Method for assessing or monitoring response to cancer treatment

Description

1 / 118 “METHOD FOR EVALUATING OR MONITORING THE RESPONSE TO CANCER TREATMENT FIELD OF THE INVENTION

[0001] The present invention relates to the field of cancer and, more particularly, to methods for evaluating or monitoring the response of an individual diagnosed with cancer and treated with an anticancer treatment to said treatment based on the detection of biomarkers in a sample from the individual. It also relates to the anticancer agents used in the treatments mentioned in the methods based on the detection of said biomarkers. It further relates to kits and their use in said methods. BACKGROUND OF THE INVENTION

[0002] Cancer is one of the leading causes 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 is tightly regulated in normal cells, where its levels are higher in proliferating cells and lower in non-proliferating cells. Abnormally high and / or dysregulated Myc activity is causally implicated in most cancers and is frequently associated with aggressive, poorly differentiated, angiogenic, and treatment-resistant tumors.

[0004] Omomyc is a dominant-negative Myc mutant comprising the b-HLH-LZ domain of Myc and harboring four amino acid substitutions in the leucine zipper of Myc (Soucek, L. et al., 1998, Oncogene 17, 2463-2472; Soucek, L. et al. (2002), Cancer Res 62: 3507-3510). The substitutions Petition 870250097700, dated 10 / 24 / 2025, p. 6 / 145 2 / 118 of the amino acids E61T, E68I, R74Q, and R75N ​​confer altered dimerization specificity to the protein, which retains the ability to bind to its natural partner Max and to form homodimers with itself, as well as heterodimers with wild-type c-, N-, and L-Myc.

[0005] Due to these properties, Omomyc is able to prevent Myc-dependent gene transactivation functions both in vitro and in vivo by abolishing Myc's ability to bind to its DNA recognition site, the E-box. At the same time, Omomyc strongly potentiates Myc-induced apoptosis in a manner dependent on the level of Myc expression and thus strengthens Myc's transrepressive activity. Omomyc, therefore, prevents Myc binding to promoter E-boxes and transactivation of target genes, while maintaining Miz-1-dependent binding to promoters and transrepression. In the presence of Omomyc, the Myc interactome is channeled towards repression and its activity changes from pro-oncogenic to tumor suppressor.

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

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

[0008] However, anticancer therapies may have limitations, such as poor response to treatment or Petition 870250097700, dated 10 / 24 / 2025, page 7 / 145 3 / 118 the emergence of innate or acquired resistance. Therefore, detecting whether a patient who has been treated with an anticancer treatment is responding to the treatment as quickly as possible is a challenge. Early detection of a good or bad response results in an increased survival rate for cancer patients, avoiding unnecessary therapeutic side effects and treatment delays, since those patients who do not respond to a treatment may be candidates for alternative treatments.

[0009] Therefore, there is a need for a non-invasive, fast, simple and economical method that allows for the early detection of a patient's response to treatment. BRIEF SUMMARY OF THE INVENTION

[0010] In a first aspect, the invention relates to an in vitro method for evaluating or monitoring the clinical response of an individual suffering from cancer to an anticancer treatment selected from the group consisting of: a) a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) a polynucleotide that codes for the polypeptide in a) or the conjugate of b); d) a vector comprising the polynucleotide of c); and e) a cell capable of secreting into the environment Petition 870250097700, dated 10 / 24 / 2025, p. 8 / 145 4 / 118 polypeptide of a) or the conjugate of b); the aforementioned method comprising: (i) determine the level of at least one biomarker selected from the group consisting of IL-17A, IFN-γ, and CD62E in a sample from the individual obtained after treatment administration, and (ii) compare the level of said at least one biomarker with a reference value, where: - an increased level of at least one biomarker compared to the reference value is indicative of a good clinical response of the individual to cancer treatment, or - A level equal to or lower than the reference value for at least one biomarker is indicative of an unsatisfactory clinical response to cancer treatment.

[0011] In a second aspect, the invention relates to an agent selected from the group consisting of: a) a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof; (b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) a polynucleotide that codes for the polypeptide in a) or the conjugate of b); Petition 870250097700, dated 10 / 24 / 2025, page 9 / 145 5 / 118 d) a vector comprising the polynucleotide according to c); and (e) a cell capable of secreting into the environment the polypeptide according to a) or the conjugate according to b); for use in the treatment of cancer in an individual, wherein the individual has been identified as a good responder to said agent by the first method of the invention.

[0012] In a third aspect, the 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 IL-17A, IFN-γ and CD62E.

[0013] In yet another aspect, the invention relates to the use of a kit of the invention or a specific reagent to determine the expression level of at least one biomarker selected from the group consisting of IL-17A, IFN-γ, CD62E, MCP-1, IL-1α, MIP-1β, TNF-α, MIP-1α, CD62P and IL-10 in the first method of the invention. DESCRIPTION OF THE INVENTION

[0014] Figure 1. Patients with stabilized disease at C3 show significantly increased levels of IFN-γ, CD62E, and IL-17A after OMO-103 infusion. The levels of the different soluble factors were measured in serum samples from patients collected at different time points after treatment with OMO-103. Levels were determined using the Luminex technique. Four patients with progressive disease (PD) and seven with stable disease (SD) were included in the analysis. A) Biomarkers increased Petition 870250097700, dated 10 / 24 / 2025, page 10 / 145 6 / 118 significantly in PD patients compared to PD patients. ns, not significant. B) All patients who underwent disease stabilization in the cycle showed a significant increase in IFN-γ (p = 0.000148), CD62E (p = 0.000338), and IL-17A (p = 0.0013) levels after OMO-103 infusion compared to PD patients at the start of cycle 3. The mean and standard error of the mean (SEM) are shown. One patient did not show this increase at C3, but showed a significant increase at C6.

[0015] Figure 2. Individual models of IFN-γ, CD62E and IL-17A can be used to identify patients with PD and PD based on their soluble factor levels after treatment with OMO-103. Individual models of IFN-γ, CD62E, and IL-17A were generated using QLattice technology. A) Models considering the maximum serum levels of the indicated cytokines and their respective ROC (Receptor Operating Characteristic) curves and their Area Under the ROC Curve (AUC) values. B) Models generated using the serum levels of all measured time points and their respective ROC curves and their AUC values. Serum levels of patients with PD are indicated as 1 (gray dots) and serum levels of PD are indicated as 0 (empty dots). Dashed lines correspond to confidence ranges (CI), with black indicating the mean, dark gray the 5% CI, and light gray the 95% CI.

