Method of predicting response to cancer treatment

Abstract

The present invention relates to a method for predicting the response of a patient suffering from cancer to an anti-cancer treatment, said method being based on the detection of biomarkers. The invention also relates to methods of treatment and methods of selecting a cancer patient to be treated, or methods of selecting a suitable therapy for a cancer patient based on the detection of said biomarkers.

Description

Technical Field

[0001] The present invention relates to the field of cancer, and more particularly, to methods for predicting the response of a subject diagnosed with cancer to anti-cancer treatment based on the detection of biomarkers in a sample from the subject. It also relates to methods for selecting a customized therapy for a subject or for selecting a subject to be treated with an anti-cancer agent, and to anti-cancer agents used in the treatment mentioned in said methods based on the detection of said biomarkers. It further relates to kits and their use in said methods. Background Art

[0002] Cancer is the leading cause of death globally, with nearly 10 million deaths in 2020. It is a large group of diseases characterized by the uncontrolled growth of abnormal cells.

[0003] Myc is tightly regulated in normal cells, with higher levels in proliferating cells and lower levels in non-proliferating cells. Abnormally high and / or dysregulated Myc activity is causally related to most cancers and is often associated with aggressive, poorly differentiated, angiogenic, and treatment-resistant tumors.

[0004] Omomyc is a dominant-negative Myc mutant that contains the b-HLH-LZ domain of Myc and has four amino acid substitutions in the leucine zipper of Myc (Soucek, L. et al., 1998, Oncogene 17, 2463-2472; Soucek, L. et al. (2002), Cancer Res 62:3507-3510). The amino acid substitutions E61T, E68I, R74Q, and R75N confer altered dimerization specificity to the protein, which retains the ability to bind to its natural partner Max, form homodimers with itself, and form heterodimers with wild-type c-, N-, and L-Myc.

[0005] Because of these properties, Omomyc is able to block Myc-dependent gene transactivation functions in vitro and in vivo by eliminating Myc's ability to bind to its DNA recognition-binding site, the E-box. At the same time, Omomyc strongly enhances Myc-induced apoptosis in a Myc expression level-dependent manner and thus potentiates Myc transrepression activity. Omomyc thus blocks Myc binding to the promoter E-box and transactivation of target genes, while retaining Miz-1-dependent binding to the promoter and transrepression. In the presence of Omomyc, the Myc interactome is redirected to repression, and its activity is transformed from pro-oncogenic activity to tumor suppressor activity.

[0006] In WO 2014 / 180889 A8, it was demonstrated that the Omomyc peptide itself is able to efficiently transduce across cell membranes and translocate into the nucleus, where it exerts its tumor-suppressive effect.

[0007] In WO 2018 / 011433 A1, it was demonstrated that Omomyc mutants in which the only cysteine has been replaced by a different amino acid are even more effective than Omomyc in the treatment of cancer.

[0008] However, anti-cancer therapies may have limitations such as unwanted side effects and the emergence of congenital or acquired drug resistance. Selecting those patients who will respond better to treatment leads to an increase in the survival rate of cancer patients, avoiding unnecessary treatment side effects and treatment delays. Those patients whose tumors have a high likelihood of showing a poor response to anti-cancer treatment may be candidates for alternative treatments.

[0009] Therefore, personalized regimens are needed to better treat the disease and to identify further useful biomarkers to predict the response of cancer patients to anti-cancer treatment. SUMMARY OF THE INVENTION

[0010] In a first aspect, the present invention relates to an in vitro method for predicting the clinical response of a subject suffering from cancer to an anti-cancer treatment selected from:

[0011] a) a polypeptide comprising the sequence SEQ ID NO:1 or a functional equivalent variant thereof;

[0012] b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO:1 or a functional equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or its functional equivalent variant;

[0013] c) a polynucleotide encoding the polypeptide of a) or the conjugate of b);

[0014] d) a vector comprising the polynucleotide according to c); and

[0015] e) a cell capable of secreting into a culture medium the polypeptide according to a) or the conjugate according to b);

[0016] The method comprises:

[0017] (i) determining the level of at least one biomarker selected from MIP-1β, CD62E, IL-8, GM-CSF and IL-1α in a sample from the subject; and

[0018] (ii) comparing the level of the at least one biomarker with a reference value,

[0019] wherein:

[0020] - A reduced level of said at least one biomarker relative to said reference value indicates a good clinical response of the subject to said anti-cancer treatment, or

[0021] - An equal or increased level of said at least one biomarker relative to said reference value indicates a poor clinical response of the subject to said anti-cancer treatment.

[0022] In a second aspect, the present invention relates to an in vitro method for selecting a customized therapy for a subject suffering from cancer, comprising:

[0023] (i) determining the level of at least one biomarker selected from MIP-1β, CD62E, IL-8, GM-CSF and IL-1α in a sample from said subject; and

[0024] (ii) comparing the level of said at least one biomarker with a reference value,

[0025] wherein:

[0026] - A reduced level of said at least one biomarker relative to said reference value indicates that the therapy to be selected comprises an agent selected from:

[0027] a) a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof;

[0028] b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of said polypeptide or functionally equivalent variant thereof;

[0029] c) a polynucleotide encoding the polypeptide of a) or the conjugate of b);

[0030] d) a vector comprising the polynucleotide according to c); and

[0031] e) a cell capable of secreting the polypeptide according to a) or the conjugate according to b) into a culture medium; or alternatively,

[0032] - An equal or increased level of said at least one biomarker relative to said reference value indicates that the therapy to be selected does not comprise an agent selected from:

[0033] a) a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof;

[0034] b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of said polypeptide or functionally equivalent variant thereof;

[0035] c) A polynucleotide that encodes the polypeptide of a) or the conjugate of b);

[0036] d) A vector that contains the polynucleotide according to c); and

[0037] e) A cell that is capable of secreting the polypeptide according to a) or the conjugate according to b) into a culture medium.

[0038] In a third aspect, the present invention relates to an in vitro method for selecting a subject suffering from cancer for treatment with an agent selected from the following:

[0039] a) A polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof;

[0040] b) A conjugate that contains a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or its functionally equivalent variant;

[0041] c) A polynucleotide that encodes the polypeptide of a) or the conjugate of b);

[0042] d) A vector that contains the polynucleotide according to c); and

[0043] e) A cell that is capable of secreting the polypeptide according to a) or the conjugate according to b) into a culture medium;

[0044] The method comprises:

[0045] (i) Determining the level of at least one biomarker selected from MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α in a sample from the subject; and

[0046] (ii) Comparing the level of the at least one biomarker with a reference value, wherein if a reduced level of the at least one biomarker relative to the reference value is detected, the patient is selected for the treatment.

[0047] In a fourth aspect, the present invention relates to an agent selected from the following for treating cancer in a subject:

[0048] a) A polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof;

[0049] b) A conjugate that contains a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or its functionally equivalent variant;

[0050] c) A polynucleotide that encodes the polypeptide of a) or the conjugate of b);

[0051] d) a vector comprising the polynucleotide according to c); and

[0052] e) a cell capable of secreting the polypeptide according to a) or the conjugate according to b) into a culture medium;

[0053] wherein the subject has been identified as a good responder to the agent by the first method of the present invention, or wherein the treatment has been selected by the second method of the present invention.

[0054] In another aspect, the present invention relates to a kit comprising reagents specifically for determining the expression level of at least one biomarker selected from MIP-1β, CD62E, IL-8, GM-CSF and IL-1α.

[0055] In yet another aspect, the present invention relates to the use of the kit of the present invention or the reagents specifically for determining the expression level of at least one biomarker selected from MIP-1β, CD62E, IL-8, GM-CSF and IL-1α in the first, second and third methods of the present invention. Description of the Drawings

[0056] Figure 1 . Patients responsive to OMO-103 showed low levels of CD62E, IL-8, MIP-1β, GM-CSF and IL-1α at baseline. Levels of different cytokines and chemokines were measured in patient serum samples collected at pretreatment using Luminex technology. A) Patients responsive to treatment and showing stable disease (SD) at cycle 3 showed significantly lower levels of CD62E, IL-8, MIP-1β, GM-CSF and IL-1α compared to patients showing progressive disease (PD). B) Levels of different cytokines and chemokines measured at pretreatment, in pg / ml. Mean values and standard deviations are shown.

[0057] Figure 2 . CD62E, IL-8, MIP-1β, GM-CSF and IL-1α are excellent predictors of response to OMO-103. Receiver operating characteristic (ROC) curve analysis of various cytokines for predicting response to OMO-103. A) Individual ROC curve plots. B) ROC-AUC scores for each individual cytokine / chemokine. The dashed line indicates an AUC score of 0.5 and the dash line indicates an AUC score of 0.8, and this threshold indicates excellent predictive ability.

[0058] Figure 3. Several combinations of two cytokines (CD62E + IL-8, MCP-1 + MIP-1β, MIP-1β + ICAM-1, MIP-1β + CD62E, MIP-1β + IFN-γ, MIP-1β + IL-1β, and MIP-1β + IL-12) are excellent predictors of the response to OMO-103 and can be used to grade SD and PD patients at pretreatment. A cytokine / chemokine combination model is generated using QLattice technology. A) shows plots of the CD62E + IL-8, MCP-1 + MIP-1β, MIP-1β + ICAM-1, MIP-1β + CD62E, MIP-1β + IFN-γ, MIP-1β + IL-1β, and MIP-1β + IL-12 combination models and how they grade patients. Stable disease (SD) is represented by empty dots / zeros, and progressive disease (PD) is represented by gray / ones. The gray lines correspond to the confidence intervals (CI), the dark line represents the mean, the medium gray line represents the 5% CI, and the light gray line represents the 95% CI. B) Receiver operating characteristic (ROC) curve analysis of the seven combination models. Detailed Description of the Invention

[0059] The inventors of the present invention have found, surprisingly, that in pretreatment serum samples of subjects suffering from cancer who respond well to treatment with a polypeptide (comprising Omomyc), the levels of the biomarkers MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α are low.

[0060] Therefore, these biomarkers have the potential value to predict a good response of cancer patients to treatment based on Omomyc or its functional equivalent variants. Based on these findings, the inventors of the present invention have developed the methods of the present invention in their different embodiments, which will now be described in detail.

[0061] The results provided in the examples of the present invention clearly show that low levels of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α are significantly associated with a good response to OMO-103 treatment. Therefore, these results suggest that subjects suffering from cancer with low levels of these biomarkers in pretreatment samples are candidates for treatment based on Omomyc or its functional equivalent variants.

[0062] The Predictive Method of the Present Invention

[0063] In a first aspect, the present invention relates to an in vitro method for predicting the clinical response of a subject suffering from cancer to an anticancer treatment selected from:

[0064] a) a polypeptide comprising the sequence SEQ ID NO:1 or a functional equivalent variant thereof;

[0065] b) A conjugate comprising a polypeptide comprising the sequence SEQ ID NO:1 or a functional equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or its functional equivalent variant;

[0066] c) A polynucleotide encoding the polypeptide of a) or the conjugate of b);

[0067] d) A vector comprising the polynucleotide according to c); and

[0068] e) A cell capable of secreting the polypeptide according to a) or the conjugate according to b) into a culture medium;

[0069] The method comprises:

[0070] (i) Determining the level of at least one biomarker selected from MIP-1β, CD62E, IL-8, GM-CSF and IL-1α in a sample from the subject; and

[0071] (ii) Comparing the level of the at least one biomarker with a reference value,

[0072] wherein:

[0073] - A decreased level of the at least one biomarker relative to the reference value indicates a good clinical response of the subject to the anti-cancer treatment, or

[0074] - An equal or increased level of the at least one biomarker relative to the reference value indicates a poor clinical response of the subject to the anti-cancer treatment.

[0075] The term "in vitro" as used herein denotes the fact that the experimental protocol or method is not carried out on the body of a human or animal subject, but on a sample isolated from the subject and already present in laboratory tools such as test tubes, culture dishes or plates.

[0076] The term "predict" as used herein denotes determining the likelihood that a subject suffering from cancer will respond favorably to a therapy using an anti-cancer treatment. In particular, the term "predict" as used herein relates to an individual assessment of the expected response of a subject suffering from cancer if the tumor is treated with an anti-cancer treatment based on Omomyc or a functional equivalent variant thereof as defined in items (a) to (e) of the first aspect of the present invention.

[0077] As used herein, the term "clinical response" refers to the response of a subject suffering from cancer to an anti-cancer treatment with Omomyc or a functionally equivalent variant thereof as defined in items (a) to (e) of the first aspect of the present invention. Standard norms that can be used concomitantly to evaluate the response to an anti-cancer therapy (Eisenhauer, E.A., et al. 2009. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 45(2):228-247) include response, stability, and progression. The term "RECIST" as used herein refers to "Response Evaluation Criteria in Solid Tumours", and it is a standard method for measuring how well a cancer patient responds to treatment. It is based on whether the tumour shrinks, stays the same, or grows. To use RECIST, there must be at least one tumour that can be measured by x-ray, computed tomography (CT) scan, or magnetic resonance imaging (MRI) scan. The types of responses a patient may have are complete response (CR), partial response (PR), progressive disease (PD), and stable disease (SD). In a preferred embodiment, the RECIST criteria for tumour response assessment have been used.

[0078] In the context of the present invention, subjects who achieve a complete or partial response and subjects who exhibit stable disease are considered "good responders" or have a good clinical response.

[0079] As used herein, "complete response" (or complete remission) (CR) is the disappearance of all detectable malignant disease, i.e., the disappearance of all target lesions, where the short axis of any pathological lymph node (whether target or non-target) must shrink to <10 mm.

[0080] "Partial response" (PR) is defined herein as at least a 30% reduction in the sum of the diameters of the target lesions, referenced to the sum of the baseline diameters.

[0081] The term "stable", "stable disease", or "stable disease" (SD) as used herein refers to neither sufficient shrinkage to meet the criteria for partial response nor sufficient increase to meet the criteria for progressive disease, referenced to the smallest sum of diameters in the study.

[0082] In the context of the present invention, subjects who exhibit progressive disease are considered "poor responders" or have a poor clinical response.

[0083] As used herein, the term "progression" or "progressive disease" (PD) is defined as at least a 20% increase in the sum of the diameters of target lesions, referenced to the smallest sum in the study (including the baseline sum if the smallest in the study). In addition to the 20% relative increase, the sum must also show an absolute increase of at least 5 mm. The appearance of one or more new lesions is also considered progression.

[0084] Those skilled in the art will understand that a poor response to anti-cancer treatment does not mean that the subject has no response at all or that the treatment has no effect on the subject. For example, compared to patients who have not undergone any treatment, anti-cancer treatment can reduce the growth of lesions in poor responders. However, compared to untreated patients, the effect achieved in the subject does not meet the conditions considered stable under the criteria used in the present invention. For example, a patient treated with anti-cancer therapy may have a 20% increase in the sum of the diameters of target lesions, but if the patient does not receive treatment, the increase may be 30%.

[0085] As will be understood by those skilled in the art, such an assessment is not generally intended to be correct for all (i.e., 100%) of the subjects to be identified. However, the term requires that the prediction provide correct results for a statistically significant proportion of the subjects. Using various well-known statistical evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney test, etc., those skilled in the art can readily determine whether a proportion is statistically significant. See Dowdy and Wearden, Statistics for Research, John Wiley and Sons, New York 1983 for details. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p-value is preferably 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001 or less. More preferably, at least 60%, at least 70%, at least 80% or at least 90% of the subjects in a population can be appropriately identified by the method of the present invention.

[0086] Any other parameter widely accepted for comparing the efficacy of alternative treatments can be used to determine the response to treatment and includes, but is not limited to:

[0087] ● No disease progression , which as used herein, describes the proportion of subjects with complete remission who have no disease recurrence during the study period.

[0088] ● Disease-free survival (DFS) , which as used herein, is understood to be the length of time after treatment of the disease that the subject survives without signs of the disease.

[0089] ● Objective response , as used herein in the context of the present invention, describes the proportion of treated subjects in whom a complete or partial response was observed.

[0090] ● Tumor control , as used herein in the context of the present invention, refers to the proportion of treated subjects in whom a complete response, partial response, minor response, or stable disease was observed for ≥6 months.

[0091] ● Progression-free survival , as used herein, is defined as the time from the start of treatment to the first measurement of cancer growth.

[0092] ● Time to progression (TTP) , as used herein, refers to the time after treatment of a disease until the disease begins to worsen. The term "progression" has been defined previously.

[0093] ● 6-month progression-free survival rate Or the "PFS6" rate, as used herein, refers to the percentage of subjects who are progression-free within the first six months after the start of treatment.

[0094] ● Median survival , as used herein, refers to the time at which half of the subjects participating in the study are still alive.

[0095] ● Overall survival , as used herein, represents the length of time that a patient diagnosed with a disease, such as cancer, remains alive from the date of diagnosis or the start of treatment of the disease.

[0096] ● Recurrence , as used herein, represents the appearance of cancer after treatment and after a period of time during which the cancer was not detected.

[0097] ● Metastasis-free survival , as used herein, represents the length of time that a patient survives after the end of cancer treatment without any signs or symptoms of cancer metastasis.

[0098] ● Reduction of circulating tumor cells , as used herein, represents a decrease in the concentration of circulating tumor cells in the blood or lymph of a patient with metastatic cancer. A decrease in circulating tumor cells is related to the efficacy of treatment for metastatic cancer.

[0099] ● Response of circulating markers , as used herein, represents a change in the concentration of a protein or nucleic acid related to a specific cancer in the blood or lymph after treatment for the specific cancer.

[0100] The method of the invention is carried out in subjects suffering from cancer, i.e., subjects who have previously been diagnosed with cancer.

[0101] As used herein, the term "subject" or "patient" denotes all animals classified as mammals and includes, but is not limited to, domestic and farm animals, primates and humans (e.g., humans), non-human primates, cattle, horses, pigs, sheep, goats, dogs, cats or rodents. Preferably, the subject is a male or female of any age or race. In the context of the present invention, the subject is a subject suffering from cancer or previously diagnosed with cancer. In a preferred embodiment, the subject is a mammal, preferably a human.

[0102] The subjects tested in the first method of the invention have previously been diagnosed with cancer. As used herein, the term "diagnosis" denotes the determination and / or identification of a disease in a subject, i.e., an opinion reached regarding the disease state of the subject, i.e., a diagnostic opinion. Thus, it can also be regarded as an attempt to classify an individual according to their disease status. Those skilled in the art will understand that the diagnosis of cancer, although preferably correct, need not be correct for 100% of the subjects to be diagnosed or evaluated. However, the term requires that a statistically significant proportion of the subjects identified as such are suffering from cancer. Methods for determining whether the proportion of subjects is statistically significant have been disclosed above with respect to the prediction method.

[0103] The method of the invention is applicable to any type of cancer.

[0104] The term "cancer" denotes a group of diseases characterized by uncontrolled cell division (or increased survival or resistance to apoptosis) and by the ability of the cells to invade other adjacent tissues (invasion) and spread through lymphatic and blood vessels to other areas of the body where the cells do not normally occur (metastasis), circulate in the bloodstream and then invade normal tissues elsewhere in the body. Tumors are classified as benign or malignant according to their ability to spread by invasion and metastasis: benign tumors are tumors that cannot spread by invasion or metastasis, i.e., they grow only locally; while malignant tumors are tumors that are able to spread by invasion and metastasis. Biological processes known to be associated with cancer include angiogenesis, immune cell infiltration, cell migration and metastasis.

