Methods for predicting the response to cancer treatment

Abstract

The present invention relates to a method for predicting the response of cancer patients to anti-cancer treatment based on the detection of biomarkers. The present invention also relates to a treatment method, a method for selecting cancer patients to be treated, or a method for selecting a treatment method suitable for cancer patients, based on the detection of the biomarkers.

Description

Field of Invention

[0001] This invention relates to the field of cancer, and more specifically, to a method for predicting the response of a subject diagnosed with cancer to anti-cancer treatment based on the detection of biomarkers in a sample of the subject. The invention also relates to a method for selecting a customized treatment appropriate to the subject, or a method for selecting a subject to receive treatment with an anti-cancer drug, and to an anti-cancer drug used in the treatment referred to in the method based on the detection of the biomarker. Furthermore, the invention relates to a kit in the method and its use. Background of the Invention

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

[0003] Myc activity is strictly regulated in normal cells, with higher levels in proliferating cells and lower levels in non-proliferating cells. Abnormally elevated or dysregulated Myc activity is associated with most cancers and is often linked to highly aggressive, poorly differentiated, angiogenic, and treatment-resistant tumors.

[0004] Omomyc is a dominant-negative Myc mutant containing the b-HLH-LZ domain of Myc and has four amino acid substitutions in the Myc leucine zipper (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 ​​alter the protein's dimerization specificity, but its ability to bind to its natural partner Max, homodimerize with itself, and heterodimerize with wild-type c-Myc, N-Myc, and L-Myc is maintained.

[0005] These properties allow Omomyc to inhibit Myc-dependent gene transcriptional activation both in vitro and in vivo by inhibiting Myc's binding to the E-box, the DNA recognition binding site. Simultaneously, Omomyc potently enhances Myc-induced apoptosis in a Myc expression level-dependent manner, thereby strengthening Myc's transcriptional repressive activity. Thus, Omomyc inhibits Myc's binding to the promoter E-box and the transcriptional activation of target genes, while maintaining Miz-1-dependent promoter binding and transcriptional repression. In the presence of Omomyc, the Myc interactome is induced to repression, and its activity shifts from pro-tumorogenic to tumor-suppressive.

[0006] In WO2014 / 180889A8, it has been demonstrated that the omomicupeptide itself efficiently permeates the cell membrane and translocates to the nucleus, where it exerts its tumor-suppressing effect.

[0007] In WO2018 / 011433A1, a variant of omomik in which only cysteine ​​is replaced with another amino acid was demonstrated to be even more effective than omomik in cancer treatment.

[0008] However, chemotherapy has limitations, such as undesirable side effects and the development of congenital or acquired resistance. By selecting patients who are more likely to respond to treatment, unnecessary treatment side effects and treatment delays can be avoided, improving the survival rate of cancer patients. Patients with tumors that are likely to be less responsive to chemotherapy may be candidates for alternative therapies.

[0009] Therefore, personalized approaches are needed to better treat the disease, and more useful biomarkers need to be identified to predict cancer patients' responses to anti-cancer treatment. [Overview of the project]

[0010] In a first embodiment, the present invention relates to an in vitro method for predicting the clinical response of a subject with cancer to an anti-cancer treatment selected from the following: a) A polypeptide containing the sequence of sequence number 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) Cells that can secrete the polypeptide described in a) or the conjugate described in b) into a culture medium. Selected from the group consisting of, The method is as follows: (i) Determine 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 at least one biomarker to a reference value, Here, - A decrease in the level of at least one biomarker relative to the reference value indicates a favorable clinical response to the anti-cancer treatment of the subject, or - Equivalent or increased levels of the at least one biomarker relative to the reference value indicate a poor clinical response in the subject to the anti-cancer treatment. Regarding things that include doing so.

[0011] In a second aspect, the present invention relates to an in vitro method for selecting a customized treatment for a subject suffering from cancer, (i) Determine 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 at least one biomarker to a reference value. Includes, Here, - A decrease in the level of at least one biomarker relative to the reference value indicates that the selected treatment includes a drug selected from the group consisting of: a) A polypeptide containing the sequence of sequence number 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) Cells capable of secreting the polypeptide described in a) or the conjugate described in b) into a culture medium: or - Equivalent or increased levels of at least one of the biomarkers relative to the reference value indicate that the selected treatment does not include any drugs selected from the group consisting of: a) A polypeptide containing the sequence of sequence number 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) Cells that can secrete the polypeptide described in a) or the conjugate described in b) into a culture medium. relates to.

[0012] In a third aspect, the invention is an in vitro method for selecting a subject suffering from cancer for treatment with a drug selected from the group consisting of: a) a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide comprising the sequence of 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; c) a polynucleotide encoding the polypeptide of a) or the conjugate of b); d) a vector comprising the polynucleotide of c); and e) a cell capable of secreting the polypeptide of a) or the conjugate of b) into a medium, the method 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, and if a decrease in the level of the at least one biomarker relative to the reference value is detected, the patient is selected for the treatment.

[0013] In a fourth aspect, the invention relates to a drug selected from the group consisting of: A drug selected from the group consisting of: a) a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide comprising the sequence of 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; c) a polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) A cell capable of secreting the polypeptide described in a) or the conjugate described in b) into a medium, which is for use in treating cancer in a subject, where the subject has been identified as a good responder to the agent by the first method of the present invention or the treatment has been selected by the second method of the present invention.

[0014] In a further aspect, the present invention relates to a kit comprising a reagent specific 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.

[0015] In yet another aspect, the present invention relates to the use of the kit or reagent of the present invention specific 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 in the first, second and third methods of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] <00,00111>Patients who showed a response to OMO-103 treatment were shown to have low levels of CD62E, IL-8, MIP-1β, GM-CSF, IL-1α at baseline. Using patient serum samples collected before treatment, the levels of various cytokines and chemokines were measured using the Luminex method. A) Patients who responded to treatment and showed stable disease (SD) at cycle 3 had significantly lower levels of CD62E, IL-8, MIP-1β, GM-CSF, IL-1α compared to patients who showed progressive disease (PD). B) Levels (pg / ml) of various cytokines and chemokines measured before treatment. Mean values and standard deviations are shown. [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 individual cytokines to predict response to OMO-103. A) Graph of individual ROC curves. B) ROC-AUC score for each cytokine / chemokine. The dotted line indicates an AUC score of 0.5, and the dashed line indicates an AUC score of 0.8, which is a threshold indicating excellent predictive power. [Figure 3] Multiple 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 response to OMO-103 and can be used to stratify patients with SD and PD before treatment. The cytokine / chemokine combination models were created using QLattice technology. A) Plots of the combination models 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 shown, along with their patient stratification. Stable disease (SD) is indicated by a white circle / zero, and progressive disease (PD) is indicated by gray / 1. The gray lines correspond to confidence intervals (CI), with dark lines representing the mean, medium gray representing the 5% confidence interval (CI), and clear gray representing the 95% confidence interval (CI). B) Receiver operating characteristic (ROC) curve analysis of seven combined models. [Modes for carrying out the invention]

[0017] The inventors of this invention surprisingly discovered that pre-treatment serum samples from cancer patients who responded well to treatment with polypeptides containing omomiku showed low levels of the biomarkers MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α.

[0018] Therefore, these biomarkers have potential value in predicting a favorable response to Omomyc or functionally equivalent variant-based therapies in cancer patients. Based on these findings, the inventors have developed the methods of the present invention in various embodiments, which are described in detail below.

[0019] The results shown in the embodiments of the present invention clearly demonstrate a significant correlation between low levels of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α and a favorable response to treatment with OMO-103. Therefore, these results suggest that cancer patients exhibiting low levels of these biomarkers in pretreatment samples are candidates for treatment based on Omomyc or its functionally equivalent variants.

[0020] Prediction method of the present invention In a first embodiment, the present invention relates to an in vitro method for predicting the clinical response of a subject with cancer to an anti-cancer treatment selected from the following: a) A polypeptide containing the sequence of sequence number 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) Cells that can secrete the polypeptide described in a) or the conjugate described in b) into a culture medium. Selected from the group consisting of, The method is as follows: (i) Determine 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 at least one biomarker to a reference value, Here, - A decrease in the level of at least one biomarker relative to the reference value indicates a favorable clinical response to the anti-cancer treatment of the subject, or - Equivalent or increased levels of the at least one biomarker relative to the reference value indicate a poor clinical response in the subject to the anti-cancer treatment. This includes doing so.

[0021] The term "in vitro" refers to the fact that an experimental protocol or method is performed on a sample that is isolated from the subject and already present in an experimental tool such as a test tube, dish, or plate, rather than on the body of a human or animal subject.

[0022] As used herein, the term “prediction” refers to determining the likelihood that a patient with cancer will respond well to treatment with an anticancer drug. In particular, as used herein, the term “prediction” refers to an individual assessment of the expected response of a patient with cancer when a tumor is treated with an anticancer drug based on Omomyc or a functionally equivalent variant as defined in items (a) to (e) of the first aspect of the present invention.

[0023] As used herein, the term “clinical response” refers to the response of a cancer-affected subject to an anti-cancer therapy based on Omomyc or a functionally equivalent variant, as defined in items (a) through (e) of the first aspect of the present invention. Standard criteria available herein for evaluating the response to anti-cancer therapy (Eisenhauer, EA, et al. 2009. New response assessment criteria in solid tumors: revised RECIST guideline (version 1.1). Eur J Cancer 45(2): 228-247) include response, stabilization, and progression. As used herein, the term “RECIST” refers to “Response Assessment Criteria for Solid Tumors,” a standard method for evaluating the response of cancer patients to treatment. This is evaluated based on whether the tumor shrinks, remains unchanged, or grows. To use RECIST, at least one tumor that can be measured by radiography, computed tomography (CT) scan, or magnetic resonance imaging (MRI) scan is required. The types of patient responses are complete response (CR), partial response (PR), progressive disease (PD), and stable disease (SD). In a preferred embodiment, RECIST criteria are used to assess tumor response.

[0024] In this invention, subjects who achieved a complete or partial response, and subjects who showed stabilization of their condition, were considered "favorable responders" or subjects who showed a favorable clinical response.

[0025] As used herein, “complete response” (or complete remission) (CR) means the disappearance of all detectable malignancies, i.e., the disappearance of all target lesions, and the reduction in the short diameter of pathological lymph nodes (whether targeted or non-targeted) to less than 10 mm.

[0026] In this specification, “partial response” (PR) means a reduction of at least 30% in the total diameter of the target lesions relative to the total diameter at baseline.

[0027] As used herein, the terms “stabilization,” “disease stabilization,” or “disease stability” (SD) are defined as the absence of a reduction in the smallest total diameter during the study that is sufficient to qualify for a partial response, or an increase in the smallest total diameter that is sufficient to qualify for a progressive disease.

[0028] In this invention, subjects exhibiting progressive disease are considered to be "poor responders" or those with poor clinical response.

[0029] As used herein, the terms “progression” or “progressive disease” (PD) mean a 20% or greater increase in the total diameter of target lesions relative to the minimum total in the study (including the baseline total if that is the minimum total in the study). In addition to a 20% relative increase, the total must show an absolute increase of at least 5 mm. The appearance of one or more new lesions is also considered progression.

[0030] As those skilled in the art will understand, a poor response to anticancer therapy does not mean that the subject does not respond at all or that the treatment is ineffective. For example, anticancer therapy may reduce the growth of lesions in a poorly responding subject compared to an untreated patient. However, the effect achieved in the subject compared to an untreated patient does not constitute stabilization under the criteria used in this invention. For example, a patient who has received anticancer therapy may have a 20% increase in the total diameter of target lesions, whereas if the patient remained untreated, this increase could have reached 30%.

[0031] As those skilled in the art will understand, such evaluations are not typically intended to be correct for all (i.e., 100 percent) of the subjects being identified. However, the term requires that the predictions yield correct results for a statistically significant portion of the subjects. Whether a portion is statistically significant can be easily determined by those skilled in the art using various well-known statistical evaluation tools, such as determining confidence intervals, p-values, Student's t-tests, and Mann-Whitney tests. Details are provided in *Statistics for Research* by Dowdy and Wearden (John Wiley and 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%, at least 97%, at least 98%, or at least 99%. P-values ​​are preferably less than or equal to 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001. More preferably, the method of the present invention can appropriately identify at least 60%, at least 70%, at least 80%, or at least 90% of the target population.

[0032] Other parameters widely accepted for comparing the effectiveness of alternative therapies can be used to determine the response to treatment, and these include, but are not limited to, the following: · disease-free progression This represents the percentage of subjects who were in complete remission and did not experience a relapse of the disease during the study period. · Disease-free survival (DFS) This is understood as the length of time a subject survives without showing signs of the disease after treatment. · Objective response rate This represents the percentage of treated subjects in whom a complete response rate or partial response rate was observed. · Tumor control This refers to the proportion of treated patients who achieved a complete response, partial response, minor response, or stable condition for six months or more. · Progression-free survivalThis is defined as the time from the start of treatment to the first measurement of cancer growth. · Time to progression (TTP) and This refers to the period after treatment for a disease until the disease begins to worsen. The term "exacerbation" has already been defined. · 6-month progression-free survival rate Alternatively, the "PFS6" rate is related to the proportion of subjects who remained progression-free during the first six months after the initiation of treatment. · Median survival time This refers to the time during which half of the subjects participating in the study are still alive. · overall survival This refers to the period from the date of diagnosis or the start of treatment for a disease such as cancer until the patient is still alive. · recurrence In this specification, this refers to the appearance of cancer after a period of time has elapsed since treatment during which cancer was not detected. · Metastasis-free survival This refers to the period after cancer treatment is completed during which a patient survives without any signs or symptoms of cancer metastasis. · Reduction of circulating tumor cells This refers to a decrease in the concentration of circulating tumor cells in the blood or lymph fluid of patients with metastatic cancer. A decrease in circulating tumor cells is associated with the effectiveness of treatment for metastatic cancer. · Circulation marker response This refers to changes in the concentration of a specific cancer-related protein or nucleic acid in the blood or lymph after treatment for that particular cancer.

[0033] The method of the present invention is performed on subjects suffering from cancer, that is, subjects previously diagnosed with cancer.

[0034] As used herein, the terms “subject” or “patient” refer to all animals classified as mammals, including but not limited to livestock, farm animals, primates, and humans (e.g., humans, non-human primates, cattle, horses, pigs, sheep, goats, dogs, cats, or rodents). Subjects are preferably male or female, regardless of age or race. In the present invention, a subject is a subject who has cancer or has been diagnosed with cancer in the past. In a preferred embodiment, the subject is a mammal, preferably a human.

[0035] The subjects examined in the first method of the present invention have been previously diagnosed with cancer. As used herein, the term “diagnosis” refers to the determination and / or identification of a disease in a subject, i.e., an opinion regarding the condition of the subject, i.e., a diagnostic opinion. Thus, it can also be considered an attempt to classify individuals according to their condition. As those skilled in the art will understand, a diagnosis of cancer is preferably, but not necessarily, 100% correct in the subjects being diagnosed or evaluated. However, this term requires that a statistically significant proportion of the subjects identified as having cancer actually have cancer. A method for determining whether a portion of the subjects are statistically significant is disclosed above in relation to the prediction method.

[0036] The method of the present invention is suitable for various types of cancer.

[0037] The term "cancer" refers to a group of diseases characterized by uncontrolled cell division (or increased survival rate or resistance to apoptosis), the ability of cells to invade other neighboring tissues (invasion), and the ability of cells to spread to other parts of the body where they are not normally present via lymphatic vessels or blood vessels (metastasis). Tumors are classified as benign or malignant depending on whether they spread by invasion and metastasis. Benign tumors are tumors that cannot spread by invasion or metastasis, meaning they grow only locally. Malignant tumors, on the other hand, are tumors that can spread by invasion and metastasis. Biological processes known to be associated with cancer include angiogenesis, immune cell infiltration, cell migration, and metastasis.