[0016] Figure 3. Models encompassing combinations of two cytokines are excellent predictors of response to OMO-103 and can be used to stratify patients with SD and PD. Soluble factor combination models were generated using QLattice technology. Petition 870250097700, dated 10 / 24 / 2025, page 11 / 145 7 / 118 considering the levels of soluble factors at all times after OMO-103 infusion. Graphs of the IL-17A + MCP-1, IL-17A + IL-Iα, IL-17A + MIP-Iβ, IL-17A + TNFα, IL-17A + MIP-Iα, IL-17A + CD62P, IL-17A + IL10, and IL-17A + CD62E combination models and how they stratify patients are shown. Stable Disease (SD) is indicated by empty points and Progressive Disease (PD) is indicated by gray. The gray lines correspond to the confidence intervals (CI), with dark indicating the mean, medium gray the 5% CI, and light gray the 95% CI. The ROC (Receiver Operating Characteristic) curve analysis of the combination models and the AUC are shown below each combination model. DETAILED DESCRIPTION OF THE INVENTION

[0017] The inventors of the present invention have discovered that, surprisingly, post-treatment serum levels of the biomarkers IL-17A, IFN-γ, and CD62E increase in cancer patients who respond better to therapy with an Omomyc-containing polypeptide after having been treated with said therapy.

[0018] Therefore, these biomarkers have potential value for early assessment of whether a cancer patient who has received Omomyc-based treatment or a functional equivalent has responded to treatment. This allows a patient to be classified as a good or poor responder quickly, simply, economically, and non-invasively, soon after treatment and without the need to wait for a CT scan performed by experienced professionals. In addition, these biomarkers can also be used to monitor the response to said treatment and detect when a patient begins or stops responding. Petition 870250097700, dated 10 / 24 / 2025, page 12 / 145 8 / 118

[0019] Based on these findings, the inventors developed the methods of the present invention in their different embodiments, which will be described in detail below.

[0020] The results provided in the examples of the present invention clearly show a significant association of high post-treatment levels of IL-17A, IFN-γ and CD62E with a good response to treatment with OMO-103.

[0021] Thus, these results suggest that individuals with cancer and high levels of these biomarkers in a post-treatment sample are candidates for treatment based on Omomyc or a functionally equivalent variant. METHODS FOR EVALUATING OR MONITORING THE RESPONSE TO THE INVENTION

[0022] In a first aspect, the invention relates to an in vitro method for evaluating or monitoring the clinical response of an individual suffering from cancer to an anticancer treatment selected from the group consisting of: a) a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof; (b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) a polynucleotide that codes for the polypeptide in a) or the conjugate of b); d) a vector comprising the polynucleotide of Petition 870250097700, dated 10 / 24 / 2025, page 13 / 145 9 / 118 c); and (e) a cell capable of secreting into the environment the polypeptide of a) or the conjugate of b); the aforementioned method comprising: (i) determine the level of at least one biomarker selected from the group consisting of IL-17A, IFN-γ, and CD62E in a sample from the individual obtained after treatment administration, and (ii) compare the level of said at least one biomarker with a reference value, where: - an increased level of at least one biomarker compared to the reference value is indicative of a good clinical response of the individual to cancer treatment, or - A level equal to or lower than the reference value for at least one biomarker is indicative of a poor clinical response to cancer treatment.

[0023] The term “in vitro”, as used in this document, refers to the fact that an experimental protocol or method is not performed on the body of a human or animal individual, but on isolated samples from said individual and already present in a laboratory instrument, such as a test tube, plate or dish.

[0024] The expression “evaluate or monitor the clinical response” to a treatment refers to the possibility of determining the response to an anticancer treatment of an individual with cancer who has been treated with said treatment. In particular, the term “evaluate” or “monitor”, Petition 870250097700, dated 10 / 24 / 2025, page 14 / 145 10 / 118 as used in this document refers to an individual assessment or monitoring of the response of an individual with cancer after treatment with an anticancer treatment based on Omomyc or a functionally equivalent variant, as defined in items (a) to (e) of the first aspect of the invention. Specifically, monitoring the response to a cancer treatment allows detecting whether a good responder to treatment has been converted into a poor responder, or whether a poor responder to treatment is converted into a good responder.

[0025] The term clinical response, as used herein, refers to the response of an individual with cancer to an anticancer treatment based on Omomyc or a functionally equivalent variant, as defined in items (a) to (e) of the first aspect of the invention. The standard criteria (Eisenhauer, EA et al. 2009. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 45(2): 228-247) which may be used here to assess response to an anticancer therapy include response, stabilization, and progression. The term RECIST, as used here, refers to Response Evaluation The Criteria for Infectious and Solid Tumors (RECIST) is a standard way to measure a cancer patient's response to treatment. It is based on the reduction, maintenance, or increase in tumor size. To use RECIST, there must be at least one tumor that can be measured on X-rays, computed tomography (CT) scans, or magnetic resonance imaging (MRI). The types of response a patient may present are complete response (CR), partial response (PR), progressive disease (PD), and stable disease (SD). In one modality Petition 870250097700, dated 10 / 24 / 2025, page 15 / 145 11 / 118 preferred, the RECIST criteria were used to assess tumor response.

[0026] In the context of the present invention, individuals who achieved a complete or partial response and individuals who presented with stable disease were considered good responders or clinically good responders.

[0027] A complete response (or complete remission) (CR), as used herein, is the disappearance of all detectable malignant disease, i.e., the disappearance of all target lesions, where any pathological lymph nodes (whether target or non-target) must show a reduction in the short axis to <10 mm.

[0028] A “partial response” (PR) is defined here as a decrease of at least 30% in the sum of the diameters of the target lesions, taking the sum of the baseline diameters as a reference.

[0029] The term “stabilization”, “disease stabilization” or “stable disease” (SD), as used herein, is considered when there is not sufficient reduction to qualify as a partial response, nor sufficient increase to qualify as progressive disease, taking as a reference the smallest sum of diameters during the study.