[0105] The term “cancer” includes, but is not limited to, leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute granulocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic granulocytic leukemia, chronic lymphocytic leukemia), hairy cell leukemia, polycythemia vera, lymphoma (e.g., Hodgkin's disease or non-Hodgkin's disease), CNS lymphoma, AIDS-related leukemia, Waldenström macroglobulinemia, multiple myeloma, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, Kaposi's sarcoma, colon cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, bladder cancer, breast cancer, biliary tract cancer, esophageal cancer, stomach / gastric cancer, ovarian cancer (including those originating from epithelial cells, stromal cells, germ cells, and mesenchymal cells), prostate cancer, oral cancer (including squamous cell carcinoma), basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchioloalveolar carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, teratoma, choriocarcinoma, endometrial / uterine / cervical cancer, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, intraepithelial neoplasia (including Bowen's disease and Paget's disease), glioma, glioblastoma, mixed glioma, optic nerve glioma, subependymal tumor, metastatic brain tumor, pituitary tumor, primitive neuroectodermal (PNET) tumor, juvenile pilocytic astrocytoma (JPA), brainstem glioma, astrocytoma, pineal tumor, rhabdoid tumor, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0106] The term "cancer" also includes, but is not limited to, cancers of the head and neck, leukemia, cancer of the heart, cancer of the esophagus, cancer of the small intestine, cancer of the spleen, cancer of the kidney, cancer of the brain, choriocarcinoma, cancer of the skin, cancer of the bone, cancer of the bone marrow, blood cancer, cancer of the thymus, cancer of the womb, cancer of the liver, sarcoma, liposarcoma, fibrosarcoma, Merkel cell carcinoma, Kaposi's sarcoma, cancer of the testis (including germ cell tumors such as seminoma, non-seminoma (teratoma, choriocarcinoma), stromal tumors, and gonadoblastoma); cancer of the thyroid, including thyroid adenocarcinoma and medullary carcinoma; cancer of the kidney, including adenocarcinoma and nephroblastoma; cholangiocarcinoma, glioblastoma, hematological neoplasms (including acute lymphocytic and myeloid leukemia, T-cell acute lymphoblastic leukemia / lymphoma, hairy cell leukemia, chronic myeloid leukemia, multiple myeloma, AIDS-related leukemia, and adult T-cell leukemia / lymphoma), intraepithelial neoplasms (including Bowen's disease and Paget's disease), lymphoma (including Hodgkin's disease and lymphocytic lymphoma), cancer of the mouth (including squamous cell carcinoma), adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germ cell tumor, glioma, hemangioendothelioma, angiosarcoma, hematoma, hepatoblastoma, medulloblastoma, melanoma, cancer of the parotid gland, neuroblastoma, cholangiocarcinoma of the liver, cancer of the adrenal gland, osteosarcoma, retinoblastoma, rhabdomyosarcoma, and teratoma. In addition, the term includes acral lentiginous melanoma, actinic keratosis adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytoma, adenocarcinoma of Bartholin gland, basal cell carcinoma, bronchioloalveolar carcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymosarcoma, Ewing's sarcoma, focal nodular hyperplasia, germ cell tumor, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma, insulinoma, intraepithelial neoplasia, squamous intraepithelial neoplasia, invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma, malignant melanoma, malignant mesothelial tumor, meduloblastoma, medulloepithelioma, mucoepidermoid carcinoma, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, papillary serous adenocarcinoma, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, serous carcinoma, microcytic carcinoma, soft tissue carcinoma, tumor secreting somatostatin, squamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vasoactive intestinal peptide tumor, nephroblastoma, intracerebral carcinoma, rectal carcinoma, astrocytoma, microcytic carcinoma and non-microcytic carcinoma, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer.

[0107] In another embodiment, the cancer includes, but is not limited to, mesothelioma, hepatobiliary (liver and bile duct) cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, gastrointestinal cancer (gastric cancer, colorectal cancer, and duodenal cancer), uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myelogenous leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell cancer, renal pelvic cancer, non-Hodgkin's lymphoma, spinal axis tumor, brainstem glioma, pituitary adenoma, adrenocortical carcinoma, gallbladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the foregoing cancers.

[0108] In certain embodiments, the cancer is selected from hepatocellular carcinoma, ovarian cancer, ovarian epithelial cancer, or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hilar cholangiocarcinoma; soft tissue and bone synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing's sarcoma; anaplastic thyroid cancer; adrenocortical adenoma; pancreatic cancer; pancreatic ductal carcinoma, or pancreatic adenocarcinoma; gastrointestinal / stomach (GIST) cancer; lymphoma; head and neck squamous cell carcinoma (SCCHN); salivary gland cancer; glioma, or brain cancer; neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST); Waldenström macroglobulinemia; or medulloblastoma.

[0109] In certain embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial cancer, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), hilar cholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid cancer, adrenocortical adenoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST), Waldenström macroglobulinemia, or medulloblastoma.

[0110] In a preferred embodiment, the cancer is a solid tumor.

[0111] Examples of solid tumors are sarcomas, carcinomas, or lymphomas. Solid tumors typically comprise abnormal masses of tissue and generally do not include cysts or fluid regions. In certain embodiments, the cancer is selected from renal cell carcinoma or kidney cancer; hepatocellular carcinoma (HCC) or hepatoblastoma or liver cancer; melanoma; breast cancer; colorectal cancer or colorectal carcinoma; colon cancer; rectal cancer; anal cancer; lung cancer, such as non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC); ovarian cancer, ovarian epithelial cancer, ovarian carcinoma, or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hilar cholangiocarcinoma; soft tissue and bone synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing sarcoma; anaplastic thyroid cancer; adrenocortical carcinoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / gastric (GIST) cancer; lymphoma; head and neck squamous cell carcinoma (SCCHN); salivary gland cancer; glioma, or brain cancer; neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST); Waldenström macroglobulinemia; or medulloblastoma. In one embodiment, the solid tumor is selected from non-small cell lung cancer (NSCLC), breast cancer, and colorectal cancer. In a particular embodiment of the invention, the solid cancer is selected from pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), salivary gland cancer, sarcoma, triple negative breast cancer, and pleural mesothelioma. In a particular embodiment of the invention, the solid cancer is selected from pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), salivary gland cancer, and sarcoma.

[0112] In certain embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial cancer, ovarian carcinoma, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), hilar cholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid cancer, adrenocortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST), Waldenström macroglobulinemia, or medulloblastoma.

[0113] In certain embodiments, the cancer is hepatocellular carcinoma (HCC). In certain embodiments, the cancer is hepatoblastoma. In certain embodiments, the cancer is colon cancer. In certain embodiments, the cancer is rectal cancer. In certain embodiments, the cancer is ovarian cancer or ovarian carcinoma. In certain embodiments, the cancer is ovarian epithelial carcinoma. In certain embodiments, the cancer is fallopian tube cancer. In certain embodiments, the cancer is papillary serous cystadenocarcinoma. In certain embodiments, the cancer is uterine papillary serous carcinoma (UPSC). In certain embodiments, the cancer is cholangiocarcinoma. In certain embodiments, the cancer is soft tissue and bone synovial sarcoma. In certain embodiments, the cancer is rhabdomyosarcoma. In certain embodiments, the cancer is osteosarcoma. In certain embodiments, the cancer is anaplastic thyroid cancer. In certain embodiments, the cancer is adrenocortical carcinoma. In certain embodiments, the cancer is pancreatic cancer or pancreatic ductal carcinoma. In certain embodiments, the cancer is pancreatic adenocarcinoma. In certain embodiments, the cancer is glioma. In certain embodiments, the cancer is malignant peripheral nerve sheath tumor (MPNST). In certain embodiments, the cancer is neurofibromatosis-1 associated MPNST. In certain embodiments, the cancer is Waldenstrom's macroglobulinemia. In certain embodiments, the cancer is medulloblastoma.

[0114] In certain embodiments, the cancer is a virus-related cancer, including human immunodeficiency virus (HIV)-associated solid tumors, human papillomavirus (HPV)-16 positive incurable solid tumors, and adult T-cell leukemia, which is caused by human T-cell leukemia virus type I (HTLV-I) and is a highly aggressive form of CD4+ T-cell leukemia characterized by clonal integration of HTLV-I in leukemic cells; and virus-related tumors in gastric cancer, nasopharyngeal cancer, cervical cancer, vaginal cancer, vulvar cancer, head and neck squamous cell carcinoma, and Merkel cell carcinoma.

[0115] Other cancers will be known to those of ordinary skill in the art.

[0116] The term cancer includes primary tumors as well as metastatic tumors. In one embodiment, the cancer is a primary tumor. As used herein, the term "primary tumor" refers to a tumor that originates in the site or organ in which it is present and has not metastasized to that site from another site. Thus, in one embodiment, the cancer to be treated is a non-metastatic cancer. In another embodiment, the cancer is a metastatic cancer or cancer metastasis. In the context of the present invention, "metastasis" is understood to mean the spread of cancer from the organ in which it is located to a different organ.

[0117] The anti-cancer treatment of the present invention is capable of reducing cell proliferation, regardless of whether the cancer shows increased Myc protein expression or activity. In one embodiment, the cancer to be prevented or treated is Myc-induced cancer. In another embodiment, the cancer to be prevented or treated is not Myc-induced cancer.

[0118] The term "treat" ("treat" or "treatment") refers to therapeutic treatment, as well as prophylactic or preventive methods, where the goal is to prevent or reduce an undesired physiological change or disease, such as cancer. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, shortening of disease duration, stabilization of the pathological state (especially without deterioration), delay in disease progression, improvement and remission (partial and complete) of detectable and non-detectable pathological states. "Treatment" can also refer to an extension of the survival period compared to the expected survival period if treatment is not applied. Subjects in need of treatment include subjects suffering from cancer.

[0119] The authors of the present invention have found several biomarkers that are significantly reduced in subjects who will respond better to treatment with OMO-103, which is a polypeptide containing Omomyc and having SEQ ID NO:4. All Omomyc-based agents act on the same target. Thus, the biomarkers of the present invention will also be significantly reduced in good responders to treatment with any other Omomyc-based agent, which is administered as a polypeptide, a conjugate with other chemical moieties, a nucleic acid or a vector for gene therapy, or as a cell capable of secreting the polypeptide or conjugate into the culture medium in cell therapy.

[0120] Thus, in the context of the present invention, the methods of the present invention allow prediction of the clinical response to anti-cancer treatments selected from:

[0121] a) a polypeptide having the sequence SEQ ID NO:1 or a functional equivalent variant thereof;

[0122] b) a conjugate containing a polypeptide having the sequence SEQ ID NO:1 or a functional equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or its functional equivalent variant;

[0123] c) a polynucleotide encoding the polypeptide of a) or the conjugate of b);

[0124] d) a vector containing the polynucleotide according to c); and

[0125] e) a cell capable of secreting the polypeptide according to a) or the conjugate according to b) into the culture medium.

[0126] In the context of the first method of the present invention, the term "anticancer treatment" refers to any treatment involving exposure of a subject who is treated with a method for inducing cancer cell death selected from items (a) to (e) above. Preferably, the anticancer treatment is administered intravenously; preferably as a 30-minute intravenous infusion. Preferably, the anticancer treatment is administered once a week.

[0127] In a preferred embodiment, the anticancer treatment comprises a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof, more preferably a polypeptide comprising the sequence SEQ ID NO:1.

[0128] The terms "polypeptide" and "peptide" are used interchangeably herein to denote a polymer of amino acids of any length. The polypeptides of the present invention may contain modified amino acids and it may be interrupted by non-amino acids. In a preferred embodiment, the polypeptide is formed solely of amino acids. Preferably, the polypeptide of item (a) of the anticancer treatment has a length between 80 and 500 amino acids, more preferably between 80 and 300 amino acids, more preferably between 80 and 250 amino acids, more preferably between 80 and 150 amino acids, even more preferably between 80 and 130 amino acids, preferably between 90 and 130 amino acids, preferably not more than 125 amino acids, more preferably not more than 100 amino acids. In a preferred embodiment, the polypeptide has a length between 90 and 98 amino acids, preferably between 90 and 95 amino acids, more preferably 91 amino acids.

[0129] The term "amino acid" denotes natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to natural amino acids. In addition, the term "amino acid" includes D- and L-amino acids (stereoisomers). Preferably, the amino acids are L-amino acids.

[0130] The term "natural amino acid" or "naturally occurring amino acid" encompasses the 20 naturally occurring amino acids; those amino acids that are often post-translationally modified in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; and other rare amino acids, including, but not limited to, 2-aminoadipic acid, hydroxylysine, alloisoleucine, norvaline, norleucine, and ornithine.

[0131] As used herein, the term "non-natural amino acid" or "synthetic amino acid" refers to a carboxylic acid or a derivative thereof in which the amine group replaces the position "a" and is structurally related to a natural amino acid. Exemplary non-limiting examples of modified or uncommon amino acids include 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminoheptanedioic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, halogenated (alio) hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyl-lysine, N-methylvaline, norvaline, norleucine, ornithine, etc.

[0132] The polypeptides of the present invention may also comprise non-amino acid moieties, e.g., hydrophobic moieties attached to the peptide (various straight-chain, branched-chain, cyclic, polycyclic or heterocyclic hydrocarbons and hydrocarbon derivatives); various protecting groups attached to the termini of the compounds to reduce degradation. Suitable protecting functional groups are described in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and 7, 1991.

[0133] Chemical (non-amino acid) groups that may be included in the polypeptides to improve various physiological properties such as reduced degradation or clearance; reduced rejection by various cellular pumps, improved various modes of administration, increased specificity, increased affinity, increased stability, bioavailability, solubility, reduced toxicity, etc.

[0134] "Mimetics" include molecules that mimic the peptide structure and retain the functional properties of the peptide structure. Protocols for designing peptide mimetics, derivatives and analogs are known in the art.

[0135] In one embodiment, the polypeptide of the present invention is a polypeptide consisting of the sequence SEQ ID NO:1 or a polypeptide consisting of a functional equivalent variant of SEQ ID NO:1, preferably a polypeptide consisting of the sequence SEQ ID NO:1.

[0136] SEQ ID NO:1 corresponds to

[0137] TEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA (SEQ ID NO:1)

[0138] The polypeptide of SEQ ID NO:1 corresponds to the Omomyc protein sequence. The term "Omomyc" as used herein refers to a polypeptide consisting of a mutant form of the bHLHZip domain of Myc carrying the E61T, E68I, R74Q, and R75N mutations (where the numbering of the mutation positions is given relative to the sequence of the Myc region corresponding to amino acids 365 - 454 of the polypeptide defined under accession number NP_002458 in the NCBI database released on March 15, 2015). The sequence of c-Myc provided under accession number NP_002458 in the NCBI database is shown below (SEQ ID NO:2), with the region from which Omomyc is derived underlined:

[0139] 1 MDFFRVVENQ QPPATMPLNV SFTNRNYDLD YDSVQPYFYC DEEENFYQQQ QQSELQPPAP

[0140] 61 SEDIWKKFEL LPTPPLSPSR RSGLCSPSYV AVTPFSLRGD NDGGGGSFST ADQLEMVTEL

[0141] 121 LGGDMVNQSF ICDPDDETFI KNIIIQDCMW SGFSAAAKLV SEKLASYQAA RKDSGSPNPA

[0142] 181 RGHSVCSTSS LYLQDLSAAA SECIDPSVVF PYPLNDSSSP KSCASQDSSA FSPSSDSLLS

[0143] 241 STESSPQGSP EPLVLHEETP PTTSSDSEEE QEDEEEIDVV SVEKRQAPGK RSESGSPSAG

[0144] 301 GHSKPPHSPL VLKRCHVSTH QHNYAAPPST RKDYPAAKRV KLDSVRVLRQ ISNNRKCTSP

[0145] 361 RSSD TEENVKRRTHNVLERQ RRNELKRSFF ALRDQIPELE NNEKAPKVVI LKKATAYILS

[0146] 421 VQAEEQKLIS EEDLLRKRRE QLKHKLEQLR NSCA (SEQ ID NO:2)

[0147] Omomyc also contains the M2 domain of c-Myc, which has the sequence RQRRNELKRSF (SEQ ID NO:3) (see Dang and Lee, Mol. Cell. Biol., 1988, 8:4048-4054) (double underlined), and which corresponds to the nuclear localization signal.

[0148] Omomyc is characterized in that it shows an increased ability to dimerize with all three oncogenic Myc proteins (c-Myc, N-Myc, and L-Myc). Omomyc can be derived from the bHLHZip domain of any Myc protein known in the art, provided that the mutations leading to tumor suppression are retained. Thus, Omomyc that can be used in the present invention can be derived from any mammalian species, including but not limited to domestic and farm animals (cattle, horses, pigs, sheep, goats, dogs, cats, or rodents), primates, and humans. Preferably, the Omomyc protein is derived from the human Myc protein (accession number NP_002458, released on March 12, 2019).

[0149] The term "Myc" as used herein refers to a family of transcription factors including c-Myc, N-Myc, and L-Myc. Myc proteins activate the expression of many genes by binding to the consensus sequence CACGTG (enhancer-box sequence or E-box and recruiting histone acetyl-transferase or HAT). However, Myc can also act as a transcriptional repressor. By binding to the Miz-1 transcription factor and displacing the p300 co-activator, it inhibits the expression of Miz-1 target genes. Myc also has a direct role in controlling DNA replication.

[0150] The Myc b-HLH-LZ or Myc basic region helix-loop-helix leucine zipper domain refers to the region that determines the dimerization of Myc with the Max protein and the binding to Myc-target genes. This region corresponds to amino acids 365-454 of human Myc and is characterized by two α helices linked by a loop (Nair, S.K., and Burley, S.K., 2003, Cell, 112:193-205).

[0151] In a preferred embodiment, the polypeptide of the invention is a polypeptide comprising, consisting of, or consisting essentially of SEQ ID NO:4 as shown below.

[0152] MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA (SEQ ID NO:4)

[0153] In this context, "consisting essentially of" means that the particular molecule will not contain any additional sequences that would alter the activity of SEQ ID NO:4.

[0154] Preferably, the polypeptide consists of SEQ ID NO:4.

[0155] In a preferred embodiment, the anti-cancer treatment comprises the use of a polypeptide consisting of SEQ ID NO:4.

[0156] The term "functionally equivalent variant" means any polypeptide produced by the insertion or addition of one or more amino acids and / or the deletion of one or more amino acids and / or the conservative substitution of one or more amino acids relative to the polypeptide of SEQ ID NO:1, and / or any polypeptide produced by chemical modification of the polypeptide of SEQ ID NO:1 and that substantially retains the tumor suppressor activity of SEQ ID NO:1. Preferably, the functionally equivalent variant means any polypeptide produced by the insertion or addition of one or more amino acids and / or the deletion of one or more amino acids and / or the conservative substitution of one or more amino acids relative to the polypeptide of SEQ ID NO:1 and that substantially retains the tumor suppressor activity of SEQ ID NO:1; more preferably produced by the insertion or addition of one or more amino acids relative to the polypeptide of SEQ ID NO:1.

[0157] Those skilled in the art will understand that retention of tumor suppressor activity requires that the variant be able to dimerize with Myc and / or its obligate partner p21 / p22Max and inhibit Myc activity, that it be able to translocate across the cell membrane and that it be able to translocate across the nuclear membrane. In certain embodiments, the functionally equivalent variants of the polypeptides of the invention homodimerize less than Omomyc, or are not forced to form homodimers by the formation of disulfide bridges. Specifically, there is less disulfide bridge formation in the homodimeric form of certain embodiments of the polypeptides of the invention than in the polypeptide Omomyc.