[0038] The term "cancer" encompasses various types of cancer, including leukemia (acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, etc.), pilocytic cell leukemia, polycythemia vera, lymphoma (Hodgkin's disease or non-Hodgkin's disease, etc.), CNS lymphoma, AIDS-related leukemia, and Waldenström macroglobulin cancer. This includes conditions such as hematoma, multiple myeloma, heavy chain disease, solid tumors including sarcomas and carcinomas (such as fibrosarcoma), myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, meningiosarcoma, lymphangiosarcoma, intralymphatic sarcoma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, Kaposi's sarcoma, colorectal cancer, pancreatic cancer, lung cancer, colon cancer, bladder cancer, breast cancer, biliary tract cancer, esophageal cancer, gastric cancer, and conditions arising from epithelial cells, stromal cells, germ cells, and mesenchymal cells. Ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, oral cancer including papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial cancer, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, teratoma, choriocarcinoma, endometrial cancer / cervical cancer, seminomas, fetal 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, mixed glioma, optic nerve glial This includes, but is not limited to, tumors, subependymal tumors, metastatic brain tumors, pituitary tumors, primitive neuroectodermal tumors (PNETs), juvenile pilocytic astrocytoma (JPA), brainstem glioma astrocytoma, pineal tumors, rhabdoid tumors, glioblastoma pleomorphonum (GBM, also called glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, Schwann cell tumor, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0039] The term "cancer" includes a wide range of cancers, including head and neck cancer, leukemia, heart cancer, esophageal cancer, small intestine cancer, spleen cancer, kidney cancer, brain tumor, choriocarcinoma, skin cancer, bone cancer, bone marrow, blood, thymus, uterus, liver cancer, sarcoma, liposarcoma, fibrosarcoma, Merkel cell carcinoma, Kaposi's sarcoma, seminomas, non-seminomas (teratomas, choriocarcinomas), stromal tumors, and germ tumors such as germ cell tumors; testicular cancer including thyroid adenocarcinoma and medullary carcinoma; kidney cancer including adenocarcinoma and Wilms' tumor; bile duct cancer, glioblastoma, hematopoietic cancer including acute lymphoblastic leukemia and myeloid leukemia. Scars, T-cell acute lymphoblastic leukemia / lymphoma, pilocytic cell leukemia, chronic myeloid leukemia, multiple myeloma, AIDS-related leukemia and adult T-cell leukemia / lymphoma, neoplasms in situ including Bowen's disease and Paget's disease, lymphomas including Hodgkin's disease and lymphocytic lymphoma, oral cancer including squamous cell carcinoma, adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germ cell tumor, glioma, hemangioendothelioma, angiosarcoma, hematoma, hepatoblastoma, medulloblastoma, melanoma, parotid gland cancer, neuroblastoma, hepatobiliary tract cancer, adrenal cancer, osteosarcoma, retinoblastoma, rhabdomyosarcoma, teratoma. Furthermore, this term includes black melanoma, actinic keratosis adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytic cell tumor, Bartholin's adenocarcinoma, basal cell carcinoma, bronchial adenocarcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Ewing's sarcoma, focal nodular hyperplasia, germ cell tumor, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma, and insulinoma. This includes, but is not limited to, neoplasia in situ, squamous intraepithelial neoplasia, invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma, malignant melanoma, malignant mesothelioma, medulloblastoma, medullary epithelioma, 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, somatostatin-secreting tumors, squamous cell carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, VIPoma, Wilm's tumor, intracranial carcinoma, rectal cancer, astrocytoma, microcytic and non-microcytic carcinomas, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer.

[0040] In another embodiment, cancers include, but are not limited to, mesothelioma, hepatobiliary cancer (liver and bile duct), bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, melanoma of the skin or eyeball, ovarian cancer, colon cancer, rectal cancer, cancer of the anal region, gastric cancer, digestive system cancer (stomach, colorectal, duodenum), uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, This includes parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphoid lymphoma, bladder cancer, kidney cancer or ureteral cancer, renal cell carcinoma, renal pelvis cancer, non-Hodgkin lymphoma, spinal axial tumor, brainstem glioma, pituitary adenoma, adrenocortical carcinoma, gallbladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the aforementioned cancers.

[0041] In some embodiments, the cancer is selected from hepatocellular carcinoma, ovarian cancer, ovarian epithelial carcinoma, or fallopian tube cancer, papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC), prostate cancer, testicular cancer, gallbladder cancer, hepatobiliary cancer, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, undifferentiated 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 type 1-associated malignant peripheral nerve sheath tumor (MPNST), Waldenström macroglobulinemia, or medulloblastoma.

[0042] In some embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial carcinoma, fallopian tube cancer, papillary serous cystadenocarcinoma, papillary serous carcinoma of the uterus (UPSC), hepatobiliary carcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, undifferentiated thyroid cancer, adrenocortical adenoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis type 1-associated malignant peripheral nerve sheath tumor (MPNST), Waldenström macroglobulinemia, or medulloblastoma.

[0043] In a preferred embodiment, the cancer is a solid tumor.

[0044] Examples of solid tumors include sarcomas, carcinomas, or lymphomas. Solid tumors generally consist of a mass of abnormal tissue that typically does not contain cysts or fluid areas. In some embodiments, cancer is selected from renal cell carcinoma, or kidney cancer, hepatocellular carcinoma (HCC), or hepatoblastoma, or liver cancer, melanoma, breast cancer, colorectal cancer, 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 carcinoma, or fallopian tube cancer, papillary serous cystadenomatous carcinoma or uterine papillary serous carcinoma (UPSC), prostate cancer, testicular cancer, gallbladder cancer, hepatobiliary cancer, soft tissue and bone synovial sarcomas. Rhabdomyosarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, anaplastic thyroid carcinoma, adrenocortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma or pancreatic adenocarcinoma, gastrointestinal / stomach (GIST) cancer, lymphoma, head and neck squamous cell carcinoma (SCCHN), salivary gland carcinoma, glioma or brain tumor, neurofibromatosis type 1-associated malignant peripheral nerve sheath tumor (MPNST), Waldenström macroglobulinemia, or medulloblastoma. In one embodiment, the solid tumor is selected from the group consisting of non-small cell lung cancer (NSCLC), breast cancer, and colorectal cancer. In a particular embodiment of the present invention, the solid tumor is selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), salivary gland carcinoma, sarcoma, triple-negative breast cancer, and pleural mesothelioma. In certain embodiments of the present invention, the solid tumor is selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), salivary gland carcinoma, and sarcoma.

[0045] In some embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial carcinoma, ovarian cancer, fallopian tube cancer, papillary serous cystadenocarcinoma, papillary serous carcinoma of the uterus (UPSC), hepatobiliary carcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, undifferentiated thyroid cancer, adrenocortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis type 1-associated malignant peripheral nerve sheath tumor (MPNST), Waldenström macroglobulinemia, or medulloblastoma.

[0046] In some embodiments, the cancer is hepatocellular carcinoma (HCC). In some embodiments, the cancer is hepatoblastoma. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is rectal cancer. In some embodiments, the cancer is ovarian cancer or ovarian carcinoma. In some embodiments, the cancer is epithelial ovarian cancer. In some embodiments, the cancer is fallopian tube cancer. In some embodiments, the cancer is papillary serous cystadenoma. In some embodiments, the cancer is papillary serous carcinoma of the uterus (UPSC). In some embodiments, the cancer is hepatobiliary carcinoma. In some embodiments, the cancer is soft tissue and bone synovial sarcoma. In some embodiments, the cancer is rhabdomyosarcoma. In some embodiments, the cancer is osteosarcoma. In some embodiments, the cancer is undifferentiated thyroid cancer. In some embodiments, the cancer is adrenocortical carcinoma. In some embodiments, the cancer is pancreatic cancer or pancreatic ductal carcinoma. In some embodiments, the cancer is pancreatic adenocarcinoma. In some embodiments, the cancer is glioma. In some embodiments, the cancer is malignant peripheral nerve sheath tumor (MPNST). In some embodiments, the cancer is neurofibromatosis type 1-associated MPNST. In some embodiments, the cancer is Waldenström macroglobulinemia. In some embodiments, the cancer is medulloblastoma.

[0047] In some embodiments, cancers are virus-associated cancers including human immunodeficiency virus (HIV)-associated solid tumors, incurable solid tumors positive for human papillomavirus (HPV)-16, and adult T-cell leukemia, which is a highly aggressive form of CD4+ T-cell leukemia caused by human T-cell leukemia virus type 1 (HTLV-I) and characterized by clonal integration of HTLV-I in leukemia cells. Furthermore, virus-associated tumors in gastric cancer, nasopharyngeal cancer, cervical cancer, vaginal cancer, vulvar cancer, head and neck squamous cell carcinoma, and Merkel cell carcinoma are also included.

[0048] Other types of cancer are also well known to those skilled in the art.

[0049] The term cancer includes both primary and metastatic tumors. In one embodiment, cancer is a primary tumor. As used herein, the term “primary tumor” refers to a tumor that originates in the site or organ where cancer is present and has not metastasized from another site to that site. Thus, in one embodiment, the cancer being treated is a non-metastatic cancer. In another embodiment, cancer is a metastatic cancer or cancer metastasis. In this invention, “metastasis” means the spread of cancer from the organ in which it originated to another organ.

[0050] The anti-cancer treatment of the present invention can reduce cell proliferation regardless of whether the cancer exhibits increased expression or activity of the Myc protein. In one embodiment, the cancer to be prevented or treated is a Myc-induced cancer. In another embodiment, the cancer to be prevented or treated is not a Myc-induced cancer.

[0051] The terms “to treat” or “treatment” refer to therapeutic actions, as well as preventive or mitigating methods, whose purpose is to prevent or mitigate undesirable physiological changes or diseases, such as cancer. Beneficial or desirable clinical outcomes include, but are not limited to, symptom relief, shortening of disease duration, stabilization of the condition (specifically, no worsening), slowing of disease progression, improvement of the condition, and remission (partial and complete) (both detectable and undetectable). “Treatment” may also mean extending survival compared to the predicted survival without treatment. People who need treatment include cancer patients.

[0052] The inventors have discovered several biomarkers that are significantly reduced in subjects who respond well to treatment with OMO-103, a polypeptide containing Omomyc and having SEQ ID NO: 4. All Omomyc-based drugs act on the same target. Therefore, the biomarkers of the present invention are also significantly reduced in subjects who respond well to treatment with other Omomyc-based drugs. These drugs are administered as polypeptides, conjugates with other chemical sites, nucleic acids, gene therapy vectors, or, in cell therapy, as cells that secrete the polypeptide or conjugate into the culture medium.

[0053] Therefore, in the context of the present invention, the method of the present invention makes it possible to predict the clinical response to an anti-cancer treatment selected from the group consisting of the following: a) A polypeptide containing the sequence of sequence number 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or its functionally equivalent variant; c) Polynucleotides encoding the polypeptide of a) or the conjugate of b); d) A vector containing the polynucleotide described in c); and e) Cells that can secrete the polypeptide described in a) or the conjugate described in b) into a culture medium.

[0054] In the first method of the present invention, the term “anti-cancer therapy” refers to any therapy that involves exposing the subject to treatment to a method used to induce the death of cancer cells selected from any of the items listed in (a) to (e) above. Preferably, the anti-cancer therapy is administered intravenously, preferably as a 30-minute intravenous infusion. Preferably, the anti-cancer therapy is administered once a week.

[0055] In a preferred embodiment, the anticancer therapy comprises a polypeptide containing the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof, and more preferably a polypeptide containing the sequence of SEQ ID NO: 1.

[0056] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to polymers of amino acids of any length. Polypeptides of the present invention may contain modified amino acids, which may be interrupted by non-amino acids. In preferred embodiments, polypeptides are formed exclusively by amino acids. Preferably, polypeptides forming item (a) of anticancer therapy have a length of 80 to 500 amino acids, more preferably 80 to 300 amino acids, more preferably 80 to 250 amino acids, more preferably 80 to 150, even more preferably 80 to 130 amino acids, preferably 90 to 130 amino acids, preferably 125 amino acids or less, and more preferably 100 amino acids or less. In preferred embodiments, polypeptides have a length of 90 to 98 amino acids, preferably 90 to 95 amino acids, and more preferably 91 amino acids.

[0057] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Furthermore, the term "amino acid" includes both D- and L-amino acids (stereoisomers). Preferably, the amino acid is an L-amino acid.

[0058] The term “natural amino acids” or “naturally occurring amino acids” includes 20 naturally occurring amino acids; for example, hydroxyproline, phosphoserine, and phosphothreonine, which are often post-translationally modified in vivo; and other non-ordinary amino acids, including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine.

[0059] As used herein, the terms “non-natural amino acid” or “synthetic amino acid” refer to carboxylic acids or derivatives thereof that are structurally related to natural amino acids by being substituted with an amine group at the position of “a”. Non-exclusive examples of modified or rare amino acids include 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, ariohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyllysine, N-methylvaline, norvaline, norleucine, and ornithine.

[0060] The polypeptides of the present invention may also include, for example, hydrophobic moieties attached to the peptide (various linear, branched, cyclic, polycyclic, or heterocyclic hydrocarbons and hydrocarbon derivatives); and non-amino acid moieties such as various protecting groups attached to the ends of the compound 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.

[0061] Chemical (non-amino acid) groups present in polypeptides may be included to improve various physiological properties, such as reducing degradation or clearance; reducing repulsion by various cell pumps; improving various administration methods; increasing specificity; increasing affinity; increasing stability; and reducing bioavailability, solubility, and toxicity.

[0062] "Mimetics" include molecules that mimic the chemical structure of a peptide and retain the functional properties of a peptide. Approaches for designing peptide analogs, derivatives, and mimetics are known in the art.

[0063] In one embodiment, the polypeptide of the present invention is a polypeptide consisting of the sequence of SEQ ID NO: 1 or a polypeptide consisting of a functionally equivalent variant of SEQ ID NO: 1, and preferably a polypeptide consisting of the sequence of SEQ ID NO: 1.

[0064] Sequence ID 1 is, TEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA(Sequence ID 1) It corresponds to.

[0065] The polypeptide sequence of Sequence ID No. 1 corresponds to the Omomyc protein sequence. The term "Omomyc," as used herein, refers to the polypeptide consisting of mutant versions of the bHLHZip domain of Myc with E61T, E68I, R74Q, and R75N ​​mutations (where the mutation site numbering is given with respect to the sequence of the Myc region corresponding to amino acids 365-454 of the polypeptide defined in the NCBI database, accession number NP_002458, released March 15, 2015). The sequence of c-Myc provided to the NCBI database with accession number NP_002458 is shown below (Sequence ID No. 2), where the region from which Omomyc originates is underlined. [ka]

[0066] Omomyc also contains the M2 domain of c-Myc with the sequence RQRRNELKRSF (sequence number 3) (see Dang and Lee, Mol. Cell. Biol., 1988, 8:4048-4054) (double underlined above), which corresponds to a nuclear localization signal.

[0067] Omomyc is characterized by its increased dimerization ability with all three oncogene Myc proteins (c-Myc, N-Myc, and L-Myc). Omomyc may be derived from the bHLHZip domain of any Myc protein known in the art, provided that the mutations that result in tumor suppressor effects are conserved. Therefore, Omomyc that can be used in the present invention may be derived from any mammalian species, including but not limited to domesticated animals and livestock (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 March 12, 2019).

[0068] 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 through 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 replacing p300 coactivators, it inhibits the expression of Miz-1 target genes. Myc also plays a direct role in the regulation of DNA replication.

[0069] The Myc b-HLH-LZ or helix-loop-helix leucine zipper domain in the Myc basic region refers to the region that determines Myc dimerization with the Max protein and binding to Myc target genes. This region corresponds to amino acids 365-454 of human Myc and is characterized by two alpha helices connected by a loop (Nair, SK and Burley, SK, 2003, Cell, 112:193-205).

[0070] In a preferred embodiment, the polypeptide of the present invention is a polypeptide comprising, consisting of, or essentially comprising SEQ ID NO: 4 shown below. MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCA(Sequence ID 4)

[0071] In this context, "essentially derived from" means that the defined molecule does not contain any additional sequences that would alter the activity of SEQ ID NO: 4.

[0072] Preferably, the polypeptide consists of Sequence ID No. 4.

[0073] In a preferred embodiment, the anticancer treatment includes the use of the polypeptide comprising SEQ ID NO: 4.

[0074] The term "functionally equivalent variant" refers to any polypeptide obtained from 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 with respect to the polypeptide of SEQ ID NO: 1, and / or from the chemical modification of the polypeptide of SEQ ID NO: 1, and which substantially preserves the tumor suppressor activity of SEQ ID NO: 1. Preferably, a functionally equivalent variant refers to any polypeptide obtained from 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 that point to the polypeptide of SEQ ID NO: 1, and which substantially preserves the tumor suppressor activity of SEQ ID NO: 1; more preferably, any polypeptide obtained from the insertion or addition of one or more amino acids with respect to the polypeptide of SEQ ID NO: 1.

[0075] Those skilled in the art will understand that preservation of tumor suppressor activity requires that the variants dimerize with Myc and / or its obligate partner p21 / p22Max, inhibit Myc activity, migrate across the cell membrane, and migrate across the nuclear envelope. In some embodiments, functionally equivalent variants of the polypeptide of the present invention are less homodimerized than Omomyc, or are not forced into homodimerization by disulfide crosslinking. In particular, disulfide crosslinking in the homodimerized form of certain embodiments of the polypeptide of the present invention is less than in polypeptide Omomyc.