[0030] In the context of the present invention, individuals who exhibit progressive disease are considered “poor responders” or have a poor clinical response.

[0031] The term “progression” or “progressive disease” (PD), as used herein, is defined as an increase of at least 20% in the sum of the diameters of Petition 870250097700, dated 10 / 24 / 2025, page 16 / 145 12 / 118 target lesions, taking as a reference the lowest sum in the study (this includes the baseline sum, if it is the lowest in the study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. The appearance of one or more new lesions is also considered progression.

[0032] As a person skilled in the art will understand, having an unsatisfactory response to anticancer treatment does not mean that the individual has no response or that the treatment has no effect on him. For example, anticancer treatment may reduce lesion growth in a patient with an unsatisfactory response when compared to a patient who does not undergo any treatment. But the effect achieved in said individual, when compared to an untreated patient, does not qualify to be considered stabilization according to the criteria used in the present invention. For example, a patient treated with anticancer therapy may have a 20% increase in the sum of the diameters of the target lesions, but this increase could have been 30% if the patient had not been treated.

[0033] As will be understood by those versed in the technique, such an assessment is generally not intended to be correct for all (i.e., 100%) of the individuals to be identified. The term, however, requires that the prediction provide correct results for a statistically significant portion of the individuals. Whether a portion is statistically significant can be determined without further ado by the expert in the technique using various statistical assessment tools. Petition 870250097700, dated 10 / 24 / 2025, page 17 / 145 13 / 118 known, for example, determination of confidence intervals, determination of p-value, Student's t-test, Mann-Whitney test, etc. Details can be found in Dowdy and Wearden, Statistics for Research, 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 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, or less. More preferably, at least 60%, at least 70%, at least 80%, or at least 90% of the individuals in a population can be adequately identified by the method of the present invention.

[0034] Any other widely accepted parameter for comparing the effectiveness of alternative treatments may be used to determine the response to a treatment, including, without limitation: • disease-free progression, which, as used in this document, describes the proportion of individuals in complete remission who did not experience disease recurrence during the study period. • disease-free survival (DFS), which, as used in this document, is understood as the period of time after treatment of a disease during which an individual survives without signs of the disease. • objective response, which, as used in the present invention, describes the proportion of treated individuals in whom a complete or partial response is observed. • tumor control, which, as used Petition 870250097700, dated 10 / 24 / 2025, page 18 / 145 14 / 118 in this document refers to the proportion of treated individuals in whom a complete response, partial response, mild response, or stable disease > 6 months is observed. • Progression-free survival, which, as used in this document, is defined as the time from the start of treatment until the first measurement of cancer growth. • Time to progression (TTP), as used in this document, refers to the time after treatment of a disease until it begins to worsen. The term progression has already been defined previously. • progression-free survival at six months or PFS6 rate, which, as used in this document, refers to the percentage of individuals who are progression-free in the first six months after the start of therapy. • median survival, which, as used in this document, refers to the time that half of the individuals included in the study are still alive. • Overall survival, which, as used in this document, refers to the period of time between the date of diagnosis or the start of treatment for a disease, such as cancer, during which patients diagnosed with the disease are still alive. • recurrence, which, as used in this document, refers to the appearance of cancer after treatment and after a period in which the cancer was not detected. • metastasis-free survival, as used Petition 870250097700, dated 10 / 24 / 2025, page 19 / 145 15 / 118 in this document refers to the period after the end of cancer treatment in which the patient survives without any signs or symptoms of cancer metastasis. • Decrease in circulating tumor cells, as used in this document, refers to a decrease in the concentration of circulating tumor cells in the blood or lymph of a patient with metastatic cancer. A decrease in circulating tumor cells is associated with the effectiveness of a therapy against metastatic cancer. • The response of circulating markers, as used in this document, refers to the change in the concentration in the blood or lymph of proteins or nucleic acids associated with a specific cancer after treatment for that specific cancer.

[0035] The method of the invention is performed on an individual suffering from cancer, that is, on an individual who has been previously diagnosed with cancer.

[0036] The term individual or patient, as used in this document, refers to all animals classified as mammals, including but not limited to domestic and farm animals, primates, and humans, for example, human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents. Preferably, the individual is a human being, male or female, of any age or race. In the context of the present invention, the individual is an individual suffering from cancer or previously diagnosed with cancer. In a preferred embodiment, the individual is a mammal, preferably a human being.

[0037] The individuals tested in the first Petition 870250097700, dated 10 / 24 / 2025, page 20 / 145 16 / 118 method of the invention were previously diagnosed with cancer. The term diagnosed, as used in this document, refers to the determination and / or identification of a disease in an individual, i.e., the opinion reached on the disease state of an individual, i.e., the diagnostic opinion. As such, it can also be considered an attempt to classify individuals depending on their disease condition. As will be understood by those skilled in the art, the diagnosis of cancer, although preferred, does not need to be correct for 100% of individuals to be diagnosed or evaluated. The term, however, requires that a statistically significant proportion of individuals identified as such suffer from cancer. Methods for determining whether a proportion of individuals is statistically significant have been disclosed above in relation to the method of evaluating or monitoring the response.

[0038] The methods of the invention are suitable for any type of cancer.

[0039] The term cancer refers to a group of diseases characterized by uncontrolled cell division (or by increased survival or resistance to apoptosis) and the ability of these cells to invade neighboring tissues (invasion) and spread to other areas of the body where the cells are not normally located (metastasis) through lymphatic and blood vessels, circulate through the bloodstream, and then invade normal tissues in other parts of the body. Depending on their ability to spread by invasion and metastasis, tumors are classified as benign or malignant: benign tumors are tumors that cannot spread by Petition 870250097700, dated 10 / 24 / 2025, page 21 / 145 17 / 118 Invasion or metastasis, meaning they grow only locally; while malignant tumors are tumors capable of spreading through invasion and metastasis. Biological processes known to be related to cancer include angiogenesis, immune cell infiltration, cell migration, and metastasis.

