Methods for predicting response to treatment

Abstract

The present invention relates to a method for predicting the outcome of a treatment for a metabolic-related disease in a subject, wherein the treatment comprises administering to the subject 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 the polypeptide; and a fatty acid or lipid or a salt thereof; the method comprises the steps of: a) evaluating the control of a cancer gene in the subject after administering the complex; and b) correlating the control of the gene with the outcome of the treatment, wherein the success of the treatment is inversely proportional to the level of control of the gene.

Description

[Technical Field]

[0001] The present invention relates to a method for predicting response to treatment, in particular to cancer treatment using 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 lipid or a salt thereof. [Background technology]

[0002] HAMLET( h uman a lpha-lactalbumin m ade le thal to tHAMLET (HAMLET) is the first member of a class of tumoricidal unfolded protein-lipid complexes consisting of partially unfolded α-lactalbumin and oleic acid. It was originally 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) and was found to be biologically active, particularly antibacterial. Other methods for preparing active complexes have since been developed, such as heating α-lactalbumin from various sources and oleic acid together in solution. However, HAMLET and related complexes, such as BAMLET derived from bovine α-lactalbumin, have been shown to not only possess antiviral activity but also kill transformed cells, such as tumor or papilloma cells. HAMLET kills many types of tumor cells in vitro, and this tumoricidal activity is maintained in vivo, as shown in human glioblastoma xenografts and animal models of bladder cancer. Topical application of HAMLET eliminated or reduced skin papillomas, killed bladder cancer cells but not healthy cells in the surrounding tissue, and reduced tumor size. Tumor cell sensitivity to HAMLET reflects oncogenic transformation and varies depending on the cellular glycolytic state (Storm P, et al. (2011). Oncogene). ShRNA silencing of c-Myc or Ras pathway members conferred resistance to HAMLET, and c-Myc expression levels paralleled HAMLET sensitivity. Furthermore, glucose deprivation sensitized tumor cells to HAMLET, and HAMLET sensitivity was altered by shRNA targeting glycolytic enzymes. Furthermore, HAMLET has been shown to have a significant impact on global metabolism through rapid metabolic paralysis of tumor cells and the potential for shifting glycolytic flux to the pentose phosphate pathway.

[0003] Conjugates such as HAMLET have already proven therapeutic in the treatment of various established cancers (WO 2005 / 082406) and in the prophylactic treatment of colon cancer (WO 2014 / 023976). In addition, the inventors have recently shown that such conjugates are useful in the treatment of cancers for which the conjugates are not directly applicable (e.g., oral application for cancers other than those of the gastrointestinal tract) and in the treatment of metabolic-related diseases.

[0004] The inventors have now observed that responses to treatment with the conjugate are surprisingly rapid and easily identifiable. Surprisingly, it is possible to very quickly assess which subjects will respond well to treatment and which will not and would benefit from alternative or additional treatments, such as surgery. This is a very important discovery, allowing subjects who respond well to avoid surgery and subjects who require additional treatment to receive treatment as soon as possible. Summary of the Invention

[0005] The present invention provides a method for predicting the outcome of a cancer treatment in a subject, wherein the treatment comprises administering to the subject 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 the polypeptide; and a fatty acid or lipid or a salt thereof; the method comprising the steps of: a) evaluating the control of a cancer gene in the subject after administering the complex; and b) correlating the control of the gene with the outcome of the treatment, wherein the success of the treatment is inversely proportional to the level of control of the gene.

[0006] The present invention also provides a method for predicting the outcome of a treatment for a metabolic-related disease in a subject, wherein the treatment comprises administering to the subject 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 the polypeptide; and a fatty acid or lipid or a salt thereof; the method comprising the steps of: a) evaluating the control of a cancer gene in the subject after administering the complex; and b) correlating the control of the gene with the outcome of the treatment, wherein the success of the treatment is inversely proportional to the level of control of the gene.

[0007] (cancer) The cancer may be any cancer. For example, the cancer may be found in one or more of the nasal cavity, digestive tract (e.g., one or more of the oral cavity, stomach, colon, and intestine), central nervous system (e.g., brain, spine), lung, kidney, vagina, bladder, liver, skin, breast, prostate, and / or ovary. In particular, the cancer may be found in the bladder. The cancer may be primary or metastatic.

[0008] (Metabolic diseases) Metabolic disease is the disease that is affected by or that affects metabolism.For example, metabolic disease can be one or more of insulin resistance, type II diabetes, metabolic syndrome, non-alcoholic fatty acid liver disease, liver cirrhosis, and hypertension.It can also be the condition that arises from the problem in the process of insulin resistance or sensitivity, lipid metabolism and / or glucose metabolism regulation or adjustment.

[0009] Such metabolic disorders may be associated with the presence of cancer, may occur secondary to cancer, or may be unrelated to cancer.

[0010] (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 lipid or a salt thereof.

[0011] In one embodiment the polypeptide has the sequence of naturally occurring alpha-lactalbumin, preferably human or bovine alpha-lactalbumin, more preferably bovine alpha-lactalbumin.

[0012] In one embodiment, the α-helical domain is the α1 (residues 1-39) or α2 (residues 81-123) domain of human α-lactalbumin, of SEQ ID NO: 3 or SEQ ID NO: 4; KQFTK X ELSQLLKDIDGYGGIALPELI X TMFHTSGYDTQ (SEQ ID NO: 3) LDDDITDDIM X AKKILDIKGIDYWLAHKAL X TEKLEQWL X EKL (SEQ ID NO: 4) Here, X is an amino acid residue other than cysteine.

[0013] In one embodiment, the conjugate consists of a peptide of up to about 45, 42 or 40 amino acids, in particular 39 amino acids, preferably corresponding to the α1 domain of human α-lactalbumin.

[0014] In one embodiment, the functional variant consists of a sequence lacking disulfide bonds, hi one embodiment, the functional variant consists of a sequence in which the cysteine ​​residues in native alpha-lactalbumin have been changed to other amino acid residues, preferably alanine residues.

[0015] In one embodiment, the fatty acid or lipid or salt thereof is a fatty acid or salt thereof, hi one embodiment, the fatty acid or salt thereof is oleic acid or oleate.

[0016] In one embodiment, the polypeptide has the sequence of bovine alpha-lactalbumin and the fatty acid or salt thereof is oleic acid or oleate.

[0017] The polypeptide present in the complex may have the sequence of alpha-lactalbumin or a variant thereof as described above.

[0018] The complex may be referred to as a biologically active complex.In this specification, "biologically active" means that the complex has a biological activity that is different from or stronger than that of each individual component.In particular, the complex can selectively induce cell death, especially in tumor cells, and / or have bactericidal or antiviral effects that are not found in native proteins, such as those containing α-lactalbumin monomers, but other therapeutic effects can also be utilized.

