-catenin signaling may drive tumor development, a process that appears to be affected by BAMLET administration in drinking water. The tumoricidal effects of BAMLET and the related complexes HAMLET and α1-oleic acid are not limited to specific cancer types. Therapeutic efficacy against bladder cancer and skin papillomas has been demonstrated in controlled studies and animal models.

[0120] Unexpectedly, the PD-1 signaling pathway was inhibited by BAMLET treatment, and PD-1 protein levels were significantly reduced in treated mice compared with the placebo group. The findings suggest that, in addition to directly affecting tumors, BAMLET may inhibit the PD-1 feedback loop and suppress T cell responses against tumors. Apart from inhibiting PD-1-associated human leukocyte antigen (HLA) antigens, there was no evidence of a more global effect of BAMLET on immune responses in intestinal tissue. The functional impact of this observation is unclear, as the effectiveness of immune checkpoint inhibitors, such as anti-PD-1 / PD-L1 therapy, is limited to the treatment of colorectal cancer and microsatellite instability-high (MSI-H) tumors. For patients with microsatellite stable (MSS) colorectal cancer, response rates are only 5%–10% in approximately 90% of patients.

[0121] Healthy C57BL / 6 mice showed a marked lack of response to BAMLET treatment, highlighting the conjugate's affinity for tumor tissue rather than healthy tissue. There was no evidence of toxicity, as evidenced by macroscopic or behavioral changes and the lack of BAMLET retention in the intestines of healthy mice. The beneficial effects suggested that BAMLET may support physiological processes in normal tissues. These included effects on lipid metabolism, glucose metabolism, and insulin resistance. The results highlight the functional diversity of α-lactalbumin. Evolution may favor proteins that adapt their structure to solve multiple functional challenges. Oleate-binding proteins may partially unfold, exposing functional domains distinct from the native protein and achieving different but equally essential functions to support tissue development. The structural and functional diversity of α-lactalbumin may be essential for maintaining intestinal health and possibly other organs, making oleate conjugates worth exploring as preventative and therapeutic tools in oncology.

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Claims

1. 1. A complex for use in treating or preventing cancer, comprising a polypeptide having the sequence of naturally occurring alpha-lactalbumin or a functional variant thereof, or a peptide of up to 50 amino acids comprising the alpha-helical domain of said polypeptide, and a fatty acid or a lipid or salt thereof, The complex is for administration at a first site and the cancer is at a second site.

2. 1. A conjugate comprising a polypeptide having the sequence of naturally occurring alpha-lactalbumin or a functional variant thereof, or a peptide of up to 50 amino acids comprising the alpha helix domain of said polypeptide, and a fatty acid or a lipid or salt thereof, for use as a checkpoint inhibitor, in particular an inhibitor of PD-1, or for use in the prevention or treatment, in particular the treatment, of PD-L1 positive cancers or other cancers amenable to PD-1 targeting.

3. A complex for use in preventing, reducing or treating metastasis, comprising a polypeptide having the sequence of naturally occurring alpha-lactalbumin or a functional variant thereof, or a peptide of up to 50 amino acids comprising the alpha helix domain of said polypeptide, and a fatty acid or a lipid or salt thereof.

4. A complex for use in tumor surveillance and / or modification of the tumor environment, comprising a polypeptide having the sequence of naturally occurring alpha-lactalbumin or a functional variant thereof, or a peptide of up to 50 amino acids comprising the alpha-helical domain of said polypeptide, and a fatty acid or a lipid or salt thereof.

5. 5. The conjugate for use according to claim 2, 3 or 4, wherein the conjugate is for administration at a first site and the cancer is at a second site.

6. A conjugate comprising a polypeptide having the sequence of naturally occurring alpha-lactalbumin or a functional variant thereof, or a peptide of up to 50 amino acids comprising the alpha helix domain of said polypeptide, for use in the treatment or prevention of a metabolic-related condition such as insulin resistance, type II diabetes, metabolic syndrome, non-alcoholic fatty acid liver disease, cirrhosis, or hypertension.

7. 1. A method for preventing or treating cancer, comprising:

1. A method comprising the step of administering to a subject having cancer, or prone to developing cancer, or at a high risk of developing cancer, an effective amount of a complex comprising a polypeptide having the sequence of naturally occurring alpha-lactalbumin or a functional variant thereof, or a peptide of up to 50 amino acids comprising the alpha-helical domain of said polypeptide, and a fatty acid or a lipid or salt thereof, or an effective amount of a pharmaceutical composition comprising said complex, The method, wherein the complex or composition is for administration to a first site and the cancer is at a second site.

8. 1. A method of preventing or treating a PD-L1 positive cancer or other cancer amenable to PD-1 targeting, comprising: A method comprising the step of administering to a subject in need thereof an effective amount of a complex comprising a polypeptide having the sequence of naturally occurring alpha-lactalbumin or a functional variant thereof, or a peptide of up to 50 amino acids comprising the alpha-helical domain of said polypeptide, and a fatty acid or a lipid or salt thereof, or an effective amount of a pharmaceutical composition comprising said complex.

9. 1. A method for preventing or treating metastatic cancer, comprising: A method comprising the step of administering to a subject in need thereof an effective amount of a complex comprising a polypeptide having the sequence of naturally occurring alpha-lactalbumin or a functional variant thereof, or a peptide of up to 50 amino acids comprising the alpha-helical domain of said polypeptide, and a fatty acid or a lipid or salt thereof, or an effective amount of a pharmaceutical composition comprising said complex.