[0040] The term cancer includes, without limitation, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), hairy cell leukemia, polycythemia vera, lymphoma (e.g., Hodgkin's disease or non-Hodgkin's disease), CNS lymphoma, AIDS-associated leukemias, Waldenstrom's macroglobulinemia, multiple myeloma, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, mendotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, Kaposi's sarcoma, colon carcinoma, pancreatic cancer, lung cancer, colon cancercolorectal cancer, bladder cancer, breast cancer, biliary tract cancer, esophageal cancer, stomach / gastric cancer, ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; prostate cancer, oral cancer, including squamous cell carcinoma, carcinoma, Petition 870250097700, dated 10 / 24 / 2025, page 22 / 145 18 / 118 basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, teratoma, choriocarcinoma, endometrial / uterine / cervical cancer, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, intraepithelial neoplasias, including Bowen's disease and Paget's disease, neuroglioma, glioma, mixed glioma, optic nerve glioma, subependymoma, metastatic brain tumor, pituitary tumors, primitive neuroectodermal tumor (PNET), juvenile pilocytic astrocytoma (JPA), brainstem glioma, astrocytoma, pineal tumor, tumor rhabdoid, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma and retinoblastoma).

[0041] The term “cancer” also includes, without limitation, head and neck cancer, leukemia, heart cancer, esophageal cancer, small bowel cancer, spleen cancer, kidney cancer, brain cancer, choriocarcinoma, skin cancer, bone cancer, bone marrow cancer, blood cancer, thymus cancer, uterine cancer, liver cancer, sarcoma, liposarcoma, fibrosarcoma, Merkel cell carcinoma, Kaposi's sarcoma, testicular cancer including germ cell tumors such as seminoma, non-seminoma (teratomas, Petition 870250097700, dated 10 / 24 / 2025, page 23 / 145 19 / 118 choriocarcinomas), stromal tumors and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullary carcinoma; renal cancer including adenocarcinoma and Wilms' tumor; cholangiocarcinoma, glioblastoma, hematological malignancies, including acute lymphocytic and myeloid leukemia, acute lymphoblastic T-cell leukemia / lymphoma, hairy cell leukemia, chronic myeloid leukemia, multiple myeloma, AIDS-associated leukemias and adult T-cell leukemia / lymphoma, intraepithelial neoplasias, including Bowen's disease and Paget's disease, lymphomas, including Hodgkin's disease and lymphocytic lymphomas, oral cancer, including squamous cell carcinoma, adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioma, hemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, medulloblastoma, melanoma, parotid gland cancer, neuroblastoma, hepatobiliary cancer, adrenal cancer, osteosarcoma, retinoblastoma,rhabdomyosarcoma and teratoma. In addition, this term includes acral lentiginous melanoma, actinic keratosis adenocarcinoma, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, astrocytic tumors, Bartholin's gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, 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 tumors, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma. Petition 870250097700, dated 10 / 24 / 2025, page 24 / 145 20 / 118 insulinoma, intraepithelial neoplasia, squamous cell intraepithelial neoplasia, invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma, malignant melanoma, malignant mesothelial tumor, medulloblastoma, medulloepithelioma, mucoepidermoid carcinoma, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, papillary serous adenocarcinoma, pituitary tumors, plasmacytoma, pseudosarcoma, lung blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, serous carcinoma, microcytic carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vipoma, Wilms' tumor, intracerebral cancer, rectal cancer, astrocytoma, microcytic cancer and non-microcytic cancer, metastatic melanoma, cancer androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer.

[0042] Cancer includes, in another category, without limitation, mesothelioma, hepatobiliary (hepatic and biliary duct), bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, gastrointestinal cancer (gastric, colorectal and duodenal), uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid gland cancer, parathyroid gland cancer, cancer of Petition 870250097700, dated 10 / 24 / 2025, page 25 / 145 21 / 118 adrenal gland, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphomas, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis carcinoma, non-Hodgkin lymphoma, spinal axis tumors, brainstem glioma, pituitary adenoma, adrenocortical cancer, gallbladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the above cancers.

[0043] In some modalities, cancer is selected from hepatocellular carcinoma, ovarian cancer, epithelial ovarian cancer, fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hepatocholangiocarcinoma; synovial sarcoma of soft and bone tissues; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing's sarcoma; anaplastic thyroid cancer; adrenocortical adenoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / stomach cancer (GIST); lymphoma; squamous cell carcinoma of the head and neck (SCCHN); salivary gland cancer; glioma or brain cancer; malignant peripheral nerve sheath tumors (MPNST) associated with neurofibromatosis-1; Waldenstrom's macroglobulinemia or medulloblastoma.

[0044] In some modalities, the cancer is selected from hepatocellular carcinoma (HCC), Petition 870250097700, dated 10 / 24 / 2025, page 26 / 145 22 / 118 hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, epithelial ovarian cancer, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), hepatocholangiocarcinoma, synovial sarcoma of soft and bone tissues, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid cancer, adrenocortical adenoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, malignant peripheral nerve sheath tumors associated with neurofibromatosis-1 (MPNST), Waldenstrom's macroglobulinemia or medulloblastoma.

[0045] In a preferred embodiment, cancer is a solid tumor.

[0046] Examples of solid tumors are sarcoma, carcinoma, or lymphoma. Solid tumors generally comprise an abnormal mass of tissue that typically does not include cysts or fluid-filled areas. In some modalities, the cancer is selected from renal cell carcinoma, or kidney cancer; hepatocellular carcinoma (HCC) or hepatoblastoma, or liver cancer; melanoma; breast cancer; colorectal carcinoma, or 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 cancer, ovarian carcinoma, or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; Hepatocholangiocarcinoma; synovial sarcoma of soft tissue and bone; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Petition 870250097700, dated 10 / 24 / 2025, p. 27 / 145 23 / 118 Ewing's sarcoma; anaplastic thyroid cancer; adrenocortical carcinoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / stomach cancer (GIST); lymphoma; squamous cell carcinoma of the head and neck (SCCHN); salivary gland cancer; glioma or brain cancer; malignant peripheral nerve sheath tumors associated with neurofibromatosis1 (MPNST); Waldenstrom's 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, preferably from NSCLC and colorectal cancer.In a particular embodiment of the invention, the solid cancer 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; more particularly, it is selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), salivary gland carcinoma, sarcoma, and pleural mesothelioma; even more preferably, it is selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), sarcoma, and salivary gland carcinoma.