[0019] The term "variant" refers to a protein or polypeptide that has a similar biological function but differs from the nucleic acid sequence from which it is derived by the substitution of one or more amino acids in the amino acid sequence with other amino acids. Amino acid substitutions may be considered "conservative" substitutions, in which an amino acid is replaced with a different amino acid having substantially similar properties. Non-conservative substitutions are those in which an amino acid is replaced with an amino acid of a different type.

[0020] "Conservative substitution" means replacing one amino acid with another amino acid of the same class:

[0021] [Table 1]

[0022] As is well known to those skilled in the art, conservative substitutions may alter the primary structure of a peptide without significantly altering the activity of the peptide, because the side chain of the amino acid inserted into the sequence can form similar bonds and contacts as the side chain of the replaced amino acid, even when the substitutions are in regions critical to determining the peptide's three-dimensional structure.

[0023] Non-conservative substitutions are possible as long as they do not interfere with the function of the DNA-binding domain polypeptide. Generally speaking, fewer non-conservative substitutions can be made without altering the biological activity of the polypeptide.

[0024] 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 underlying protein. For example, in determining whether a polypeptide variant falls within the scope of the invention, one of ordinary skill in the art will determine whether a complex comprised of the variant retains the biological activity (e.g., tumor cell killing) of the complex formed with the unfolded form of the native protein, and whether the polypeptide retains 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 activity of the native protein.

[0025] A variant of a polypeptide may comprise or consist essentially of an amino acid sequence having at least 70%, for example at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98% or 99% identity to a native protein sequence, such as an alpha-lactalbumin or lysozyme sequence.

[0026] The level of sequence identity is preferably determined using the BLASTP computer program, with the native protein sequence as the base sequence. That is, the native protein sequence is the base sequence when determining the percentage identity. The BLAST software is publicly available at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi (accessed March 12, 2009).

[0027] In certain embodiments, polypeptide is α-lactalbumin, such as human, bovine or ovine α-lactalbumin.While these variants as described above can be useful in the present invention, it may be preferable to use native protein in the product, especially for use as a dietary supplement.In certain embodiments, human α-lactalbumin is used.In another embodiment, α-lactalbumin is bovine α-lactalbumin.The sequence of a wide range of α-lactalbumin is known in the literature, and is shown, for example, in Watanabe et al., J. Vet Med Sci, (2000) 62(11); 1217-1219.

[0028] 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 rendering the recombinant protein lacking intramolecular disulfide bridges, the molecule is three-dimensionally non-native, rendering its original intrinsic biological activity completely inactive. This is achieved by changing the cysteine ​​residues in native 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 other sources of α-lactalbumin, including bovine or bovine α-lactalbumin, can also be used to obtain the recombinant protein.

[0029] In certain embodiments, the polypeptide is a recombinant protein having the sequence of native mature alpha-lactalbumin, but in which 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 have been mutated to other amino acids that do not form disulfide bonds, such as alanine. Thus, particular proteins that may be utilized in accordance with the present invention include the protein of SEQ ID NO: 1.

[0030] [Table 2]

[0031] Bold indicates the position of the cysteine ​​mutation in native human α-lactalbumin.

[0032] As reported in WO 2010 / 079362, additional amino acid residues, for example up to 20 amino acids, can be added to the N-terminus 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 (SEQ ID NO: 2 shown below) is used in the conjugates of the invention.

[0033] [Table 3]

[0034] The polypeptides used in the complexes are preferably in pure form and are preferably prepared using conventional peptide synthesis methods or by recombinant expression. In particular, the DNA encoding the desired recombinant alpha-lactalbumin can be inserted into a suitable expression vector, such as a plasmid, and then used to transform host cells, for example prokaryotic cells such as E. coli or eukaryotic cells, such as certain insect cells, in a conventional manner.

[0035] Suitable fatty acids or lipids include those known to provide biologically active complexes. These include, for example, the fatty acids described in WO 2008 / 058547. When salts are used, they are preferably water-soluble salts. Specific examples of suitable salts include alkali metal salts 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 is pharmaceutically acceptable.

[0036] Particular examples of fatty acids or lipids for use in the present invention are those having 4 to 30, such as 6 to 28, for example 8 to 26 carbon atoms. In particular embodiments, the fatty acids or lipids have 10 to 24, such as 12 to 22, for example 14 to 20 carbon atoms. In particular, the fatty acids or lipids have 16, 17, 18 or 20 carbon atoms. The fatty acids may be saturated or unsaturated.

[0037] 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 in which 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. A specific example is a C18:1 fatty acid or a salt thereof of formula CH3(CH2)7CH=CH(CH2)7COOH or CH3(CH2)7CH=CH(CH2)7COO-. In one embodiment, the fatty acid or a salt thereof is oleic acid or an oleate.

[0038] The complexes may be prepared using methods similar to those described in, for example, WO 99 / 26979, WO 2008 / 138348, WO 2010 / 131237, WO 2014 / 023976, WO 2018 / 210759, and WO 2022 / 073982, the contents of which are incorporated herein by reference. It has been found that complexes can be prepared by contacting unfolded α-lactalbumin or a derivative thereof with a cofactor, particularly oleic acid or a salt thereof, under ion exchange conditions such as those found in an ion exchange column, but also 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.

[0039] However, these methods generally focus on recreating the conditions under which proteins become unfolded and complex with oleate ions. Such studies have focused on using pure proteins, including recombinant variants of the base protein, to facilitate the production of active complexes. However, such starting materials increase production costs.

[0040] It is known that complexes obtained using α-lactalbumin derived from non-human milk, particularly BAMLET obtained using bovine α-lactalbumin, exhibit qualitatively similar effects to HAMLET on cells, particularly tumor cells (see, for example, Rammer et al. (2010) Mol. Cancer Ther. 9(1) 24-32). Therefore, the effects shown below using BAMLET will also be observed when HAMLET or a composition based on HAMLET is used instead of BAMLET.

[0041] (Treatment method) The conjugate is administered by an appropriate route and in an appropriate dosage.

[0042] (Methods for predicting treatment outcomes) The method can be carried out using any suitable procedure for assessing oncogene regulation, for example, by measuring one or more markers of oncogene regulation in a sample obtained from a subject.

[0043] The one or more markers may be any marker of oncogene regulation, such as genetic or molecular markers. In particular, the markers may be DNA or RNA, especially RNA.

[0044] The sample may be any sample in which oncogene regulation can be measured, such as urine, saliva, blood, cerebrospinal fluid (CSF), menstrual blood, cervicovaginal fluid, semen, synovial fluid, pleural fluid, pericardial fluid, amniotic fluid, nasal discharge, otic fluid, gastric juice, or feces. Typically, the sample is a bodily fluid. In one embodiment, the sample is a urine sample.

[0045] Ideally, the sample is taken from or near the site of the cancer being treated, but it can also be taken from a site distant from the cancer being treated. For example, if the cancer is bladder cancer, the sample can be a urine sample. If the cancer is oral cancer, the sample can be saliva. If the cancer is central nervous system cancer, the sample can be cerebrospinal fluid.