[0076] When used herein, "low homodimerization" refers to a lower ability of the polypeptide of the present invention to form obligate homodimers, even under reducing conditions. In preferred embodiments, the 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%, and at least 95% lower than the ability of Omomyc to form homodimers.

[0077] Reducing conditions, as used herein, refer to the presence of a reducing agent, which is a compound that donates electrons to another chemical species in a redox reaction. Non-limiting examples of reducing agents include DTT (dithiothreitol), β-mercaptoethanol, or TCEP (tris(2-carboxyethyl)phosphine). The amount of homodimers being the same in vitro, and the differences between functionally equivalent variants and Omomyc, may only exist in cells in the presence of a heterodimerizing partner, where the absence of disulfide allows for potentially higher heterodimer formation.

[0078] As a non-limiting example of thermal denaturation monitored by circular dichroism, several assays may be used to determine the homodimerization of peptides, which may be detected by quantification of folding and thermal stability.

[0079] Preferred functionally equivalent variants include polypeptides that are essentially derived from the polypeptide of SEQ ID NO: 1. In this context, "essentially derived from" means that the defined molecule does not contain any additional sequences that would alter the activity of SEQ ID NO: 1.

[0080] In a preferred embodiment, a functionally equivalent variant of SEQ ID NO: 1 is a polypeptide obtained by the insertion or addition of one or more amino acids with respect to the polypeptide of SEQ ID NO: 1. In a particular embodiment, a functionally equivalent variant is obtained by the insertion of fewer than 10 amino acids, more preferably fewer than 5 amino acids, and more preferably by the insertion of one amino acid. In a preferred embodiment, it is obtained by the insertion of one amino acid, which is methionine.

[0081] In another embodiment, a functionally equivalent variant of SEQ ID NO: 1 is a polypeptide obtained from the deletion of one or more amino acids with respect to the polypeptide of SEQ ID NO: 1. In one embodiment, a functionally equivalent variant is obtained from the deletion of fewer than 10 amino acids, more preferably fewer than 5 amino acids, and more preferably from the deletion of one amino acid.

[0082] Preferred functional variants of the targeted peptide exhibit amino acid sequence identity higher than approximately 25% with respect to the peptide of SEQ ID NO: 1, for example, 25%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of identity between two polypeptides is determined using computer algorithms and methods widely known to those skilled in the art. The identity between two amino acid sequences is preferably determined using the previously described BLASTP algorithm (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 by the entire length of the polypeptide of SEQ ID NO: 1, the entire length of the variant, or both.

[0083] Functionally equivalent variants of the polypeptide of the present invention may also include post-translational modifications, such as glycosylation, acetylation, isoprenylation, myristoylation, and proteolytic processing.

[0084] In another embodiment, preferred functional variants of the targeted peptide contain amino acids in which one or more positions within the polypeptide of the present invention are conserved substitutions of amino acids present in the proteins listed above. A “conservative amino acid substitution” is obtained by replacing one amino acid with another having similar structure and / or chemical properties. For example, the following six groups each contain amino acids that are conserved substitutions with respect to 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 those skilled in the art and has been 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).

[0085] In a preferred embodiment, it will be understood that a functionally 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 locations where the mutations should occur in the functionally equivalent variant can be determined by multiple sequence alignment of different Myc sequences, and can be identified by alignment of positions 61, 68, 74, and 75 in the sequence of Omomyc derived from human c-Myc. In one embodiment, a functionally 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.

[0086] In another embodiment, a functionally equivalent variant of Omomyc contains mutations at positions corresponding to E61, E68, R74, and R75 in the sequence of Omomyc, where E61 is mutated to E61A or E61S, E68 is mutated to E68L, E68M, or E68V, R74 is mutated to R74N, and R75 is mutated to R75Q.

[0087] Multiple sequence alignment is an extension of pairwise alignment, which incorporates more than two sequences at a time. The multiple alignment method aligns all sequences in a given queryset. Preferred multiple sequence alignment programs (and their algorithms) are ClustalW, Clustal2W, or ClustalW XXL (see Thompson et al. (1994) Nucleic Acids Res 22:4673-4680). When c-Myc and variant sequences from different organisms are compared (aligned) as described herein, those skilled in the art can easily identify the positions within the sequences corresponding to the E61T, E68I, R74Q, and R75N ​​positions found in Omomyc and introduce them into Omomyc variant mutations corresponding to the E61T, E68I, R74Q, and R75N ​​mutations found in Omomyc derived from human c-Myc.

[0088] Suitable assays for determining whether a polypeptide can be considered a functionally equivalent variant of Omomyc include, but are not limited to, the following: - Assays that measure the ability of polypeptides to form dimeric complexes with Max and Myc, e.g., assays based on reporter gene expression as described by Soucek et al. (Oncogene, 1998, 17:2463-2472), as well as PLA (protein ligation assay) or co-immunoprecipitation. - Assays that measure the ability of polypeptides to bind to Myc / Max recognition sites (CACGTG sites) in DNA, such as the electrophoretic mobility shift assay (EMSA) described by Soucek et al. (see above). - Assays that measure the ability to respond to Myc-induced transactivation, such as the assay described by Soucek et al. (see above) that is based on the expression of a reporter gene under the control of a Myc / Max-specific DNA binding site. - An assay based on the ability of polypeptides to inhibit the growth of cells expressing the myc oncogene, as described by Soucek et al. (see above). - Assays that measure the ability of polypeptides to enhance myc-induced apoptosis, such as the assay described by Soucek et al. (Oncogene, 1998:17, 2463-2472). Alternatively, any assay commonly known in the art for evaluating apoptosis in cells, such as Hoechst staining, propidium iodide (PI) or annexin V staining, trypan blue, DNA laddering / fragmentation, and TUNEL may be used.

[0089] In a preferred embodiment, a polypeptide is considered a functionally equivalent variant of Omomyc if it exhibits activity that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of that of native Omomyc in one or more of the above assays.

[0090] In a specific embodiment, a functionally equivalent variant of the polypeptide of SEQ ID NO: 1 comprises the polypeptide of SEQ ID NO: 1, where 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 sulfurized amino acid, or a dicarboxyl amino acid or an amide thereof, 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, it is an amino acid selected from serine, threonine, and alanine, preferably selected from serine and alanine.

[0091] Preferred functionally equivalent variants of SEQ ID NO: 1 having a non-cysteine ​​residue X at position 89 of SEQ ID NO: 1 are disclosed in the following table.

[0092] [Table 1]

[0093] Therefore, in a preferred embodiment, a functionally equivalent variant of the polypeptide of SEQ ID NO: 1 is selected from the group consisting of 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 functionally equivalent variant is SEQ ID NO: 4.

[0094] In addition, functionally equivalent variants of Omomyc can also be transduced into cells after the variant has come into contact with the cells. It will be understood that functionally equivalent variants of Omomyc contain a protein transduction domain found in native Omomyc or another functional protein transduction domain.

[0095] In a preferred embodiment, a polypeptide is considered a functionally equivalent variant of SEQ ID NO: 1 if it can transduce target cells with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the efficiency of SEQ ID NO: 1.

[0096] In addition, functionally equivalent variants of Sequence ID No. 1 can also translocate to the nucleus of target tumor cells.

[0097] In a preferred embodiment, a polypeptide is considered a functionally equivalent variant of SEQ ID NO: 1 if it can move to the nucleus of a target tumor cell with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the efficiency of SEQ ID NO: 1.

[0098] A preferred assay for determining whether a polypeptide is a functionally equivalent variant of Sequence ID No. 1 with respect to its ability to traverse the cell membrane and move to the nucleus involves dual labeling of the cell with a polypeptide-specific reagent and a dye that specifically labels the cell nucleus (such as DAPI or Hoechst dye). Detection of the polypeptide of the present invention can be performed by confocal microscopy or fluorescence microscopy.

[0099] In another preferred embodiment, the anticancer therapy is a conjugate comprising a polypeptide containing the sequence of 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.

[0100] As used herein, the term "conjugate" refers to two or more compounds that are covalently linked together such that the functions of each compound are preserved in the conjugate.

[0101] 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 enriched in hydrophobic amino acids and hydrophobic chemical moieties.

[0102] In a preferred embodiment, the conjugate described in the present 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 cellular uptake of the polypeptide or a functionally equivalent variant of the polypeptide.

[0103] In one embodiment, the chemical moiety that promotes the uptake of polypeptides into cells is a lipid or a fatty acid.

[0104] Fatty acids are generally molecules that contain a carbon chain having an acidic portion (e.g., a carboxylic acid) at the end of the chain. The carbon chain of a fatty acid may be of any length, however, it is preferable that the carbon chain length 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 derived therefrom. In certain embodiments, the carbon chain length is 4 to 18 carbon atoms in the chain portion of the fatty acid. In certain embodiments, the carbon chain of a fatty acid may contain an odd number of carbon atoms, however, in certain embodiments, an even number of carbon atoms in the chain may be preferred. Fatty acids that contain only single bonds in their carbon chain are called saturated, while fatty acids that contain at least one double bond in their chain are called unsaturated. Fatty acids may be branched, but in preferred embodiments of the present invention, they are 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.

[0105] In a preferred embodiment, the chemical moiety that facilitates cellular uptake of the polypeptide containing the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof is a cell-permeable peptide sequence, in which case the conjugate would comprise a fusion protein comprising the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof and the cell-permeable peptide sequence.

[0106] The term "fusion protein" refers to a protein created by genetic technology, consisting of two or more functional domains derived from different proteins. Fusion proteins may also be obtained by conventional means, for example, by gene expression of the nucleotide sequence encoding the fusion protein in a suitable cell. It will be understood that the cell-permeable peptide refers to a cell-permeable peptide that is different from the cell-permeable peptide that forms part of the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant of SEQ ID NO: 1.

[0107] The term “cell-permeable peptide sequence” is used herein interchangeably with “CPP,” “protein transduction domain,” or “PTD.” This refers to a peptide chain of variable length that directs the transport of proteins within a cell. The process of delivery to the cell generally occurs by endocytosis, although peptides can also be translocated into the cell by direct membrane translocation. CPPs typically have an amino acid composition containing high relative abundances of positively charged amino acids, such as lysine or arginine, or sequences containing an alternating pattern of polar / charged amino acids and nonpolar hydrophobic amino acids.

[0108] 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), and HIV-1 consisting of amino acids 49-57 (RKKRRQRRR, SEQ ID NO: 16), amino acids 48-60 (GRKKRRQRRRTPQ, SEQ ID NO: 17), and amino acids 47-57 (YGRKKRRQRRR, SEQ ID NO: 18). CPP of TAT protein; CPP of S413-PV peptide (ALWKTLLKKVLKAPKKKRKV; SEQ ID NO: 19), CPP of penetratin (RQIKWFQNRRMKWKK; SEQ ID NO: 20), CPP of SynB1 (RGGRLSYSRRRFSTSTGR; SEQ ID NO: 21), CPP of SynB3 (RRLSYSRRRF; SEQ ID NO: 22), CPP of PTD-4 (PIRRRKKLRRLK; SEQ ID NO: 23), CPP of PTD-5 (RRQRRTSKLMKR; SEQ ID NO: 24), CPP of FHV coat-(35~49) (RRRRNRTRRNRRRVR; SEQ ID NO: 25), CPP of BMV Gag-(7~25) (KMTRAQRRAAARRNRWTAR; SEQ ID NO: 26), HTLV-II CPP of Rex-(4~16) (TRRQRTRRARRNR; SEQ ID NO: 27), CPP of D-Tat (GRKKRRQRRRPPQ; SEQ ID NO: 28), CPP of R9-Tat (GRRRRRRRRRPPQ; SEQ ID NO: 29), CPP of MAP (KLALKLALKLALALKLA; SEQ ID NO: 30), CPP of SBP (MGLGLHLLVLAAALQGAWSQPKKKRKV; SEQ ID NO: 31), CPP of FBP (GALFLGWLGAAGSTMGAWSQPKKKRKV; SEQ ID NO: 32), CPP of MPG (ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cya; SEQ ID NO: 33), CPP of MPG(ENLS) (ac-GALFLGFLGAAGSTMGAWSQPKSKRKV-cya;SEQ ID NO: 34), Pep-1 CPP (ac-KETWWETWWTEWSQPKKKRKV-cya; SEQ ID NO: 35), Pep-2 CPP (ac-KETWFETWFTEWSQPKKKRKV-cya; SEQ ID NO: 36), polyarginine sequences with structural RN (where N is 4-17), GRKKRRQRRR sequence (SEQ ID NO: 37), RRRRRRLR sequence (SEQ ID NO: 38), RQRRTS Examples include the KLMKR sequence (sequence number 39); transportan GWTLNSAGYLLGKINLKALAALAKKIL (sequence number 40); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (sequence number 41); RQIKIWFQNRRMKWKK (sequence number 42); YGRKKRRQRRR sequence (sequence number 43); RKKRRQRR sequence (sequence number 44); YARAAARQARA sequence (sequence number 45); THRLPRRRRRR sequence (sequence number 46); and GGRRARRRRRR sequence (sequence number 47).

[0109] In a preferred embodiment, the cell-permeable peptide is not intrinsically contained in SEQ ID NO: 1.

[0110] In a preferred embodiment, the CPP is the CPP of the HIV-1 TAT protein consisting 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 yet another embodiment, the CPP is the GRKKRRQRRR sequence (SEQ ID NO: 37) or RRRRRRRR (SEQ ID NO: 65).

[0111] In some embodiments, the CPP is as described in WO2019 / 018898, the contents of which are incorporated herein by reference in their entirety.

[0112] In one embodiment, the cell-permeable peptide sequence is fused at the N-terminus of the polypeptide of the present invention or a functionally equivalent variant of the polypeptide. In another embodiment, the cell-permeable peptide is fused at the C-terminus of the polypeptide of the present invention or a functionally equivalent variant of the polypeptide.

[0113] In a preferred embodiment, the conjugate or fusion protein of the combination described in the present invention comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more additional cell-permeable peptides, in addition to the cell-permeable peptide itself found in the polypeptide of SEQ ID NO: 1 or a functionally equivalent variant of said polypeptide.

[0114] A preferred fusion protein of the present invention is the polypeptide Omomyc as defined below. * TAT and Omomyc * Includes LZArg.

[0115] [Table 2]

[0116] Therefore, in a preferred embodiment, the fusion protein is a polypeptide selected from SEQ ID NOs: 11 and 12.

[0117] A suitable assay for determining whether the conjugate preserves Omomyc's cell membrane mobility is, but is not limited to, an assay that measures the conjugate's ability to transduce cells in culture. This assay is based on bringing the conjugate into contact with cultured cells and detecting the presence of the conjugate at its location within the cells.

[0118] In another preferred embodiment, the conjugate of the combination of the present invention further includes an additional nuclear localization signal.

[0119] The term “nuclear localization signal” (NLS), as used herein, refers to an amino acid sequence of approximately 4 to 20 amino acid residues that plays a role in orienting a protein toward the nucleus. Typically, nuclear localization sequences are rich in basic amino acids, and exemplary sequences are well known in the art (Gorlich D. (1998) EMBO 5.17:2721-7). In some embodiments, the NLS is selected from the group consisting of 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); hnRNP A NLS (NQSSNFGPMKGGNFGGRSSGPYGGGGQYFKPRNQGGY, SEQ ID NO: 53); and rpL23a NLS (VHSHKKKKIRTSPTFTTPKTLRLRRQPKYPRKSAPRRNKLDHY, SEQ ID NO: 54). In one embodiment of the present invention, the nuclear localization signal includes the motif K(K / R)X(K / R).

[0120] In a more preferred embodiment, the nuclear localization signal is selected from the group consisting of PKKKRKV (SEQ ID NO: 48), PAAKRVKLD (SEQ ID NO: 56), and KRPAATKKAGQ AKKKK (SEQ ID NO: 49).

[0121] In another preferred embodiment, the NLS may be at the N-terminus or C-terminus of a conjugate or fusion protein comprising the polypeptide of SEQ ID NO: 1 or a functionally equivalent variant thereof.

[0122] Those skilled in the art will understand that the conjugate of the present invention may preferably further comprise a polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof, a cell-permeable peptide sequence, and / or one or more flexible peptides connecting the NLS. Therefore, in a specific embodiment, the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof is directly conjugated to the cell-permeable peptide sequence. In another specific embodiment, the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof is conjugated to the cell-permeable peptide sequence via a flexible peptide. In one embodiment, the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof is directly conjugated to the NLS. In another embodiment, the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant thereof is conjugated to the NLS via a flexible peptide.