[0158] As used herein, "less homodimerization" refers to a lower ability to form obligate homodimers of the polypeptides of the present invention even under reducing conditions. In a preferred embodiment, said ability is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% lower than the ability to form homodimers of Omomyc.

[0159] The reducing conditions used herein involve the presence of a reducing agent, which is a compound that donates electrons to another chemical substance in a redox chemical reaction. Exemplary non-limiting examples of reducing agents are DTT (dithiothreitol), β-mercaptoethanol or TCEP (tris(2-carboxyethyl)phosphine). The amount of homodimer may be the same in vitro, and the difference between the functional equivalent variant and Omomyc lies only in cells where a heterodimerization partner is present, where the absence of disulfide bonds allows for a higher degree of heterodimer formation.

[0160] Several assays can be used to determine the homodimerization of a peptide, for example, thermal denaturation monitored by circular dichroism as an exemplary non-limiting example, and thus dimerization can be detected by quantification of folding and thermal stability.

[0161] Suitable functional equivalent variants include polypeptides consisting essentially of the polypeptide of SEQ ID NO:1. In this context, "consisting essentially of" means that a particular molecule will not contain any additional sequences that would alter the activity of SEQ ID NO:1.

[0162] In a preferred embodiment, a functional equivalent variant of SEQ ID NO:1 is a polypeptide produced by the insertion or addition of one or more amino acids relative to the polypeptide of SEQ ID NO:1. In one embodiment, said functional equivalent variant is produced by an insertion of less than 10 amino acids, more preferably less than 5 amino acids, and even more preferably by an insertion of one amino acid. In a preferred embodiment, it is produced by the insertion of one amino acid, which is methionine.

[0163] In another embodiment, a functional equivalent variant of SEQ ID NO:1 is a polypeptide produced by the deletion of one or more amino acids relative to the polypeptide of SEQ ID NO:1. In one embodiment, said functional equivalent variant is produced by a deletion of less than 10 amino acids, more preferably less than 5 amino acids, and even more preferably by a deletion of one amino acid.

[0164] Suitable functional variants of the targeting peptide are those that exhibit a degree of amino acid sequence identity of about greater than 25% (such as 25%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) relative to the peptide of SEQ ID NO:1. Computer algorithms and methods widely known to those skilled in the art are used to determine the degree of identity between two polypeptides. Preferably, the identity between two amino acid sequences is determined by using the BLASTP algorithm as described above (BLAST Manual, Altschul, S., et al., NCBI / NLM / NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 1990; 215:403-410). In a preferred embodiment, sequence identity is determined over the entire length of the polypeptide of SEQ ID NO:1 or over the entire length of the variant or both.

[0165] Functional equivalent variants of the polypeptides of the present invention may also include post-translational modifications such as glycosylation, acetylation, isoprenylation, myristoylation, proteolytic processing, etc.

[0166] In another embodiment, suitable functional variants of the targeting peptide are variants in which one or more positions within the polypeptides of the present invention contain an amino acid that is a conservative substitution of an amino acid present in the above proteins. "Conservative amino acid substitutions" result from replacing one amino acid with another amino acid having similar structure and / or chemical properties. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W). The selection of such conservative amino acid substitutions is within the skill of the ordinary artisan in the art and is described, for example, by Dordo et al. (J. Mol. Biol, 1999, 217; 721-739) and Taylor et al. (J. Theor. Biol., 1986, 119:205-218).

[0167] It should be understood that, in a preferred embodiment, a functional equivalent variant of Omomyc contains mutations at positions corresponding to the mutations E61T, E68I, R74Q, and R75N found in Omomyc derived from human c-Myc. The positions at which such mutations must occur in the functional equivalent variant can be determined by multiple sequence alignment of different Myc sequences and identified by alignment with those positions corresponding to positions 61, 68, 74, and 75 within the sequence of Omomyc derived from human c-Myc. In one embodiment, a functional equivalent variant of Omomyc contains mutations at positions corresponding to the mutations E61T, E68I, R74Q, and R75N found in Omomyc derived from human c-Myc.

[0168] In another embodiment, a functional equivalent variant of Omomyc contains mutations at positions corresponding to E61, E68, R74, and R75 within the sequence of Omomyc, where E61 has been mutated to E61A or E61S; E68 has been mutated to E68L, E68M, or E68V; R74 has been mutated to R74N; and R75 has been mutated to R75Q.

[0169] Multiple sequence alignment is an extension of pair-wise alignment to include more than two sequences at a time. Multiple alignment methods align all the sequences in a given query set. Preferred multiple sequence alignment programs (and their algorithms) are ClustalW, Clusal2W, or ClustalW XXL (see Thompson et al. (1994) Nucleic Acids Res 22:4673-4680). Once the sequences of c-Myc from different organisms and the sequences of variants are aligned (compared) as described herein, a person skilled in the art can readily identify the positions within each sequence corresponding to the positions E61T, E68I, R74Q, and R75N found in Omomyc and introduce mutations in the Omomyc variant corresponding to the E61T, E68I, R74Q, and R75N mutations found in Omomyc derived from human c-Myc.

[0170] Suitable assays for determining whether a polypeptide can be considered a functional equivalent variant of Omomyc include, but are not limited to:

[0171] - Assays measuring the ability of the polypeptide to form a dimer complex with Max and Myc, such as reporter gene expression-based assays described in Soucek et al. (Oncogene, 1998, 17:2463-2472), as well as PLA (protein ligation assay) or co-immunoprecipitation.

[0172] - Assays for measuring the ability of a polypeptide to bind to the Myc / Max recognition site (CACGTG site) within DNA, such as the electrophoretic mobility shift assay (EMSA) described by Soucek et al. (supra).

[0173] - Assays for measuring the ability to inhibit Myc-induced transactivation, such as the assay based on reporter gene expression controlled by a DNA binding site specific for Myc / Max described by Soucek et al. (supra).

[0174] - Assays based on the ability of a polypeptide to inhibit the growth of cells expressing the myc oncogene, as described by Soucek et al. (supra).

[0175] - Assays for measuring the ability of a polypeptide to enhance Myc-induced apoptosis, such as those described by Soucek et al. (Oncogene, 1998:17, 2463-2472). Additionally, any assay known in the art for assessing apoptosis can be used, such as Hoechst staining, propidium iodide (PI) or annexin V staining, trypan blue, DNA laddering / fragmentation, and TUNEL.

[0176] In a preferred embodiment, a polypeptide is considered a functional equivalent variant of Omomyc if it exhibits at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the activity of native Omomyc in one or more of the above assays.

[0177] In a particular embodiment, a functional equivalent variant of the polypeptide of SEQ ID NO:1 comprises the polypeptide of SEQ ID NO:1, wherein the residue X at position 89 of SEQ ID NO:1 is not cysteine. Preferably, the residue X at position 89 of SEQ ID NO:1 is an aliphatic amino acid, or a sulfur-containing amino acid, or a dicarboxylic amino acid or its amide, or an amino acid having two basic groups, or an aromatic amino acid, or a cyclic amino acid, or a hydroxylated amino acid. More preferably, the amino acid is selected from serine, threonine, and alanine, preferably from serine and alanine.

[0178] Suitable functional equivalent variants of SEQ ID NO:1 are disclosed in the table below, which have a residue X at position 89 of SEQ ID NO:1 that is not cysteine.

[0179]

[0180] Thus, in a preferred embodiment, the functional equivalent variants of the polypeptide of SEQ ID NO:1 are selected from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. Preferably, the functional equivalent variant is SEQ ID NO:4.

[0181] In addition, the functional equivalent variants of Omomyc are also capable of transducing cells after contact of the variant with the cells. It should be understood that the functional equivalent variants of Omomyc contain the protein transduction domain found in native Omomyc or another functional protein transduction domain.

[0182] In a preferred embodiment, a polypeptide is considered a functional equivalent variant of SEQ ID NO:1 if it can transduce target cells at an efficiency that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO:1.

[0183] In addition, the functional equivalent variants of SEQ ID NO:1 are also capable of translocating to the nuclei of target tumor cells.

[0184] In a preferred embodiment, a polypeptide is considered a functional equivalent variant of SEQ ID NO:1 if it can translocate to the nuclei of target tumor cells at an efficiency that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO:1.

[0185] With respect to the ability of the polypeptide to cross cell membranes and translocate to the nucleus, suitable assays for determining whether a polypeptide is a functional equivalent variant of SEQ ID NO:1 include double-labeling cells with a reagent specific for the polypeptide and a dye that specifically labels the nuclei of the cells (such as DAPI or Hoechst dyes). Detection of the polypeptides of the present invention can be performed by confocal microscopy or by fluorescence microscopy.

[0186] In another preferred embodiment, the anti-cancer treatment is a conjugate that contains a polypeptide comprising the sequence SEQ ID NO:1 or a functional equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or its functional equivalent variant.

[0187] As used herein, the term "conjugate" refers to two or more compounds that are covalently linked together such that the function of each compound is retained in the conjugate.

[0188] The term "chemical moiety" refers to any chemical compound containing at least one carbon atom. Examples of chemical moieties include, but are not limited to, any peptide chain rich in hydrophobic amino acids and hydrophobic chemical moieties.

[0189] In a preferred embodiment, the conjugate according to the invention comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more chemical moieties that promote the cellular uptake of the polypeptide or a functional equivalent variant of the polypeptide.

[0190] In one embodiment, the chemical moiety that promotes the cellular uptake of the polypeptide is a lipid or a fatty acid.

[0191] A fatty acid is generally a molecule comprising a carbon chain having an acidic moiety (e.g., a carboxylic acid) at the chain terminus. The carbon chain of the fatty acid can be of any length, however, preferably the length of the carbon chain is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms, and any range derivable therefrom. In certain embodiments, in the chain portion of the fatty acid, the length of the carbon chain is from 4 to 18 carbon atoms. In certain embodiments, the fatty acid carbon chain can contain an odd number of carbon atoms, however, in certain embodiments, an even number of carbon atoms in the chain may be preferred. A fatty acid that contains only single bonds in its carbon chain is called saturated, while a fatty acid that contains at least one double bond in its chain is called unsaturated. The fatty acid can be branched, although in a preferred embodiment of the invention it is unbranched. Specific fatty acids include, but are not limited to, linoleic acid, oleic acid, palmitic acid, linolenic acid, stearic acid, lauric acid, myristic acid, arachidic acid, palmitoleic acid and arachidonic acid.

[0192] In a preferred embodiment, the chemical moiety that promotes the cellular uptake of the polypeptide comprising SEQ ID NO:1 or a functional equivalent variant thereof is a cell-penetrating peptide sequence, in which case the conjugate will comprise a fusion protein containing the polypeptide comprising SEQ ID NO:1 or a functional equivalent variant thereof and the cell-penetrating peptide sequence.

[0193] The term "fusion protein" refers to a protein produced by genetic engineering, which consists of two or more functional domains derived from different proteins. A fusion protein can be obtained in a conventional manner, for example, by gene expression of a nucleotide sequence encoding the fusion protein in a suitable cell. It should be understood that a cell-penetrating peptide refers to a cell-penetrating peptide different from the cell-penetrating peptide that forms part of the polypeptide comprising SEQ ID NO:1 or a functional equivalent variant of SEQ ID NO:1.

[0194] The term "cell-penetrating peptide sequence" may be used interchangeably in this specification with "CPP", "protein transduction domain", or "PTD". It refers to a peptide chain of variable length that directs the intracellular transport of proteins. The delivery process into cells typically occurs via endocytosis, but the peptide may also be internalized into cells by means of direct membrane translocation. CPPs generally have an amino acid composition that contains a relatively high abundance of positively charged amino acids such as lysine or arginine, or have a sequence with an alternating pattern of polar / charged and nonpolar hydrophobic amino acids.

[0195] Examples of CPPs that can be used in the present invention include, but are not limited to, the CPP found in the Drosophila antennapedia protein (RQIKIWFQNRRMKWKK; SEQ ID NO:13), the CPP found in the herpes simplex virus 1 (HSV-1) VP22 DNA-binding protein (DAATATRGRSAASRPTERPRAPARSASRPRRPVE; SEQ ID NO:14), the CPP of Bac-7 (RRIRPRPPRLPRPRPRPLPFPRPG; SEQ ID NO:15), the CPPs of the HIV-1 TAT protein consisting of amino acids 49-57 (RKKRRQRRR; SEQ ID NO:16), amino acids 48-60 (GRKKRRQRRRTPQ; SEQ ID NO:17), amino acids 47-57 (YGRKKRRQRRR; SEQ ID NO:18); the CPP of the S413-PV peptide (ALWKTLLKKVLKAPKKKRKV; SEQ ID NO:19), the CPP of the penetratin (RQIKWFQNRRMKWKK; SEQ ID NO:20), the CPP of SynB1 (RGGRLSYSRRRFSTSTGR; SEQ ID NO:21), the CPP of SynB3 (RRLSYSRRRF; SEQ ID NO:22), the CPP of PTD-4 (PIRRRKKLRRLK; SEQ ID NO:23), the CPP of PTD-5 (RRQRRTSKLMKR; SEQ ID NO:24), the CPP of FHV capsid-(35-49) (RRRRNRTRRNRRRVR; SEQ ID NO:25), the CPP of BMV Gag-(7-25) (KMTRAQRRAAARRNRWTAR; SEQ ID NO:26), the CPP of HTLV-II Rex-(4-16) (TRRQRTRRARRNR; SEQ ID NO:27), the CPP of D-Tat (GRKKRRQRRRPPQ; SEQ ID NO:28), the CPP of R9-Tat (GRRRRRRRRRPPQ; SEQ ID NO:29), the CPP of MAP (KLALKLALKLALALKLA; SEQ ID NO:30), the CPP of SBP (MGLGLHLLVLAAALQGAWSQPKKKRKV; SEQ ID NO:31), the CPP of FBP (GALFLGWLGAAGSTMGAWSQPKKKRKV; SEQ ID NO:32), the CPP of MPG (ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cya;(SEQ ID NO:33), the CPP of MPG(ENLS) (ac-GALFLGFLGAAGSTMGAWSQPKSKRKV-cya; SEQ ID NO:34), the CPP of Pep-1 (ac-KETWWETWWTEWSQPKKKRKV-cya; SEQ ID NO:35), the CPP of Pep-2 (ac-KETWFETWFTEWSQPKKKRKV-cya; SEQ ID NO:36), a polyarginine sequence having the structure RN (where N is between 4 and 17), the GRKKRRQRRR sequence (SEQ ID NO:37), the RRRRRRLR sequence (SEQ ID NO:38), the RRQRRTSKLMKR sequence (SEQ ID NO:39); the transporter GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:40); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:41); RQIKIWFQNRRMKWKK (SEQ ID NO:42), the YGRKKRRQRRR sequence (SEQ ID NO:43); the RKKRRQRR sequence (SEQ ID NO:44); the YARAAARQARA sequence (SEQ ID NO:45); the THRLPRRRRRR sequence (SEQ ID NO:46); the GGRRARRRRRR sequence (SEQ ID NO:47).;

[0196] In a preferred embodiment, the cell-penetrating peptide is not an endogenous peptide contained in SEQ ID NO:1.

[0197] In a preferred embodiment, the CPP is the CPP of the HIV-1 TAT protein composed of amino acids 49-57 (RKKRRQRRR, SEQ ID NO:16). In another preferred embodiment, the CPP is the GRKKRRQRRR sequence (SEQ ID NO:37) or RRRRRRLR (SEQ ID NO:38). In another embodiment, the CPP is the GRKKRRQRRR sequence (SEQ ID NO:37) or RRRRRRRR (SEQ ID NO:65).

[0198] In certain embodiments, the CPP is the CPP as described in WO2019 / 018898, the content of which is incorporated herein by reference in its entirety.

[0199] In one embodiment, the cell-penetrating peptide sequence is fused at the N-terminus of the polypeptide of the invention or a functional equivalent variant of said polypeptide. In another embodiment, the cell-penetrating peptide is fused at the C-terminus of the polypeptide of the invention or a functional equivalent variant of said polypeptide.

[0200] In a preferred embodiment, in addition to the native cell-penetrating peptide found in the polypeptide of SEQ ID NO:1 or a functional equivalent variant of said polypeptide, the conjugate or fusion protein of the combination according to the invention further comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more additional cell-penetrating peptides.

[0201] Suitable fusion proteins of the invention include the polypeptides Omomyc*TAT and Omomyc*LZArg as defined below:

[0202]

[0203] Thus, in a preferred embodiment, the fusion protein is a polypeptide selected from SEQ ID NO:11 and 12.

[0204] Suitable assays for determining whether the conjugate retains the cell membrane translocation ability of Omomyc include, but are not limited to, assays measuring the ability of the conjugate to transduce cells in culture. The assay is based on contacting the conjugate with cultured cells and detecting the presence of the conjugate at an intracellular location.

[0205] In another preferred embodiment, the conjugate of the combination of the invention further comprises an additional nuclear localization signal.

[0206] As used herein, the term "nuclear localization signal" (NLS) refers to an amino acid sequence of about 4 to 20 amino acid residues in length that is used to direct a protein to the nucleus. Generally, nuclear localization sequences are rich in basic amino acids, and exemplary sequences are well known in the art (Gorlich D. (1998) EMBO J. 17:2721-7). In certain embodiments, the NLS is selected from the SV40 large T antigen NLS (PKKKRKV, SEQ ID NO:48); nucleoplasmin NLS (KRPAATKKAGQAKKKK, SEQ ID NO:49); CBP80 NLS (RRRHSDENDGGQPHKRRK, SEQ ID NO:50); HIV-I Rev protein NLS (RQARRNRRRWE, SEQ ID NO:51); HTLV-I Rex (MPKTRRRPRRSQRKRPPT, SEQ ID NO:52); hnRNPA NLS (NQSSNFGPMKGGNFGGRSSGPYGGGGQYFKPRNQGGY, SEQ ID NO:53); rpL23a NLS (VHSHKKKKIRTSPTFTTPKTLRLRRQPKYPRKSAPRRNKLDHY, SEQ ID NO:54). In one embodiment of the invention, the nuclear localization signal comprises the motif K(K / R)X(K / R).

[0207] In an even more preferred embodiment, the nuclear localization signal is selected from PKKKRKV (SEQ ID NO:48), PAAKRVKLD (SEQ ID NO:56), and KRPAATKKAGQAKKKK (SEQ ID NO:49).

[0208] In another preferred embodiment, the NLS can be at the N-terminus or C-terminus of a conjugate or fusion protein comprising the polypeptide of SEQ ID NO:1 or a functional equivalent variant thereof.

[0209] Those skilled in the art will understand that it may be desirable for the conjugates of the present invention to further comprise one or more flexible peptides that link the polypeptide comprising SEQ ID NO:1 or a functional equivalent variant thereof, the cell-penetrating peptide sequence, and / or the NLS. Thus, in one particular embodiment, the polypeptide comprising SEQ ID NO:1 or a functional equivalent variant thereof is directly linked to the cell-penetrating peptide sequence. In another particular embodiment, the polypeptide comprising SEQ ID NO:1 or a functional equivalent variant thereof is linked to the cell-penetrating peptide sequence via a flexible peptide. In one embodiment, the polypeptide comprising SEQ ID NO:1 or a functional variant thereof is directly linked to the NLS. In another embodiment, the polypeptide comprising SEQ ID NO:1 or a functional equivalent variant thereof is linked to the NLS via a flexible peptide.