[0046] Treatment with the complex results in regulation of a significant number of genes. Regulation may refer to either upregulation, downregulation, or inhibition. The method may include assessing both upregulation and downregulation of oncogenes.

[0047] The genes downregulated or suppressed by the complex include angiogenesis-related genes such as ADIPOQ, CCL11, CCL2, CCN1, CSF3, CXCL1, CXCL2, CXCL6, CXCL8, DCN, EGR3, EREG, FGF7, FPR2, GREM1, HBEGF, HTRA1, LAMA2, LEP, LYVE1, MEOX2, MMP3, NTRK2, PROKR1, PTGS1, PTN, PTPRZ1, PTX3, RSPO3, SELE, SFRP1, SFRP2, SVEP1, TF, TNMD, and VEGFD; CHD5, CNN1, CXCL3, EFEMP1, FLNC, GC, GREB1, KLF9, NNMD, and NN. These include, but are not limited to, genes related to tumor cell migration and migration, such as MT, XIST, EYA1, SHC4, and TNS1; genes related to metastasis, such as CTHRC1, EPHA6, FBN2, P2RY6, PDLIM3, PLCXD3, PPP2R2C, PRAME, PTPRT, and SERPINB2; genes related to cell survival, such as APOD, CALB1, CRH, DDX43, DGKB, DUSP1, GADD45B, LGALS7 / LGALS7B, MFAP5, PDK4, PLAGL1, TFPI2, and ZBTB16; and genes related to inflammatory responses, such as CNTNAP2, DUOXA2, and ULBP1.

[0048] In one embodiment, the method comprises measuring the regulation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more genes associated with one or more of angiogenesis, tumor cell migration and migration, metastasis, cell survival, inflammatory response, biological death, cell movement, cell migration, cytoskeletal organization, tumor cell line cell migration, blood cell activation, cell activation, neuronal development, leukocyte migration and / or chemotaxis.

[0049] Additionally or alternatively, the method includes measuring the regulation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more genes associated with one or more of the following pathways: S100 family signaling pathway, phagosome formation, pathogen-induced cytokine storm signaling pathway, FAK signaling, CREB signaling in neurons, breast cancer regulation by Stathmin1, cardiac hypertrophy signaling (enhanced), G protein-coupled receptor signaling, IL-17 signaling, and pulmonary fibrosis idiopathic signaling pathway.

[0050] In one embodiment, the method comprises measuring the regulation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 or more cancer genes, particularly genes regulated by administration of the complex, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes selected from ADIPOQ, CALB1, EREG, CXCL2, CXCL6, GC, CSF3, PTPRZ1, DGKB, CRH, XIST, SLC5A1, CCL11, NEGR1, CLDN10, TF.

[0051] The method is performed on a subject to which the conjugate has been administered, or on a sample obtained from the subject, and generally can be performed and / or the sample obtained within 30, 45, 60, 75, 90, 105, 120, 135, 150 minutes, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 36, 48, 60, 72, 84, 96, 108, or 120 hours of administration of the conjugate.

[0052] If the conjugate is administered over a period of time, the method can generally be performed and / or samples obtained within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 36, 48, 60, 72, 84, 96, 108, or 120 hours of complete administration of the conjugate.

[0053] If the conjugate is administered multiple times, the method can generally be performed and / or samples obtained within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 36, 48, 60, 72, 84, 96, 108, or 120 hours after the first, subsequent, or final administration of the conjugate is completed.

[0054] (Correlation with cancer) The complex regulates multiple cancer genes. The inventors have found that continued or high levels of regulation of these genes after administration of the complex indicates the continued presence of cancer cells, which generally indicates a poor treatment outcome. On the other hand, lower or no regulation of cancer genes indicates fewer remaining cancer cells and more successful or complete treatment with the complex. In other words, a high level of gene regulation is associated with a poor treatment outcome, i.e., the cancer is somewhat responsive to treatment, but is likely to require further treatment, such as surgery. A low level of gene regulation is associated with a more successful treatment outcome, i.e., the cancer is likely to respond well to treatment, and further treatment, such as surgery, is not required.

[0055] (Correlation with metabolic diseases) The inventors also found that after administration of the complex, ongoing or high levels of gene regulation in the ADIPOQ network, as shown in Figures 3A and 3B, indicate a less successful treatment outcome, whereas low levels of gene regulation, or lower or no gene regulation, indicate a more successful or complete treatment with the complex.

[0056] (Treatment method) The present invention also provides a method for treating cancer, comprising administering to a subject a therapeutic 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 lipid or a salt thereof; assessing the control of cancer genes in the subject after administering said complex, correlating the control of genes with treatment outcome, wherein successful treatment outcome is inversely proportional to the level of control of said genes, and determining and / or implementing further courses of treatment.

[0057] The further course of treatment may include administration of another therapeutic agent, surgical intervention, or both.

[0058] The present invention also provides a method for treating a metabolic-related disease, comprising administering to a subject a therapeutic 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 lipid or a salt thereof; assessing the control of cancer genes in the subject after administration of said complex, correlating the control of genes with treatment outcome, wherein successful treatment outcome is inversely proportional to the level of control of said genes, and determining and / or implementing a further course of treatment.

[0059] The further course of treatment may include administration of another therapeutic agent, surgical intervention, or both.

[0060] (General Provisions) Throughout the specification and claims of this application, the words "comprise" and "contain," as well as variations thereof, such as "comprising" and "comprises," mean "including but not limited to" and do not exclude other elements, integers, or steps. Furthermore, the singular includes the plural unless the context requires otherwise: in particular, where the indefinite article is used, the specification is understood to contemplate the plural as well as the singular, unless the context requires otherwise.

[0061] Preferred features of each aspect of the invention are as 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, 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, except where such features are incompatible. [Brief explanation of the drawings]