[0123] In specific embodiments, the polypeptide of the conjugate described in the present invention is directly ligated to a cell-permeable peptide sequence and NLS.

[0124] In one embodiment, the NLS is one of the NLSs endogenously found in the Myc sequence, for example, the M1 peptide (PAAKRVKLD, SEQ ID NO: 56) or the M2 peptide (RQRRNELKRSF, SEQ ID NO: 57).

[0125] In another embodiment, the additional NLS refers to an NLS distinct from the endogenous NLS found in the polypeptide containing SEQ ID NO: 1 or a functionally equivalent variant of SEQ ID NO: 1.

[0126] In a preferred embodiment, the conjugate or fusion protein described in the present 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, and at least 10 NLS in addition to the endogenous NLS found in the polypeptide of the present invention or a functionally equivalent variant thereof.

[0127] In another specific embodiment, the polypeptide of the conjugate for use described in the present invention is linked to a cell-permeable peptide sequence through a first flexible peptide linker and to the NLS through a second flexible peptide linker.

[0128] As used herein, the terms “flexible peptide,” “spacer peptide,” or “linker peptide” refer to peptides that covalently bond to two proteins or parts but are not part of either polypeptide, and that allow one to move relative to the other without causing substantially adverse effects on the function of either protein or part. Thus, flexible linkers do not affect the tumor trace activity of polypeptide sequences, the cell permeability activity of cell-permeable peptides, or the nuclear localization ability of NLS.

[0129] Flexible peptides contain at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, at least ten amino acids, at least twelve amino acids, at least fourteen amino acids, at least sixteen amino acids, at least eighteen 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 some embodiments, the flexible peptide will allow the movement of one protein relative to another protein in order to increase the solubility of the protein and / or improve its activity. A preferred linker region is a polyglycine region, which is a GPRRRR sequence (SEQ ID NO: 58) of a combination of glycine, proline, and alanine residues.

[0130] In specific embodiments, the conjugate described in the present invention comprises a tag attached to the conjugate or C-terminal or N-terminal domain of the polypeptide or its fusion protein or variant. The tag is generally a peptide or amino acid sequence that can be used in the isolation or purification of the fusion protein. Therefore, the tag can bind to one or more ligands, for example, one or more ligands of an affinity matrix such as a chromatography support or beads having high affinity. An example of the tag is a histidine tag (His tag or HT), for example, a tag containing six histidine residues (His6 or H6), which has high affinity to nickel (Ni 2+ ) or cobalt (Co 2+ The His tag can be bound to a column. The His tag has the desirable property of being able to bind to its ligand under conditions that denature most proteins and disrupt most protein-protein interactions. Therefore, it can be used to remove H6-tagged bait proteins after disruption of bait-involved protein-protein interactions.

[0131] Non-limiting examples of tags useful for isolating or purifying conjugates, polypeptides containing SEQ ID NO: 1, their variants, or fusion proteins include: Arg-tag, FLAG-tag (DYKDDDDK; SEQ ID NO: 59), Strep-tag (WSHPQFEK, SEQ ID NO: 60), antibody-recognizable epitopes 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 Examples include amino acid sequences (K., Appl. Microbiol. Biotechnol. 2003, 60:523-525), such as AHGHRP (SEQ ID NO: 63) or PIHDHDHPHLVIHSGMTCXXC (SEQ ID NO: 64), and β-galactosidase.

[0132] The tag may be used, if desired, for the isolation or purification of the fusion protein.

[0133] In another preferred embodiment, the anticancer therapy is a polynucleotide encoding a polypeptide or fusion protein disclosed above. In a preferred embodiment, the anticancer therapy is a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof. In another embodiment, the anticancer therapy is a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that facilitates cellular uptake of the polypeptide or its functionally equivalent variant, and more preferably a polynucleotide encoding a fusion protein between a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof and a cell-permeable peptide sequence.

[0134] The terms “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” are used interchangeably to refer to polymeric forms of nucleotides of any length. Polynucleotides may contain deoxyribonucleotides, ribonucleotides, and / or analogues thereof. Nucleotides may have any three-dimensional structure and may perform any known or unknown function. The term “polynucleotide” includes, for example, single-stranded, double-stranded, and triple-helix 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 native 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.

[0135] In a preferred embodiment, the polynucleotide of the present invention is mRNA.

[0136] mRNA can be chemically synthesized, obtained by in vitro transcription, or synthesized in vivo in target cells. The nucleotide sequences forming the polynucleotides encoding the conjugate or fusion protein of the present invention are in the same correct reading frame for their expression.

[0137] In a preferred embodiment, the anticancer treatment is an mRNA encoding a polypeptide consisting of the sequence of SEQ ID NO: 1, a polypeptide consisting of a functionally equivalent variant of SEQ ID NO: 1, or a polypeptide consisting of SEQ ID NO: 4.

[0138] In another embodiment, the anti-cancer treatment is a vector containing the polynucleotide of the present invention.

[0139] The term “vector,” as used herein, refers to a nucleic acid sequence containing the required sequence such that, after transcription and translation in a cell, the polypeptide encoded by the polynucleotide of the present invention is produced. The sequence is operably ligated to an additional segment that provides its self-replication in the host cell of interest. Preferably, the vector is an expression vector, which is defined as a vector that, in addition to the self-replication region in the host cell, contains a region operably ligated to the nucleic acid of the present invention and can enhance the expression of the nucleic acid product described in the present invention. The vector of the present invention can be obtained by techniques widely known in the art.

[0140] Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensers, liposome-encapsulated DNA or RNA expression vectors, and certain eukaryotic cells, such as producing cells. Suitable vectors containing polynucleotides of the present invention include expression vectors in prokaryotes, e.g., pUC18, pUC19, pBluescript and their derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCRl, RP4, phage and "shuttle" vectors, e.g., pSA3 and pAT28; expression vectors in yeast, e.g., 2-micron plasmid-type vectors, embedded plasmids, YEP vectors, centromere plasmids and analogues; expression vectors in insect cells, e.g., pAC series and pVL series vectors; expression vectors in plants, e.g., series pIBI vectors, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE and analogues; and expression vectors in higher eukaryotic cells based on viral vectors (adenoviruses, adenovirus-related viruses, and retroviruses, and especially lentiviruses) and non-viral vectors, e.g., pSilencer 4.1-CMV(Ambion), pcDNA3, pcDNA3.1 / hyg, pHCMV / Zeo, pCR3.1, pEFl / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAXl, pZeoSV2, pCI, pSVL, pKSV-10, pBPV-1, pML2d, and pTDT1 are vectors derived from these. In preferred embodiments, the polynucleotides of the present invention are contained in vectors selected from the group consisting of pEGFP or pBabe retroviral vectors and pTRIPZ or pSLIK lentiviral vectors.

[0141] The vectors of the present invention may be used to transform, transfect, or infect cells that can be transformed, transfected, or infected by the vectors. The cells may be prokaryotic or eukaryotic.

[0142] The vector preferably comprises the polynucleotide of the present invention operationally bound to a sequence that modulates the expression of the polynucleotide of the present invention. The regulatory sequence used in the present invention may be a nuclear promoter, or alternatively, other regulatory sequences that increase the expression of enhancer sequences and / or heterologous nucleic acid sequences. In principle, any promoter can be used in the present invention, provided that the promoter is compatible with the cell in which the polynucleotide is expressed. Therefore, suitable promoters for realizing the present invention are not necessarily limited to, but include constitutive promoters, such as derivatives of eukaryotic viral genomes, such as polyomavirus, adenovirus, SV40, CMV, aerosarcoma virus, hepatitis B virus, metallothionein gene promoter, herpes simplex virus thymidine kinase gene promoter, retroviral LTR region, immunoglobulin gene promoter, actin gene promoter, EF-1 alpha gene promoter, as well as inductive promoters, such as tetracycline systems, NFκB / UV light systems, Cre / Lox systems, and heat shock gene promoters, the moduloable RNA polymerase II promoter described in WO / 2006 / 135436, and tissue-specific promoters.

[0143] In another embodiment, the anticancer treatment is a cell capable of secreting the polypeptide or conjugate of the present invention, preferably the polypeptide or fusion protein of the present invention, into a culture medium.

[0144] 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, vascular intima cells, isolated cells from different organs, preferably islets of Langerhans, hepatocytes, leukocytes, e.g., mononuclear leukocytes, mesenchymal cells, primary cultures of cells isolated from the umbilical cord or from adults (skin, lung, kidney and liver), osteoclasts, chondrocytes and other connective tissue cells. Cells from established strains, e.g., 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 recognize that cells capable of secreting the polypeptides of the present invention into culture media may be found to form microparticles or microcapsules, so that the cells have a longer useful life in patients. Suitable materials for forming the microparticles of the present invention include any biocompatible polymer material that allows for the continuous secretion of therapeutic products and acts as a support for cells. Therefore, the biocompatible polymer material may be, for example, a thermoplastic polymer or a hydrogen polymer.Among thermoplastic polymers, acrylic acid, acrylamide, 2-aminoethyl methacrylate, poly(tetrafluoroethylene-cohexafluoropropylene), methacrylate-(7-coumaroxy)ethyl ester acid, N-isopropylacrylamide, polyacrylic acid, polyacrylamide, polyamidoamine, poly(amino)-p-xylene, poly(chloroethyl vinyl ether), polycaprolactone, poly(caprolactone-co-trimethylene carbonate), poly(carbonate urea)urethane, poly(carbonate)urethane, polyethylene, polyethylene and acrylamide copolymer, polyethylene glycol, polyethylene glycol methacrylate There are copolymers containing poly(ethylene terephthalate), poly(4-hydroxybutyl acrylate), poly(hydroxyethyl methacrylate), poly(N-2-hydroxypropyl methacrylate), poly(glycolic acid lactate), poly(L-lactic acid), poly(gamma-methyl,L-glutamate), poly(methyl methacrylate), poly(propylene fumarate), poly(propylene oxide), polypyrrole, polystyrene, poly(tetrafluoroethylene), polyurethane, polyvinyl alcohol, ultra-high molecular weight polyethylene, 6-(p-vinylbenzamide)-hexanoate Np-vinylbenzyl-D-maltonamide, and two or more of the above polymers. Among the hydrogel-type polymers are alginates, agarose, collagen, starch, hyaluronic acid, bovine serum albumin, cellulose and their derivatives, pectin, chondroitin sulfate, fibrin and fibroin, and synthetic hydrogels, such as natural materials like Sepharose® and Sephadex®.

[0145] The method for predicting clinical response according to the present invention is performed on a pre-treatment sample, i.e., a patient sample obtained before the patient receives the treatment for which a response is predicted.

[0146] With regard to treatments received by a subject before the isolation of a sample in any aspect of the present invention, it is preferable that the subject has not received any anti-cancer treatment at any point in time, preferably within the past 168 hours, more preferably within the past 72 hours, even more preferably within the past 48 hours, even more preferably within the past 36 hours, even more preferably within the past 24 hours, and even more preferably within 24 hours prior to the isolation of the sample. In another preferred embodiment, the subject has not received anti-cancer treatment in the 24 hours prior to the isolation of the sample, but preferably within the past 23 hours, preferably within the past 22 hours, even more preferably within the past 21 hours, even more preferably within the past 20 hours, even more preferably within the past 15 hours, even more preferably within the past 10 hours, even more preferably within the past 5 hours, even more preferably within the past 2 hours, and even more preferably within the past 1 hour prior to the isolation of the sample. In a further preferred embodiment, the subject from which the sample is isolated has not received any anti-cancer treatment at any point in time prior to the isolation of the sample; that is, the subject has never received anti-cancer treatment.

[0147] In a preferred embodiment, the subjects have not received anti-cancer treatment within 24 hours prior to sample isolation.

[0148] In another specific embodiment, the subject from which the sample is isolated has received anticancer treatment at any point prior to the isolation of the sample, preferably within the past 168 hours, more preferably within the past 72 hours, even more preferably within the past 48 hours, even more preferably within the past 36 hours, and even more preferably within the past 24 hours prior to the isolation of the sample. If administered within 24 hours prior to the isolation of the sample, preferably within the past 24 hours prior to the isolation of the sample, the anticancer treatment is different from an anticancer treatment selected from the group consisting of items (a) to (e) of the first embodiment of the present invention.

[0149] In a preferred embodiment, the subject from which the sample was isolated had received anticancer treatment prior to the isolation of the sample, and said anticancer treatment was different from a treatment selected from the group consisting of items (a) to (e) of the first aspect of the present invention. Preferably, said anticancer treatment was selected from targeted therapies including surgery, chemotherapy, radiotherapy, hormone therapy, and immunotherapy, and combinations thereof. In a preferred embodiment, said anticancer treatment was selected from the group consisting of surgery and radiotherapy. In a more preferred embodiment, said anticancer treatment was radiotherapy.

[0150] In a preferred embodiment, the subject from which the sample is isolated has not received systemic anti-cancer treatment within 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, or at least 50 weeks prior to the isolation of the sample. In a more preferred embodiment, the subject has not received systemic anti-cancer treatment within at least 4 weeks prior to the isolation of the sample.

[0151] The timing of evaluating the response after the start of treatment is not particularly limited. Therefore, 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 cycles of treatment. 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, or longer. In certain embodiments, the response to treatment can be evaluated when the subject has received at least two, preferably at least three, anti-cancer treatments.

[0152] Furthermore, there are no particular limitations on the types and number of treatments applied to the patient before the treatment effect is assessed. Therefore, the treatment effect can be assessed after the patient has received first-line treatment, including surgery, chemotherapy, or a combination of surgery and adjuvant or neoadjuvant chemotherapy, or any type of treatment, or after the patient has received two or more treatments consecutively or simultaneously.

[0153] Accordingly, the first step of the first method of the present invention includes 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 a subject suffering from the target cancer for which a response to treatment is to be predicted.

[0154] 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 collected from a subject and contains any biological material suitable for detection at RNA or protein levels. In certain embodiments, a sample may include genetic material derived from the subject under study, such as DNA, genomic DNA (gDNA), complementary DNA (cDNA), RNA, heteronuclear RNA (hnRNA), mRNA, etc. In other specific embodiments, a sample may include proteins. A sample may include cellular and / or non-cellular material of the subject. The method can be applied to any type of biological sample from a subject, such as biopsy samples, tissues, cells, or bodily fluids (blood, plasma, serum, saliva, urine, semen, sputum, cerebrospinal fluid (CSF), tears, mucus, sweat, milk), feces, brain extracts, bone marrow, nipple aspirate, bronchial lavage, bronchoscopy, fine-needle aspiration biopsy (FNAB), solid tumor biopsy samples, buccal mucosa, or buccal pharyngeal swabs. These samples can be collected using conventional methods such as biopsy, surgical excision, or aspiration, and methods well known to those skilled in the art of the relevant medical field. Methods for collecting samples from biopsies include coarse division of the tumor, microanatomy, or other known cell separation methods. Tumor cells can also be collected by fine-needle aspiration cytology. In a preferred embodiment, samples are collected by fine-needle aspiration biopsy (FNAB). To simplify sample storage and handling, samples can be fixed in formalin and embedded in paraffin, or frozen and then immersed in a cryogenic medium that allows for rapid freezing and embedded in a cryo-solidifying medium such as OCT-Compound. Samples may be cell suspensions, cell pellets, cell slides, or frozen solid tumor biopsies.

[0155] In certain embodiments, the sample is a tumor tissue sample, preferably a biopsy of a fresh or frozen tumor tissue sample.

[0156] In certain embodiments, the sample comprises cancer cells, preferably breast cancer, ovarian cancer, prostate cancer, gastric cancer, pancreatic cancer, lung cancer, colorectal cancer, endometrial cancer, cervical cancer, bladder cancer, head and neck cancer, leukemia, sarcoma, cholangiocarcinoma, glioblastoma, multiple myeloma, and lymphoma cells. In preferred embodiments, the sample comprises cells selected from the group consisting of pancreatic cancer cells, lung cancer cells, colorectal cancer cells, salivary gland cells, and sarcoma cells. In preferred embodiments, the sample is a tumor tissue sample or a portion thereof. Preferably, the tumor tissue sample is breast cancer, ovarian tumor, prostate tumor, gastric tumor, pancreatic tumor, lung tumor, colorectal tumor, gastric / gastric 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 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.