[0210] In one particular embodiment, the polypeptide of the conjugate according to the invention is directly linked to the cell-penetrating peptide sequence and the NLS.

[0211] In one embodiment, the NLS is one of the NLSs that occur endogenously in the Myc sequence, such as the M1 peptide (PAAKRVKLD, SEQ ID NO:56) or the M2 peptide (RQRRNELKRSF, SEQ ID NO:57).

[0212] In another embodiment, the additional NLS represents an NLS that is different from the endogenous NLS found in the polypeptide comprising SEQ ID NO:1 or a functional equivalent variant of SEQ ID NO:1.

[0213] In a preferred embodiment, in addition to the endogenous NLS found in the polypeptide of the present invention or a functional equivalent variant thereof, the conjugate or fusion protein according to the invention further comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 NLSs.

[0214] In another particular embodiment, the polypeptide of the conjugate used according to the invention is linked to the cell-penetrating peptide sequence via a first flexible peptide linker and to the NLS via a second flexible peptide linker.

[0215] As used herein, the terms "flexible peptide", "spacer peptide", or "linker peptide" refer to a peptide that covalently binds two proteins or moieties but is not part of either polypeptide, which allows one to move relative to the other without causing a substantial detrimental effect on the function of the protein or the moiety. Thus, the flexible linker does not affect the tumor-tracking activity of the polypeptide sequence, the cell-penetrating activity of the cell-penetrating peptide, or the nuclear localization ability of the NLS.

[0216] The flexible peptide comprises at least 1 amino acid, at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 12 amino acids, at least 14 amino acids, at least 16 amino acids, at least 18 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids or about 100 amino acids. In certain embodiments, the flexible peptide will allow one protein to move relative to another protein to increase the solubility of the protein and / or enhance its activity. Suitable linker regions include poly-glycine regions, the GPRRR sequence (SEQ ID NO:58) which is a combination of glycine, proline and alanine residues.

[0217] In one particular embodiment, the conjugate according to the invention comprises a tag that binds to the C-terminal or N-terminal domain of the conjugate or the polypeptide or fusion protein or variant thereof. The tag is typically a peptide or amino acid sequence that can be used to isolate or purify the fusion protein. Thus, the tag is capable of binding one or more ligands with high affinity, such as one or more ligands of an affinity matrix (such as a chromatography support or beads). An example of the tag is a histidine tag (His-tag or HT), such as a tag comprising 6 histidine residues (His6 or H6), which can bind nickel (Ni 2+ ) or cobalt (Co 2+ ) columns. The His-tag has the desirable feature that it can bind its ligand under conditions that denature most proteins and disrupt most protein-protein interactions. Thus, it can be used to remove the bait protein labeled with H6 after the protein-protein interaction in which the bait has participated has been disrupted.

[0218] Additional exemplary, non-limiting examples of tags that can be used to separate or purify conjugates or polypeptides comprising SEQ ID NO:1 or variants or fusion proteins thereof include Arg-tag, FLAG-tag (DYKDDDDK; SEQ ID NO:59), Strep-tag (WSHPQFEK, SEQ ID NO:60), epitopes recognizable by antibodies such as c-myc-tag (recognized by anti-c-myc antibody), HA tag (YPYDVPDYA, SEQ ID NO:61), V5 tag (GKPIPNPLLGLDST, SEQ ID NO:62), SBP-tag, S-tag, calmodulin-binding peptide, cellulose-binding domain, chitin-binding domain, glutathione S-transferase-tag, maltose-binding protein, NusA, TrxA, DsbA, Avi-tag, etc. (Terpe K., Appl. Microbiol. Biotechnol. 2003, 60:523-525), amino acid sequences such as AHGHRP (SEQ ID NO:63) or PIHDHDHPHLVIHSGMTCXXC (SEQ ID NO:64), β-galactosidase, etc.

[0219] If desired, the tag can be used to separate or purify the fusion protein.

[0220] In another preferred embodiment, the anti-cancer treatment is a polynucleotide encoding the above polypeptide or fusion protein. In a preferred embodiment, the anti-cancer treatment is a polynucleotide encoding a polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant thereof. In another embodiment, the anti-cancer treatment is a polynucleotide encoding a conjugate comprising a polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or its functionally equivalent variant; more preferably a polynucleotide encoding a fusion protein between a polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant thereof and a cell-penetrating peptide sequence.

[0221] The terms "polynucleotide", "nucleic acid", and "nucleic acid molecule" are used interchangeably to denote a polymeric form of nucleotides of any length. The polynucleotide may contain deoxyribonucleotides, ribonucleotides, and / or their analogs. The nucleotides can have any three-dimensional structure and can perform any known or unknown function. The term "polynucleotide" includes, for example, single-stranded, double-stranded, and triple-helical molecules, genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. In addition to natural nucleic acid molecules, the nucleic acid molecules of the present invention may also include modified nucleic acid molecules. As used herein, mRNA refers to RNA that can be translated in a cell.

[0222] In a preferred embodiment, the polynucleotide of the present invention is mRNA.

[0223] mRNA can be chemically synthesized, can be obtained by in vitro transcription, or can be synthesized in vivo in a target cell. The nucleotide sequence forming the polynucleotide encoding the conjugate or fusion protein of the present invention is in the same correct reading frame in which it is expressed.

[0224] In a preferred embodiment, the anticancer treatment is mRNA encoding a polypeptide consisting of SEQ ID NO:1 or a polypeptide consisting of a functionally equivalent variant of SEQ ID NO:1 or a polypeptide consisting of SEQ ID NO:4.

[0225] In another embodiment, the anticancer treatment is a vector comprising the polynucleotide of the present invention.

[0226] The term "vector" as used herein denotes a nucleic acid sequence that contains the necessary sequences such that, after transcription and translation of the sequence in a cell, a polypeptide encoded by the polynucleotide of the present invention is produced. The sequence is operably linked to additional segments that provide for its autonomous replication in a host cell of interest. Preferably, the vector is an expression vector, which is defined as a vector that contains, in addition to a region for autonomous replication in a host cell, a region that is operably linked to the nucleic acid of the present invention and that is capable of enhancing the expression of the product of the nucleic acid according to the present invention. The vectors of the present invention can be obtained by techniques widely known in the art.

[0227] Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors complexed with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells. Suitable vectors containing the polynucleotides of the invention are vectors derived from the following vectors: expression vectors in prokaryotes such as pUC18, pUC19, pBluescript and their derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phages, and "shuttle" vectors such as pSA3 and pAT28, expression vectors in yeast such as vectors of the 2-micron plasmid type, integrating plasmids, YEP vectors, centromeric plasmids and the like, expression vectors in insect cells such as vectors of the pAC series and pVL series, expression vectors in plants, such as vectors of the pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series and the like, and vectors based on viral vectors (adenovirus, adeno-associated virus, and retroviruses and especially lentiviruses) and non-viral vectors such as pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1 / hyg, pHCMV / Zeo, pCR3.1, pEF1 / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAX1, pZeoSV2, pCI, pSVL, pKSV-10, pBPV-1, pML2d, and pTDT1 in higher eukaryotic cells. In a preferred embodiment, the polynucleotides of the invention are contained in a vector selected from the pEGFP or pBabe retroviral vectors and the pTRIPZ or pSLIK lentiviral vectors.

[0228] The vectors of the invention can be used to transform, transfect or infect cells that can be transformed, transfected or infected by the vectors. The cells can be prokaryotic cells or eukaryotic cells.

[0229] The vector preferably contains the polynucleotide of the invention operably linked to a sequence that regulates the expression of the polynucleotide of the invention. Regulatory sequences used in the present invention may be nuclear promoters, or alternatively, enhancer sequences and / or other regulatory sequences that increase the expression of heterologous nucleic acid sequences. Generally speaking, any promoter can be used in the present invention as long as the promoter is compatible with the cell in which the polynucleotide is to be expressed. Thus, promoters suitable for carrying out the present invention include, but are not necessarily limited to, constitutive promoters such as derivatives of eukaryotic viral genomes such as polyomavirus, adenovirus, SV40, CMV, avian sarcoma virus, hepatitis B virus, metallothionein gene promoter, herpes simplex virus thymidine kinase gene promoter, LTR region of retrovirus, immunoglobulin gene promoter, actin gene promoter, EF-1α gene promoter, and inducible promoters (where protein expression depends on the addition of a molecule or exogenous signal, such as the tetracycline system, NFκB / ultraviolet system, Cre / Lox system, and heat shock gene promoter), the regulatable RNA polymerase II promoter described in WO 2006 / 135436, and tissue-specific promoters.

[0230] In another embodiment, the anti-cancer treatment is a cell that is capable of secreting the polypeptide of the invention or the conjugate of the invention, preferably the polypeptide of the invention or the fusion protein of the invention, into the culture medium.

[0231] Suitable cells capable of secreting the polypeptides of the present invention include, but are not limited to, cardiomyocytes, adipocytes, endothelial cells, epithelial cells, lymphocytes (B and T cells), mast cells, eosinophils, intimal cells, primary cultures of isolated cells from different organs, preferably cells isolated from the islets of Langerhans, hepatocytes, leukocytes (including mononuclear leukocytes), stroma, umbilical cord or adults (skin, lung, kidney and liver), osteoclasts, chondrocytes and other connective tissue cells. Established cell lines such as Jurkat T cells, NIH-3T3, CHO, Cos, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 cells, C2C12 myoblasts and W138 cells are also suitable. Those skilled in the art will understand that cells capable of secreting the polypeptides of the present invention into the culture medium can be found to form micro-particles or micro-capsules, so that the cells have a longer lifespan in patients. Materials suitable for forming the micro-particles of the present invention include any biocompatible polymeric material that allows for continuous secretion of the therapeutic product and acts as a support for the cells. Thus, the biocompatible polymeric material can be, for example, a thermoplastic polymer or a hydrogel polymer. Among the thermoplastic polymers, we have acrylic acid, acrylamide, 2-aminoethyl methacrylate, poly(tetrafluoroethylene-co-hexafluoropropylene), (7-cumaroxy)ethyl methacrylate, N-isopropylacrylamide, polyacrylic acid, polyacrylamide, polyamidoamine, poly(amino)-p-xylene, poly(chloroethyl vinyl ether), polycaprolactone, poly(caprolactone-co-trimethyl carbonate), poly(urea carbonate)urethane, poly(carbonate)urethane, polyethylene, polyethylene and acrylamide copolymer, polyethylene glycol, polyethylene glycol methacrylate, polyethylene terephthalate, poly(4-hydroxybutyl acrylate), poly(2-hydroxyethyl methacrylate), poly(N-2-hydroxypropyl methacrylate), poly(lactic-co-glycolic acid), poly(L-lactic acid), poly(γ-methyl,L-glutamate), poly(methyl methacrylate), poly(allyl fumarate), poly(propylene oxide), polypyrrole, polystyrene, poly(tetrafluoroethylene), polyurethane, polyvinyl alcohol, ultra-high molecular weight polyethylene, 6-(p-vinylbenzamide)-hexanoic acid N-vinylbenzyl-D-malonamide and copolymers containing more than one of the said polymers. Among the hydrogel type polymers, we have natural substances such as alginate, agarose, collagen, starch, hyaluronic acid, bovine serum albumin, cellulose and its derivatives, pectin, chondroitin sulfate, fibrin and fibroin, and synthetic hydrogels such as and

[0232] The method for predicting clinical response of the present invention is carried out on a pre-treated sample, i.e., a patient sample obtained before the patient has received the treatment whose response is to be predicted.

[0233] Regarding the treatment received by the subject before the sample is isolated in any aspect of the present invention, preferably the subject has not received any anti-cancer treatment at any time, preferably within the most recent 168 hours, more preferably within the most recent 72 hours, more preferably within the most recent 48 hours, even more preferably within the most recent 36 hours, more preferably within the most recent 24 hours, even more preferably 24 hours before the sample is isolated. In another preferred embodiment, the subject has not received anti-cancer treatment 24 hours before the sample is isolated, but preferably within the most recent 23 hours, preferably the most recent 22 hours, more preferably within the most recent 21 hours, more preferably within the most recent 20 hours, even more preferably within the most recent 15 hours, even more preferably within the most recent 10 hours, even more preferably within the most recent 5 hours, and even more preferably within the most recent 2 hours, and even more preferably within the last 1 hour before the sample is isolated. In yet another more preferred embodiment, the subject from whom the sample is isolated has not received any anti-cancer treatment at any time before the sample is isolated, i.e., the subject has never received anti-cancer treatment.

[0234] In a preferred embodiment, the subject has not received any anti-cancer treatment within the most recent 24 hours before the sample is isolated.

[0235] In another specific embodiment, the subject from whom the sample is isolated has received anti-cancer treatment at any time during a period before the sample is isolated, preferably within the most recent 168 hours before the sample is isolated, more preferably within the most recent 72 hours, even more preferably within the most recent 48 hours, even more preferably within the most recent 36 hours, more preferably within the most recent 24 hours. If administered 24 hours before the sample is isolated, preferably within the most recent 24 hours before the sample is isolated, the anti-cancer treatment is different from the anti-cancer treatment selected from items (a) to (e) of the first aspect of the present invention.

[0236] In a preferred embodiment, the subject from whom the sample is isolated has received anti-cancer treatment before the sample is isolated, and wherein the anti-cancer treatment is different from the treatment selected from items (a) to (e) of the first aspect of the present invention. Preferably, the anti-cancer treatment is selected from surgery, chemotherapy, radiation therapy, hormone therapy, and targeted therapy (including immunotherapy) and combinations thereof. In a preferred embodiment, the anti-cancer therapy is selected from surgery and radiation therapy. In a more preferred embodiment, the anti-cancer therapy is radiation therapy.

[0237] In a preferred embodiment, the subject from whom the sample is isolated has not received systemic anti-cancer treatment for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 50 weeks prior to sample isolation. In a more preferred embodiment, the subject has not received systemic anti-cancer treatment for at least 4 weeks prior to sample isolation.

[0238] There is no particular limitation on the time point for evaluating the response after the start of treatment. Thus, the response to treatment can be determined after at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 treatment cycles. The response to treatment can be determined at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 months after the start of the first treatment, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more after the start of the first treatment. In a particular embodiment, the response to treatment can be evaluated in a subject who has received at least two, preferably at least three, previous lines of anti-cancer treatment.

[0239] In addition, there is no particular limitation on the type and number of treatment regimens applied to the patient before determining the response. Thus, the response can be determined in the patient after the patient has been treated with first-line therapy (including surgical therapy, chemotherapy or surgery) in combination with an adjuvant or neoadjuvant chemotherapy or any type of therapy, or after the patient has been treated with two or more successive or concurrent treatment regimens.

[0240] Thus, the first step of the first method of the present invention comprises determining the level of at least one biomarker selected from MIP-1β, CD62E, IL-8, GM-CSF and IL-1α in a sample from a subject suffering from cancer, the response of whom to treatment is to be predicted.

[0241] The levels of these biomarkers can be detected in any type of sample. As used herein, the term "sample" refers to any sample that can be obtained from a subject and that contains any biological material suitable for detecting RNA or protein levels. In a particular embodiment, the sample contains genetic material from a subject, such as, for example, DNA, genomic DNA (gDNA), complementary DNA (cDNA), RNA, heterogeneous nuclear RNA (hnRNA), mRNA, etc. In another particular embodiment, the sample contains protein. The sample can comprise cells and / or acellular material from the subject. The method can be applied to any type of biological sample from a subject, such as biopsy samples, tissues, cells, or biological fluids (blood, plasma, serum, saliva, urine, semen, sputum, cerebrospinal fluid (CSF), tears, mucus, sweat, milk), feces, brain extracts, bone marrow, nipple aspirates, samples obtained by bronchoalveolar lavage, bronchoscopy, fine needle aspiration biopsy (FNAB), solid tumor biopsy samples, buccal or oropharyngeal swabs, etc. The sample can be obtained by conventional methods (e.g., biopsy, surgical resection, or aspiration) using methods well known to those of ordinary skill in the relevant medical arts. Methods for obtaining a sample from a biopsy include gross dissection of the mass, or microdissection or other cell isolation methods known in the art. Additionally, tumor cells can be obtained from fine needle aspiration cytology. In a preferred embodiment, the sample is obtained by fine needle aspiration biopsy (FNAB). To simplify sample preservation and processing, these can be fixed in formalin and embedded in paraffin, or first frozen and then embedded in a cryopolymerizable medium (such as OCT-compound) by immersion in a highly cryogenic medium that allows rapid freezing. The sample can also be a cell suspension, cell pellet, cell slide, frozen solid tumor biopsy.

[0242] In a particular embodiment, the sample is a tumor tissue sample, preferably a biopsy of fresh or frozen tumor tissue.

[0243] In a particular embodiment, the sample comprises cancer cells, preferably breast cancer, ovarian cancer, prostate cancer, gastric cancer, pancreatic cancer, lung cancer, colorectal cancer, gastric / stomach cancer, endometrial / uterine / cervical cancer, bladder cancer, head and neck cancer, leukemia, sarcoma, cholangiocarcinoma, glioblastoma, multiple myeloma, lymphoma cells. In a preferred embodiment, the sample comprises cells selected from pancreatic cancer cells, lung cancer cells, colorectal cancer cells, salivary gland cells, and sarcoma cells. In a preferred embodiment, it is a tumor tissue sample or a portion thereof. Preferably, the tumor tissue sample is a breast tumor, ovarian tumor, prostate tumor, gastric tumor, pancreatic tumor, lung tumor, colorectal tumor, gastric / stomach tumor, endometrial / uterine / cervical tumor, bladder tumor, head and neck tumor, sarcoma tumor, cholangiocarcinoma tumor, glioblastoma tumor, multiple myeloma tumor, lymphoma tumor tissue or a sample of a portion thereof. In a preferred embodiment, the sample is a pancreatic tumor sample, a lung tumor sample, a colorectal tumor sample, a salivary gland tumor sample, or a sarcoma tumor sample.

[0244] In another particular embodiment, according to the method of the present invention, the sample from the subject is a biological fluid, preferably a biological fluid from an affected organ. As used herein, "biological fluid", "biological fluid sample", or "body fluid sample" refers to any physiological or pathological biological secretion or fluid produced in a subject. Such biological fluids include, but are not limited to, blood, plasma, serum, bronchoalveolar lavage fluid, urine, nasal secretions, ear secretions, urethral secretions, cerebrospinal fluid, pleural fluid, synovial fluid, peritoneal fluid, ascitic fluid, pericardial fluid, amniotic fluid, gastric juice, lymph fluid, interstitial fluid, saliva, sputum, fluid deposits, tears, mucus, sweat, milk, semen, vaginal secretions, ulcer fluid, blisters, abscesses, and other surface eruptions. Using procedures known to those skilled in the art in the prior art, such as blood draws, instillation and aspiration of fluids during bronchoscopy, cistern, ventricular or lumbar puncture, pleural puncture or thoracentesis, percutaneous puncture of joints or synovial fluid, abdominal puncture, amniocentesis, expectoration of sputum, percutaneous puncture of the peritoneum, percutaneous puncture of the pericardium, etc., or by simple collection, the sample can be obtained by conventional methods.

[0245] In a preferred embodiment, the sample is selected from blood, serum, plasma, saliva, cerebrospinal fluid (CSF), and tumor biopsy samples; more preferably selected from blood, plasma, and serum; even more preferably serum.