[0062] 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]Figure 1 shows gene expression differences in 14 individual tumors treated with 8.5 mM α1-oleate. (a) For individual tumors from patients treated with 8.5 mM α1-oleate, a complete response (CR) was defined as tumor elimination, a partial response (PR) was defined as tumor size reduction, stable disease (SD) was defined as tumor size unchanged, and a non-responder was defined as tumor size increase. A 25% change in tumor size was used as the cutoff. (b) Gene expression versus treatment outcome as defined by 3D principal component analysis (PCA) for each of the 14 individual tumors (green = SD, blue = PR, red = CR). The majority of CR and PR tumors cluster closely together with low variance. The four SD tumors cluster independently and are distant from the responding tumors. (c) Gene expression (red = upregulated, blue = downregulated) and predominance of repressed genes (z-score compared to PR and CR tumors) in each tumor distinguishing between CR, PR, and SD groups. (d) Top regulated canonical pathways in SD, PR, and CR tumors sorted by z-score (blue = repressed: z-score < -2, orange = activated: z-score > 2). Pathway analysis detected a stronger response to α1-oleate in patients with residual tumor tissue, and a repressed profile of S100 family signaling, phagosome formation, cytokine storm signaling, FAK signaling, and CREB signaling pathways was observed in SD tumors compared to PR and CR tumors. (e) Top regulated biological functions in SD, PR, and CR tumors sorted by z-score (blue = repressed: z-score < -2, orange = activated: z-score > 2). Biological function analysis revealed that SD tumors exhibited stronger suppression of cell activation, migration, migration, and biological death activation compared with PR and CR tumors. (f) Functional classification of shared and strongly suppressed genes (z score < -2, P < 0.05). The top suppressed functions included genes related to angiogenesis, tumor cell survival, migration, metastasis, and inflammation.(g) Top repressed genes in each tumor: SD showed significant repression, while PR and CR tumors showed lower and lowest repression. [Figure 2] Patients with non-muscle invasive bladder cancer received intravesical instillation of α1-oleate. Figure 2 shows the results of gene expression analysis of subjects treated with the conjugate and those treated with placebo. [Figure 3] Figure 3 shows the up- and down-regulation of genes in the ADIPOQ network after treatment with the 8.5 mM complex (a) and the 1.7 mM complex (b). [Figure 4] Figure 4 shows adiponectin protein levels in patients receiving the complex treatment and in patients receiving a placebo. [Figure 5-6] Figures 5 and 6 show the interaction of the adiponectin receptor with the complex. Figure 5C shows the superposition of the predicted α-fold binding of the α1 peptide to ADIPOR1 and ADIPOR2, indicating high structural homology between AdipoR1 and AdipoR2 and a common binding pocket for the α1 peptide in both receptor proteins. [Example]

[0063] [Effect on number, size, and appearance of tumors] (Changes in each tumor recorded during endoscopy) Macroscopic changes in tumor number or size were recorded using a flexible cystoscope equipped with white light and narrowband imaging. Changes in tumor size were assessed by comparing tumors at diagnosis and after treatment prior to transurethral resection of bladder tumors. A fully opened flexible clamp was used as the measuring device.

[0064] Significant changes were observed in treated tumors compared with placebo. Treatment was associated with changes in tumor number and tumor size.

[0065] (Tumor Histopathology) Tumor response was further assessed histopathologically. Significant changes were observed in treated tumors compared with the placebo group. A complete response was confirmed by the absence of tumor tissue in the control biopsy or the presence of minimal residual tissue stroma containing no tumor cells. In the partial response group, fragmentation and cell shedding revealed few intact papillae, and the tissue surface appeared exfoliated. Furthermore, tumor tissue surrounding the target lesion, including disappeared satellite tumors, was affected in responding patients.

[0066] (immediate tumor response) Intravesical instillation of α1-oleate induced a rapid tumor response, which was quantified by comparing each patient's pre-instillation sample with the first urine sample after instillation (≤2 hours).

[0067] (cell shedding) Cell shedding was detected in all patients, suggesting that alpha-1-oleate had a direct and rapid effect on the tumor. After alpha-1-oleate was instilled into the bladder, tumor cells were rapidly shed into the urine. A highly significant increase in cell counts was detected after infusion at each visit compared with each pre-infusion sample.

[0068] The sloughed cells were pathologically scored according to the Paris classification [20, 21]. Superficial cell sloughing and tumor fragmentation were also detected by histopathological examination of biopsy specimens obtained at surgery (Fig. 2f).

[0069] (Uptake of α1-oleate into tumor cells and tissues) When α1-oleate was instilled into the bladder, it was rapidly taken up by tumor tissue.

[0070] Tumor uptake of α1-oleate was further quantified in tissue biopsies obtained by TURB.

[0071] (DNA strand breaks) Double-stranded DNA breaks were quantified by TUNEL staining in decidual cells after α1-oleate infusion compared with pre-infusion samples. A significant increase in TUNEL staining was detected after infusion in all treated patients.

[0072] (Gene expression analysis) Tumor response was characterized in more detail by sequencing RNA extracted from tumor tissue obtained by TURB and from biopsies obtained from healthy-appearing adjacent tissue.

[0073] Heatmap analysis identified over 3500 genes as being regulated, and functional analysis of the regulated common genes predicted downregulation of cancer-related functions, including angiogenesis, tumor cell migration, and tumor invasion.

[0074] Pathway analysis further confirmed a strong inhibitory effect on cancer-related genes and pathways, including those related to the tumor microenvironment, breast cancer, and bladder cancer. Furthermore, the innate immune response was strongly downregulated, affecting cytokine storm signaling and G protein-coupled receptor signaling. Downregulation of chemokines and cytokines, including CCL2, CCL11, CCL20, CXCL1, CXCL2, and CXCL12, as well as IL1B and IL17D, was common.

[0075] Among cancer-related genes, genes regulating tumor microenvironment pathways were predominantly downregulated, including vascular endothelial growth factor D (VEGFD), transforming growth factor β genes (TGFB2, TGFB3), fibroblast growth factors (FGF1, FGF5, FGF7, FGF10), and insulin-like growth factor 1 (IGF1).

[0076] (bladder cancer gene) Bladder cancer-related genes were strongly suppressed. Early growth response protein 3 (EGR3), a biomarker of VEGF-dependent angiogenesis, was strongly downregulated, as were genes previously reported to be highly expressed in bladder cancer subtypes, including protein kinase alpha kinase 2 (ALPK2), platelet-derived growth factor receptor beta (PDGFRB), and cGMP-dependent protein kinase 1 (PRKG1). Bladder cancer-associated transcription factors, including Twist family of transcription factor 2 (TWIST2), FOS-like 1 (FOSL1), and zinc finger E-box binding homeobox 1 (ZEB1), which are markers of epithelial-mesenchymal transition and metastasis, were also strongly downregulated.

[0077] (Therapeutic effect on each tumor) For individual tumors from patients treated with α1-oleate, a complete response (CR) was defined as tumor elimination, a partial response (PR) as tumor size reduction, stable disease (SD) as tumor size retention, or a non-responder as tumor size increase (cutoff = 25% change in tumor size) (Fig. 1a). The effect of α1-oleate on gene expression was further examined for individual patients and tumors using urine samples to detect differences associated with clinical treatment response for each tumor. Principal component analysis (PCA; Fig. 1b) revealed that the majority of responding tumors formed a cluster with an overall similar gene expression profile. All stable (SD) or non-responding tumors clustered distantly from this cluster, suggesting a strong effect of α1-oleate treatment on these tumors and their differentiation from the rest of the group. Consistent with the PCA analysis, biofunction and pathway analyses also demonstrated quantitative differences between responders and non-responders, as defined by z-score intensity (Fig. 1c, d).