[0157] In another specific embodiment, the sample taken from the subject according to the method of the present invention is a biological fluid, preferably a biological fluid from an affected organ. As used herein, “biological fluid,” “biological fluid sample,” or “body fluid sample” means any biological secretion or body fluid, whether physiological or pathological, produced within the body of the subject. Such biological fluids include, but are not limited to, blood, plasma, serum, bronchoalveolar lavage fluid, urine, nasal discharge, ear discharge, urethral secretions, cerebrospinal fluid, pleural fluid, synovial fluid, ascites, pericardial fluid, amniotic fluid, gastric juice, lymph, interstitial fluid, saliva, sputum, fluid deposits, tears, mucus, sweat, lactation, semen, vaginal secretions, body fluids from ulcers, blisters, abscesses, and other surface rashes. The aforementioned sample can be obtained by conventional methods using processes known to those skilled in the art, such as blood extraction, infusion and aspiration of fluids during bronchoscopy, cystopuncture, ventricular or lumbar puncture, pleural or thoracentesis, articular or synovial percutaneous puncture, abdominal puncture, amniocentesis, expectoration, peritoneal percutaneous puncture, pericardial percutaneous puncture, or by simple collection.

[0158] In a preferred embodiment, the sample is selected from the group consisting of blood, serum, plasma, saliva, cerebrospinal fluid (CSF), and tumor biopsy samples, more preferably from the group consisting of blood, plasma, and serum, and even more preferably serum.

[0159] Blood samples are typically obtained by puncturing an artery or vein (usually the inside of the elbow or the back of the hand), and the blood sample is collected in an airtight vial or syringe. Capillary puncture of the heel or distal phalanx of the finger is usually performed for microscopic analysis. To obtain serum from a whole blood sample, if no anticoagulant is present, the sample is allowed to stand for 10 minutes to coagulate, and then centrifuged at 1,500 rpm for 10 minutes to separate the cells (precipitate) from the serum (supernatant). Next, to obtain a plasma sample, the whole blood is brought into contact with an anticoagulant and centrifuged at 3,000 rpm for 20 minutes. The precipitate obtained by the centrifugation corresponds to the forming components, and the supernatant corresponds to the plasma. The obtained serum or plasma can be transferred to a storage tube for sample analysis using the method of the present invention.

[0160] As used herein, the term "biomarker" refers to a gene product from a gene of interest, understood as a transcript or translation product of the gene of interest (i.e., mRNA transcribed from the gene or a protein encoded by the gene), the amount of which reflects a state, such as predicting the response to anti-cancer treatment in a subject with cancer.

[0161] The biomarkers of the present invention are mainly (i.e., the primary biomarkers of the present invention) MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α.

[0162] The terms "MIP-1β," "MIP-1b," "MIP1B," or "MIP-1beta" refer to "macrophage inflammatory protein-1beta," now formally known as "CCL4," "chemokine (CC motif) ligand 4," or "CC motif chemokine 4." Former names for human CCL4 include AT 744, Act-2, LAG-1, HC21, and G-26. In this specification, this term refers to the protein with UniProt accession number P13236 (version 216 of the entry as of October 12, 2022), or the gene encoding it. The term "CC motif chemokine 4" includes all isoforms described for this protein.

[0163] The term "CD62E" refers to "CD62 antigen-like family member E," also known as "SELE," "ELAM1," "ELAM-1," "LECAM2," "E-selectin," "endothelial cell-leukocyte adhesion molecule 1," or "leukocyte-endothelial cell adhesion molecule 2." In this specification, this term refers to the protein with UniProt accession number P16581 (version 235 of the entry as of October 12, 2022), or the gene that codes for it. The term "CD62 antigen-like family member E" includes all isoforms described for this protein.

[0164] The terms "IL-8" or "CXCL8" refer to "interleukin-8" or "chemokine (CXC motif) ligand 8." In this specification, these terms refer to the protein or the gene encoding it, UniProt accession number P10145 (version 237 of the entry as of October 12, 2022). The term "interleukin-8" includes all isoforms of this protein as described.

[0165] The terms "GM-CSF" or "GMCSF" refer to "granulocyte-macrophage colony-stimulating factor," also known as "CSF2" or "colony-stimulating factor 2," or "morgrammostin" or "salgrammostim." In this specification, these terms refer to the protein or the gene encoding it, UniProt accession number P04141 (version 207 of the entry as of October 12, 2022). The term "granulocyte-macrophage colony-stimulating factor" includes all isoforms of this protein described herein.

[0166] The terms "IL-1α", "IL-1a", "IL-1 alpha", "IL1A", or "IL1F1" refer to "interleukin-1 alpha", also known as "hematopoietin-1". In this specification, this term refers to the protein or the gene encoding it, UniProt accession number P01583 (version 224 of the entry as of October 12, 2022). The term "interleukin-1 alpha" includes all isoforms of this protein described herein.

[0167] As used herein, the expression “at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α” means that, according to the present invention, the expression levels of one, two, three, four, or five of the biomarkers can be determined. In a particular embodiment, the method of the present invention includes determining the expression level of one of the biomarkers. In a preferred embodiment, the biomarker to be determined is MIP-1β. In another embodiment, the biomarker to be determined is CD62E. In another embodiment, the biomarker to be determined is IL-8. In another embodiment, the biomarker to be determined is GM-CSF. In another embodiment, the biomarker to be determined is IL-1α. In another particular embodiment, the method of the present invention includes determining the expression levels of two of the biomarkers. In a preferred embodiment, the biomarkers to be determined are MIP-1β and CD62E. In another embodiment, the biomarkers to be determined are MIP-1β and IL-8. In another embodiment, the determined biomarkers are MIP-1β and GM-CSF. In another embodiment, the determined biomarkers are MIP-1β and IL-1α. In another embodiment, the determined biomarkers are CD62E and IL-8. In another embodiment, the determined biomarkers are CD62E and GM-CSF. In another embodiment, the determined biomarkers are CD62E and IL-1α. In another embodiment, the determined biomarkers are IL-8 and GM-CSF. In another embodiment, the determined biomarkers are IL-8 and IL-1α. In another embodiment, the determined biomarkers are GM-CSF and IL-1α. In another embodiment, the method of the present invention includes determining the expression levels of three of the biomarkers. In another embodiment, the method of the present invention includes determining the expression levels of four of the biomarkers. In another specific embodiment, the method of the present invention includes determining the expression levels of five biomarkers: MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α.

[0168] The method of the present invention includes any combination of key biomarkers.

[0169] In a more preferred embodiment, the method includes measuring the level of a biomarker combination selected from the group consisting of 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 the group consisting of a) a combination of biomarkers comprising MIP-1β and CD62E, and b) a combination of biomarkers comprising CD62E and IL-8.

[0170] The method of the present invention may further include the determination of other biomarkers distinct from MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α.

[0171] Accordingly, in a preferred embodiment, the method further comprises determining the expression level of an additional biomarker (i.e., a secondary biomarker) different from the primary biomarker of the present invention, and preferably the method further comprises 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.

[0172] The term "MCP-1" or "MCP1" refers to "monocyte chemotactic protein 1" or "monocyte chemotactic protein 1," also known as "CCL2" or "chemokine (CC motif) ligand 2" or "CC motif chemokine 2" or "small molecule inducible cytokine A2" or "SCYA2." In this specification, this term refers to the protein or the gene encoding it, UniProt accession number P13500 (version 246 of the entry as of October 12, 2022). The term "monocyte chemotactic protein 1" includes all isoforms described for this protein.

[0173] The term "ICAM-1" or "ICAM1" refers to "intercellular adhesion molecule 1," also known as "CD54" or "differentiation cluster 54." In this specification, this term refers to the protein with UniProt accession number P05362 (version 251 of the entry as of October 12, 2022), or the gene encoding it. The term "intercellular adhesion molecule 1" includes all isoforms of this protein described herein.

[0174] The terms “IFN-γ,” “IFN-g,” “IFN-γ,” or “IFNG” refer to “interferon-gamma.” In this specification, this term refers to the protein or the gene encoding it, UniProt accession number P01579 (version 238 of the entry as of October 12, 2022). The term “interferon-gamma” includes all isoforms of this protein described herein.

[0175] The terms "IL-1β", "IL-1b", "IL-1beta", "IL1beta", "IL1B", or "IL1F2" refer to "interleukin-1 beta," also known as "leukocyte pyrogen", "leukocyte endogenous mediator", "mononuclear cell factor", or "lymphocyte activator." In this specification, this term refers to the protein or the gene encoding it, UniProt accession number P01584 (version 249 of the entry as of October 12, 2022). The term "interleukin-1 beta" includes all isoforms described for this protein.

[0176] 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). After protein synthesis, an active heterodimer (called "p70") and a homodimer of p40 are formed. In this specification, this term refers to either the protein with UniProt accession numbers P29459 (version 190 of the entry as of October 12, 2022) and P29460 (version 231 of the entry as of October 12, 2022), or the gene encoding the protein. The protein with UniProt accession number P29459 is associated with "interleukin-12 subunit alpha," or "IL12A," or "IL-12A," or "NKSF1." The protein with UniProt accession number P29460 is associated with "interleukin-12 subunit beta," "IL12B," or "NKSF2."

[0177] In a preferred embodiment, the method of the present invention further includes determining the expression level of MCP-1. In another embodiment, the method of the present invention further includes determining the expression level of ICAM-1. In another embodiment, the method of the present invention further includes determining the expression level of IFN-γ. In another embodiment, the method of the present invention further includes determining the expression level of IL-1β. In another embodiment, the method of the present invention further includes determining the expression level of IL-12. In another embodiment, the method of the present invention further includes determining the expression levels of a combination of MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12, preferably MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12.

[0178] The method of the present invention includes any combination of secondary biomarkers.

[0179] In a preferred embodiment, the method of the present invention includes determining the level of a biomarker combination selected from the group consisting of 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 includes determining the level of a biomarker combination selected from the group consisting of 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 biomarker combination consists of the combinations defined above.

[0180] In a preferred embodiment, the method of the first aspect of the present invention is further described below: i) Determine the level of at least one additional biomarker selected from the group consisting of MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12 in the sample from the subject, and ii) A step of comparing the level of at least one biomarker with a reference value, Here, - A decrease in the level of at least one biomarker relative to the reference value indicates a favorable clinical response to anti-cancer treatment, or - The equivalent or increased level of the at least one biomarker relative to the reference value indicates a poor clinical response to anti-cancer treatment in the subject.

[0181] The method of the present invention includes any combination of primary and secondary biomarkers.

[0182] The term “level” is also understood as “expression level” or “level of expression,” and as used herein, refers to the expression level of a gene product, more specifically, the measurable amount of the gene product produced by a particular gene in a particular sample of interest. As used herein, the term “gene product” refers to a transcript or translation product, and therefore corresponds to mRNA transcribed from the gene or a protein encoded by a particular gene. When referring to the expression level of a biomarker, the gene product is the biomarker, which may be mRNA or a protein transcribed from or encoded by a selected gene of the present invention, as shown below. As will be understood by those skilled in the art, the gene expression level can be quantified by measuring the messenger RNA level of the gene or the protein encoded by the gene. In the present invention, the expression levels of 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 mRNA encoded by the gene, or by measuring the level of the protein encoded by the gene, i.e., MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, or IL-12 protein or its variants. MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12 protein variants include all physiologically relevant post-translational chemical modification forms, such as glycosylation, phosphorylation, and acetylation, as long as the protein function is maintained. This term includes the MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12 proteins of all mammals, including but not limited to livestock (cattle, horses, pigs, sheep, goats, dogs, cats, rodents), primates, and humans.Preferably, MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12 proteins are human proteins.

[0183] As those skilled in the art will understand, 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.

[0184] In a preferred embodiment, the level of at least one biomarker determined is the mRNA level.

[0185] To measure the mRNA levels of the biomarkers, biological samples can be physically, mechanically, or chemically treated to disrupt tissue or cellular structures, releasing intracellular components into aqueous solutions or organic solvents, and preparing nucleic acids for further analysis. Nucleic acids are extracted from the sample using commercially available reagents and procedures known to those skilled in the art. RNA is then extracted from frozen or fresh samples using one of the methods common in the art, for example, Sambrook, J., et al., 2001. Molecular cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, NY, Vol. 1-3. Care is preferably taken to avoid RNA degradation during the extraction process.

[0186] Expression levels can be measured using mRNA obtained from formalin-fixed, paraffin-embedded tissue samples. mRNA can first be isolated from deparaffinized preserved pathology or biopsy samples. One example of deparaffinization is 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 methanol, ethanol, propanol, and butanol. The deparaffinized sample can be rehydrated, for example, by continuous washing with a reduced concentration of the lower alcohol solution. Alternatively, the sample can be deparaffinized and rehydrated simultaneously. The sample is then lysed and the RNA is extracted. Samples can also be obtained from fresh tumor tissue, such as excised tumors. In certain embodiments, samples can be obtained from fresh tumor tissue or OCT-embedded frozen tissue.

[0187] 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 analysis, Northern blotting, RNA dot blotting, and TaqMan®; tag-based methods such as sequential gene expression analysis (SAGE) including variants such as LongSAGE and SuperSAGE; and fluorescence in situ hybridization including variants such as microarrays, nucleic acid sequence-based amplification (NASBA), Flow-FISH, qFiSH, and double-fusion fish (D-FISH).

[0188] In certain embodiments, the biomarker expression level is determined by a microarray. In another specific embodiment, the biomarker expression level is determined by quantitative PCR, preferably real-time PCR.

[0189] To normalize mRNA expression levels between different samples, the expression level of a target mRNA in a test sample can be compared to the expression level of a control RNA. In this specification, "control RNA" refers to RNA whose expression level in tumor cells is unchanged or only slightly changed compared to non-tumor-forming cells. Preferably, the control RNA is mRNA derived from a housekeeping gene, which is constitutively expressed and encodes a protein responsible for essential cellular functions. Examples of housekeeping genes used in this invention include β-2-microglobulin, ubiquitin, 18-S ribosomal protein, cyclophyllin, GAPDH, PSMB4, tubulin, and β-actin.

[0190] In one embodiment, relative gene expression levels are calculated according to the comparative threshold cycle (Ct) method, using GAPDH, β-actin, or PSMB4 as an endogenous control and a commercially available RNA control as a calibrator. The final result is calculated according to Equation 2 (ΔCt sample - ΔCt calibrator). Here, the ΔCT values ​​of the calibrator and sample are determined by subtracting the CT value of the target gene from the CT value of the control gene.

[0191] In a preferred embodiment, the level of at least one biomarker determined is at the protein level.

[0192] The expression level of the biomarker can also be measured by measuring the level of the protein encoded by the gene or a variant thereof. In the context of the present invention, substantially any conventional method can be used to quantify the level of the protein. Suitable methods for measuring gene expression levels at the protein level include, but are not limited to, conventional methods that use an antibody capable of specifically binding to the protein encoded by the gene (or a fragment thereof containing an antigenic determinant) and quantify the resulting antibody-antigen conjugate. In certain embodiments, the protein level of the biomarker of the present invention can be quantified using standard assays for determining protein expression levels, such as Western blotting or Western 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 containing specific antibodies; assays based on colloidal precipitation in the form of dipsticks or the like; or immunoassays based on Luminex technology.

[0193] Antibodies used in these assays include, for example, polyclonal serum, hybridoma supernatant, monoclonal antibodies, antibody fragments, Fv, Fab, Fab', F(ab')2, ScFv, diabodies, triabodies, tetrabodies, and humanized antibodies. Antibodies can be used in any combination, with or without labeling. Examples of usable markers include, but are not limited to, radioisotopes, enzymes, phosphors, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, and colorants. The present invention allows the use of various widely known assays employing unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies). These techniques include Western blotting or Western transfer, ELISA, RIA, competitive EIA, DAS-ELISA, immunocytochemistry and immunohistochemistry, techniques using biochips or protein microarrays (including specific antibodies), or colloidal precipitation assays in the form of dipsticks. Affinity chromatography and binding ligand assays can also be used for the detection and quantification of target proteins.

[0194] On the other hand, the level of biomarker proteins can be determined by constructing a tissue microarray (TMA) assembled from the target sample and measuring the expression level of the corresponding protein using immunohistochemistry. Immunostaining intensity is evaluated by two or more pathologists and scored using a unified and clear cutoff criterion. This maintains the reproducibility of the method. Discrepancies can be resolved by simultaneous re-evaluation. In short, the results of immunostaining can be recorded as negative expression (0) vs. positive expression, low expression (1+) vs. moderate (2+) and high expression (3+), taking into account expression in tumor cells and specific cutoff values ​​for each marker. As a general criterion, the cutoff value is selected to facilitate reproducibility and, if possible, to allow interpretation of biological events. Alternatively, immunostaining intensity can also be evaluated using imaging techniques or automated methods, such as those disclosed in Rojo, MG et al. (Folia Histochem. Cytobiol.2009; 47: 349-54) or Mulrane, L. et al. (Expert Rev. Mol. Diagn. 2008; 8: 707-25).