[0246] Blood samples are usually extracted by puncturing an artery or a vein (usually a vein from the inner elbow or from the back of the hand), and the blood samples are collected in an airtight tube or syringe. Capillary puncture can usually be performed on the heel or on the distal phalanx of the finger for analysis by micro-methods. Serum can be obtained from whole blood samples, and in the absence of an anticoagulant, the sample is allowed to precipitate for 10 minutes to coagulate it and then centrifuged at 1,500 rpm for 10 minutes to separate the cells (pellet) from the serum (supernatant). Conversely, to obtain a plasma sample, whole blood is contacted with an anticoagulant and centrifuged at 3,000 rpm for 20 minutes. The pellet from the centrifugation corresponds to the formed elements, and the supernatant corresponds to the plasma. The obtained serum or plasma can be transferred to a storage tube for sample analysis by means of the method of the present invention.

[0247] The term "biomarker" as used herein refers to a gene product from a gene of interest, understood to be a transcript or a translation product of the gene of interest (i.e., the mRNA transcribed from the gene or the protein encoded by the gene), the amount of which reflects a particular situation, such as, for example, a condition such as the prediction of the response of a subject suffering from cancer to anti-cancer treatment.

[0248] The biomarkers of the present invention are mainly (i.e., the main biomarkers of the present invention are) MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α.

[0249] The terms "MIP-1β", "MIP-1b", "MIP1B", or "MIP-1β" refer to "macrophage inflammatory protein-1β", and are now officially named "CCL4" or "chemokine (C-C motif) ligand 4" or "C-C motif chemokine 4". The former name of human CCL4 was also AT 744, Act-2, LAG-1, HC21, and G-26. The term as used herein refers to the protein with UniProt accession number P13236 (version 216 of the accession as of October 12, 2022) or the gene encoding this protein. The term "C-C motif chemokine 4" includes any isoforms that have been described for this protein.

[0250] The term "CD62E" refers to "CD62 antigen-like family member E", and is also known as "SELE", "ELAM1", "ELAM-1", "LECAM2", "E-selectin", "endothelial-leukocyte adhesion molecule 1", or "leukocyte-endothelial cell adhesion molecule 2". The term as used herein refers to the protein with UniProt accession number P16581 (version 235 of the accession as of October 12, 2022) or the gene encoding this protein. The term "CD62 antigen-like family member E" includes any isoforms that have been described for this protein.

[0251] The term "IL-8" or "CXCL8" refers to "Interleukin-8" or "Chemokine (C-X-C motif) ligand 8". The term as used herein refers to the protein having UniProt accession number P10145 (version 237 of the accession as of October 12, 2022) or the gene encoding the protein. The term "Interleukin-8" includes any isoforms that have been described for the protein.

[0252] The term "GM-CSF" or "GMCSF" refers to "Granulocyte-macrophage colony-stimulating factor", also known as "CSF2" or "Colony-stimulating factor 2", and alternatively known as "molgramostin" or "sargramostim". The term as used herein refers to the protein having UniProt accession number P04141 (version 207 of the accession as of October 12, 2022) or the gene encoding the protein. The term "Granulocyte-macrophage colony-stimulating factor" includes any isoforms that have been described for the protein.

[0253] The term "IL-1α", "IL-1a", "IL-1α", "IL1A" or "IL1F1" refers to "Interleukin-1α", also named "Erythropoietin-1". The term as used herein refers to the protein having UniProt accession number P01583 (version 224 of the accession as of October 12, 2022) or the gene encoding the protein. The term "Interleukin-1α" includes any isoforms that have been described for the protein.

[0254] The expression "at least one biomarker selected from MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α" as used herein means that, according to the present invention, the expression levels of one, two, three, four, or five of said biomarkers can be determined. In a particular embodiment, the method comprises determining the expression level of one of said biomarkers. In a preferred embodiment, the biomarker determined is MIP-1β. In another embodiment, the biomarker determined is CD62E. In another embodiment, the biomarker determined is IL-8. In another embodiment, the biomarker determined is GM-CSF. In another embodiment, the biomarker determined is IL-1α. In another particular embodiment, the method of the present invention comprises determining the expression levels of two of said biomarkers. In a preferred embodiment, the biomarkers determined are MIP-1β and CD62E. In another embodiment, the biomarkers determined are MIP-1β and IL-8. In another embodiment, the biomarkers determined are MIP-1β and GM-CSF. In another embodiment, the biomarkers determined are MIP-1β and IL-1α. In another embodiment, the biomarkers determined are CD62E and IL-8. In another embodiment, the biomarkers determined are CD62E and GM-CSF. In another embodiment, the biomarkers determined are CD62E and IL-1α. In another embodiment, the biomarkers determined are IL-8 and GM-CSF. In another embodiment, the biomarkers determined are IL-8 and IL-1α. In another embodiment, the biomarkers determined are GM-CSF and IL-1α. In another embodiment, the method of the present invention comprises determining the expression levels of three of said biomarkers. In another embodiment, the method of the present invention comprises determining the expression levels of four of said biomarkers. In another particular embodiment, the method of the present invention comprises determining the expression levels of the five biomarkers MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α.

[0255] The methods of the present invention cover any combination of the primary biomarkers.

[0256] In a more preferred embodiment, the method comprises determining the levels of a combination of biomarkers selected from: a) a combination of biomarkers comprising MIP-1β and CD62E; and b) a combination of biomarkers comprising CD62E and IL-8. More preferably, the combination is selected from a) a combination of biomarkers consisting of MIP-1β and CD62E; and b) a combination of biomarkers consisting of CD62E and IL-8.

[0257] The method of the present invention may further comprise determining other biomarkers different from MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α.

[0258] Thus, in a preferred embodiment, the method further comprises determining the expression level of additional biomarkers (i.e., secondary biomarkers) different from the primary biomarkers of the present invention. Preferably, the method further comprises determining the level of at least one additional biomarker selected from MCP-1, ICAM-1, IFN-γ, IL-1β, IL-12, and combinations thereof.

[0259] The term "MCP-1" or "MCP1" refers to "monocyte chemoattractant protein 1" or "monocyte chemotactic protein 1", and is also referred to as "CCL2" or "chemokine (C-C motif) ligand 2" or "C-C motif chemokine 2" or "small inducible cytokine A2" or "SCYA2". As used herein, the term refers to the protein having UniProt accession number P13500 (version 246 of the accession as of October 12, 2022) or the gene encoding the protein. The term "monocyte chemoattractant protein 1" includes any isoforms that have been described for the protein.

[0260] The term "ICAM-1" or "ICAM1" refers to "intercellular adhesion molecule 1", and is also referred to as "CD54" or "cluster of differentiation 54". As used herein, the term refers to the protein having UniProt accession number P05362 (version 251 of the accession as of October 12, 2022) or the gene encoding the protein. The term "intercellular adhesion molecule 1" includes any isoforms that have been described for the protein.

[0261] The term "IFN-gamma", "IFN-g", "IFN-γ", or "IFNG" refers to "interferon γ". As used herein, the term refers to the protein having UniProt accession number P01579 (version 238 of the accession as of October 12, 2022) or the gene encoding the protein. The term "interferon γ" includes any isoforms that have been described for the protein.

[0262] The terms "IL-1β", "IL-1b", "IL-1β", "IL1β", "IL1B", or "IL1F2" refer to "Interleukin-1β", also known as "leukocytic pyrogen", "leukocytic endogenous mediator", "monokine", or "lymphocyte activating factor". The term as used herein refers to the protein having UniProt accession number P01584 (version 249 of the accession as of October 12, 2022) or the gene encoding such protein. The term "Interleukin-1β" includes any isoforms that have been described for the protein.

[0263] The term "IL-12" refers to "Interleukin 12". IL-12 is a heterodimeric cytokine encoded by two separate genes: IL-12A (p35) and IL-12B (p40). An active heterodimer (referred to as "p70") and a homodimer of p40 are formed after protein synthesis. The term as used herein refers to either the protein having UniProt accession numbers P29459 (version 190 of the accession as of October 12, 2022) and P29460 (version 231 of the accession as of October 12, 2022) or the gene encoding such protein. The protein having UniProt accession number P29459 relates to "Interleukin-12 subunit α" or "IL12A" or "IL-12A" or "NKSF1". The protein having UniProt accession number P29460 relates to "Interleukin-12 subunit β" or "IL12B" or "NKSF2".

[0264] In a preferred embodiment, the method of the invention further comprises determining the expression level of MCP-1. In another embodiment, the method further comprises determining the expression level of ICAM-1. In another embodiment, the method further comprises determining the expression level of IFN-γ. In another embodiment, the method further comprises determining the expression level of IL-1β. In another embodiment, the method further comprises determining the expression level of IL-12. In another embodiment, the method of the invention further comprises determining the expression level of a combination of MCP-1, ICAM-1, IFN-γ, IL-1β, IL-12, preferably the expression levels of MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12.

[0265] The method of the invention encompasses any combination of secondary biomarkers.

[0266] In a preferred embodiment, the method of the present invention comprises determining the levels of a combination of biomarkers selected from: a) a combination of biomarkers comprising MIP-1β and MCP-1; b) a combination of biomarkers comprising MIP-1β and ICAM-1; c) a combination of biomarkers comprising MIP-1β and CD62E; d) a combination of biomarkers comprising MIP-1β and IFN-γ; e) a combination of biomarkers comprising MIP-1β and IL-1β; f) a combination of biomarkers comprising MIP-1β and IL-12; and g) a combination of biomarkers comprising CD62E and IL-8. Preferably, the method comprises determining the levels of a combination of biomarkers selected from: a) a combination of biomarkers comprising MIP-1β and MCP-1; and b) a combination of biomarkers comprising CD62E and IL-8. In a preferred embodiment, the combination of biomarkers consists of the combinations defined above.

[0267] In a preferred embodiment, the method of the first aspect of the present invention further comprises:

[0268] i) determining the levels of at least one additional biomarker selected from MCP-1, ICAM-1, IFN-γ, IL-1β and IL-12 in a sample from the subject; and

[0269] ii) comparing the levels of the at least one biomarker with a reference value, wherein:

[0270] - a reduced level of the at least one biomarker relative to the reference value indicates a good clinical response of the subject to the anti-cancer treatment, or

[0271] - an equal or increased level of the at least one biomarker relative to the reference value indicates a poor clinical response of the subject to the anti-cancer treatment.

[0272] The method of the present invention encompasses any combination of primary and secondary biomarkers.

[0273] The term "level" is also understood as "expression level" or "level of expression" and is used herein to denote the expression level of a gene product, more specifically the measurable amount of a gene product produced by a particular gene in a particular sample of a subject. The term "gene product" as used herein denotes a transcriptional product or a translational product and thus corresponds to the mRNA transcribed from said gene or to the protein encoded by a particular gene. When referring to the expression level of a biomarker, it should be understood that the gene product is the biomarker, which, as shown below, can be an mRNA or a protein transcribed or encoded from a selected gene of the present invention. As will be understood by those skilled in the art, gene expression levels can be quantified by measuring the messenger RNA level of the gene or of the protein encoded by the gene. In the context of the present invention, the expression level of the genes encoding MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β or IL-12 can be determined by measuring the level of the mRNA encoded by said gene, or by measuring the level of the protein encoded by said gene (i.e. the MIP-1β, CD62E, IL-8, GM-CSF and IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β or IL-12 protein) or of a variant thereof. Variants of the MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β and IL-12 proteins include all physiologically relevant post-translational chemically modified forms of said proteins, such as, for example, glycosylation, phosphorylation, acetylation, etc., provided that the functionality of said protein is maintained. The term encompasses MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β and IL-12 proteins of any mammalian species, including, but not limited to, domestic and farm animals (cattle, horses, pigs, sheep, goats, dogs, cats or rodents), primates and humans. Preferably, the MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β and IL-12 proteins are human proteins.

[0274] As will be understood by those skilled in the art, the expression level of the biomarker can be measured by determining the mRNA expression level of the gene encoding the biomarker or by determining the level of the protein encoded by the gene.

[0275] In a preferred embodiment, the level of at least one biomarker determined is the mRNA level.

[0276] To measure the mRNA level of the biomarker, the biological sample can be processed to physically, mechanically, or chemically disrupt tissue or cell structure, releasing intracellular components into an aqueous or organic solution to prepare nucleic acids for further analysis. Nucleic acids are extracted from the sample using commercially available reagents by procedures known to those skilled in the art. Then, RNA is extracted from frozen or fresh samples by any typical method in the art, such as Sambrook, J., et al., 2001. Molecular cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, N.Y., Volumes 1-3. Preferably, care is taken to avoid degradation of the RNA during the extraction process.

[0277] Using mRNA obtained from formalin-fixed, paraffin-embedded tissue samples, the expression level can be determined. mRNA can be isolated from archived pathology samples or biopsy samples that are first deparaffinized. An exemplary method of deparaffinization involves washing the paraffinized sample with an organic solvent such as xylene. The deparaffinized sample can be rehydrated with an aqueous solution of a lower alcohol. Suitable lower alcohols include, for example, methanol, ethanol, propanol, and butanol. For example, the deparaffinized sample can be rehydrated by successive washes with solutions of decreasing concentration of the lower alcohol. Alternatively, the sample is simultaneously deparaffinized and rehydrated. The sample is then lysed and RNA is extracted from the sample. Samples can also be obtained from fresh tumor tissue such as an excised tumor. In a particular embodiment, samples can be obtained from fresh tumor tissue or from OCT-embedded frozen tissue.

[0278] Suitable methods for determining gene expression levels at the mRNA level include, but are not limited to, standard assays for determining mRNA expression levels, such as qPCR, RT-PCR, RNA protection assays, Northern blotting, RNA dot blotting, tag-based methods such as serial analysis of gene expression (SAGE) (including variants such as LongSAGE and SuperSAGE), microarrays, nucleic acid sequence-based amplification (NASBA), fluorescence in situ hybridization (including variants such as Flow-FISH, qFiSH, and dual-fusion fish (D-FISH), etc.

[0279] In a particular embodiment, the expression level of the biomarker is determined by microarray. In another particular embodiment, the expression level of the biomarker is determined by quantitative PCR, preferably real-time PCR.

[0280] To normalize mRNA expression values between different samples, the expression level of the mRNA of interest in the test sample may be compared to the expression of a control RNA. As used herein, "control RNA" refers to an RNA whose expression level does not change or only changes in limited amounts in tumor cells relative to non-tumorigenic cells. Preferably, the control RNA is mRNA derived from a housekeeping gene and encodes a protein that is constitutively expressed and performs essential cellular functions. Examples of housekeeping genes used in the present invention include β-2-microglobulin, ubiquitin, 18-S ribosomal protein, cyclophilin, GAPDH, PSMB4, tubulin, and β-actin.

[0281] In one embodiment, GAPDH, β-actin, or PSMB4 is used as an endogenous control and a commercial RNA control is used as a calibrator, and relative gene expression quantification is calculated according to the comparative threshold cycle (Ct) method. The final result is determined according to the formula 2-(ΔCt sample - ΔCt calibrator), where the ΔCT values of the calibrator and the sample are determined by subtracting the CT value of the target gene from the CT value of the control gene.

[0282] In a preferred embodiment, the level of at least one biomarker determined is the protein level.

[0283] The expression level of the biomarker can also be measured by determining the level of the protein or its variant encoded by the gene. In fact, any conventional method can be used in the context of the present invention to quantify the level of the protein. Suitable methods for determining gene expression levels at the protein level include, but are not limited to, conventional methods such as using an antibody having the ability to specifically bind to the protein (or a fragment thereof containing an antigenic determinant) encoded by the gene and subsequently quantifying the resulting antibody-antigen complex. In a particular embodiment, the protein level of the biomarker of the present invention can be quantified by using standard assays for determining protein expression levels such as Western blotting or protein transfer, immunoassays such as ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemistry and immunohistochemistry techniques, techniques based on the use of protein biochips or microarrays including specific antibodies, or assays based on colloidal precipitation (in the form of dipsticks, for example) or immunoassays based on luminex technology.

[0284] The antibodies to be employed in these assays can be, for example, polyclonal sera, hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab’, and F(ab’)2, ScFv, diabodies, triabodies, tetra-bodies, and humanized antibodies. Meanwhile, the antibodies can be labeled or unlabeled. Exemplary, but non-exclusive, examples of markers that can be used include radioisotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, colorants, etc. There are a variety of well-known assays that can be used in the present invention, which use unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies); these techniques include Western blotting or protein transfer, ELISA, RIA, competitive EIA, DAS-ELISA, immunocytochemistry and immunohistochemistry techniques, techniques based on the use of biochips or protein microarrays including specific antibodies, or assays based on colloidal precipitation (in the form of, for example, dipsticks). Other methods for detecting and quantifying the levels of proteins of interest include techniques such as affinity chromatography, ligand-binding assays, etc.

[0285] On the other hand, the determination of biomarker protein levels can be carried out as follows: construct a tissue microarray (TMA) containing assembled subject samples, and determine the expression levels of the corresponding proteins by immunohistochemical techniques. The immunostaining intensity can be evaluated by two or more different pathologists and scored using uniform and well-defined cut-off criteria to maintain the reproducibility of the method. Discrepancies can be resolved by simultaneous re-evaluation. Briefly, the results of immunostaining can be recorded as negative expression (0) versus positive expression, and low expression (1+) versus medium (2+) and high (3+) expression, taking into account the expression in tumor cells and the specific cut-off values for each biomarker. As a general criterion, the cut-off values are selected to facilitate reproducibility and, whenever possible, translate biological events. Alternatively, by using imaging techniques and automated methods, such as those disclosed in Rojo, M.G. et al. (Folia Histochem. Cytobiol. 2009; 47:349-54) or Mulrane, L. et al. (Expert Rev. Mol. Diagn. 2008; 8:707-25), the immunostaining intensity can be evaluated.

[0286] Alternatively, in another specific embodiment, the levels of biomarker proteins are determined by Western blotting. Western blotting is based on the detection of a specific protein that has been previously resolved by gel electrophoresis under denaturing conditions and immobilized on a membrane (usually nitrocellulose) by incubation with an antibody-specific developing system (such as a chemiluminescent agent).

[0287] In a particular embodiment, the expression level is determined as a protein level. In a more specific embodiment, the protein level is determined by ELISA, Western blot, or immunoassay.

[0288] In a preferred embodiment, the level is determined by immunoassay. As used herein, the term "immunoassay" refers to a biochemical assay that measures the presence or concentration of a molecule in solution by using an antibody or an antigen-binding fragment thereof that specifically recognizes the molecule. In a preferred embodiment, the immunoassay is an immunoassay based on antibodies conjugated to Luminex beads, particularly using Luminex technology, and more particularly using the ProcartaPlex TM kit for the immunoassay.

[0289] As previously described, variants of the protein can be used to measure the expression levels of MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12 in order to practice the method of the present invention.

[0290] Thus, variants of the MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12 proteins can be variants in which: (i) one or more amino acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and such replaced amino acid residues may or may not be amino acid residues encoded by the genetic code; (ii) one or more modified amino acid residues are present, e.g., residues modified by the attachment of a substituent group; (iii) the protein is an isoform or alternative splice variant of the protein of the present invention; and / or (iv) a fragment of the protein. The fragment includes a protein produced by proteolytic cleavage (including multi-site proteolysis) of the original sequence. From the teachings herein, variants are considered to be within the scope of those skilled in the art.

[0291] Variants according to the invention include amino acid sequences that are at least 60%, 70%, 80%, 90%, 95% or 96% similar or identical to the original amino acid sequence. As is known in the art, "similarity" between two proteins is determined by comparing the amino acid sequence of one protein and its conservative amino acid substitutions with the sequence of a second protein. Computer algorithms and methods widely known to those skilled in the art are used to determine the degree of identity between two proteins. Preferably, the identity between two amino acid sequences is determined by using the BLASTP algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990)].