[0078] A group of 297 genes was strongly downregulated (logFC ≤ -10; Figure 1e) in all treated tumors. Functional analysis identified these genes as being related to angiogenesis, tumor cell migration, migration, survival, and metastasis. 31 / 297 of the downregulated genes were bladder cancer-related genes, including PRAME nuclear receptor transcriptional regulator (PRAME) and protein tyrosine phosphatase receptor type T (PTPRT), which were strongly downregulated (logFC ≤ -10). Similarly, bladder cancer genes previously reported to play important roles in angiogenesis, including CXC motif chemokine ligand 2 (CXCL2), neurotrophic receptor tyrosine kinase (NTRK2), protein tyrosine phosphatase receptor type Z1 (PTPRZ1), and transferrin (TF), were also downregulated (logF ≤ 10) (Fig. 1f). The robust tumor-wide downregulation of these molecules suggests that α1-oleate affects all tumors, regardless of clinical response category.

[0079] [overview] Selecting appropriate treatment endpoints is particularly challenging for incurable diseases. New molecular tools may complement clinical observations and enhance resolution of disease response to understand the potential of new drug candidates. In this study, we combined clinical and molecular tools to characterize tumor response to α1-oleate in patients with NMIBC. In addition to changes in tumor size, tumor response was defined by urinary shedding of tumor cells and tumor debris, tumor uptake of α1-oleate, and evidence of tumor cell-specific apoptosis. Gene expression analysis of tumor RNA visually confirmed interesting differences between groups defined as CR, PR, and NR. Using tumor cell shedding, apoptosis, and suppression of cancer-related genes as endpoints, all patients in this study were defined as responders to α1-oleate.

[0080] Materials and Methods [Peptide synthesis and α1-oleic acid conjugate formation] Peptide synthesis and preparation of the investigational drug were performed under GMP conditions, and the conjugate was diluted to final concentration in PBS. The placebo group received PBS (sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, water for injection) and appeared identical to the active treatment.

[0081] [Research Plan] After diagnosis and informed consent, subjects received six intravesical instillations of alpha-1-oleate or placebo over the course of one month prior to scheduled transurethral resection (TURB). Safety follow-up was performed 52 days after the first instillation.

[0082] [Primary endpoint] - Safety as adverse event profile (duration: from informed consent signing (day 1) to end of study (day 52)): incidence of adverse events and classification in terms of severity, causality, and treatment outcome. -Efficacy as cell shedding (period: 1-22 days): Change in cell shedding in urine (number of epithelial cells per mL of urine). - Change from baseline in papillary tumor characteristics (time period: pre-treatment (baseline) and day 30 relative to planned surgery): Bladder tumors are characterized by in vivo imaging during cystoscopy.

[0083] [Secondary endpoints] Histopathological scoring of the tumor using established parameters for scoring Grade and Stage / Invasiveness. -Pre- and post-infusion urinary cytology using the Paris scoring system. -Uptake of alpha 1-oleate by tumor cells, as defined by staining with specific antibodies. -Apoptotic response of tissue to α1-oleate as defined by TUNEL staining. - Tumor response to α1-oleate as defined by RNA-seq analysis. - Proteomic analysis of urinary markers has not been completed. - The long-term effects of study treatment have not been evaluated.

[0084] [Adverse event profile] Adverse events (AEs) were collected from the signing of informed consent until the end of the study (FU1 Visit, Day 52). All diagnoses, symptoms, signs, or findings on the first day after the first dose of study drug were recorded as AEs or severe AEs (SAEs). (S)AEs related to the study procedure were coded by preferred term and primary system organ classification under MedDRA throughout the study. All adverse events recorded during the study were listed in the subject data list, and overall summaries were created for treatment-emergent (S)AEs, early study discontinuation due to AEs, treatment-related AEs, and the number (percentage) of subjects experiencing an (S)AE. The number of subjects experiencing each adverse event was tallied, regardless of the number of adverse events reported by each subject. The severity of each adverse event was also tallied and assessed as the most severe recorded adverse event.

[0085] [Cell shedding] To quantify cell and cell cluster shedding in urine, samples were collected from each patient before and after administration of α-1-oleate or placebo (Visits 1–6). Cell shedding was quantified by counting the total number of epithelial cells in one unit of uncentrifuged urine under a light microscope using a hemocytometer. Changes in cell shedding were quantified at each visit by comparing cell counts in samples obtained before and after each infusion. Cell clusters were scored on a scale of 0–2 (0 = no clusters, 2 = highest number of clusters) based on examination of these samples by an experienced pathologist.

[0086] [Characteristics of papillary tumors, tumor size] To determine whether α1-oleate treatment affected tumor size, all subjects underwent outpatient cystoscopy at Visit 0. Tumor reexamination was performed at Visit 7 before scheduled surgery. High-quality photographs were collected endoscopically using a flexible cystoscope (Olympus) before TURB resection according to the recommendations of the EAU guidelines

[23] . Changes in tumor size were assessed within individuals using paired images.

[0087] [Histopathological scoring] Tumor biopsies taken at surgery were evaluated histopathologically using established parameters for scoring grade and stage / invasiveness. Tissue samples were analyzed by a designated uropathologist. Both grading systems (WHO 1973 and 2004 / 2016) were used. Biopsies of healthy tissue distant from the tumor were also taken for comparison.

[0088] [Urine cytology] Urine cells were centrifuged at 113g for 5 minutes onto L-lysine-coated microscope slides (Cytospin 3, Shandon), fixed, and stored at room temperature until further analysis. Urine cytology was evaluated using the Paris System for Reporting Urinary Cytology 2016 and defined as follows: 1. Not diagnostic / insufficient; 2. Negative for high-grade urothelial carcinoma; 3. Atypical urothelial cells; 4. Suspected high-grade urothelial carcinoma; 5. High-grade urothelial carcinoma; 6. Low-grade urothelial neoplasm; 7. Positive for other malignancies and miscellaneous lesions.

[0089] [Immunohistochemistry] Tumor cell uptake of α-1-oleate was quantified by staining with a specific antibody. Cells on cytospin slides were washed (TBS, 10 min), permeabilized (0.25% Triton X-100 in TBS, 20 min, room temperature), and blocked (5% normal goat serum in TBS, 1 h, room temperature) before addition of anti-human α-lactalbumin rabbit polyclonal antibody (1:50 in 5% normal goat serum, 4°C, overnight, Mybiosource, Cat#MBS175270). Slides were washed (TBS, 2 x 5 min) and stained with Alexa-568-conjugated secondary antibody (1:200, 1 h, room temperature, ThermoFisher). Nuclei were counterstained with DRAQ5 (1:1000, 15 min) followed by a final wash (TBS, 2 x 5 min). Slides were mounted (Fluoromount aqueous mounting medium) and images were taken with a laser scanning confocal microscope (Carl Zeiss). Fluorescence intensity was quantified using ImageJ, and net fluorescence was calculated after subtracting the secondary antibody background.

[0090] For other markers, biopsy samples were first deparaffinized in xylene, rehydrated in reduced ethanol concentrations (100%, 95%, 70%, and 50%), and then washed with deionized water. Slides were then immersed in target retrieval solution (Dako, S1699) and boiled for 20 minutes. They were then permeabilized with 0.25% Triton in PBS for 30 minutes at room temperature. A blocking solution containing 5% goat serum in PBS was added, followed by the addition of the primary antibody (anti-human α-lactalbumin rabbit polyclonal antibody; Mybiosource, Cat# MBS175270) for 1 hour at room temperature.