[0047] In some modalities, the cancer is selected from among hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, cancer of Petition 870250097700, dated 10 / 24 / 2025, page 28 / 145 24 / 118 ovary, epithelial ovarian cancer, ovarian carcinoma, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), hepatocholangiocarcinoma, synovial sarcoma of soft and bone tissues, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid cancer, adrenocortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, malignant peripheral nerve sheath tumors associated with neurofibromatosis-1 (MPNST), Waldenstrom's macroglobulinemia or medulloblastoma.

[0048] In some modalities, the cancer is hepatocellular carcinoma (HCC). In some modalities, the cancer is hepatoblastoma. In some modalities, the cancer is colon cancer. In some modalities, the cancer is rectal cancer. In some modalities, the cancer is ovarian cancer, or ovarian carcinoma. In some modalities, the cancer is epithelial ovarian cancer. In some modalities, the cancer is fallopian tube cancer. In some modalities, the cancer is papillary serous cystadenocarcinoma. In some modalities, the cancer is uterine papillary serous carcinoma (UPSC). In some modalities, the cancer is hepatocholangiocarcinoma. In some modalities, the cancer is synovial sarcoma of soft and bone tissue. In some modalities, the cancer is rhabdomyosarcoma. In some modalities, the cancer is osteosarcoma. In some modalities, the cancer is anaplastic thyroid cancer. In some forms, the cancer is adrenocortical carcinoma.In some forms, the cancer is pancreatic cancer, or pancreatic ductal carcinoma. In some forms, the cancer is pancreatic adenocarcinoma. In some forms... Petition 870250097700, dated 10 / 24 / 2025, page 29 / 145 25 / 118 the cancer is glioma. In some modalities, the cancer is malignant peripheral nerve sheath tumors (MPNST). In some modalities, the cancer is MPNST associated with neurofibromatosis1. In some modalities, the cancer is Waldenstrom's macroglobulinemia. In some modalities, the cancer is medulloblastoma.

[0049] In some modalities, a cancer is a virus-associated cancer, including solid tumors associated with human immunodeficiency virus (HIV), incurable solid tumors positive for human papillomavirus (HPV)-16, adult T-cell leukemia, which is caused by human T-cell leukemia virus type I (HTLV-I). It is a highly aggressive form of CD4+ T-cell leukemia characterized by clonal integration of HTLV-I into leukemic cells; as well as virus-associated tumors in gastric cancer, nasopharyngeal carcinoma, cervical cancer, vaginal cancer, vulvar cancer, squamous cell carcinoma of the head and neck, Merkel cell carcinoma.

[0050] Other cancers will be known to someone with common knowledge of the technique.

[0051] The term cancer includes both primary and metastatic tumors. In one embodiment, the cancer is a primary tumor. The term “primary tumor,” as used in this document, refers to a tumor that originated in the site or organ where it is present and has not metastasized to that site from another location. Therefore, in one embodiment, the cancer to be treated is a non-metastatic cancer. In another embodiment, the cancer is a metastatic cancer or cancer metastasis. In the context of the present Petition 870250097700, dated 10 / 24 / 2025, page 30 / 145 26 / 118 invention, metastasis is understood as the spread of cancer from the organ where it originated to a different organ.

[0052] The anticancer treatment of the invention is capable of decreasing 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.

[0053] The term treat or treatment refers to a therapeutic treatment, as well as a prophylactic or preventive method, where the objective is to prevent or reduce an undesirable physiological alteration of a disease, such as cancer. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction in disease duration, stabilization of the pathological state (specifically not deteriorated), delay in disease progression, improvement of the pathological state and remission (partial and total), both detectable and undetectable. Treatment can also mean prolonging survival, compared to the survival expected if treatment is not applied. Those who need treatment include those suffering from cancer.

[0054] The authors of the present invention have discovered several biomarkers that are significantly increased in post-treatment samples from individuals who respond well to treatment with OMO-103, which is a polypeptide comprising Omomyc and possessing SEQ ID NO: 4. All Omomyc-based agents act on the same target. Therefore, the biomarkers of the invention will be Petition 870250097700, dated 10 / 24 / 2025, page 31 / 145 27 / 118 significantly increased also in good responders to treatment with any other Omomyc-based agent, whether administered as a polypeptide, a conjugate with other chemical fractions, a nucleic acid or a vector for gene therapy or in cell therapy as a cell capable of secreting the polypeptide or conjugate into the medium.

[0055] Therefore, in the context of the present invention, the method of the invention allows evaluating or monitoring the clinical response to an anticancer treatment selected from the group consisting of: a) a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof; (b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) a polynucleotide that codes for the polypeptide in a) or the conjugate of b); d) a vector comprising the polynucleotide of c); and e) a cell capable of secreting into the environment the polypeptide of a) or the conjugate of b).

[0056] The term “anticancer treatment”, in the context of the first method of the invention, refers to any treatment comprising exposing the individual to be treated to a method used to induce the death of cancer cells selected from items (a) to (e) cited above. Preferably, the anticancer treatment is Petition 870250097700, dated 10 / 24 / 2025, page 32 / 145 28 / 118 administered intravenously; preferably as an intravenous infusion over 30-45 minutes. Preferably, the anticancer treatment is administered once a week.

[0057] In a preferred embodiment, the anticancer treatment comprises a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof, more preferably a polypeptide comprising the sequence SEQ ID NO: 1.

[0058] The terms The terms polypeptide and peptide are used interchangeably in this document to refer to amino acid polymers of any length. The polypeptide of the invention may comprise modified amino acids and may be interrupted by non-amino acids. In a preferred embodiment, the polypeptide is formed exclusively from amino acids. Preferably, the polypeptide of item (a) of the anticancer treatment has a length between 80 and 500 amino acids, more preferably between 80 and 300 amino acids, more preferably between 80 and 250 amino acids, more preferably between 80 and 150, even more preferably between 80 and 130 amino acids, preferably between 90 and 130 amino acids, preferably not more than 125 amino acids, more preferably not more than 100 amino acids. In a preferred embodiment, the polypeptide has a length between 90 and 98 amino acids, preferably between 90 and 95 amino acids, more preferably 91 amino acids.