[0292] The protein can be post-translationally modified. For example, post-translational modifications falling within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding, and proteolytic processing, etc. In addition, the protein can include non-natural amino acids formed by post-translational modification or by the introduction of non-natural amino acids during translation.

[0293] In a particular embodiment, the variant is a mammalian variant, preferably a human variant, more preferably having at least 60%, 70%, 80%, 90%, 95% or 96% similarity or identity to the original amino acid sequence.

[0294] The second step of the first method of the present invention comprises comparing the level of said at least one biomarker obtained in step (i) with a reference value.

[0295] The term "reference value" as used herein refers to a laboratory value used as a reference for values / data obtained from a sample collected from a subject. The reference value or reference level can be an absolute value, a relative value, a value with an upper or lower limit, a range of values, an average value, a median value, a mean value, or a value compared to a specific control or baseline value. The reference value can be based on a single sample value, such as, for example, a value obtained from a sample from a subject, but at an earlier time point. The reference value can be based on a large number of samples, for example, based on a group of patients considered to be representative, such as values obtained from a population of subjects age-matched to the chronological age of the patient subjects under study, or based on a sample bank that includes or does not include the sample to be tested. Appropriate reference values are indicated in the context of the method of the present invention for predicting the response of a cancerous subject receiving anti-cancer therapy.

[0296] In the context of the method of the present invention for predicting the response of a subject suffering from cancer to an anti-cancer treatment, the preferred reference values can be the MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12 expression levels determined in a sample from a subject who is suffering from cancer or who already has cancer, where the subject shows a good clinical response to treatment with an anti-cancer therapy, and the expression levels have been determined at the time the patient received the treatment. In another embodiment, the reference values are the MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12 expression levels determined in a sample from a subject who is suffering from cancer or who already has cancer, where the subject shows a poor clinical response to treatment with an anti-cancer therapy, and the expression levels have been determined at the time the patient received the treatment. In another embodiment, the reference values are the MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12 expression levels determined in a sample from a pool of subjects who are suffering from cancer or who already have cancer, where several of the subjects show a good response to treatment with an anti-cancer therapy and others show a poor response, and the expression levels have been determined at the time the patients received the treatment. In another embodiment, the reference values are the MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12 expression levels in healthy patients (i.e., patients who have not been diagnosed with the type of cancer for which the response to treatment needs to be predicted).

[0297] In one embodiment, the reference values can be obtained by determining the median value of the expression levels of each biomarker measured in a set of samples from subjects suffering from cancer but in whom the biomarker has not decreased or from normal tissue. In one embodiment, the reference values can be obtained by determining the median value of the expression levels of each biomarker measured in a set of samples from subjects suffering from cancer but in whom the biomarker has not increased or from normal tissue.

[0298] The collection of samples from which the reference levels are derived will preferably consist of a mixture of subjects suffering from the same type of cancer or of tissues from normal individuals not affected by cancer.

[0299] Alternatively, the use of a reference value for determining whether the expression level of a biomarker is "increased" or "decreased" can correspond to the median value of the expression level of each biomarker measured in an RNA sample obtained by pooling equal amounts of RNA from each sample obtained from a subject suffering from cancer but not having a reduced level of the biomarker of the present invention, preferably from subjects suffering from the same type of cancer. In another embodiment, the reference value can correspond to the median value of the expression level of each biomarker measured in an RNA sample obtained by pooling equal amounts of RNA from each sample obtained from a subject suffering from cancer but not having an increased level of the biomarker of the present invention, preferably from subjects suffering from the same type of cancer.

[0300] In the present invention, the "reference value" can be any cut-off point established according to the ROC methodology. Once this cut-off point is determined, the level of the biomarker expressed in a biological fluid or tumor tissue from a subject can be compared to this cut-off point, and thus, if below this cut-off point it is designated as a "low" expression level, and if above this cut-off point it is designated as a "high" expression level. Thus, in a preferred embodiment, the expression level of each biomarker in the combination is compared to a predetermined cut-off value for each said gene, where the predetermined cut-off value for each gene preferably corresponds to the expression level of the gene associated with the highest specificity at the desired sensitivity in an ROC curve calculated based on the expression level of the biomarker.

[0301] Once the reference value is established, the level of the biomarker expressed in a sample can be compared to the reference value and thereby an expression level of "increased", "decreased" or "equal" can be designated. For example, an increase in the expression level that is at least 1.1-fold, 1.5-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or even more compared to the reference value is considered an "increased" expression level. On the other hand, a decrease in the expression level that is at least 0.9-fold, 0.75-fold, 0.2-fold, 0.1-fold, 0.05-fold, 0.025-fold, 0.02-fold, 0.01-fold, 0.005-fold or even lower compared to the reference value is considered a "decreased" expression level.

[0302] This comparison allows the detection of a decrease in the expression of one or more of said biomarkers relative to said reference value. Stating a "decreased level" of one or more biomarkers selected from MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12 means that the expression of a given biomarker is decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or more relative to said reference value.

[0303] Stating an "increased level" of one or more biomarkers selected from MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12 means that the expression of a given biomarker is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or more relative to said reference value.

[0304] If the difference in the expression level relative to the reference value is less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001% or less, the expression level can be considered "equal" to the reference value.

[0305] When comparing the levels of one or more biomarkers selected from MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α with the reference value, the method of the present invention allows predicting whether a subject will show a good clinical response or a poor clinical response to an anti-cancer treatment using items (a) to (e) of the first aspect of the present invention. Thus, a subject having a decreased level of said at least one biomarker relative to the reference value will have a good clinical response to said anti-cancer treatment.

[0306] As used herein, the expression "good clinical response" means that the subject will show a favorable response to the anti-cancer treatment of items (a) to (e) according to the first aspect of the present invention, which is understood by those skilled in the art as a complete response, partial response or stabilization of the disease.

[0307] In a preferred embodiment, the good clinical response is the stabilization of the disease.

[0308] Alternatively, a patient having an equal or increased level of the at least one biomarker relative to the reference value will have a poor clinical response to the anti-cancer treatment.

[0309] As used herein, the expression "poor clinical response" means that the response of the subject is not as favorable as just shown. Poor clinical response includes prediction of disease progression. It can also include prediction of recurrence, mortality, or the need for a change or administration of a new medical treatment.

[0310] In a preferred embodiment, the poor clinical response is the progression of the disease.

[0311] As will be understood by those skilled in the art, such probability assessments, while preferred, may not generally be correct for 100% of the subjects to be analyzed. However, the term requires that a statistically significant proportion of the subjects can be identified as having a tendency to respond to the chemotherapy treatment. Using various well-known statistical evaluation tools, such as determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney test, etc., those skilled in the art can readily determine whether the proportion is statistically significant. For details, see Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-value is preferably 0.1, 0.05, 0.01, 0.005 or 0.0001. More preferably, at least 60%, at least 70%, at least 80% or at least 90% of the subjects in the population can be appropriately identified by the method of the present invention.

[0312] In a preferred embodiment, the anti-cancer treatment is initiated after predicting the clinical response of a subject suffering from cancer to the treatment by the first method of the present invention.

[0313] The selective method of the present invention

[0314] The authors of the present invention have observed that a decrease in the level of MIP-1β, CD62E, IL-8, GM-CSF or IL-1α relative to a reference value indicates a good clinical response to OMO-103 treatment. Thus, the detection of low levels of said biomarker can be a marker useful for selecting patients for treatment with a therapy based on Omomyc or a functional equivalent variant thereof. In this way, a subject can be directly administered an appropriate therapy while avoiding less effective therapies or the side effects associated therewith.

[0315] Thus, in a second aspect, the present invention relates to an in vitro method for selecting a customized therapy for a subject suffering from cancer, comprising:

[0316] (i) determining the level of at least one biomarker selected from MIP-1β, CD62E, IL-8, GM-CSF and IL-1α in a sample from said subject; and

[0317] (ii) comparing the level of said at least one biomarker with a reference value,

[0318] wherein:

[0319] - a decreased level of said at least one biomarker relative to said reference value indicates that the therapy to be selected comprises an agent selected from:

[0320] a) a polypeptide comprising SEQ ID NO:1 or a functional equivalent variant thereof;

[0321] b) a conjugate comprising a polypeptide comprising SEQ ID NO:1 or a functional equivalent variant thereof and a chemical moiety that facilitates cellular uptake of said polypeptide or functional equivalent variant thereof;

[0322] c) a polynucleotide encoding the polypeptide of a) or the conjugate of b);

[0323] d) a vector comprising the polynucleotide according to c); and

[0324] e) a cell capable of secreting the polypeptide according to a) or the conjugate according to b) into a culture medium; or alternatively,

[0325] - an equal or increased level of said at least one biomarker relative to said reference value indicates that the therapy to be selected does not comprise an agent selected from:

[0326] a) a polypeptide comprising SEQ ID NO:1 or a functional equivalent variant thereof;

[0327] b) A conjugate comprising a polypeptide comprising the sequence SEQ ID NO:1 or a functional equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or its functional equivalent variant;

[0328] c) A polynucleotide encoding the polypeptide of a) or the conjugate of b);

[0329] d) A vector comprising the polynucleotide according to c); and

[0330] e) A cell capable of secreting the polypeptide according to a) or the conjugate according to b) into a culture medium.

[0331] As used herein, the term "selective customized therapy" or "selective personalized therapy" refers to the selection of an appropriate and optimal therapy based on the specific medical characteristics of a patient (arising from their personal background). In the context of the present invention, such characteristics consist of the levels of biomarkers in a sample isolated from the patient.

[0332] In the context of the second method of the present invention, the term "therapy" refers to any anti-cancer treatment that involves exposing a subject to any method for inducing cancer cell death. The method is not limited to the administration of chemical agents and may include surgery, chemotherapy, radiation therapy, hormone therapy, targeted therapy (including immunotherapy), or a combination thereof.

[0333] When referring to cancer, the term "surgery" refers to major surgery in which at least a portion of the primary tumor and / or at least a portion of at least one metastasis is removed.

[0334] As used herein, the term "chemotherapy" refers, without limitation to chemical structure, to any therapy applicable to cancer and / or treatment consisting of the administration of one or more anti-cancer agents, administered alone or in combination with other compounds.

[0335] As used herein, the terms "radiation therapy", "radiotherapy", or "ionizing radiation" refer to a therapy using ionizing radiation, typically as part of cancer treatment to control or kill tumor cells and usually delivered by a linear accelerator. Gamma rays, X-rays, and the higher ultraviolet portion of the electromagnetic spectrum are ionizing radiation, while the lower spectral portion below ultraviolet, including visible light (including almost all types of lasers), infrared, microwaves, and radio waves, are considered non-ionizing radiation. Radiation therapy can treat various types of cancer if they are confined to one area of the body. Radiation therapy is commonly applied to cancerous tumors because it can control cell growth. The mode of action of ionizing radiation is to damage the DNA of cancerous tissue, resulting in cell death.

[0336] The term "targeted therapy" as used herein refers to a therapy that blocks the growth of cancer cells as follows: interfering with specific target molecules required for carcinogenesis and tumor growth, rather than simply interfering with all rapidly dividing cells. Since most agents used in targeted therapy are biopharmaceuticals, the term biological therapy is sometimes synonymous with targeted therapy (and thus distinguished from chemotherapy, i.e., cytotoxic therapy) when used in the context of cancer therapy. However, the modalities can be combined. Another form of targeted therapy involves the use of nanoengineered enzymes to bind to tumor cells so that the body's natural cell degradation processes can digest the cells, effectively clearing them from the body. Many targeted therapies are examples of immunotherapy.

[0337] The term "immunotherapy" as used herein refers to cancer immunotherapy, which includes regimens that alter the host immune system and / or utilize components of the immune system as cancer treatment. Non-limiting examples of immunotherapy include the nonspecific immunostimulants BCG and levamisole; the cytokines interferon-α and interleukin-2; the monoclonal antibodies anti-PD1, anti-PDL1, anti-CTLA-4, rituximab, ofatumumab, alemtuzumab, trastuzumab, bevacizumab, cetuximab, and panitumumab; the radioactively labeled antibodies Y-90 ibritumomab tiuxetan and I-131 tositumomab; the immunotoxins denileukin diftitox and the antibody gemtuzumab ozogamicin; non-myeloablative allografts with donor lymphocyte infusion; and the therapy sipuleucel-T based on anti-prostate cancer cells. Other examples of immunotherapy are such as DC vaccines, CART-T cells, or NK therapy. Some immunotherapies involve removing immune cells from the blood or tumor, culturing those specific to the tumor, and returning them to the patient, where they attack the tumor; alternatively, immune cells can be genetically engineered to express tumor-specific receptors, cultured, and returned to the patient. Cell types that can be used in this way are natural killer cells, lymphokine-activated killer cells, cytotoxic T cells, and dendritic cells.

[0338] The first step of the second method of the present invention comprises determining the level of at least one biomarker selected from MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α in a sample from the subject; and the second step comprises comparing the level of the at least one biomarker with a reference value.

[0339] When comparing the levels of one or more biomarkers selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α with a reference value, the method of the present invention allows for determining an appropriate treatment for a tested subject. Thus, a subject having a reduced level of said at least one biomarker relative to said reference value should be treated with a therapy comprising an agent selected from the group consisting of items (a) to (e) of the first aspect of the present invention.

[0340] As used herein, an "anticancer agent" is an agent that at least partially inhibits the development or progression of cancer or stabilizes the disease, including completely or partially inhibiting cancer-related symptoms, even if only for a short period.

[0341] On the other hand, a subject having an equal or increased level of said at least one biomarker relative to said reference value should be treated with an alternative therapy, i.e., a therapy that does not comprise an agent selected from the group consisting of:

[0342] a) a polypeptide comprising the sequence SEQ ID NO:1 or a functional equivalent variant thereof;

[0343] b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO:1 or a functional equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or its functional equivalent variant;

[0344] c) a polynucleotide encoding the polypeptide of a) or the conjugate of b);

[0345] d) a vector comprising the polynucleotide according to c); and

[0346] e) a cell capable of secreting the polypeptide according to a) or the conjugate according to b) into a culture medium.

[0347] In the context of the second method of the present invention, the expression "alternative therapy" means any therapy different from a therapy comprising an agent defined in items (a) to (e) of the first method of the present invention. Alternative therapies include, but are not limited to, surgery, chemotherapy, radiation therapy, hormone therapy, and targeted therapy (including immunotherapy) and combinations thereof.

[0348] Exemplary, non-limiting examples of alternative therapies include surgery and treatment with the following agents: anticancer agents such as chemotherapeutic agents, including anthracyclines such as doxorubicin and daunorubicin, and taxanes such as paclitaxel TMand docetaxel, vinca alkaloids such as vincristine and vinblastine, 5-fluorouracil (5-FU), leucovorin, irinotecan, idarubicin, mitomycin C, oxaliplatin, raltitrexed, tamoxifen, cisplatin, carboplatin, methotrexate, actinomycin D, mitoxantrone, bleomycin sulfate or mithramycin; toxins such as ricin A-chain, saponin, diphtheria A-chain, active non-binding fragments of diphtheria toxin, Pseudomonas aeruginosa exotoxin A-chain, abrin A-chain, modecin A-chain, alpha-sarcin, Leurites fordii A-protein, gyromitrin, Phytolaca americana (PAPI, PAPII and PAP-S) proteins, momordica inhibitor, curcin, croton toxin, saponaria inhibitor, euphorbia factor L1, mitogelin, enlimicin, phenomycin, enomycin and trichothecene; enzymes such as alkaline phosphatase that activates etoposide and doxorubicin; carboxypeptidase G2 that activates nitrogen mustard; beta-lactamase that activates doxorubicin, paclitaxel and mitomycin; cytokines such as TNF factor alpha, INF-gamma, GM-GSF factor or IL-2; radioisotopes such as 131 I, 90 Y, 177 Lu, 188 Re, 67 Cu, 211 At, 213 Bi, 125 I, 111In; anti-angiogenic agents such as paclitaxel, 2-methoxyestradiol, prinomastat, batimastat, BAY 12-9566, carboxyamidotriazole, CC-1088, dextromethorphan acetate, dimethylxanthenone acetic acid, endostatin, IM-862, marimastat, penicillamine, PTK787 / ZK 222584, RPI.4610, squalamine lactate, SU5416, thalidomide, combretastatin, tamoxifen, COL-3, neovastat, BMS-275291, SU6668, anti-VEGF antibodies, Medi-522 (Vitaxin II), CAI, interleukin 12, IM862, amiloride, angiostatin, Kl-3 angiostatin, Kl-5 angiostatin, captopril, DL-α-difluoromethylornithine, DL-α-difluoromethylornithine HCl, endostatin, fumagillin, herbimycin A, 4-hydroxyphenyl retinamide, juglone, laminin, laminin hexapeptide, laminin pentapeptide, fumagillin A, medroxyprogesterone, minocycline, placental ribonuclease inhibitor, suramin, thrombospondin, antibodies against angiogenic factors (e.g., Avastin, Erbitux, Vectibix, Herceptin); low molecular weight tyrosine kinase inhibitors of angiogenic growth factors (e.g., Tarceva, Nexavar, Sutent, Iressa); mTOR inhibitors (e.g., Torisel); interferon α, β and γ, IL-12, matrix metalloproteinase inhibitors (e.g., COL3, marimastat, batimastat); ZD6474, SU11248, vitaxin; PDGFR inhibitors (e.g., Gleevec); NM3 and 2-ME2; cyclic peptides such as cilengitide; antiproliferative agents such as (i) antimetabolites such as folic acid antimetabolites (aminopterin, methotrexate, edatrexate, trimetrexate, nolatrexed, lometrexol, pemetrexed, raltitrexed, piritrexim, pteropterin, folinic acid, 10-propargyl-5,8-dideazafolate (PDDF, CB3717)), purine analogs (cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine) and pyrimidine analogs (capecitabine, cytarabine or cytosine arabinoside, decitabine, fluorouracil, 5-fluorouracil, doxifluridine, floxuridine and gemcitabine); (ii) natural products, such as antitumor antibiotics and mitotic inhibitors such as vinca alkaloids such as vindesine, vincristine, vinblastine, vinorelbine; taxanes such as paclitaxel (Taxol TM ), docetaxel (Taxotere TM); colchicine (NSC 757), thiocolchicine (NSC 361792), colchicine derivatives (e.g., NSC 33410) and demecolcine (NSC 406042); halichondrin B (NSC 609395); dolastatin 10 (NSC 376128); maytansine (NSC 153858); rhizoxin (NSC 332598); epothilone A, epothilone B; discodermolide; estramustine; nocodazole; (iii) hormones and their antagonists such as tamoxifen, toremifene, anastrozole, arzoxifene, lasofoxifene, raloxifene, nafoxidine, fulvestrant, aminoglutethimide, testolactone, atamestane, exemestane, formestane, letrozole, goserelin, leuprorelin (leuprorelin or leuprolide), buserelin, histrelin, megestrol acetate and fluoxymesterone; (iv) biological agents such as viral vectors, interferon α and interleukin; (v) platinum-based compounds such as carboplatin, cisplatin [cis-diammine dichloroplatinum, (CDDP)], oxaliplatin, iproplatin, nedaplatin, triplatin tetranitrate, tetraplatin, satraplatin (JM216), JM118 [cis-ammine dichloro (II)], JM149 [cis-ammine dichloro (cyclohexylamine) trans-dihydroxo platinum (IV)], JM335 [trans-ammine dichloro dihydroxo platinum (IV)], transplatin, ZD0473, cis, trans, cis-Pt(NH3)(C6H11NH2)(OOCC3H7)2Cl, malanate-1,2-diaminocyclohexane platinum (II), 5-sulphosalycilate-trans-(1,2-diaminocyclohexane) platinum (II) (SSP), poly-[(trans-1,2-diaminocyclohexane) platinum]-carboxyamilose (poly-PLAT) and 4-hydroxy-sulphonylphenylacetate (trans-1,2-diaminocyclohexane) platinum (II) (SAP), etc., and (vi) DNA-alkylating drugs such as nitrogen mustard, nitrosourea, ethyleneimine derivatives, alkyl sulphonates and triazenes, including, but not limited to, cyclophosphamide (Cytoxan TM) Busulfan, Inprosulfan, Piposulfan, Pipobroman, Melphalan (L-Sarcolysin), Chlorambucil, Nitrogen Mustard, Uramustine (Uramustine or Uracil Mustard), Neomustine, Cholesteryl Benzoate, Trofosfamide, Ifosfamide, Carmustine (BCNU), Lomustine (CCNU), Chlorozotocin, Fotemustine, Nimustine, Remustine, Semustine (Methylcyclohexylnitrosourea), Streptozocin, Thiotepa, Triethylenemelamine, Triethylenethiophosphoramine, Procarbazine, Altretamine, Dacarbazine, Mitozolomide, and Temozolomide; Targeted therapies or immunotherapies such as anti-PD1 or anti-CTLA4.