[0091] Slides were then washed with 0.025% PBS-T and stained with anti-rabbit goat Alexa Fluor-488 (Invitrogen, A11034) or anti-rabbit goat Alexa Fluor-568 (Invitrogen, A11036) secondary antibodies (1:200, 1 hour at room temperature). Nuclei were counterstained with DAPI for 15 minutes, followed by a final wash in PBS and mounting with Fluoromount aqueous mounting medium (Sigma, F4680). After drying, images were captured using a confocal microscope (Zeiss LSM 900) or a Hamamatzu Nanozoomer scanner, and fluorescence intensity was quantified using ImageJ.

[0092] [Detection of apoptosis by TUNEL staining] 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). Tissue sections were deparaffinized in xylene and then serially dehydrated with ethanol (100, 95, 75, 50%). Dehydrated sections were fixed (4% PFA, 15 min), permeabilized (DNase-free proteinase K solution, 20 μg / mL, 15 min), and incubated with TdT-containing TUNEL reaction mixture at 37°C for 60 min. After the TUNEL reaction, the sections were incubated in Click-iT reaction mixture at 37°C for 30 min. Sections were counterstained with DAPI (1 μg / mL, 5 min), mounted in Fluoromount aqueous mounting medium, and analyzed under a fluorescence microscope (Zeiss). Fluorescence intensity was quantified using ImageJ, and net mean fluorescence intensity was calculated after subtraction of background fluorescence.

[0093] [RNA-seq analysis of tissue biopsies] RNA was extracted from tissues stabilized in RNAlater using the AllPrep DNA / RNA / miRNA Universal Kit. Disruption was performed using a QIAshredder homogenizer in a TissueLyser system and CK28 Precellys tubes. RNA sample quantity and quality were assessed using a NanoDrop and Agilent 2100 Bioanalyzer. RNA samples were prepared using the Illumina TruSeq Stranded mRNA Library Prep Kit (20020594), and libraries were multiplexed and sequenced using NextSeq500 / 550 High Output Kits (v2.5 2 × 75 cycles) with an average of 22 million reads per sample. Raw sequencing data was demultiplexed using bcl2fastq (version 2.18), and abundance predictions were performed using RSEM (1.3) using Human Genome Release 37 / Ensemble75. Samples were thoroughly QCed and analyzed for RNA-seq-specific biases (e.g., GC bias, transcriptome complexity, alignment quality), as well as dimension reduction (i.e., PCA) and visualization using MA-plots. Differential expression analysis was performed using R (version 3.4) with the limma and DESeq2 packages. Tumors were compared between treatment and placebo groups, and gene expression ratios (fold changes) were calculated. Relative expression levels were analyzed, and genes with absolute fold changes >2.0 and P <0.05 were considered differentially expressed. Heat maps were generated using Gitools 2.1.1 software. Differentially expressed genes were functionally characterized using Ingenuity Pathway Analysis version 57662101 (IPA, Qiagen) software.

[0094] [Statistical analysis] Regarding efficacy, the sample size was based on the analysis of tumor cell changes evaluated in a previous study (Reference 16). A sample size of 20 patients per group was determined to be appropriate to achieve a significance level (α) of 0.05 and a power of 90%. The null hypothesis was H0: mean change in cell shedding = 0, and the alternative hypothesis was HA : Mean change in cell shedding >0. Gaussian distribution was determined by the D'Agostino-Pearson normality test. For data that followed a Gaussian distribution, a Student t-test was used. Other data sets were analyzed with the Mann-Whitney U test. Correlations were determined by Spearman correlation. Kinetic data were analyzed using a two-way repeated measures analysis of variance (ANOVA) test. All statistical analyses were performed using Prism version 6.02 (GraphPad Software). A P value of <0.05 was considered statistically significant. All images were generated by the research team.

[0095] [Chemical substances] Sodium oleate (Sigma-Aldrich, Cat# O7501), Click-iT TUNEL Alexa Fluor 488 Imaging Assay Kit (ThermoFisher Scientific, Cat# C10245), DRAQ5 (Abcam, Cat# ab108410), Fluoromount (Sigma-Aldrich, Cat# F4680), DNA / RNA / miRNA Universal Kit (Qiagen, Cat# 80224).

[0096] [Preparation of BAMLET and α-1-oleate] The BAMLET conjugate was prepared by mixing bovine α-lactalbumin (Sigma, Cat# L5385) and oleic acid (Sigma, Cat# O1008). α1 was synthesized using Fmoc solid-phase chemistry (Mimotopes). The sequence of α1 is: aa 1-39 Ac-KQFTKAELSQLLKDIDGYGGIA-LPELIATMFHTSGYDTQ-OH.

[0097] [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 empirical Bayes ANOVA, and genes with absolute fold changes greater than 1.5 or 2.0 were considered differentially expressed. Heatmaps were generated using Graphpad Prism 9, and differentially expressed genes were analyzed using Ingenuity Pathway Analysis software (IPA, Qiagen).

[0098] [Involvement of adiponectin and its receptors ADIPOR1 and ADIPOR2 in the tumoricidal function of α1-HAMLET] [background] The primary function of adipose tissue is to store energy in the form of fat, accounting for 20–25% of total body weight in healthy individuals. Adipose tissue cells (adipocytes) secrete bioactive molecules called adipocytokines (also known as adipokines). The most abundant adipocytokine is adiponectin (APN). In humans, APN has 244 amino acids and a molecular mass of 28 kDa. Circulating APN oligomers exist in trimer (low molecular weight), hexamer (medium molecular weight), and multimer (high molecular weight) forms, the latter of which is the most biologically active form. APN also exists in small amounts in plasma as a globular form (the result of proteolysis).

[0099] Adiponectin is well known as a homeostatic factor that regulates glucose levels, lipid metabolism, and insulin sensitivity through its anti-inflammatory, anti-fibrotic, and antioxidant properties. All of these metabolic processes are mediated by three receptors: AdipoR1, AdipoR2, and T-cadherin. The AMPK and PPAR pathways are activated through adiponectin binding to AdipoR1 and AdipoR2, respectively. AdipoR1 and AdipoR2 share seven highly structurally homologous transmembrane domains and intracellular N- and extracellular C-termini, which are opposite to those of classical G protein-coupled receptors (GPCRs).

[0100] [investigation] In an ongoing randomized, placebo-controlled, phase II clinical trial in patients with non-muscle-invasive bladder cancer (NMIBC), bladder instillation of the tumoricidal peptide-lipid complex α1-Hamlet showed promising therapeutic effects. Patient tumor tissue samples (obtained at visit 6 after treatment) were analyzed. Gene expression profiling of these samples revealed that the ADIPOQ gene, encoding the adiponectin protein, was highly significantly downregulated (FC=-5353.7) in patients treated with 8.5 mM α1-H compared with placebo, and was also the second most downregulated gene. The regulatory effect and pathway analysis of the top-regulated molecules using a network are shown in Figure 2.