[0059] The term amino acid refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Furthermore, the Petition 870250097700, dated 10 / 24 / 2025, page 33 / 145 29 / 118 The term amino acid includes D- and L-amino acids (stereoisomers). Preferably, amino acids are L-amino acids.

[0060] The term natural amino acids or naturally occurring amino acids comprises the 20 naturally occurring amino acids; those amino acids that are frequently modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other uncommon amino acids, including, among others, 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine and ornithine.

[0061] As used herein, the term non-natural amino acid or synthetic amino acid refers to a carboxylic acid, or a derivative thereof, substituted at the α position by an amine group and structurally related to a natural amino acid. Illustrative but not limiting examples of modified or unusual 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, Netylsparagine, hydroxylysine, alliohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyl-lysine, N-methylvaline, norvaline, norleucine, ornithine, etc.

[0062] The polypeptide of the present invention may also comprise non-amino acid fractions, such as, Petition 870250097700, dated 10 / 24 / 2025, page 34 / 145 30 / 118 for example, hydrophobic moieties (various linear, branched, cyclic, polycyclic or heterocyclic hydrocarbons and hydrocarbon derivatives) attached to peptides; various protecting groups that are attached to the compound's terminals to decrease 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.

[0063] Chemical groups (non-amino acid) present in the polypeptide may be included to improve various physiological properties, such as decreased degradation or clearance; decreased repulsion by various cellular pumps; improvement of various administration modes; increased specificity; increased affinity; increased stability, increased bioavailability, increased solubility; decreased toxicity and the like.

[0064] Mimetics include molecules that mimic the chemical structure of a peptide structure and retain the functional properties of the peptide structure. Approaches to the development of peptide analogs, derivatives, and mimetics are known in the art.

[0065] In one embodiment, the polypeptide of the invention is a polypeptide consisting of the sequence SEQ ID NO: 1 or a polypeptide consisting of a functionally equivalent variant of SEQ ID NO: 1, preferably it is a polypeptide consisting of the sequence SEQ ID NO: 1.

[0066] SEQ ID NO: 1 corresponds to TEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQA ETQKLISEIDLLRKQNEQLKHKLEQLRNSCA (SEQ ID NO: 1)

[0067] The polypeptide with sequence SEQ ID NO: 1 Petition 870250097700, dated 10 / 24 / 2025, page 35 / 145 31 / 118 corresponds to the Omomyc protein sequence. The term Omomyc, as used in this document, refers to a polypeptide consisting of a mutated version of the bHLHZip domain of Myc bearing the E61T, E68I, R74Q, and R75N ​​mutations (where the numbering of the mutated positions is given relative to the sequence of the Myc region corresponding to amino acids 365-454 of the polypeptide, as defined under accession number NP_002458 in the NCBI database, version of March 15, 2015). The c-Myc sequence provided in the NCBI database under accession number NP_002458 is shown below (SEQ ID NO: 2), where the region from which Omomyc derives is shown underlined: MDFFRVVENQ QPPATMPLNV SFTNRNYDLD YDSVQPYFYC DEEENFYQQQ QQSELQPPAP SEDIWKKFEL LPTPPLSPSR RSGLCSPSYV AVTPFSLRGD NDGGGGSFST ADQLEMVTEL 121 LGGDMVNQSF ICDPDDETFI KNIIIQDCMW SGFSAAAKLV SEKLASYQAA RKDSGSPNPA 181 RGHSVCSTSS LYLQDLSAAA SECIDPSVVF PYPLNDSSSP KSCASQDSSA FSPSSDSLLS 241 STESSPQGSP EPLVLHEETP PTTSSDSEEE QEDEEEIDVV SVEKRQAPGK RSESGSPSAG 301 GHSKPPHSPL VLKRCHVSTH QHNYAAPPST RKDYPAAKRV KLDSVRVLRQ ISNNRKCTSP 361 RSSDTEENVK RRTHNVLERQ RRNELKRSFF ALRDQIPELE NNEKAPKVVI LKKATAYILS 421 VQAEEQKLIS EEDLLLRKRRE QLKHKLEQLR NSCA (SEQ ID NO: 2)

[0068] Omomyc also contains the M2 domain of cMyc, possessing the sequence RQRRNELKRSF (SEQ ID NO: 3) (see Dang and Lee, Mol.Cell. Biol., 1988, 8:4048-4054) (double underline above), which corresponds to a nuclear localization signal.

[0069] Omomyc is characterized by exhibiting enhanced dimerization capacity with all three oncogenic Myc proteins (c-Myc, N-Myc, and L-Myc). Omomyc Petition 870250097700, dated 10 / 24 / 2025, p. 36 / 145 32 / 118 can be derived from the bHLHZip domain of any Myc protein known in the art, provided that the mutations resulting in the tumor suppressor effect are preserved. Thus, the Omomyc that can be used in the present invention can be derived from any mammalian species, including, but not limited to, domestic and farm animals (cows, 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, publication of March 12, 2019).

[0070] The term Myc, as used herein, refers to a family of transcription factors that includes c-Myc, N-Myc, and L-Myc. The Myc protein activates the expression of many genes by binding to the CACGTG consensus sequence (Enhancer Box or E-box sequences and recruitment of histone acetyltransferases or HATs). However, Myc can also act as a transcriptional repressor. By binding to the transcription factor Miz-1 and displacing the p300 coactivator, it inhibits the expression of Miz-1 target genes. Myc also plays a direct role in controlling DNA replication.

[0071] The b-HLH-LZ domain or basic region of Myc, helix-loop-helix, leucine zipper, refers to a region that determines the dimerization of Myc with the Max protein and the binding to Myc target genes. This region corresponds to amino acids 365-454 of human Myc and is characterized by two alpha helices connected by a loop (Nair, SK, & Burley, SK, 2003, Cell, 112: 193-205).

[0072] In a preferred embodiment, the polypeptide of the invention is a polypeptide comprising or consisting essentially of the SEQ ID NO: 4 shown below. Petition 870250097700, dated 10 / 24 / 2025, page 37 / 145 33 / 118 MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQ AETQKLISEIDLLRKQNEQLKHKLEQLRNSCA (SEQ ID NO: 4)

[0073] In this context, “consisting essentially of” means that the specified molecule would not contain any additional sequences that would alter the activity of SEQ ID NO: 4.