[0349] In a third aspect, the present invention relates to an in vitro method of selecting a subject suffering from cancer for treatment with an agent selected from the group consisting of:

[0350] a) A polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant thereof;

[0351] b) A conjugate comprising a polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or its functionally equivalent variant;

[0352] c) A polynucleotide encoding the polypeptide of a) or the conjugate of b);

[0353] d) A vector comprising the polynucleotide according to c); and

[0354] e) A cell capable of secreting the polypeptide according to a) or the conjugate according to b) into a culture medium;

[0355] The method comprises:

[0356] (i) Determining the level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α in a sample from the subject; and

[0357] (ii) Comparing the level of the at least one biomarker with a reference value, wherein if a decreased level of the at least one biomarker relative to the reference value is detected, the patient is selected for the treatment.

[0358] The first step of the third method of the present invention comprises determining the level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α in a sample from the subject; and the second step comprises comparing the level of the at least one biomarker with a reference value.

[0359] When comparing the levels of one or more biomarkers selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α with a reference value, the method of the present invention allows for the selection of patients to receive treatment. In particular, a subject having a reduced level of said at least one biomarker relative to said reference value is selected for treatment with any one of items (a) to (e) of the first aspect of the present invention.

[0360] Any therapeutic treatment and pharmaceutical form of treatment of the present invention can be used as defined in the context of the fourth aspect of the present invention.

[0361] Those skilled in the art will understand that the specific embodiments developed in the first aspect of the present invention can also be applied to the second and third aspects of the present invention.

[0362] All terms have been described in detail above in the context of the first aspect of the present invention and are used with the same meaning in the context of the second and third aspects of the present invention.

[0363] The therapeutic method of the present invention

[0364] The results obtained in the present invention indicate that low levels of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α in samples of cancer-suffering patients taken before the start of treatment indicate a high likelihood of successful therapeutic treatment with the anti-cancer treatment defined in items (a) to (f) of the first aspect of the present invention. This indicates that this may be the best treatment available for such patients. For patients with low levels of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α, the anti-cancer treatment of items (a) to (f) of the first aspect of the present invention would be a choice as a first-line treatment.

[0365] In a fourth aspect, the present invention relates to a medicament selected from the group consisting of for treating cancer in a subject, the group consisting of:

[0366] a) a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof;

[0367] b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of said polypeptide or its functionally equivalent variant;

[0368] c) a polynucleotide encoding the polypeptide of a) or the conjugate of b);

[0369] d) a vector comprising the polynucleotide according to c); and

[0370] e) A cell that is capable of secreting into the culture medium the polypeptide according to a) or the conjugate according to b);

[0371] wherein the subject has been identified as a good responder to the agent according to the method of the first aspect of the invention, or wherein the treatment has been selected by the second method of the invention.

[0372] Alternatively, the invention relates to the use of an agent selected from the group consisting of:

[0373] a) A polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof;

[0374] b) A conjugate that contains a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or its functionally equivalent variant;

[0375] c) A polynucleotide that encodes the polypeptide of a) or the conjugate of b);

[0376] d) A vector that contains the polynucleotide according to c); and

[0377] e) A cell that is capable of secreting into the culture medium the polypeptide according to a) or the conjugate according to b);

[0378] The drug for treating a subject suffering from cancer, wherein the subject has been identified as a good responder to the agent according to the method of the first aspect of the invention, or wherein the treatment has been selected by the second method of the invention.

[0379] Alternatively, the invention relates to a method for treating cancer in a subject, the method comprising administering to the subject an agent selected from the group consisting of:

[0380] a) A polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof;

[0381] b) A conjugate that contains a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or its functionally equivalent variant;

[0382] c) A polynucleotide that encodes the polypeptide of a) or the conjugate of b);

[0383] d) A vector that contains the polynucleotide according to c); and

[0384] e) A cell that is capable of secreting into the culture medium the polypeptide according to a) or the conjugate according to b);

[0385] wherein a subject has been identified as a good responder to the agent by a method according to the first aspect of the invention, or wherein the treatment has been selected by a second method of the invention.

[0386] The agent preferably forms part of a medicament, i.e., a composition comprising a therapeutically effective amount of the agent, which agent comprises at least one pharmaceutically acceptable excipient or carrier. The medicament may comprise more than one agent, i.e., more than one agent defined under items (a) to (e) of the first aspect of the invention or additional agents useful for treating cancer.

[0387] The term "therapeutically effective amount" as used herein means an amount of the agent sufficient to provide the desired effect and will generally be determined by reasons such as the characteristics of the compound itself and the therapeutic effect to be achieved. It will also depend on the subject to be treated, the severity of the cancer disease suffered by the subject, the dosage form selected, the route of administration, etc.

[0388] The expression "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" as used herein means any compound or combination of compounds that is substantially non-toxic to the subject at the doses and concentrations employed and is compatible with the other components of the medicament. Thus, an excipient is an inactive substance formulated together with the active ingredient of the medicament for the purpose of bulking up the composition containing the active ingredient. Excipients can also be used for various purposes of enhancing treatment, such as promoting the absorption or dissolution of the active ingredient, or other pharmacokinetic considerations. Excipients can also be used during the preparation process to assist in the handling of the active substances involved, such as by promoting powder flowability or non-stick properties, and to assist in in vitro stability such as preventing denaturation during the expected storage period.

[0389] In a preferred embodiment, the anti-cancer treatment is administered intravenously; preferably as a 30-minute intravenous infusion. In one embodiment, the anti-cancer therapy is administered once a week.

[0390] In a preferred embodiment, the agent is a polypeptide comprising SEQ ID NO:1 or a functional equivalent variant thereof; more preferably a polypeptide consisting of SEQ ID NO:4.

[0391] All embodiments of the first, second, and third aspects of the invention are also applicable to the fourth aspect of the invention.

[0392] All terms have been previously defined in the context of the first, second, and third aspects of the invention and are used in the same meaning in the context of the fourth aspect of the invention.

[0393] The kits of the invention and their uses

[0394] The inventors of the present invention have found that the levels of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α can be determined by immunoassay to predict the response of a subject suffering from cancer to anti-cancer treatment with OMO-103.

[0395] In another aspect, the present invention relates to a kit comprising reagents specifically for determining the expression level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α.

[0396] In another aspect, the present invention relates to the use of a kit comprising reagents specifically for determining the expression level of at least one biomarker selected from MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α, or to the use of reagents specifically for determining the expression level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12, preferably selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α, in a sample from a subject suffering from cancer for predicting the clinical response of said subject to treatment with any of the agents defined in items (a) to (e) of the first aspect of the present invention. In a preferred embodiment, the kit or reagent is used in the method according to the first aspect of the present invention.

[0397] In another aspect, the present invention relates to the use of a kit comprising reagents specifically for determining the expression level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α, or to the use of reagents specifically for determining the expression level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12, preferably selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α, in a sample from a subject suffering from cancer for selecting a therapy for said subject, particularly a therapy using any of the agents defined in items (a) to (e) of the first aspect of the present invention. In a preferred embodiment, the kit or reagent is used in the method according to the second aspect of the present invention.

[0398] In another aspect, the present invention relates to the use of a kit comprising reagents specifically for determining the expression level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α, or to the use of reagents specifically for determining the expression level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12, preferably selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α, in a sample from a subject suffering from cancer for selecting a cancer patient for treatment with any one of the medicaments defined in items (a) to (e) of the first aspect of the present invention. In a preferred embodiment, the kit or the reagents are used in the method according to the third aspect of the present invention.

[0399] In the context of the present invention, a "kit" is understood to be a product containing different reagents required to achieve the different uses of the present invention, the reagents being packaged to allow their transport and storage. Materials suitable for packaging the components of the kit include crystals, plastics (such as polyethylene, polypropylene, polycarbonate, etc.), bottles, vials, paper, sachets, etc. In addition, the kit used in the present invention may contain instructions regarding the simultaneous, sequential, or separate use of the different components in the kit. The instructions may be in the form of printed material or in the form of an electronic support capable of storing instructions that are easy to read or understand, such as, for example, an electronic storage medium (such as a disk, magnetic tape) or an optical medium (such as a CD-ROM, DVD) or an audio material. Additionally or alternatively, the medium may contain an Internet address providing the instructions.

[0400] In a preferred embodiment, the kit further comprises reagents specifically for determining the expression level of at least one additional biomarker selected from the group consisting of MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12 and combinations thereof.

[0401] In a more preferred embodiment, the kit comprises reagents specifically for determining the expression level of MIP-1β and at least one additional reagent specifically for determining the expression level of a biomarker selected from the group consisting of CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12.

[0402] As used herein, the term "reagent" refers to any compound or composition that can be used to detect the level of any biomarker of the present invention, such as the level of any one of the biomarkers MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α, or the level of any one of the biomarkers MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12. In a preferred embodiment, the reagent is a reagent for detecting the level of MIP-1β. In another embodiment, the reagent is a reagent for detecting the level of CD62E. In another embodiment, the reagent is a reagent for detecting the level of IL-8. In another embodiment, the reagent is a reagent for detecting the level of GM-CSF. In another embodiment, the reagent is a reagent for detecting the level of IL-1α. In another embodiment, the reagent is a reagent for detecting the level of one or more biomarkers selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12, i.e., for detecting its gene, protein, or variant thereof. The reagent may optionally include a reagent for detecting one or more housekeeping genes or the proteins encoded by the housekeeping genes.

[0403] The expression "reagent specifically for determining the expression level of a biomarker" as used herein refers to any compound or collection of compounds that allows the specific determination of the expression level of a gene or protein by detection methods well known to those skilled in the art. Specifically, the term "specific reagent" as used herein refers to any reagent that can be used for the specific quantification of one of the biomarkers of the present invention.

[0404] In one embodiment, the reagent specifically for determining the expression level of a biomarker is a nucleic acid capable of specifically hybridizing to any one of the genes MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12. In a preferred embodiment, the nucleic acid is an oligonucleotide that specifically hybridizes to the biomarker.

[0405] As used herein, the term "oligonucleotide" refers to a nucleic acid of preferably at least 10 nucleotides, preferably at least 15 nucleotides, preferably at least 20 nucleotides, preferably at least 25 nucleotides and preferably not more than 100 nucleotides, said nucleotides including polyribonucleotides, polydeoxyribonucleotides and combinations thereof. The term "oligonucleotide" also refers to a molecule formed by conventional nucleotides linked by conventional phosphodiester bonds and variants thereof, including modifications in purines or pyrimidines or in ribose or deoxyribose, said modifications being designed to increase the stability of the oligonucleotide. Alternatively or additionally, the oligonucleotide may contain modified bonds such as phosphotriesters, phosphorothioates, methylphosphonates, etc., or may be a peptide nucleic acid (PNA). Methods for synthesizing oligonucleotides are well known to those skilled in the art. The oligonucleotide may be a primer or a probe. In one embodiment, the oligonucleotide is a probe. In another embodiment, the oligonucleotide is a primer, preferably a pair of primers. In one embodiment, the present invention provides a set of one or more pairs of oligonucleotide primers designed to specifically amplify the biomarker of the method of the present invention.

[0406] Nucleic acids capable of specifically hybridizing to the MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β and IL-12 genes are, for example, one or more pairs of primer oligonucleotides for specifically amplifying a fragment of the mRNA (or its corresponding cDNA) of said genes.

[0407] As will be appreciated by those skilled in the art, the oligonucleotide primers and probes of the kits of the present invention can be used in all techniques for gene expression profiling (RT-PCR, SAGE, TaqMan, real-time PCR, FISH, NASBA, etc.).

[0408] In another embodiment, the reagent specifically for determining the expression level of the biomarker is a compound that specifically binds to the biomarker protein, particularly selected from antibodies, aptamers and fragments thereof.

[0409] In a preferred embodiment, the reagent specifically for determining the expression level of the biomarker is an antibody or a fragment thereof capable of specifically binding (i.e., specifically recognizing) the MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β or IL-12 protein or a variant thereof (including a fragment containing an antigenic determinant).

[0410] Antibodies or fragments thereof that are capable of detecting an antigen and specifically binding to a protein or its variant are, for example, monoclonal and polyclonal antibodies, antibody fragments, Fv, Fab, Fab’, and F(ab’)2, ScFv, diabodies, triabodies, tetra-bodies, and humanized antibodies. The antibodies of the kits of the present invention can be used in conventional methods for detecting protein expression levels, such as Western blotting or protein transfer, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemistry and immunohistochemistry techniques, techniques based on the use of biochips including specific antibodies, protein microarrays, or assays based on colloidal precipitation (in the form of, for example, dipsticks), etc. In a preferred embodiment, the antibody is used in an immunoassay. Preferably, the immunoassay is based on luminex technology, more preferably ProcartaPlex TM Kit

[0411] The reagents, in particular the probes and antibodies, can be immobilized on a solid support (such as a membrane, plastic, or glass), optionally treated to facilitate immobilization of the probe or antibody on the support.

[0412] The kits of the present invention optionally contain additional reagents for detecting a housekeeping gene, a polypeptide encoded by the housekeeping gene, or mRNA encoded by the housekeeping gene. The availability of the additional reagents allows normalization of measurements made in different samples (such as test samples and control samples) to exclude differences in biomarker expression due to different amounts of total protein in the samples rather than actual differences in relative expression levels. Housekeeping genes used herein refer to genes encoding proteins that are constitutively expressed and perform essential cellular functions. Preferred housekeeping genes for use in the present invention include β-2-microglobulin, ubiquitin, 18-S ribosomal protein, cyclophilin, PSMB4, GAPDH, tubulin, and β-actin.

[0413] In a preferred embodiment, the kits mentioned in any of the above uses further contain reagents specifically for detecting or determining the expression level of a gene or protein, the expression of which determines whether the cells in the sample are proliferating. Preferably, the kit contains an antibody capable of specifically recognizing a protein, the expression of which determines whether the cells in the sample are proliferating, and the protein is preferably selected from geminin, KI-67, proliferating cell nuclear antigen, cyclin A2.

[0414] The kit of the present invention further contains other reagents that allow the determination of the expression level of the biomarker but are not specifically for said biomarker, such as reagents for extracting RNA materials, etc., such as primers for synthesizing the corresponding cDNA by means of RT, reagents for amplifying DNA such as DNA polymerase, dNTP, buffer, etc.

[0415] In another preferred embodiment, any reagent mentioned in this section, especially any oligonucleotide or antibody mentioned in this section, more preferably any antibody, each accounts for at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% of the total amount of the reagents forming the kit of the present invention (preferably the total amount of the reagents forming said kit that are specifically for determining the expression level of one or more biomarkers).

[0416] In a more preferred embodiment, the reagents specifically for determining the expression level of at least one biomarker account for at least 10%, at least 20%, at least 30%, at least 40%, at least 50% of the total amount of the reagents forming said kit. Preferably, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% of the reagents specifically for determining the biomarker contained in said kit are reagents specifically for determining the expression level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β and IL-12.

[0417] All specific embodiments disclosed for the method of the present invention are applicable to the kit of the present invention and its uses.

[0418] All terms have been previously defined in the context of the first, second, third and fourth aspects of the present invention, and these terms are used with the same meaning in the context of the kit of the present invention and its uses.

[0419] The present invention also relates to the following aspects:

[0420] 1. An in vitro method for predicting the clinical response of a subject suffering from cancer to an anticancer treatment selected from the group consisting of:

[0421] a) a polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant thereof;

[0422] b) A conjugate comprising a polypeptide having the sequence SEQ ID NO:1 or a functional equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or its functional equivalent variant;

[0423] c) A polynucleotide encoding the polypeptide of a) or the conjugate of b);

[0424] d) A vector comprising the polynucleotide according to c); and

[0425] e) A cell capable of secreting the polypeptide according to a) or the conjugate according to b) into a culture medium;

[0426] The method comprises:

[0427] (i) Determining the level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α in a sample from the subject; and

[0428] (ii) Comparing the level of the at least one biomarker with a reference value,

[0429] Wherein:

[0430] - A reduced level of the at least one biomarker relative to the reference value indicates a good clinical response of the subject to the anti-cancer treatment, or

[0431] - An equal or increased level of the at least one biomarker relative to the reference value indicates a poor clinical response of the subject to the anti-cancer treatment.

[0432] 2. The method according to aspect 1, wherein the good clinical response is stabilization of the disease.

[0433] 3. The method according to any one of aspects 1 or 2, wherein the poor clinical response is progression of the disease.

[0434] 4. An in vitro method for selecting a customized therapy for a subject suffering from cancer, comprising:

[0435] (i) Determining the level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α in a sample from the subject; and

[0436] (ii) Comparing the level of the at least one biomarker with a reference value,

[0437] Wherein:

[0438] - A reduced level of said at least one biomarker relative to said reference value indicates that the therapy to be selected comprises an agent selected from the group consisting of:

[0439] a) a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof;

[0440] b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of said polypeptide or functionally equivalent variant thereof;

[0441] c) a polynucleotide encoding the polypeptide of a) or the conjugate of b);

[0442] d) a vector comprising the polynucleotide according to c); and

[0443] e) a cell capable of secreting the polypeptide according to a) or the conjugate according to b) into a culture medium; or alternatively,

[0444] - An equal or increased level of said at least one biomarker relative to said reference value indicates that the therapy to be selected does not comprise an agent selected from the group consisting of:

[0445] a) a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof;

[0446] b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of said polypeptide or functionally equivalent variant thereof;

[0447] c) a polynucleotide encoding the polypeptide of a) or the conjugate of b);

[0448] d) a vector comprising the polynucleotide according to c); and

[0449] e) a cell capable of secreting the polypeptide according to a) or the conjugate according to b) into a culture medium.

[0450] 5. An in vitro method of selecting a subject suffering from cancer for treatment with an agent selected from the group consisting of:

[0451] a) a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof;

[0452] b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of said polypeptide or functionally equivalent variant thereof;

[0453] c) a polynucleotide encoding the polypeptide of a) or the conjugate of b);

[0454] d) a vector comprising the polynucleotide according to c); and

[0455] e) a cell capable of secreting the polypeptide according to a) or the conjugate according to b) into a culture medium;

[0456] The method comprises:

[0457] (i) determining the level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α in a sample from the subject; and

[0458] (ii) comparing the level of the at least one biomarker with a reference value, wherein if a decreased level of the at least one biomarker relative to the reference value is detected, the patient is selected for the treatment.