[0101] Network analysis of ADIPOQ revealed that 78 ADIPOQ-dependent molecules were strongly regulated, of which 66 were downregulated and 12 were upregulated. Interestingly, ADIPOQ and its related molecules regulated several pathways, including the molecular mechanisms of cancer, the tumor microenvironment, and adipogenic signaling (Figure 3).

[0102] To determine the adiponectin protein concentration in the patients' urine samples, an adiponectin ELISA test (Proteintech) was performed. Signal (absorbance) values ​​were measured at 480 nm and interpolated to concentration (pg / mL) values ​​using linear regression statistical analysis. Measured urinary adiponectin concentrations were significantly higher in the α1-oleate group than in the placebo group (P < 0.001, Mann-Whitney t-test) (Figure 4). The graph shows the urinary adiponectin concentrations in both groups. Samples were collected before, after, 3, and 6. The lines indicate the median change in urinary adiponectin concentration (Figure 4). A statistically significant positive correlation was observed between the number of cells (shedding) in the urine samples and the measured adiponectin levels using linear regression (P = 0.002) (Figure 4). Statistical analysis was performed using GraphPad Prism.

[0103] [Adiponectin receptor] Previously reported crystal structures of AdipoR1 and AdipoR2 proteins were intriguing, revealing that oleic acid occupies a major cavity in the protein structure. Furthermore, AdipoR1 and AdipoR2 possess an adiponectin-independent function of maintaining membrane fluidity in many types of human cells. The inventors hypothesized that α1-H, which contains an oleic acid moiety, may interact with AdipoR1 and AdipoR2 receptors at the membrane level, providing valuable information for understanding the tumoricidal mechanism of α1-H. Furthermore, the highly membrane-active nature of α1-oleate suggests that the combined action of α1-oleate and adiponectin receptors may be essential for initiating the broad-spectrum effects of α1-oleate on tumor cells, leading to tumor cell death (Figure 5).

[0104] To predict whether the α1 peptide would have affinity for the adipoQ receptor, we used in silico modeling. The Alphafold software, which predicts protein-protein interactions, suggested that there is a common binding pocket region for α1 (shown in green) near the intracellular N-terminal domains (shown in cyan and orange) of both adiponectin receptors (Figure 5).

[0105] <Clause> 1. A method for predicting the outcome of a cancer treatment in a subject, wherein the treatment comprises administering to the subject 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 the polypeptide; and a fatty acid or lipid or a salt thereof; the method further comprises the steps of: a) assessing the control of a cancer gene in the subject after administering the complex; and b) correlating the control of the gene with the outcome of the treatment, wherein the success of the treatment is inversely proportional to the level of control of the gene. 2. In the method of paragraph 1, the cancer is found in one or more of the following: the nasal cavity, gastrointestinal tract (e.g., one or more of the oral cavity, stomach, colon, intestine), central nervous system (e.g., brain, spine), lung, kidney, vagina, bladder, liver, skin, breast, prostate, and / or ovary. 3. The method of claim 2, wherein the cancer is bladder cancer. 4. A method for predicting the outcome of a treatment for a metabolic-related disease in a subject, wherein the treatment comprises administering to the subject 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 the polypeptide; and a fatty acid or lipid or a salt thereof; the method further comprises the steps of: a) assessing the control of an oncogene in the subject after administering the complex; and b) correlating the control of the gene with the outcome of the treatment, wherein a successful outcome of the treatment is inversely proportional to the level of control of the gene. 5. The method according to any one of items 1 to 4, comprising assessing the level of cancer gene control by measuring a cancer gene control marker in a sample obtained from the subject. 6. The method of claim 5, wherein the sample is a sample of a body fluid. 7. The method of claim 6, wherein the sample is a urine sample. 8. The method according to any one of items 5 to 7, wherein the marker is RNA or DNA. 9. The method of claim 8, wherein the marker is RNA. 10. The method of any one of claims 1 to 9, wherein one or more of said oncogenes are regulated by said complex. 11. The method of any one of paragraphs 1 to 10, wherein the one or more oncogenes comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more oncogenes, and the genes comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more genes selected from ADIPOQ, CALB1, EREG, CXCL2, CXCL6, GC, CSF3, PTPRZ1, DGKB, CRH, XIST, SLC5A1, CCL11, NEGR1, CLDN10, and TF. 12. The method of claim 11, wherein the one or more oncogenes comprises ADIPOQ. 13. The method of paragraph 11 or 12, wherein the one or more oncogenes includes CALB1. 14. The method according to any one of items 11 to 13, wherein the one or more oncogenes include EREG. 15. The method of any one of paragraphs 11 to 14, wherein the one or more oncogenes include CXCL2. 16. The method of any one of paragraphs 11 to 15, wherein the one or more oncogenes include CXCL6. 17. The method of any one of paragraphs 11 to 16, wherein the one or more cancer genes include GC. 18. The method of any one of paragraphs 11 to 17, wherein the one or more oncogenes include CSF3. 19. The method of any one of paragraphs 11 to 18, wherein the one or more oncogenes includes PTPRZ1. 20. The method of any one of paragraphs 11 to 19, wherein the one or more oncogenes include DGKB. 21. The method of any one of paragraphs 11 to 20, wherein the one or more oncogenes comprises CRH. 22. The method of any one of paragraphs 11 to 21, wherein the one or more oncogenes include XIST. 23. The method of any one of paragraphs 11 to 22, wherein the one or more oncogenes comprises SLC5A1. 24. The method of any one of paragraphs 11 to 23, wherein the one or more oncogenes includes CCL11. 25. The method of any one of paragraphs 11 to 24, wherein the one or more oncogenes includes NEGR1. 26. The method of any one of paragraphs 11 to 25, wherein the one or more oncogenes includes CLDN10. 27. The method of any one of paragraphs 11 to 26, wherein the one or more oncogenes comprises a TF. 28. A method for treating cancer, comprising administering to a subject a therapeutic 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 lipid or a salt thereof; assessing the control of cancer genes in the subject after administration of said complex; correlating the gene control with treatment results using the method of any one of items 1 to 3 or 5 to 27; and determining the course of further treatment. 29. A method for treating melanoma, comprising administering to a subject a therapeutic 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 lipid or a salt thereof, evaluating the control of cancer genes in the subject after administration of said complex, correlating the gene control with the treatment outcome using a method described in any one of items 4 to 27, and determining the course of further treatment.