[0195] Alternatively, in another specific embodiment, the level of a biomarker protein is measured by Western blotting. Western blotting is a method for detecting proteins that have been separated by gel electrophoresis under denaturing conditions and immobilized, usually on a nitrocellulose membrane, by incubation with a specific antibody and a chromogenic system (e.g., a chemiluminescent system).

[0196] In certain embodiments, the expression level is measured as a protein level. In more specific embodiments, the protein level is measured by ELISA, Western blotting, or immunoassay.

[0197] In a preferred embodiment, the level is measured by an immunoassay. As used herein, the term “immunoassay” refers to a biochemical test that measures the presence or concentration of a molecule in a solution using an antibody or antigen-binding fragment that can specifically recognize the molecule. In a preferred embodiment, the immunoassay is an immunoassay based on an antibody bound to Luminex beads, specifically an immunoassay performed using Luminex technology, more specifically an immunoassay performed using a ProcartaPlex® kit.

[0198] As described above, in order to carry out the method of the present invention, the expression levels of MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12 can be measured using the protein variants.

[0199] Therefore, variants of the MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12 proteins are those that (i) have one or more amino acid residues substituted with conserved or non-conserved amino acid residues (preferably conserved amino acid residues) (the substituted amino acid residues may or may not be encoded by the genetic code), (ii) have one or more modified amino acid residues (e.g., residues modified by the addition of substituents), (iii) the protein is an isoform or alternative splice variant of the protein of the present invention, and / or (iv) is a fragment of the protein. Fragments include proteins produced by proteolysis (including multi-site proteolysis) of the original sequence. Variations are considered to be within the scope of those skilled in the art from the teachings herein.

[0200] The variants according to the present invention contain an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, or 96% similar or identical to the original amino acid sequence. As is well known in the art, the "similarity" between two proteins is determined by comparing the amino acid sequence of one protein and its conserved amino acid substitutions with the sequence of the other protein. The degree of identity between two proteins is determined using computer algorithms and methods widely known to those skilled in the art. The identity between two amino acid sequences is preferably determined using the BLASTP algorithm [BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)].

[0201] Proteins can undergo post-translational modifications. For example, post-translational modifications within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding, and proteolysis processing. Furthermore, proteins may contain unnatural amino acids formed by post-translational modifications or by unnatural amino acids introduced during translation.

[0202] In certain embodiments, the variant is a mammalian variant, preferably a human variant, and more preferably has at least 60%, 70%, 80%, 90%, 95%, or 96% similarity or identity with the original amino acid sequence.

[0203] A second step of the first method of the present invention includes comparing the level of the at least one biomarker obtained in step (i) with a reference value.

[0204] In this specification, the term “reference value” refers to a clinical laboratory value used as a baseline for values / data obtained using samples taken from subjects. A reference value or reference level may be an absolute value, a relative value, a value with an upper or lower limit, a range of values, a mean, a median, a mean value, or a value compared to a specific control or baseline value. A reference value may be based on individual sample values, e.g., values ​​obtained from samples of the subject being tested, at an earlier point in time. A reference value may be based on a large number of samples, e.g., values ​​obtained from a patient population considered representative, e.g., an age-matched population of subjects matched to the age of the patients under study, or on a sample pool that includes or excludes the samples being tested. Appropriate reference values ​​are shown in the context of the present invention’s method for predicting the response of cancer patients undergoing anti-cancer therapy.

[0205] In the context of the method of the present invention, preferred reference values ​​for predicting the response of subjects with cancer to anticancer therapy may be MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12. These are expression levels measured in samples of subjects who have or have had cancer but have shown a good clinical response to anticancer therapy, and these expression levels were measured at the time the patient was receiving treatment. In another embodiment, the reference values ​​are the expression levels of MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12 measured in samples of subjects who have or have had cancer but have shown a poor clinical response to anticancer therapy, and these expression levels were measured at the time the patient was receiving treatment. In another embodiment, the reference values ​​are the expression levels of MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12 measured in samples taken from at least a subset of subjects who responded well and poorly to anticancer therapy in a pool of subjects who have or have had cancer, and these expression levels were measured at the time the patients were receiving treatment. In another embodiment, the reference values ​​are 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 healthy patients, i.e., patients who have not been diagnosed with a type of cancer for which a response to treatment is desired.

[0206] In one embodiment, the reference value is obtained by determining the median expression level of each biomarker measured in a group of subjects with cancer but no decrease in biomarkers, or in a group of samples taken from normal tissue.

[0207] The collection of samples used to derive the baseline level preferably consists of a mixture of tissues from subjects with the same type of cancer, or from normal individuals who do not have cancer.

[0208] Alternatively, the use of a reference value to determine whether the biomarker expression level is "increased" or "decreased" can correspond to the median expression level of each biomarker measured in an RNA sample obtained by pooling equal amounts of RNA from each sample obtained from subjects with cancer in which the levels of the biomarkers of the present invention have not decreased, preferably from subjects with the same type of cancer. In another embodiment, the reference value can correspond to the median expression level of each biomarker measured in an RNA sample obtained by pooling equal amounts of RNA from each sample obtained from subjects with cancer in which the levels of the biomarkers of the present invention have not increased, preferably from subjects with the same type of cancer.

[0209] In the present invention, the “reference value” may be any cutoff point set according to the ROC method. Once this cutoff point is set, the level of the marker expressed in the target biological fluid or tumor tissue can be compared to this cutoff point, and thus a “low” expression level can be assigned if it is below this cutoff point, and a “high” expression level if it is above this cutoff point. Therefore, in a preferred embodiment, the expression level of each biomarker in the combination is compared to a predetermined cutoff value for each gene. Here, the predetermined cutoff value for each gene preferably corresponds to the expression level of the gene that correlates with the highest specificity at the desired sensitivity in the ROC curve calculated based on the biomarker expression levels.

[0210] Once this reference value is established, the expression levels of biomarkers in a sample can be compared to this reference value and classified as "increased," "decreased," or "equal." For example, if the expression level increases by at least 1.1 times, 1.5 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or more compared to the reference value, the expression level is considered "increased." On the other hand, a decrease in expression level below the reference value by at least 0.9 times, 0.75 times, 0.2 times, 0.1 times, 0.05 times, 0.025 times, 0.02 times, 0.01 times, 0.005 times, or less compared to the reference value is considered a "decrease" in expression level.

[0211] This comparison allows for the detection of a decrease in the expression of one or more of the biomarkers relative to a reference value. The expression "decreased 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-gamma, IL-1β, and IL-12 means a decrease of 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 in the expression of a given biomarker compared to a reference value.

[0212] The expression "increased 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-gamma, IL-1β, and IL-12 means that the expression of a given biomarker 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%, and 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%, at least 160%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, and less 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 380%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 480%, at least 500%, at least 520%, at least 530%, at least 540%, at least 5 This means an increase of 50%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000%, at least 1100%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 18, at least 120%, at least 130%, at least 140%, and at least 150% or more.

[0213] If the expression level differs from the reference value by 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 "equivalent" to the reference value.

[0214] By 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 reference values, the method of the present invention makes it possible to predict whether a subject will show a good or poor clinical response to anticancer treatment according to items (a) to (e) of the first aspect of the present invention. Accordingly, subjects in which the level of at least one of the biomarkers is reduced compared to the reference value will show a good clinical response to anticancer treatment.

[0215] As used herein, the expression “good clinical response” means that the subject shows a favorable response to anticancer treatment according to items (a) to (e) of the first aspect of the present invention, which will be understood by those skilled in the art as a complete response, partial response, or stabilization of the disease.

[0216] In a preferred embodiment, a good clinical response is disease stabilization.

[0217] Alternatively, patients whose levels of at least one of the biomarkers are equal to or elevated relative to a reference value are likely to have a poor clinical response to anti-cancer treatment.

[0218] As used herein, the term "poor clinical response" means that the response to the subject is not as favorable as described above. Poor clinical response may include cases where disease progression is predicted, or where relapse, death, or the need for a change or administration of new treatment is predicted.

[0219] In a preferred embodiment, poor clinical response is disease progression.

[0220] As those skilled in the art will understand, such probability assessments, while preferable, are not typically accurate for 100% of the subjects being analyzed. However, this term requires that a statistically significant portion of the subjects be identified as having a predisposition to respond to chemotherapy. Whether a portion is statistically significant can be easily determined by those skilled in the art using various well-known statistical assessment tools, such as confidence interval calculation, p-value calculation, Student's t-test, and Mann-Whitney test. Details are described in "Statistics for Research" by Dowdy and Wearden (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%, and at least 95%. P-values ​​are preferably 0.1, 0.05, 0.01, 0.005, or 0.0001. More preferably, the method of the present invention can adequately identify at least 60%, at least 70%, at least 80%, or at least 90% of the subject population.

[0221] In a preferred embodiment, the first method of the present invention is used to predict the clinical response of a patient with cancer to anticancer treatment, and then to initiate anticancer treatment.

[0222] Selection method according to the present invention The inventors observed that decreased levels of MIP-1β, CD62E, IL-8, GM-CSF, or IL-1α compared to reference values ​​indicated a favorable clinical response to treatment with OMO-103. Therefore, the detection of low levels of these biomarkers may be a useful indicator for selecting patients for treatment based on Omomyc or its functionally equivalent variants. This would allow patients to proceed directly to appropriate treatment while avoiding less effective treatments and associated side effects.

[0223] Therefore, in a second embodiment, the present invention relates to an in vitro method for selecting a customized treatment for a subject suffering from cancer, (i) Determine 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 at least one biomarker to a reference value. Includes, Here, - A decrease in the level of at least one biomarker relative to the reference value indicates that the selected treatment includes a drug selected from the group consisting of: a) A polypeptide containing the sequence of sequence number 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) Cells capable of secreting the polypeptide described in a) or the conjugate described in b) into a culture medium: or - Equivalent or increased levels of at least one of the biomarkers relative to the reference value indicate that the selected treatment does not include any drugs selected from the group consisting of: a) A polypeptide containing the sequence of sequence number 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) Cells that can secrete the polypeptide described in a) or the conjugate described in b) into a culture medium.

[0224] In this specification, the terms “select a customized treatment” or “select a personalized treatment” refer to selecting an appropriate and optimal treatment based on specific medical characteristics resulting from the patient’s personal background. In the context of this invention, such characteristics consist of levels of biomarkers in a sample isolated from the patient.

[0225] In the context of the second method of the present invention, the term “treatment” means any anti-cancer treatment, which includes exposing a subject to treatment to any method used to induce the death of cancer cells. The method is not limited to the administration of chemicals and may consist of targeted therapies, including surgery, chemotherapy, radiotherapy, hormone therapy, immunotherapy, or a combination thereof.

[0226] In relation to cancer, the term "surgery" refers to a major surgical procedure that removes at least a portion of the primary tumor and / or at least a portion of at least one metastasis.

[0227] As used herein, the term "chemotherapy" refers to a variety of treatments consisting of the administration of one or more anticancer agents, alone or in combination with other compounds, that are suitable for the treatment and / or management of cancer, without any restrictions on their chemical structure.

[0228] As used herein, the terms “radiotherapy,” “radiotherapy,” or “ionizing radiation” generally refer to therapies that use ionizing radiation to control or kill tumor cells as part of cancer treatment, usually delivered by a linear accelerator. Gamma rays, X-rays, and the wavelengths of the electromagnetic spectrum above ultraviolet are ionizing radiation, while the spectrum below ultraviolet, i.e., visible light (including almost all types of laser light), infrared, microwaves, and radio waves, are all considered non-ionizing radiation. Radiotherapy can bring about a cure for many types of cancer when localized to one area of ​​the body. Radiotherapy is often applied to cancerous tumors because it can control cell growth. Ionizing radiation works by damaging the DNA of cancerous tissue, causing cell death.

[0229] As used herein, the term “targeted therapy” refers to treatments that inhibit the growth of cancer cells by inhibiting specific target molecules necessary for carcinogenesis and tumor growth, rather than simply inhibiting all rapidly dividing cells. Because most of the drugs used in targeted therapy are biopharmaceuticals, the term “biological therapy” can be synonymous with targeted therapy when used in the context of cancer treatment (and thus distinguished from chemotherapy, i.e., cytotoxic therapy). However, these treatments can be combined. Another form of targeted therapy uses nanoengineered enzymes to bind to tumor cells, digesting them through the body’s natural cytolytic processes and effectively eliminating them from the body. Many targeted therapies are examples of immunotherapy.

[0230] The term "immunotherapy" refers to cancer immunotherapy, which consists of methods that modify the host's immune system and / or methods that utilize components of the immune system as cancer treatment. Non-specific examples of immunotherapy include the nonspecific immunostimulants BCG and levamisole, the cytokines interferon-α and interleukin-2, the monoclonal antibodies anti-PD1, anti-PDL-1, anti-CTLA-4, rituximab, ofatumumab, alemtuzumab, trastuzumab, bevacizumab, cetuximab, and panitumumab, the radiolabeled antibodies Y-90 ibritumomab tiuxetan and I-131 tocitumomab, the immunotoxin denileukin difutitox and the antibody gemtuzumab ozogamicin, and non-myeloablative allografting by infusion of donor lymphocytes. Anti-prostate cancer cell therapy cypleuric acid T. Other immunotherapies include dendritic cell vaccines, CART-T cell therapy, and NK cell therapy. Some immunotherapies involve collecting immune cells from blood or tumors, culturing tumor-specific cells, and returning them to the patient to attack the tumor. Another method involves culturing immune cells that have been genetically modified to express tumor-specific receptors and returning them to the patient. The types of cells that can be used in this method include natural killer cells, lymphokine-activated killer cells, cytotoxic T cells, and dendritic cells.

[0231] The first step of the second method of the present invention includes 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 includes comparing the level of the at least one biomarker with a reference value.

[0232] 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 reference values, the method of the present invention makes it possible to determine appropriate treatment for the subject. Accordingly, subjects whose levels of at least one of the biomarkers are reduced compared to reference values ​​should be given treatment comprising a drug selected from the group consisting of items (a) to (e) of the first embodiment of the present invention.

[0233] In this specification, “anticancer agent” means a drug that stabilizes a disease, including inhibiting at least partially the development or progression of cancer, or inhibiting all or part of the symptoms associated with cancer.

[0234] On the other hand, subjects whose levels of at least one biomarker are equivalent to or elevated compared to a reference value should choose alternative therapy, i.e., a therapy that does not include drugs selected from the following group: a) A polypeptide containing the sequence of sequence number 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or its functionally equivalent variant; c) Polynucleotides encoding the polypeptide of a) or the conjugate of b); d) A vector containing the polynucleotide described in c); and e) Cells that can secrete the polypeptide described in a) or the conjugate described in b) into a culture medium.

[0235] In the context of the second method of the present invention, the term “alternative therapy” refers to any therapy that is different from therapies that include the agents defined in items (a) through (e) of the first method of the present invention. Alternative therapies include, but are not limited to, surgery, chemotherapy, radiotherapy, hormone therapy, targeted therapies including immunotherapy, and combinations thereof.