10. 1. A method for tumor surveillance and / or modification of the tumor environment, comprising: A method comprising the step of administering to a subject in need thereof an effective amount of a complex comprising a polypeptide having the sequence of naturally occurring alpha-lactalbumin or a functional variant thereof, or a peptide of up to 50 amino acids comprising the alpha-helical domain of said polypeptide, and a fatty acid or a lipid or salt thereof, or an effective amount of a pharmaceutical composition comprising said complex.

11. 11. The method of claim 8, 9 or 10, wherein the conjugate or pharmaceutical composition is administered at a first site and the cancer is at a second site.

12. 1. A method of treating a metabolic-related condition, such as insulin resistance, type II diabetes, metabolic syndrome, non-alcoholic fatty acid liver disease, cirrhosis, or hypertension, comprising: A method comprising the step of administering to a subject in need thereof an effective amount of a complex comprising a polypeptide having the sequence of naturally occurring alpha-lactalbumin or a functional variant thereof, or a peptide of up to 50 amino acids comprising the alpha-helical domain of said polypeptide, and a fatty acid or a lipid or salt thereof, or an effective amount of a pharmaceutical composition comprising said complex.

13. The conjugate for use according to any one of claims 1 to 6 or the method according to any one of claims 7 to 12, wherein said conjugate or pharmaceutical composition is for administration, i.e. is administered, to one of the nasal passages, gastrointestinal tract, brain, lungs, kidneys, vagina, bladder, liver, skin, breast, prostate, and / or ovaries.

14. 14. The complex for use according to any one of claims 1 to 5 or 13, or the method according to any one of claims 7 to 10 or 13, wherein the cancer is found in one or more of the nasal passages, digestive tract, brain, lung, kidney, vagina, bladder, liver, skin, breast, prostate, and / or ovaries.

15. The conjugate for use according to any one of claims 1 to 5, 13 or 14, or the method according to any one of claims 7 to 10, 13 or 14, wherein the administration site of the conjugate or pharmaceutical composition and the site of the cancer are different.

16. The complex for use according to claim 15, or the method according to claim 15, wherein the administration site of the complex or pharmaceutical composition and the site of the cancer are separate from each other.

17. The conjugate for use according to claim 2, or the method according to claim 8, wherein the cancer is a primary cancer.

18. The complex for use according to claim 2, or the method according to claim 8, wherein the cancer is metastasis.

19. The conjugate for use according to any one of claims 2, 3 or 17, or the method according to any one of claims 8, 9 or 18, wherein the administration site of the conjugate or pharmaceutical composition and the site of the cancer are the same.

20. The conjugate for use according to claim 6 or the method according to claim 12, wherein the metabolic-related condition is secondary to cancer or is found in a subject who has or has had cancer.

21. The conjugate for use according to claim 6 or the method according to claim 12, wherein the metabolic-related condition is unrelated to cancer or is found in subjects who do not have or have never had cancer.

22. A complex for use according to any one of claims 1 to 6, 13 to 21 or a method according to any one of claims 7 to 21, wherein the polypeptide has the sequence of naturally occurring alpha-lactalbumin, in particular human or bovine alpha-lactalbumin.

23. A complex for use according to any one of claims 1 to 6, 13 to 22 or a method according to any one of claims 7 to 22, wherein said polypeptide has the sequence of bovine alpha-lactalbumin.

24. the alpha helix domain is the α1 (residues 1-39) or α2 (residues 81-123) domain of human α-lactalbumin, SEQ ID NO: 3 or SEQ ID NO: 4; KQFTK XELSQLKDIDGYGGIALPELI XTMFHTSGYDTQ (SEQ ID NO: 3) LDDDITDIM XAKKILDIKGIDYWLAHKALXTEKLEQWL XEKL (SEQ ID NO: 4) where X is an amino acid residue other than cysteine. A complex for use according to any one of claims 1 to 6, 13 to 23, or a method according to any one of claims 7 to 23.

25. A conjugate for use according to any one of claims 1 to 6, 13 to 24 or a method according to any one of claims 7 to 24, wherein said functional variant consists of a sequence lacking disulfide bonds.

26. A conjugate for use according to any one of claims 1 to 6, 13 to 25 or a method according to any one of claims 7 to 25, wherein said functional variant consists of a sequence in which the cysteine ​​residue in natural alpha-lactalbumin is changed to another amino acid residue, preferably an alanine residue.

27. The complex for use according to any one of claims 1 to 6, 13 to 26, or the method according to any one of claims 7 to 26, wherein the fatty acid or lipid or salt thereof is a fatty acid or a salt thereof, in particular oleic acid or an oleate.

28. A complex for use according to any one of claims 1 to 6, 13 to 27, or a method according to any one of claims 7 to 27, wherein the polypeptide has the sequence of bovine alpha-lactalbumin and the fatty acid or its salt is oleic acid or oleate.

29. The complex for use according to any one of claims 1 to 6, 13 to 28 or the method according to any one of claims 7 to 27, wherein the complex or pharmaceutical composition is in the form of a drink, a food, an additive or ingredient for a drink or food, or another nutritional composition.

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