[0106] [References] 1. Hakansson, A., et al., Apoptosis induced by a human milk protein. Proc Natl Acad Sci USA, 1995. 92(17): p. 8064-8. 2. Svensson, M., et al., Conversion of alpha-lactalbumin to a protein inducing apoptosis. Proc Natl Acad Sci USA, 2000. 97(8): p. 4221-6. 3. Pettersson-Kastberg, J., et al., alpha-Lactalbumin, engineered to be nonnative and inactive, kills tumor cells when in complex with oleic acid: a new biological function resulting from partial unfolding. J Mol Biol, 2009. 394(5): p. 994-1010. 4. Fischer, W., et al., Human alpha-lactalbumin made lethal to tumor cells (HAMLET) kills human glioblastoma cells in brain xenografts by an apoptosis-like mechanism and prolongs survival. Cancer Res, 2004. 64(6): p. 2105-12. 5. Gustafsson, L., et al., Treatment of skin papillomas with topical alpha-lactalbumin-oleic acid. N Engl J Med, 2004. 350(26): p. 2663-72. 6. Mossberg, A.K., et al., HAMLET treatment delays bladder cancer development. J Urol, 2010. 183(4): p. 1590-7. 7. Puthia, M., et al., Prevention and treatment of colon cancer by peroral administration of HAMLET (human alpha-lactalbumin made lethal to tumour cells). Gut, 2014. 63(1): p. 131-42. 8. Mossberg, A.K., et al., Bladder cancers respond to intravesical instillation of HAMLET (human alpha-lactalbumin made lethal to tumor cells). Int J Cancer, 2007. 121(6): p. 1352-9. 9. Brisuda, A., et al., Bladder cancer therapy using a conformationally fluid tumoricidal peptide complex. Nat Commun, 2021. 12(1): p. 3427. 10. Ho, C.S., et al., Low resolution solution structure of HAMLET and the importance of its alpha-domains in tumoricidal activity. PLoS One, 2012. 7(12): p. e53051. 11. Antoni, S., et al., Bladder Cancer Incidence and Mortality: A Global Overview and Recent Trends. Eur Urol, 2017. 71(1): p. 96-108. 12. van Rhijn, B.W., et al., Recurrence and progression of disease in non-muscle-invasive bladder cancer: from epidemiology to treatment strategy. Eur Urol, 2009. 56(3): p. 430-42. 13. Hu, Q., et al., Recent advances of cocktail chemotherapy by combination drug delivery systems. Adv Drug Deliv Rev, 2016. 98: p. 19-34. 14. Sharma, P. and J.P. Allison, Immune checkpoint targeting in cancer therapy: toward combination strategies with curative potential. Cell, 2015. 161(2): p. 205-14. 15. Ourfali, S., et al., Recurrence Rate and Cost Consequence of the Shortage of Bacillus Calmette-Guerin Connaught Strain for Bladder Cancer Patients. Eur Urol Focus, 2021. 7(1): p. 111-116. 16. de Jong, J.J., et al., Hyperthermic Intravesical Chemotherapy for BCG Unresponsive Non-Muscle Invasive Bladder Cancer Patients. Bladder Cancer, 2018. 4(4): p. 395-401. 17. Colombo, R., et al., Multicentric study comparing intravesical chemotherapy alone and with local microwave hyperthermia for prophylaxis of recurrence of superficial transitional cell carcinoma. J Clin Oncol, 2003. 21(23): p. 4270-6. 18. Arends, T.J., et al., Results of a Randomised Controlled Trial Comparing Intravesical Chemohyperthermia with Mitomycin C Versus Bacillus Calmette-Guerin for Adjuvant Treatment of Patients with Intermediate- and High-risk Non-Muscle-invasive Bladder Cancer. Eur Urol, 2016. 69(6): p. 1046-52. 19. Hien, T.T., et al., Bladder cancer therapy without toxicity-A dose-escalation study of alpha1-oleate. International Journal of Cancer, 2020. 147(9): p. 2479-2492. 20. Barkan, G.A., et al., The Paris System for Reporting Urinary Cytology: The Quest to Develop a Standardized Terminology. Acta Cytol, 2016. 60(3): p. 185-97. 21. VandenBussche, C.J., A review of the Paris system for reporting urinary cytology. Cytopathology, 2016. 27(3): p. 153-6. 22. Gono, K., Narrow Band Imaging: Technology Basis and Research and Development History. Clin Endosc, 2015. 48(6): p. 476-80. 23. Babjuk, M., et al., EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder: update 2016. Eur. Urol., 2017. 71(3): p. 447-461.

Claims

1. A method for predicting the outcome of a cancer treatment in a subject, wherein the treatment comprises administering to the subject 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 the polypeptide; and a fatty acid or lipid or a salt thereof; the method further comprises the steps of: a) evaluating the control of a cancer gene in the subject after administering the complex; and b) correlating the control of the gene with the outcome of the treatment, wherein the success of the treatment is inversely proportional to the level of control of the gene.

2. 10. The method of claim 1, wherein the cancer is found in one or more of the nasal cavity, gastrointestinal tract (e.g., one or more of the oral cavity, stomach, colon, intestine), central nervous system (e.g., brain, spine), lung, kidney, vagina, bladder, liver, skin, breast, prostate, and / or ovary.

3. 3. The method of claim 2, wherein the cancer is bladder cancer.

4. A method for predicting the outcome of a cancer treatment in a subject, wherein the treatment comprises administering to the subject 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 the polypeptide; and a fatty acid or lipid or a salt thereof; the method further comprises the steps of: a) assessing the regulation of ADIPOQ network genes in the subject after administering the complex; and b) correlating the regulation of the genes with the outcome of the treatment, wherein a successful outcome of the treatment is inversely proportional to the level of regulation of the genes.

5. The method according to any one of claims 1 to 4, comprising assessing the level of oncogene regulation by measuring an oncogene regulation marker in a sample obtained from the subject.

6. The method of claim 5 , wherein the sample is a sample of a bodily fluid.

7. 7. The method of claim 6, wherein the sample is a urine sample.

8. The method according to any one of claims 5 to 7, wherein the marker is RNA or DNA.

9. 9. The method of claim 8, wherein the marker is RNA.

10. 10. The method of any one of claims 1 to 9, wherein one or more of said oncogenes are regulated by said complex.

11. 11. The method of any one of claims 1-10, wherein the one or more cancer genes comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more cancer genes, and the genes comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more genes selected from ADIPOQ, CALB1, EREG, CXCL2, CXCL6, GC, CSF3, PTPRZ1, DGKB, CRH, XIST, SLC5A1, CCL11, NEGR1, CLDN10, and TF.

12. A method for treating cancer, comprising administering to a subject a therapeutic 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 lipid or a salt thereof; evaluating the control of cancer genes in the subject after administration of said complex; correlating the gene control with treatment results using the method of any one of claims 1 to 3 or 5 to 11; and determining a further course of treatment.

13. A method for treating a metabolic-related disease, comprising administering to a subject a therapeutic 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 lipid or a salt thereof; assessing the regulation of ADIPOQ network genes in the subject after administration of said complex; and correlating the regulation of the genes with treatment outcome using the method of any one of claims 4 to 11, and determining the course of further treatment.

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