[0236] Non-limiting examples of alternative therapies include surgery, treatment with anti-cancer agents such as chemotherapeutic agents including anthracycline antibiotics such as doxorubicin and daunorubicin, taxanes such as taxol (trademark) and 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, brequinar or mitramycin, etc. Toxins such as lysin A chain, saponin, diphtheria A chain, active non-binding fragment of diphtheria toxin, Pseudomonas aeruginosa exotoxin A chain, abrin A chain, modeccin A chain, α-sarcin, Leurites fordii A protein, dianthin protein, American yam (PAPI, PAPII and PAP-S) protein, Momordica charantia inhibitor, cucurbitacin, crocin, Saponaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, trichothecene, etc. Enzymes such as alkaline phosphatase that activates etoposide and doxorubicin, carboxypeptidase G2 that activates nitrogen mustard, etc. Beta-lactamase that activates doxorubicin, paclitaxel, mitomycin, cytokines such as TNF factor alpha, INF-gamma, GM-GSF factor, IL-2, 131 I、 90 Y、 177 Lu、 188 Re、 67 Cu、 211 At、 213 Bi、 125 I、 111Radioactive isotopes such as In. Paclitaxel, 2-methoxyestradiol, prinomast, bacimast, BAY 12-9566, carboxamide triazole, CC-1088, dextromethorphan acetate, dimethylxanthenone acetate, endostatin, IM-862, marimast, penicillamine, PTK787 / ZK 222584, RPI.4610, squalamine lactate, SU5416, thalidomide, combretastatin, tamoxifen, COL-3, neovastat, BMS-275291, SU6668, anti-VEGF antibody, Medi-522 (vitaxin II), CAI, interleukin-12, IM862, amiloride, angiostatin, Kl-3 angiostatin and other anti-angiogenic agents Kl-5 Angiostatin, captopril, DL-α-difluoromethylornithine, DL-α-difluoromethylornithine HCl, endostatin, fumagiline, harbimycin A, 4-hydroxyphenylretinamide, juglon, laminin, laminin hexapeptide, laminin pentapeptide, lavendustin A, medroxyprogesterone, minocycline, placental ribonuclease inhibitors, suramin, thrombospondin, antibodies against pro-angiogenic factors (e.g., Avastin, Erbitux, Vectibix, Herceptin), low molecular weight tyrosine kinase inhibitors of pro-angiogenic growth factors (e.g., Tarceva, Nexavar, Sutent, Iressa), mTOR inhibitors (e.g., Tricel).Interferon α, β, and γ, IL-12, matrix metalloproteinase inhibitors (e.g., COL3, Marimast, Batymast), ZD6474, SUL1248, Vitaxin, PDGFR inhibitors (e.g., Gleevec), NM3 and 2-ME2, cyclopeptides such as silendides, antiproliferative agents, e.g., (i) folate antimetabolites (aminopterin, denopterin, methotrexate, edatrexate, trimethrexate, noratexed, lometrexol, pemetrexed, larcitrexed, pyritrexime, pteropterin, leucovorin, 10-propargyl-5,8-dideaza folate). (ii) Acids (PDDF, CB3717), purine analogs (cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine), pyrimidine analogs (capecitabine, cytarabine or alla-C, decitabine, fluorouracil, 5-fluorouracil, doxifluridine, floxuridine, gemcitabine) (ii) Products such as natural product antitumor antibiotics and mitotic inhibitors, e.g., vinca alkaloids (vindesine, vincristine, vinblastine, vinorelbine, etc.), taxanes (paclitaxel (Taxol®), docetaxel (Taxotere®), colchicine (NSC) 757), thiocolchicine (NSC 361792), colchicine derivatives (e.g., NSC 33410), allocolchicine (NSC 406042), halichondrin B (NSC 609395), dorastatin 10 (NSC 376128), meitansine (NSC 153858), lyzoxin (NSC 332598), epotilon A, epotilon B, discodermorid, estramustine, nocodazole. (iii) (iv) Hormone and its antagonists such as tamoxifen, toremifene, anastrozole, alzoxifen, rasofoxifen, raloxifene, naphoxidine, fulvestrant, aminoglutethimide, testolactone, atamestan, exemestane, fadrozol, formestan, letrozole, goserelin, leuprorelin or leuprolide, buserelin, histrelin, megestrol, and fluoxymesterone. (iv) Biological agents such as viral vectors, interferon alfa, and interleukins.(v) Platinum compounds, e.g., carboplatin, cisplatin [cis-diaminedichloroplatin (CDDP)], oxaliplatin, iproplatin, nedaplatin, triplatin tetranitrate, tetraplatin, satraplatin (JM216), JM118 [cis-amminedichloro (II)], JM149 [cis-amminedichloro(cyclohexylamine)transdihydroxoplatin (IV)], JM335 [trans-amminedichlorodihydroxoplatin (IV)], transplatin, ZD0473, cis, trans, cis-Pt(NH3)(C6H11NH2)(OOCC3H7)2Cl, maranate-l,2-diaminocyclohexanoplatin (II), 5-sulfosalicylate-trans-(l,2-diaminocyclohexane)platin (II) (SSP) Poly-[(Trans-1,2-diaminocyclohexane)platinum]carboxyamylose (POLY-PLAT) and 4-hydroxysulfonylphenylacetic acid (Trans-1,2-diaminocyclohexane)platinum(II) (SAP), etc., and (vi) DNA alkylating agents such as nitrogen mustard, nitrosourea, ethyleneimine derivatives, alkyl sulfonates and triazenes. These include, but are not limited to, cyclophosphamide (Cytoxan) (trademark), busulfan, improsulfan, pigosulfan, pipobromane, melphalan (L-sarcolicin), chlorambucil, mechloretamine or mustine, uramustine or uracil mustard, nobembitine, fenesterine, trophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), chlorozotosine, fotemustine, etc. Targeted therapies or immunotherapies such as nimustine, ranimustine, semustine (methyl CCNU), streptozosin, thiotepa, triethylenemelamine, triethylenethiophosphoramine, procarbazine, altretamine, dacarbazine, mitozolomide and temozolomide, anti-PD1 or anti-CTLA4.

[0237] Regarding in vitro methods for selecting cancer patients for treatment involving drugs selected from the following group: a) A polypeptide containing the sequence of sequence number 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) Cells that can secrete the polypeptide described in a) or the conjugate described in b) into a culture medium, The method includes the following: (i) Determine 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) The level of the at least one biomarker is compared to a reference value, and if a decrease in the level of the at least one biomarker relative to the reference value is detected, the patient is selected for the treatment.

[0238] The first step of the third method of the present invention includes 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 includes comparing the level of the at least one biomarker with a reference value.

[0239] By 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 reference values, the method of the present invention makes it possible to select patients for treatment. In particular, subjects in whom the level of at least one of the biomarkers is reduced compared to the reference value are selected for treatment according to any of items (a) to (e) of the first aspect of the present invention.

[0240] The treatment method and pharmaceutical form of the treatment method of the present invention can be used as defined in the context of the fourth aspect of the present invention.

[0241] Those skilled in the art will understand that the specific embodiments developed in the first aspect of the present invention are also applicable to the second and third aspects of the present invention.

[0242] All terms are described in detail above in the context of the first aspect of the present invention and are used in the same meaning in the context of the second and third aspects of the present invention.

[0243] Therapeutic method of the present invention The results obtained in the present invention indicate that low levels of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α in the specimens of cancer patients taken before the start of treatment are likely to result in successful treatment by the anticancer treatment defined in items (a) to (f) of the first aspect of the present invention. This suggests that it may be the best treatment method available for such patients. The anticancer treatment including items (a) to (f) of the first aspect of the present invention will be selected as the first-choice treatment for patients with low levels of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α.

[0244] In a fourth aspect, the present invention relates to a drug selected from the group consisting of: a) a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide comprising the sequence of 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; c) a polynucleotide encoding the polypeptide of a) or the conjugate of b); d) a vector comprising the polynucleotide described in c); and e) a cell capable of secreting the polypeptide of a) or the conjugate of b) into a medium The invention relates to a target cancer, wherein the target has been identified as a good responder to the drug by the first method of the present invention, or the treatment has been selected by the second method of the present invention.

[0245] Alternatively, the present invention relates to the use of a drug selected from the group consisting of: a) A polypeptide containing the sequence of sequence number 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) Cells that can secrete the polypeptide described in a) or the conjugate described in b) into a culture medium, Use for the manufacture of a drug for treating a subject suffering from cancer, wherein the subject is identified as a good responder to the drug by a method according to a first aspect of the present invention, or the treatment is selected by a second aspect of the present invention.

[0246] Alternatively, the present invention relates to a method for treating cancer in a subject, comprising administering a drug selected from the following group to the subject: a) A polypeptide containing the sequence of sequence number 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) Cells that can secrete the polypeptide described in a) or the conjugate described in b) into a culture medium. And, Here, the subject is identified as a good responder to the drug by a method according to the first aspect of the present invention, or the treatment is selected by a second method of the present invention.

[0247] Preferably, the agent forms part of a pharmaceutical composition comprising a therapeutically effective amount of the agent, which includes at least one pharmaceutically acceptable excipient or carrier. The agent may include any of the multiple agents defined in items (a) to (e) of the first aspect of the present invention, or additional agents useful for the treatment of cancer.

[0248] In this specification, "therapeutic dose" refers to the amount of drug sufficient to produce the desired effect, and is generally determined by various factors, including the properties of the compound itself and the therapeutic effect to be achieved. It also depends on the target patient, the severity of the cancer they have, the selected dosage form, and the route of administration.

[0249] As used herein, the terms “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” refer to any compound or combination of compounds that is essentially non-toxic to the subject at the dose and concentration used and is compatible with other components of the pharmaceutical. Therefore, an excipient is an inert substance that is compounded with the active ingredient of a pharmaceutical to increase the volume of the composition containing the active ingredient. Excipients also serve to enhance various therapeutic effects, such as improving the absorption and solubility of the active ingredient and other pharmacokinetic considerations. Excipients also facilitate the handling of the active ingredient in question during the manufacturing process, such as by improving the flowability and non-stickiness of the powder, and contribute to improved in vitro stability, such as preventing denaturation over the expected shelf life.

[0250] In a preferred embodiment, the anticancer therapy is administered intravenously, preferably as an intravenous infusion over 30 minutes. In one embodiment, the anticancer therapy is administered once a week.

[0251] In a preferred embodiment, the drug is a polypeptide comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof, and more preferably a polypeptide comprising SEQ ID NO: 4.

[0252] All embodiments of the first, second, and third aspects of the present invention are also applicable to the fourth aspect of the present invention.

[0253] All terms have already been defined in the context of the first, second, and third aspects of the present invention and are used in the same sense in the context of the fourth aspect of the present invention.

[0254] The present invention's kit and its applications The inventors of this invention have found that by measuring the levels of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α using immunoassay, they can predict the response of cancer patients to anti-cancer treatment with OMO-103.

[0255] In another embodiment, the present invention relates to a kit comprising a reagent specific 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.

[0256] In another aspect, the present invention relates to the use of a kit comprising a specific reagent 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 the use of a specific reagent 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 at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF and IL-1α. The kit or reagent in a sample from a subject suffering from cancer is used to predict the clinical response of the 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.

[0257] In another aspect, the present invention relates to the use of a kit comprising a reagent specific 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 in a sample from a subject suffering from cancer, or the use of a reagent specific 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 at least one biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF and IL-1α, for selecting a treatment method for the subject, particularly a treatment method 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.

[0258] In another embodiment, the present invention relates to a kit comprising a specific reagent 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 in a sample of a cancer-affected subject, in order to select cancer patients to be treated with any of the agents defined in items (a) to (e) of the first aspect of the present invention, or the use of a specific reagent 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 MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α. In a preferred embodiment, the kit or reagent is used in a method according to the third aspect of the present invention.

[0259] In the present invention, "kit" is understood to mean a product in which various reagents necessary for carrying out various applications of the present invention are packaged for transport and storage. Suitable materials for packaging the components of the kit include crystal, plastics (such as polyethylene, polypropylene, and polycarbonate), bottles, vials, paper, and envelopes. Furthermore, the kit used in the present invention may include instructions for using the various components contained in the kit simultaneously, sequentially, or individually. Such instructions may be in the form of printed material, or in the form of electronic media capable of storing readable or understandable instructions, such as electronic storage media (such as magnetic disks and tapes), optical media (such as CD-ROMs and DVDs), or audio material. In addition, or alternatively, such media may include an internet address providing such instructions.

[0260] The system further includes specific reagents for determining the expression levels of at least one additional biomarker selected from the group consisting of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α.

[0261] In a more preferred embodiment, the kit includes a reagent specific for determining the expression level of MIP-1β and at least additional reagents specific 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.

[0262] As used herein, the term “reagent” refers to any compound or composition that can be used to detect any level of any of the biomarkers of the present invention, for example, the level of any of the biomarkers MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α, or the level of any 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., a reagent for detecting their genes, proteins, or variants thereof. The reagent may optionally include a reagent for detecting one or more housekeeping genes, or proteins encoded by said housekeeping genes.

[0263] In this specification, the expression “specific reagent for determining the expression level of a biomarker” refers to any compound or group of compounds that enables the specific determination of the expression level of a gene or protein by detection methods well known to those skilled in the art. In particular, the term “specific reagent” in this specification refers to any reagent that can be used to specifically quantify any of the biomarkers of the present invention.

[0264] In one embodiment, the specific reagent for determining the expression level of a biomarker is a nucleic acid that can specifically hybridize with any of the MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, and IL-12 genes. In a preferred embodiment, the nucleic acid is an oligonucleotide that specifically hybridizes with the biomarker.

[0265] In this specification, the term “oligonucleotide” refers to nucleic acids, preferably 10 or more nucleotides, preferably 15 or more nucleotides, preferably 20 or more nucleotides, preferably 25 or more nucleotides, and preferably 100 or less, and includes polyribonucleotides, polydeoxyribonucleotides, and combinations thereof. The term “oligonucleotide” also refers to molecules formed by conventional nucleotides linked by conventional phosphodiester bonds, as well as their variants, including purine or pyrimidine modifications, or ribose or deoxyribose modifications designed to enhance the stability of the oligonucleotide. Alternatively or additionally, oligonucleotides may include modification bonds such as phosphotriesters, phosphorothioates, methylphosphonates, or peptide nucleic acids (PNAs). Methods for synthesizing oligonucleotides are well known to those skilled in the art. Oligonucleotides can be primers or probes. In one embodiment, the oligonucleotide is a probe. In another embodiment, the oligonucleotide is a primer, preferably a primer pair. In one embodiment, the present invention provides a set of one or more oligonucleotide primers designed to specifically amplify a biomarker of the method of the present invention.

[0266] Nucleic acids that can specifically hybridize with 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 fragments of the mRNA (or corresponding cDNA) of the said genes.

[0267] As those skilled in the art will understand, the oligonucleotide primers and probes of the present invention kit can be used with any gene expression profiling technique (RT-PCR, SAGE, TaqMan, Real Time-PCR, FISH, NASBA, etc.).

[0268] In another embodiment, the specific reagent for determining the expression level of the biomarker is a compound that specifically binds to the biomarker protein, and is specifically selected from the group consisting of antibodies, aptamers, and fragments thereof.

[0269] In a preferred embodiment, the specific reagent for determining the expression level of a biomarker is an antibody or fragment thereof that can specifically bind to (i.e., specifically recognize) the MIP-1β, CD62E, IL-8, GM-CSF, IL-1α, MCP-1, ICAM-1, IFN-γ, IL-1β, or IL-12 protein or its variants (including fragments thereof containing antigenic determinants).

[0270] Antibodies or fragments thereof that can detect antigens and specifically bind to proteins or their variants include, for example, monoclonal and polyclonal antibodies, antibody fragments, Fv, Fab, Fab', F2, (ab')ScFv, diabodies, triabodies, tetrabodies, and humanized antibodies. The antibodies in the kit of the present invention can be used in conventional methods for detecting protein expression levels, such as Western blotting or Western transfer, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzymatic immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemistry and immunohistochemistry, biochip technology, protein microarrays containing specific antibodies, or colloidal precipitation assays using formats such as dipsticks. In a preferred embodiment, the antibody is used in an immunoassay. Preferably, the immunoassay is based on Luminex technology, and more preferably a ProcartaPlex® kit.

[0271] The reagents, specifically the probe and antibody, can be immobilized on a solid support such as a membrane, plastic, or glass, and optionally, the probe or antibody can be treated to facilitate its immobilization on the support.

[0272] The kit of the present invention optionally includes additional reagents for detecting housekeeping genes, polypeptides encoded by housekeeping genes, or mRNA encoded by said housekeeping genes. By using the additional reagents, measurements obtained from different samples can be normalized (e.g., the test sample and the control sample) to eliminate the possibility that differences in biomarker expression are due to differences in the total amount of protein in the samples rather than differences in relative expression levels. In this specification, “housekeeping gene” refers to a gene that encodes a protein that is constitutively expressed and performs essential cellular functions. Housekeeping genes preferably used in the present invention include β-2-microglobulin, ubiquitin, 18-S ribosomal protein, cyclophyllin, PSMB4, GAPDH, tubulin, and β-actin.

[0273] In a preferred embodiment, the kit referred to in any of the above applications further comprises a specific reagent for detecting or determining the expression level of a gene or protein that determines whether or not cells in the sample are proliferating. Preferably, the kit comprises an antibody capable of specifically recognizing a protein that determines whether or not cells in the sample are proliferating, preferably selected from the group consisting of geminin, KI-67, proliferating cell nuclear antigen, and cyclin A2.

[0274] The kit of the present invention includes other reagents that can determine the expression level of a biomarker but are not specific to the biomarker, such as reagents for extracting RNA material, and DNA amplification reagents such as primers for synthesizing the corresponding cDNA by RT, DNA polymerase, dNTPs, and buffers.

[0275] In another preferred embodiment, any of the reagents mentioned in this section, particularly any oligonucleotides or antibodies mentioned in this section, more preferably any antibody, constitute 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 reagents constituting the kit of the present invention, preferably the total amount of specific reagents determining the expression level of one or more biomarkers constituting the kit.

[0276] In a more preferred embodiment, specific reagents for determining the expression level of at least one biomarker constitute at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of the total amount of reagents constituting the kit. Preferably, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of the specific reagents for determining biomarkers included in the kit are specific reagents 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.