[0074] Preferably, the polypeptide consists of SEQ ID NO: 4.

[0075] In a preferred embodiment, the anticancer treatment comprises the use of a polypeptide consisting of SEQ ID NO: 4.

[0076] The term “functional equivalent variant” refers to any polypeptide resulting 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 resulting 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, the functional equivalent variant refers to any polypeptide resulting 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 which substantially preserves the tumor suppressor activity of SEQ ID NO: 1; more preferably resulting from the insertion or addition of one or more amino acids with respect to the polypeptide of SEQ ID NO: 1.

[0077] The person skilled in the art will understand that preserving tumor suppressor activity requires that the Petition 870250097700, dated 10 / 24 / 2025, p. 38 / 145 The 34 / 118 variant can dimerize with Myc and / or its obligate partner p21 / p22Max and inhibit the activity of Myc, which is capable of translocating across the cell membrane and through the nuclear envelope. In some embodiments, the functionally equivalent variant of the polypeptide of the invention homodimerizes less than Omomyc or is not forced into homodimers by disulfide bridge formation. In particular, disulfide bridge formation in the homodimeric form of certain embodiments of the polypeptide of the invention is less than in the Omomyc polypeptide.

[0078] “Less homodimerization”, as used in this document, refers to the lower ability of the polypeptide of the invention to form obligate homodimers, even under reducing conditions. In a preferred embodiment, 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%, at least 95% lower than the ability of Omomyc to form homodimers.

[0079] Reducing conditions, as used in this document, refer to the presence of a reducing agent, a compound that donates an electron to another chemical species in a redox chemical reaction. Illustrative and non-limiting examples of reducing agents are DTT (dithiothreitol), b-mercaptoethanol, or TCEP (tris(2-carboxyethyl)phosphine). It is possible that the number of homodimers is the same in vitro and that the difference between the functionally equivalent variant and Omomyc is present only in cells in Petition 870250097700, dated 10 / 24 / 2025, page 39 / 145 35 / 118 presence of heterodimerization partners, where the absence of the disulfide allows for a potentially greater formation of heterodimers.

[0080] Several assays can be used to determine the homodimerization of a peptide, by way of illustrative and non-limiting example, by thermal denaturation monitored by circular dichroism, so that dimerization can be detected by means of folding and quantification of thermal stability.

[0081] Suitable functional equivalent variants include polypeptides consisting essentially of the polypeptide with SEQ ID NO: 1. In this context, consisting essentially of means that the specified molecule would not contain any additional sequences that would alter the activity of SEQ ID NO: 1.

[0082] In a preferred embodiment, the functional equivalent variant of SEQ ID NO: 1 is a polypeptide resulting from the insertion or addition of one or more amino acids relative to the polypeptide of SEQ ID NO: 1. In one embodiment, the functional equivalent variant results from the insertion of less than 10 amino acids, more preferably less than 5 amino acids, most preferably resulting from the insertion of one amino acid. In a preferred embodiment, it results from the insertion of an amino acid that is methionine.

[0083] In another embodiment, the functional equivalent variant of SEQ ID NO: 1 is a polypeptide resulting from the deletion of one or more amino acids relative to the polypeptide of SEQ ID NO: 1. In one embodiment, the functional equivalent variant results from the deletion of less than 10 amino acids, more preferably less than 5 amino acids, Petition 870250097700, dated 10 / 24 / 2025, p. 40 / 145 36 / 118 most preferably results from the deletion of an amino acid.

[0084] Suitable functional variants of the target peptide are those that exhibit a degree of identity with respect to the peptide of SEQ ID NO:1 of approximately more than 25% amino acid sequence identity, such as 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 algorithms and computer methods widely known to those skilled in the art. The identity between two amino acid sequences is preferably determined using the BLASTP algorithm as previously described (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, the sequence identity is determined along the entire length of the polypeptide of SEQ ID NO: 1 or along the entire length of the variant of both.

[0085] Functionally equivalent variants of the polypeptide of the invention may also include post-translational modifications such as glycosylation, acetylation, isoprenylation, myristoylation, proteolytic processing, etc.

[0086] In another embodiment, suitable functional variants of the target peptide are those in which one or more positions within the polypeptide of the invention contain an amino acid that is a conservative substitution of the amino acid present in the protein mentioned above. Conservative amino acid substitutions result from the substitution of one amino acid for another with properties Petition 870250097700, dated 10 / 24 / 2025, p. 41 / 145 37 / 118 similar structural and / or chemical characteristics. For example, the following six groups contain amino acids that are conservative substitutions with each other: 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 competence of a person skilled in the art and is described, for example, by Dordo et al., (J. Mol. Biol., 1999, 217,-721-739) and Taylor et al., (J. Theor. Biol., 1986, 119:205-218).

[0087] It should be understood that, in a preferred embodiment, the functional equivalent variants of Omomyc contain mutations at positions corresponding to the E61T, E68I, R74Q, and R75N ​​mutations found in human c-Myc-derived Omomyc. The position where such mutations should occur in the functional equivalent variant can be determined by a multi-sequence alignment of different Myc sequences, identified by the alignment of those positions corresponding to positions 61, 68, 74, and within the human c-Myc-derived Omomyc sequence. In one embodiment, the functional equivalent variants of Omomyc contain mutations at positions corresponding to the E61T, E68I, R74Q, and R75N ​​mutations found in human c-Myc-derived Omomyc.

[0088] In another embodiment, the functional equivalent variants of Omomyc contain mutations at positions corresponding to E61, E68, R74 and R75 within the Omomyc sequence, where E61 has been mutated to E61A or E61S; Petition 870250097700, dated 10 / 24 / 2025, p. 42 / 145 38 / 118 E68 was mutated to E68L, E68M, or E68V; R74 was mutated to R74N; and R75 mutated to R75Q.

[0089] A multi-sequence alignment is an extension of pairwise alignment to incorporate more than two sequences at a time. Multi-sequence alignment methods align all sequences in a given query set. A preferred multi-sequence alignment program (and its algorithm) is ClustalW, Clusal2W, or ClustalW XXL (see Thompson et al. (1994) Nucleic Acids Res 22: 4673-4680). Because c-Myc sequences from different organisms and variants are compared (aligned) as described here, the subject matter expert can easily identify the positions within each sequence corresponding to the E61T, E68I, R74Q, and positions. R75N mutations found in Omomyc are introduced into the Omomyc variant, corresponding to the E61T, E68I, R74Q, and R75N ​​mutations found in Omomyc derived from human c-Myc.