[0459] 6. The method according to any one of aspects 1 to 5, wherein the subject has not received any anti-cancer treatment within the most recent 24 hours prior to sample isolation.

[0460] 7. The method according to aspect 6, wherein the subject has not received systemic anti-cancer treatment for at least 4 weeks prior to sample isolation.

[0461] 8. The method according to any one of aspects 1 to 7, wherein the sample is blood, serum, or plasma.

[0462] 9. The method according to aspect 8, wherein the sample is a serum sample.

[0463] 10. The method according to any one of aspects 1 to 9, wherein the level of the at least one biomarker determined is the mRNA level.

[0464] 11. The method according to any one of aspects 1 to 9, wherein the level of the at least one biomarker determined is the protein level.

[0465] 12. The method according to aspect 11, wherein the level of at least one biomarker protein is determined by immunoassay.

[0466] 13. The method according to any one of aspects 1 to 12, wherein the anti-cancer treatment comprises using a polypeptide comprising SEQ ID NO:1 or a functional equivalent variant thereof.

[0467] 14. The method according to aspect 13, wherein the anti-cancer treatment comprises using a polypeptide consisting of SEQ ID NO:4.

[0468] 15. The method according to any one of aspects 1 to 14, wherein the cancer is a solid tumor.

[0469] 16. A method according to any one of aspects 1 to 15, wherein the subject from whom the sample has been isolated has not received any anti-cancer treatment prior to the isolation of the sample.

[0470] 17. A method according to any one of aspects 1 to 15, wherein the subject from whom the sample has been isolated has received anti-cancer treatment prior to the isolation of the sample, and wherein the anti-cancer treatment is different from the treatment selected from the group consisting of:

[0471] a) a polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant thereof;

[0472] b) a conjugate comprising a polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof;

[0473] c) a polynucleotide encoding the polypeptide of a) or the conjugate of b);

[0474] d) a vector comprising the polynucleotide according to c); and

[0475] e) a cell capable of secreting the polypeptide according to a) or the conjugate according to b) into a culture medium.

[0476] 18. A method according to any one of aspects 1 to 17, the method further comprising determining the level of at least one additional biomarker selected from the group consisting of MCP-1, ICAM-1, IFN-γ, IL-1β, IL-12 and combinations thereof.

[0477] 19. A method according to any one of aspects 1 to 18, wherein the method comprises determining the level of a combination of biomarkers selected from the group consisting of:

[0478] a) a combination of biomarkers comprising MIP-1β and MCP-1;

[0479] b) a combination of biomarkers comprising MIP-1β and ICAM-1;

[0480] c) a combination of biomarkers comprising MIP-1β and CD62E;

[0481] d) a combination of biomarkers comprising MIP-1β and IFN-γ;

[0482] e) a combination of biomarkers comprising MIP-1β and IL-1β;

[0483] f) a combination of biomarkers comprising MIP-1β and IL-12; and

[0484] g) A combination of biomarkers comprising CD62E and IL-8.

[0485] 20. The method according to aspect 19, wherein the method comprises determining the levels of a combination of biomarkers selected from the group consisting of:

[0486] a) A combination of biomarkers comprising MIP-1β and MCP-1; and

[0487] b) A combination of biomarkers comprising CD62E and IL-8.

[0488] 21. The method according to any one of aspects 19 or 20, wherein the method comprises determining the levels of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α.

[0489] 22. An agent selected from the group consisting of agents for treating cancer in a subject, the group consisting of:

[0490] a) A polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof;

[0491] b) A conjugate comprising a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or its functionally equivalent variant;

[0492] c) A polynucleotide encoding the polypeptide of a) or the conjugate of b);

[0493] d) A vector comprising the polynucleotide according to c); and

[0494] e) A cell capable of secreting the polypeptide according to a) or the conjugate according to b) into a culture medium;

[0495] wherein the subject has been identified as a good responder to the agent by the method as defined in any one of aspects 1 to 3 or 6 to 21.

[0496] 23. An agent selected from the group consisting of agents for treating cancer in a subject, the group consisting of:

[0497] a) A polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof;

[0498] b) A conjugate comprising a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that promotes cellular uptake of the polypeptide or its functionally equivalent variant;

[0499] c) A polynucleotide encoding the polypeptide of a) or the conjugate of b);

[0500] d) a vector comprising the polynucleotide according to c); and

[0501] e) a cell capable of secreting the polypeptide according to a) or the conjugate according to b) into a culture medium;

[0502] wherein the treatment has been selected by a method as defined in any one of aspects 4 or 6 to 21.

[0503] 24. An agent for use according to any one of aspects 22 or 23, wherein the agent is a polypeptide comprising SEQ ID NO:1 or a functional equivalent variant thereof.

[0504] 25. An agent for use according to aspect 24, wherein the agent is a polypeptide consisting of SEQ ID NO:4.

[0505] 26. A kit comprising reagents specifically for determining the expression level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF and IL-1α.

[0506] 27. The kit according to aspect 26, the kit further comprising reagents specifically for determining the expression level of at least one additional biomarker selected from the group consisting of MCP-1, ICAM-1, IFN-γ, IL-1β, IL-12 and combinations thereof.

[0507] 28. The kit according to aspect 27, wherein the kit comprises reagents specifically for determining the expression level of MIP-1β and at least one additional reagent specifically for determining the expression level of a biomarker selected from the group consisting of CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β and IL-12.

[0508] 29. The kit according to any one of aspects 26 to 28, wherein the reagents are antibodies capable of specifically recognizing at least one biomarker.

[0509] 30. The kit according to any one of aspects 26 to 29, wherein the reagents specifically for determining the expression level of at least one biomarker account for at least 10% of the total amount of the reagents forming the kit.

[0510] 31. Use of a kit according to any one of aspects 26 to 30 or a reagent specifically for determining the expression level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β and IL-12 in the method as defined in any one of aspects 1 to 21.

[0511] The present invention will be described by the following examples, which should be considered merely exemplary and not limiting the scope of the present invention.

[0512] Examples

[0513] Experimental details

[0514] The product administered was OMO-103 (SEQ ID NO: 4), which corresponds to the Omomyc peptide sequence further comprising methionine at the N-terminal end. OMO-103 is the final drug product, consisting of a mixture of active pharmaceutical ingredient (API) and excipients, and is used for intravenous infusion in clinical practice.

[0515] Patients had not received systemic anti-cancer treatment for at least 4 weeks before sample isolation and had not received any anti-cancer treatment for at least 24 hours before sample isolation.

[0516] Patients had different types of solid tumors, including pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), salivary gland cancer, sarcoma, triple-negative breast cancer and pleural mesothelioma.

[0517] Blood samples of patients included in the Phase I of the OMO-103 clinical trial were collected at pre-treatment, just before the patients were about to receive the first OMO-103 infusion. OMO-103 was administered as a 30-minute intravenous infusion at doses of 2.88 mg / kg, 4.32 mg / kg, 6.48 mg / kg and 9.72 mg / kg, respectively. Then, serum was separated using standard procedures and stored frozen at -80 °C until use. The cytokine and chemokine levels were measured using the Inflammation 20-Plex Human ProcartaPlex TM Kit (Invitrogen) by Luminex technology. The cytokine and chemokine levels were inferred from the standard curves using ProcartaPlex Analyst software.

[0518] After three treatment cycles (9 weeks), the response of patients to OMO-103 was measured by CT scan using the RECIST 1.1 criteria (Eisenhauer, E.A., et al. (2009). New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1), Eur J Cancer 45:228-247). Based on the RECIST results, patients were then classified as progressive disease (PD) and stable disease (SD). A total of 18 patients were evaluable for cytokine and chemokine analysis, 11 patients were classified as PD, and 7 patients were classified as SD.

[0519] To determine whether the levels of different cytokines and chemokines at pretreatment were different between PD and SD patients, univariate analysis was performed. The Mann-Whitney U test was used to determine whether the cytokine / chemokine levels between the two groups were significantly different ( Figure 1 ).

[0520] By using the area under the receiver-operator characteristic curve (ROC-AUC), the diagnostic accuracy of various cytokines and chemokines for identifying SD patients was estimated ( Figure 2 ). As a general measure of the predictive ability of each individual cytokine / chemokine, the ROC-AUC was used. Generally, an AUC of 0.5 indicates no difference, an AUC of 0.7 to 0.8 is considered acceptable, an AUC of 0.8 to 0.9 is considered excellent, and an AUC exceeding 0.9 is considered outstanding, as it is known that when the AUC is 1, the model is a perfect classifier (DW Hosmer, S Lemeshow (2000). Applied Logistic Regression, 2nd Edition, Chapter 5, John Wiley and Sons, New York, NY (2000), pp. 160-164). Analysis was performed by fitting a logistic regression model to each individual cytokine, with RECIST (C3) as the binary output variable. The Python package of Scikit-learn (Pedragosa F. et al. (2011). Scikit-learn: Machine Learning in Python. Journal of Machine Learning Research, 12, pp. 2825-2830) was used to fit the model.

[0521] Using the QLattice modeling technique (Abzu), a combination of cytokines / chemokines that can correctly distinguish between PD and SD patients was discovered (René, K et al. (2021). An approach to symbolic regression using Feyn. arXiv:2014.05417 [cs.LG]; Wilsup, C. et al. (2021). Symbolic regression outperforms other models for small datasets. arXiv:2013.15147 [cs.LG]; Christensen, N. et al. (2022) Identifying interactions in omics data for clinical biomarker discovery using symbolic regression, Bioinformatics, Volume 38, Issue 15: 3749-3758). QLattice is a symbolic regression algorithm that searches for combinations of input variables and mathematical functions that provide predictions for the output variable. It uses an evolutionary scheme for the search and selects the best model by minimizing an error metric computed between the predictions and the true outputs. QLattice runs within a leave-one-out cross-validation loop, i.e., the algorithm is run 16 times, excluding one patient at each iteration. Two models appear at each iteration: MCP-1 + MIP-1β and CD62E + IL-8. CD62E + MIP-1β and ICAM-1 + MIP-1β appear 15 times; IFN-γ + MIP-1β and IL-1β + MIP-1β appear 14 times; and IL-12 + MIP-1β appears 13 times. ROC-AUC was also calculated for all combined models to determine their prediction accuracy and ability ( Figure 3 ). The parameter values of all 16 models discovered were used to estimate the confidence interval bands of the models.

[0522] Results

[0523] Patients responsive to OMO-103 treatment who showed disease stabilization at cycle 3 showed significantly lower levels of CD62E, IL-8, MIP-1β, GM-CSF, and IL-1α at baseline compared to the levels shown by patients with progressive disease. Figure 1 )

[0524] To investigate the predictive ability of the identified cytokines and chemokines for response to OMO-103, receiver operating characteristic (ROC) curve analysis was performed. Figure 2)。The ROC-AUCs for the five significantly decreased cytokines / chemokines were 0.87 (for CD62E), 0.91 (for IL-8), 0.97 (for MIP-1β), 0.82 (for GM-CSF), and 0.79 (for IL-1α), respectively. All values were close to 0.8 or above, indicating that they represent excellent predictors of SD outcomes and have very strong prognostic capabilities.

[0525] Finally, the QLattice modeling technique was used to find combinations of cytokines that could stratify and distinguish which patients responded to treatment at baseline, showing stabilization of the disease. This analysis showed that different cytokine / chemokine combinations could correctly classify SD and PD patients: CD62E + IL-8, MIP-1β and MCP-1, MIP-1β + ICAM-1, MIP-1β + CD62E, MIP-1β + IFN-γ, MIP-1β + IL-1β, and MIP-1β + IL-12( Figure 3 A).

[0526] For the CD62E + IL-8 combination model, when IL-8 > 8.0 pg / ml, the probability of a patient developing PD independent of CD62E was higher than 80%, and when CD62E > 33,880 pg / ml, the probability of a patient developing PD independent of IL-8 was higher than 80%. On the other hand, for IL-8 below 8.0 pg / ml, as CD62E increased, the critical threshold of IL-8 decreased, and vice versa for CD62E below 33,880 pg / ml. For the combination of MIP-1β and MCP-1, when MIP-1β > 65 pg / ml, the probability of a patient developing PD independent of MCP-1 was 80%, and when MIP-1β was below 65 pg / ml, as MCP-1 increased, the critical threshold of MIP-1β decreased.

[0527] For the ICAM-1 + MIP-1β model, when MIP-1β > 55.96 pg / ml, the probability of patients developing PD independently of the ICAM-1 level is higher than 80%. On the other hand, when MIP-1β < 55.96 pg / ml, this critical threshold decreases as the ICAM-1 level increases. For the CD62E + MIP-1β model, when MIP-1β > 117.06 pg / ml, the probability of patients developing PD independently of the CD62E level is higher than 80%. On the other hand, when MIP-1β < 117.06 pg / ml, this critical threshold decreases as the CD62E level increases. For the IFN-γ + MIP-1β model, when MIP-1β > 81.78 pg / ml, the probability of patients developing PD independently of the IFN-γ level is higher than 80%. On the other hand, when MIP-1β < 81.78 pg / ml, this critical threshold decreases as the IFN-γ level increases. For the IL-1β + MIP-1β model, when MIP-1β > 91.18 pg / ml, the probability of patients developing PD independently of the IL-1β level is higher than 80%. On the other hand, when MIP-1β < 91.18 pg / ml, this critical threshold decreases as the IL-1β level increases. For the IL-12 + MIP-1β model, when MIP-1β > 91.18 pg / ml, the probability of patients developing PD independently of the IL-12 level is higher than 80%. On the other hand, when MIP-1β < 91.18 pg / ml, this critical threshold decreases as the IL-12 level increases.

[0528] Again, we studied the predictive ability of the identified cytokine / chemokine combinations by ROC curve analysis. The results showed that the ROC curves of the models CD62E + IL-8 and MIP-1β + MCP-1 had an AUC very close to 1 (CD62E + IL-8, AUC = 0.97; MIP-1β + MCP-1, AUC = 1), which means that both models are excellent predictors of the SD result ( Figure 3 B). The AUC and thus the predictive ability of the combined models were improved compared to one of the various cytokines / chemokines.

[0529] Other combinations that are prominent predictors of the SD outcome are: MIP-1β + ICAM-1, MIP-1β + CD62E, MIP-1β + IFN-γ, MIP-1β + IL-1β, and MIP-1β + IL-12. The AUC of these models is 1 (MIP-1β + IFN-γ, AUC = 1) or very close to 1 (MIP-1β + ICAM-1, AUC = 0.98; MIP-1β + CD62E, AUC = 0.96; MIP-1β + IL-1β, AUC = 0.91; MIP-1β + IL-12, AUC = 0.95)( Figure 3 B).

[0530] Taken together, all these results clearly indicate that these cytokines, either as individual markers or in combination with other cytokines, can be used as early predictive biomarkers of response to OMO-103 treatment.

Claims

1. An in vitro method for predicting the clinical response of a subject suffering from cancer to an anti-cancer treatment selected from the group consisting of: a) a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or its functionally equivalent variant; c) a polynucleotide encoding the polypeptide of a) or the conjugate of b); d) a vector comprising the polynucleotide according to c); and e) a cell capable of secreting into the culture medium the polypeptide according to a) or the conjugate according to b); The method comprises: (i) determining the level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF and IL-1α in a sample from the subject; and (ii) comparing the level of the at least one biomarker with a reference value, wherein: - a level of the at least one biomarker that is reduced relative to the reference value indicates a good clinical response of the subject to the anti-cancer treatment, or - a level of the at least one biomarker that is equal to or increased relative to the reference value indicates a poor clinical response of the subject to the anti-cancer treatment.

2. The method according to claim 1, wherein the good clinical response is stabilization of the disease and the poor clinical response is progression of the disease.

3. An in vitro method for selecting a customized therapy for a subject suffering from cancer, comprising: (i) determining the level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF and IL-1α in a sample from the subject; and (ii) comparing the level of the at least one biomarker with a reference value, wherein: - a level of the at least one biomarker that is reduced relative to the reference value indicates that the therapy to be selected comprises an agent selected from the group consisting of: a) a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or its functionally equivalent variant; c) a polynucleotide encoding the polypeptide of a) or the conjugate of b); d) a vector comprising the polynucleotide according to c); and e) a cell capable of secreting into the culture medium the polypeptide according to a) or the conjugate according to b); or alternatively, - a level of the at least one biomarker that is equal to or increased relative to the reference value indicates that the therapy to be selected does not comprise an agent selected from the group consisting of: a) a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or its functionally equivalent variant; c) a polynucleotide encoding the polypeptide of a) or the conjugate of b); d) a vector comprising the polynucleotide according to c); and e) a cell capable of secreting into a culture medium the polypeptide according to a) or the conjugate according to b).

4. An in vitro method of selecting a subject suffering from cancer for treatment with an agent selected from the group consisting of: a) a polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or its functionally equivalent variant; c) a polynucleotide encoding the polypeptide of a) or the conjugate of b); d) a vector comprising the polynucleotide according to c); and e) a cell capable of secreting into a culture medium the polypeptide according to a) or the conjugate according to b); the method comprising: (i) determining in a sample from the subject the level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α; and (ii) comparing the level of the at least one biomarker to a reference value, wherein if a decreased level of the at least one biomarker relative to the reference value is detected, the patient is selected for the treatment.

5. The method according to any one of claims 1 to 4, wherein the sample is blood, serum, or plasma.

6. The method according to any one of claims 1 to 5, wherein the level of the at least one biomarker determined is an mRNA level or a protein level.

7. The method according to claim 6, wherein the level of at least one biomarker protein is determined by immunoassay.

8. The method according to any one of claims 1 to 7, wherein the anti-cancer treatment comprises using the polypeptide consisting of SEQ ID NO:

4.

9. The method according to any one of claims 1 to 8, the method further comprising determining the level of at least one additional biomarker selected from the group consisting of MCP-1, ICAM-1, IFN-γ, IL-1β, IL-12, and combinations thereof.

10. An agent selected from the group consisting of: a) a polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide comprising SEQ ID NO:1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or its functionally equivalent variant; c) a polynucleotide encoding the polypeptide of a) or the conjugate of b); d) a vector comprising the polynucleotide according to c); and e) a cell capable of secreting into a culture medium the polypeptide according to a) or the conjugate according to b); the agent for treating cancer in a subject, wherein the subject has been identified as a good responder to the agent by a method as defined in any one of claims 1 or 2 or 6 - 10, or wherein the treatment has been selected by a method as defined in any one of claims 3 or 5 - 9.

11. A kit, which comprises reagents specifically for determining the expression level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF and IL-1α.

12. The kit according to claim 11, wherein the kit further comprises reagents specifically for determining the expression level of at least one additional biomarker selected from the group consisting of MCP-1, ICAM-1, IFN-γ, IL-1β, IL-12 and combinations thereof.

13. The kit according to any one of claims 11 or 12, wherein the reagent is an antibody capable of specifically recognizing at least one of the biomarkers.

14. The kit according to any one of claims 11-13, wherein the reagent specifically for determining the expression level of at least one biomarker accounts for at least 10% of the total amount of the reagents forming the kit.

15. Use of the kit according to any one of claims 11-14 or a reagent specifically for determining the expression level of at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β and IL-12 in the method as defined in any one of claims 1 to 9.

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