[0277] All specific embodiments disclosed in the method of the present invention are applicable to the kit of the present invention and its use.

[0278] All terms have already been defined in the context of the first, second, third, and fourth aspects of the present invention and are used in the same sense in the context of the kit and its use.

[0279] The present invention also relates to the following embodiments.

[0280] 1. An in vitro method for predicting the clinical response of a patient with cancer to an anti-cancer treatment selected from the following: a) A polypeptide containing the sequence of sequence number 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) Cells that can secrete the polypeptide described in a) or the conjugate described in b) into a culture medium. Selected from the group consisting of, The method is as follows: (i) Determine 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 at least one biomarker to a reference value, Here, - A decrease in the level of at least one biomarker relative to the reference value indicates a favorable clinical response to the anti-cancer treatment of the subject, or - A method wherein an equivalent 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. 2. In the method described in Embodiment 1, a good clinical response is defined as stabilization of the disease. 3. The method according to embodiment 1 or 2, wherein poor clinical response is disease progression. 4. An in vitro method for selecting a customized treatment for a patient suffering from cancer, (i) Determine 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 at least one biomarker to a reference value. Includes, Here, - A decrease in the level of at least one biomarker relative to the reference value indicates that the selected treatment includes a drug selected from the group consisting of: a) A polypeptide containing the sequence of sequence number 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) Cells capable of secreting the polypeptide described in a) or the conjugate described in b) into a culture medium: or - Equivalent or increased levels of at least one of the biomarkers relative to the reference value indicate that the selected treatment does not include any drugs selected from the group consisting of: a) A polypeptide containing the sequence of sequence number 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and a) e) Cells capable of secreting the polypeptide described in a) or the conjugate described in b) into a culture medium. 5. In vitro methods for selecting cancer patients for treatment including drugs selected from the following groups: a) A polypeptide containing the sequence of sequence number 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) Cells that can secrete the polypeptide described in a) or the conjugate described in b) into a culture medium, The method includes the following: (i) Determine 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) The level of the at least one biomarker is compared to a reference value, and if a decrease in the level of the at least one biomarker relative to the reference value is detected, the patient is selected for the treatment. 6. A method according to any of embodiments 1 to 5, wherein the subject has not received any anti-cancer treatment within 24 hours prior to the isolation of the sample. 7. The method according to embodiment 6, wherein the subject has not received systemic anti-cancer treatment within at least four weeks prior to the isolation of the sample. 8. The method according to any one of embodiments 1 to 7, wherein the sample is blood, serum, or plasma. 9. The method according to embodiment 8, wherein the sample is a serum sample. 10. A method according to any one of embodiments 1 to 9, wherein the level of at least one biomarker determined is at the mRNA level. 11. A method according to any one of embodiments 1 to 9, wherein the level of at least one biomarker determined is at the protein level. 12. The method according to embodiment 11, wherein the level of at least one biomarker protein is determined by an immunoassay. 13. A method according to any one of embodiments 1 to 12, wherein the anticancer treatment comprises the use of a polypeptide containing the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof. 14. The method according to embodiment 13, wherein the anticancer treatment includes the use of a polypeptide comprising SEQ ID NO: 4. 15. The method according to any one of embodiments 1 to 14, wherein the cancer is a solid tumor. 16. The method according to any of embodiments 1 to 15, wherein the subject from which the sample was isolated had not received any anti-cancer treatment prior to the isolation of the sample. 17. The method according to any of embodiments 1 to 15, wherein the subject from which the sample was isolated had received anticancer treatment prior to the isolation of the sample, and the anticancer treatment is different from the treatment selected from the group consisting of: a) A polypeptide containing the sequence of sequence number 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) Cells that can secrete the polypeptide described in a) or the conjugate described in b) into a culture medium. 18. A method according to any one of embodiments 1 to 17, 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. 19. A method according to any one of embodiments 1 to 18, comprising determining the level of a combination of biomarkers selected from the following group: a) A combination of biomarkers containing MIP-1β and MCP-1. b) A combination of biomarkers including MIP-1β and ICAM-1. c) A combination of biomarkers including MIP-1β and CD62E. d) A combination of biomarkers including MIP-1β and IFN-γ. e) A combination of biomarkers containing MIP-1β and IL-1β. f) A combination of biomarkers including MIP-1β and IL-12. g) A combination of biomarkers containing CD62E and IL-8. 20. A method according to embodiment 19, comprising determining the level of a combination of biomarkers selected from the following group: a) A combination of biomarkers containing MIP-1β and MCP-1. b) A combination of biomarkers containing CD62E and IL-8. 21. A method according to embodiment 19 or 20, comprising determining the levels of MIP-1β, CD62E, IL-8, GM-CSF, and IL-1α. 22. Drugs selected from the following groups: a) A polypeptide containing the sequence of sequence number 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) A cell capable of secreting the polypeptide described in a) or the conjugate described in b) into a culture medium, The subject is intended for use in the treatment of cancer in the subject, and the subject is identified as a good responder to the drug by a method defined in any of embodiments 1-3 or 6-21. 23. Drugs selected from the following groups: a) A polypeptide containing the sequence of sequence number 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) A cell capable of secreting the polypeptide described in a) or the conjugate described in b) into a culture medium, It is intended for use in the treatment of cancer in the subject, and the treatment is selected by a method defined in any one of embodiments 4 or 6-21. 24. A drug for use according to either embodiment 22 or 23, wherein the drug is a polypeptide containing the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof. 25. A drug for use as described in embodiment 24, wherein the drug is a polypeptide comprising SEQ ID NO: 4. 26. A kit containing specific reagents 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. 27. The kit according to embodiment 26 further comprises a reagent specific 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. 28. A kit according to embodiment 27, wherein the kit comprises a reagent specific for determining the expression level of MIP-1β and at least additional reagents specific 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. 29. A kit according to any one of embodiments 26 to 28, wherein the reagent is an antibody capable of specifically recognizing at least one biomarker. 30. A kit according to any one of embodiments 26 to 28, wherein a reagent specific for determining the expression level of at least one biomarker constitutes at least 10% of the total amount of reagents comprising the kit. 31. A method as defined in any one of embodiments 1 to 21, using a kit as described in any one of embodiments 26 to 30, or a reagent specific 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. [Examples]

[0281] The present invention will be illustrated by the following embodiments, but these embodiments are for illustrative purposes only and do not limit the scope of the present invention.

[0282] Experiment details The administered product was OMO-103 (SEQ ID NO: 4), which corresponds to the Omomyc peptide sequence containing methionine at the N-terminus. OMO-103 is a final drug consisting of a mixture of active pharmaceutical ingredients (APIs) and excipients, and is used for intravenous infusion in clinical settings.

[0283] The patient had not received any systemic anti-cancer treatment within at least four weeks prior to sample isolation, nor had received any anti-cancer treatment within at least 24 hours prior to sample isolation.

[0284] The patients had various 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.

[0285] Blood samples were collected from patients participating in Phase I of the OMO-103 clinical trial, immediately prior to the first administration of OMO-103. OMO-103 was administered by intravenous infusion over 30 minutes at doses of 2.88 mg / kg, 4.32 mg / kg, 6.48 mg / kg, and 9.72 mg / kg. Serum was then separated using standard procedures and stored frozen at -80°C until use. Cytokine and chemokine concentrations were measured using the Luminex method with the Inflammation 20-Plex Human ProcartaPlex® kit (Invitrogen). Cytokine and chemokine concentrations were extrapolated from standard curves using ProcartaPlex Analyst software.

[0286] Patient responses to OMO-103 were measured by CT scans after three cycles (9 weeks) of treatment, based on RECIST 1.1 criteria (Eisenhauer, EA, et al. (2009). New response assessment criteria in solid cancers: revised RECIST guideline (version 1.1), Eur J Cancer 45: 228-247). Based on the RECIST results, patients were classified into advanced (PD) and stable (SD) stages. A total of 18 patients were evaluated for cytokine and chemokine analysis, with 11 classified as PD and 7 as SD.

[0287] Univariate analysis was performed to determine whether there were differences in pre-treatment cytokine and chemokine concentrations between PD patients and SD patients. The Mann-Whitney U test was used to determine whether there was a significant difference in cytokine / chemokine concentrations between the two groups (Figure 1).

[0288] The diagnostic accuracy of individual cytokines and chemokines for identifying SD patients was estimated using the area under the receiver operating characteristic curve (ROC-AUC) (Figure 2). ROC-AUC was used as a measure to comprehensively compare the predictive power of individual cytokines / chemokines. Generally, an AUC of 0.5 is considered undiscriminatory, 0.7-0.8 is acceptable, 0.8-0.9 is excellent, and 0.9 or higher is extremely good. It is known that when the AUC is 1, the model becomes a perfect classifier (DW Hosmer, S Lemeshow (2000). Applied Logistic Regression, 2nd Ed. Chapter 5, John Wiley and Sons, New York, NY (2000), pp. 160-164). Using RECIST(C3) as the binary output variable, a logistic regression model was individually fitted to each cytokine and analyzed. The Python package 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.

[0289] Cytokine / chemokine combinations that can correctly stratify patients between PD and SD were found using QLattice modeling technology (Abzu) (Rene, 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 of output variables. The algorithm employs an evolutionary approach to perform the search and selects the optimal model by minimizing the calculated error metric between the prediction and the actual output. QLattice was run within a leave-one-out cross-validation loop. That is, the algorithm was run 16 times, with one patient excluded in each iteration. Two models appeared in each iteration: MCP-1+MIP-1β and CD62E+IL-8. CD62E+MIP-1β and ICAM-1+MIP-1β appeared 15 times, IFN-gamma+MIP-1β and IL-1β+MIP-1β appeared 14 times, and IL-12+MIP-1β appeared 13 times. The ROC-AUC for all combination models was also calculated to determine predictive accuracy and power (Figure 3). Confidence interval bands for the models were estimated using parameter values ​​for all 16 models found.

[0290] result Patients who responded to OMO-103 treatment and showed disease stabilization in the third cycle had significantly lower levels of CD62E, IL-8, MIP-1β, GM-CSF, and IL-1α at baseline compared to patients whose disease progressed (Figure 1).

[0291] To investigate the predictive ability of identified cytokines and chemokines to respond to OMO-103, receiver operating characteristic (ROC) curve analysis was performed (Figure 2). The ROC-AUC values ​​for the five cytokines / chemokines that showed a significant decrease 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. All values ​​were close to or above 0.8, suggesting that these are excellent predictors of SD outcomes and possess very strong prognostic ability.

[0292] Finally, using QLattice modeling techniques, we identified cytokine combinations that could stratify and identify patients who responded to treatment and showed disease stabilization at baseline. This analysis revealed that various cytokine / chemokine combinations could correctly classify SD patients from PD patients. These combinations included 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 3A).

[0293] In the CD62E+IL-8 combination model, when IL-8 > 8.0 pg / ml, patients have a greater than 80% chance of developing PD regardless of CD62E, and when CD62E > 33,880 pg / ml, patients have a greater than 80% chance of developing PD regardless of IL-8. On the other hand, when IL-8 is less than 8.0 pg / ml, an increase in CD62E lowers the critical threshold for IL-8, and the opposite occurs when CD62E is less than 33,880 pg / ml. In the MIP-1β and MCP-1 combination, when MIP-1β > 65 pg / ml, patients have an 80% chance of developing PD regardless of MCP-1, and when MIP-1β is less than 65 pg / ml, an increase in MCP-1 lowers the critical threshold for MIP-1β.

[0294] In the ICAM-1+MIP-1β model, if MIP-1β > 55.96 pg / ml, the probability of the patient developing PD exceeds 80%, regardless of ICAM-1 levels. On the other hand, if MIP-1β < 55.96 pg / ml, this critical threshold decreases with increasing ICAM-1 levels. In the CD62E+MIP-1β model, if MIP-1β > 117.06 pg / ml, the probability of the patient developing PD exceeds 80%, regardless of CD62E levels. On the other hand, if MIP-1β < 117.06 pg / ml, this critical threshold decreases with increasing CD62E levels. In the IFN-gamma+MIP-1β model, if MIP-1β > 81.78 pg / ml, the probability of the patient developing PD exceeds 80%, regardless of IFN-gamma levels. On the other hand, when MIP-1β < 81.78 pg / ml, this critical threshold decreases with increasing IFN-γ levels. In the IL-1β + MIP-1β model, when MIP-1β > 91.18 pg / ml, the probability of the patient developing PD exceeds 80%, regardless of IL-1β levels. On the other hand, when MIP-1β < 91.18 pg / ml, this critical threshold decreases with increasing IL-1β levels. In the IL-12 + MIP-1β model, when MIP-1β > 91.18 pg / ml, the probability of the patient developing PD exceeds 80%, regardless of IL-12 levels. On the other hand, when MIP-1β < 91.18 pg / ml, this critical threshold decreases with increasing IL-12 levels.

[0295] The predictive power of the identified cytokine / chemokine combinations was re-examined using ROC curve analysis. The results showed that the AUC of the ROC curves for CD62E+IL-8 and MIP-1β+MCP-1 was very close to 1 (CD62E+IL-8, AUC = 0.97; MIP-1β+MCP-1, AUC = 1). This indicates that both models are excellent predictors. The predictive accuracy of the SD outcome (Figure 3B) improved. The AUC, and therefore the predictive power, of the combination models improved compared to individual cytokines / chemokines.

[0296] Other combinations that are excellent predictors of SD outcomes are MIP-1β+ICAM-1, MIP-1β+CD62E, MIP-1β+IFN-gamma, MIP-1β+IL-1β, and MIP-1β+IL-12. The AUC of these models is 1 (MIP-1β+IFN-gamma, 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 3B).

[0297] Taken together, these results clearly demonstrate that these cytokines, both individually and in combination with other cytokines, can be used as early predictive biomarkers for response to OMO-103 therapy.

Claims

1. An in vitro method for predicting the clinical response of a patient with cancer to an anti-cancer treatment selected from the following: a) A polypeptide containing the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) Cells that can secrete the polypeptide described in a) or the conjugate described in b) into a culture medium. Selected from the group consisting of, The method in question is as follows: (i) Determine 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 to a reference value, Here, - A decrease in the level of at least one biomarker relative to the reference value indicates a favorable clinical response to the anti-cancer treatment for the subject, or - A method wherein an equivalent 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.

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

3. An in vitro method for selecting a customized treatment for a patient with cancer, (i) Determine 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 at least one biomarker to a reference value. Includes, Here, - A decrease in the level of at least one of the biomarkers relative to the reference value indicates that the selected treatment includes a drug selected from the group consisting of: a) A polypeptide containing the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) Cells capable of secreting the polypeptide described in a) or the conjugate described in b) into a culture medium: or, - Equivalent or increased levels of at least one of the biomarkers relative to the reference value indicate that the selected treatment does not include any drugs selected from the group consisting of: a) A polypeptide containing the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) Cells capable of secreting the polypeptide described in a) or the conjugate described in b) into a culture medium, The way of doing so.

4. An in vitro method for selecting a subject with cancer for treatment including a drug selected from the following group, a) A polypeptide containing the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) Cells capable of secreting the polypeptide described in a) or the conjugate described in b) into a culture medium, The method includes the following: (i) Determine 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) A method comprising comparing the level of the at least one biomarker to a reference value, wherein if a decrease in the level of the at least one biomarker relative to the reference value is detected, the patient is selected for the treatment.

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

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

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

8. The method according to any one of claims 1 to 7, wherein the anti-cancer treatment includes the use of a polypeptide comprising SEQ ID NO:

4.

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

10. Drugs selected from the following group: a) A polypeptide containing the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof; b) A polypeptide comprising the sequence of Sequence ID No. 1 or a functionally equivalent variant thereof, and a conjugate comprising a chemical moiety that promotes cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) A polynucleotide encoding the polypeptide of a) or the conjugate of b); d) A vector comprising the polynucleotide described in c); and e) A cell capable of secreting the polypeptide described in a) or the conjugate described in b) into a culture medium, A drug for use in the treatment of a target cancer, wherein the target is identified as a good responder to the drug by the method of claim 1 or 2 or any one of claims 6 to 10, or the treatment is selected by the method of claim 3 or any one of claims 5 to 9.

11. A kit comprising a specific reagent 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, further comprising a reagent specific 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 claim 11 or 12, wherein the reagent is an antibody capable of specifically recognizing at least one biomarker.

14. The kit according to any one of claims 11 to 13, wherein a reagent specific for determining the expression level of at least one biomarker constitutes at least 10% of the total amount of reagents comprising the kit.

15. The method according to any one of claims 1 to 9, using a reagent specific 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, or using a kit according to any one of claims 11 to 14.

Images