[0090] Suitable assays to determine whether a polypeptide can be considered a functionally equivalent variant of Omomyc include, without limitation: - Assays that measure the ability of the polypeptide to form dimeric complexes with Max and Myc, such as assays based on the expression of a reporter gene, as described in Soucek et al. (Oncogene, 1998, 17: 2463- 2472), as well as PLA (protein binding assay) or coimmunoprecipitation. - Assays that measure the ability of the polypeptide to bind to the Myc / Max recognition site on DNA (the CACGTG site), such as the electrophoretic mobility displacement assay (EMSA) described in Soucek et al. Petition 870250097700, dated 10 / 24 / 2025, p. 43 / 145 39 / 118 (above) - Assays that measure the ability to repress Myc-induced transactivation, such as the assay based on the expression of a reporter gene under the control of Myc / Max-specific DNA binding sites, as described by Soucek et al. (supra.). - Assays based on the ability of the polypeptide to inhibit the growth of cells expressing the myc oncogene, as described by Soucek et al. (supra.). - Assays that measure the polypeptide's ability to enhance myc-induced apoptosis, such as the assays described by Soucek et al. (Oncogene, 1998: 17, 2463-2472). In addition, any assay commonly known in the art for evaluating apoptosis in a cell can be used, such as Hoechst staining, propidium iodide (PI) or annexin V staining, trypan blue, DNA laddering / fragmentation, and TUNEL.

[0091] In a preferred embodiment, a polypeptide is considered a functionally equivalent variant of Omomyc if it exhibits activity in one or more of the above assays that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of native Omomyc.

[0092] In a particular embodiment, the functional equivalent variant of the polypeptide of SEQ ID NO: 1 comprises the polypeptide of SEQ ID NO: 1, wherein residue X at position 89 of SEQ ID NO: 1 is not a cysteine. Preferably, residue X at position 89 of SEQ ID NO: 1 is an aliphatic amino acid, or a sulfur-containing amino acid, or a dicarboxylic amino acid or amides thereof, or an amino acid with two basic groups, or an amino acid Petition 870250097700, dated 10 / 24 / 2025, p. 44 / 145 40 / 118 aromatic, 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.

[0093] Suitable functional equivalent variants of SEQ ID NO: 1 with an X residue at position 89 of SEQ ID NO: 1 that is not a cysteine ​​are disclosed in the following table. TABLE 1: SEQ ID NO SEQUÊNCIA SEQ ID NO: 5 TEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSV QAETQKLISEIDLLRKQNEQLKHKLEQLRNSXA (em que X é qualquer aa diferente de Cys) SEQ ID NO: 6 MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILS VQAETQKLISEIDLLRKQNEQLKHKLEQLRNSXA (em que X é qualquer aa diferente de Cys) SEQ ID NO: 7 TEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSV QAETQKLISEIDLLRKQNEQLKHKLEQLRNSSA SEQ ID NO: 8 MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILS VQAETQKLISEIDLLRKQNEQLKHKLEQLRNSSA SEQ ID NO: 9 TEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSV QAETQKLISEIDLLRKQNEQLKHKLEQLRNSAA SEQ ID NO: 10 MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILS VQAETQKLISEIDLLRKQNEQLKHKLEQLRNSAA

[0094] Asim, some modalities preferably, an equivalent variant of the function of placing the SEQ ID NO: 1 into one part of the group that consists of the SEQ ID NO: 4, the SEQ ID NO: 5, the SEQ ID NO: 6, the SEQ ID NO: 7, the SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. Preferably, the functionally equivalent variant is SEQ ID NO: 4.

[0095] In addition, equivalent variants Petition 870250097700, dated 10 / 24 / 2025, page 45 / 145 41 / 118 functional Omomyc variants are also capable of transducing cells after contact of the variant with said cell. It should be understood that functional equivalent variants of Omomyc contain either the protein transduction domain found in native Omomyc or another functional protein transduction domain.

[0096] In a preferred embodiment, a polypeptide is considered a functionally equivalent variant of SEQ ID NO: 1 if it is capable of transducing a target cell with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% efficiency of SEQ ID NO: 1.

[0097] Furthermore, functionally equivalent variants of SEQ ID NO: 1 are also capable of translocating to the nucleus of the target tumor cell.

[0098] In a preferred embodiment, a polypeptide is considered a functionally equivalent variant of SEQ ID NO: 1 if it is capable of translocating to the nucleus of target tumor cells with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% efficiency of SEQ ID NO: 1.

[0099] Suitable assays to determine whether a polypeptide is a functionally equivalent variant of SEQ ID NO: 1 in terms of its ability to translocate across the cell membrane and into the nucleus include double labeling of a cell with a reagent specific for the polypeptide and with a dye that specifically labels the cell nucleus (such as DAPI or Hoechst dye). Detection of the polypeptide of the invention can be performed by confocal microscopy or fluorescence microscopy.

[00100] In another preferred mode, the Petition 870250097700, dated 10 / 24 / 2025, page 46 / 145 42 / 118 anticancer treatment is a conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant and a chemical moiety that facilitates cellular uptake of the polypeptide or the functionally equivalent variant.

[00101] The term conjugate, as used in this document, refers to two or more compounds that are covalently linked together, such that the function of each compound is retained in the conjugate.

[00102] The term chemical fraction refers to any chemical compound containing at least one carbon atom. Examples of chemical fractions include, among others, any peptide chain enriched in hydrophobic amino acids and hydrophobic chemical fractions.

[00103] In preferred embodiments, the conjugate according to the invention comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more chemical fractions that facilitate cellular uptake of the polypeptide or the functional equivalent variant of said polypeptide.

[00104] In one embodiment, the chemical fraction that facilitates cellular uptake of the polypeptide is a lipid or a fatty acid.

[00105] A fatty acid is generally a molecule comprising a carbon chain with an acidic moiety (e.g., carboxylic acid) at one end of the chain. The carbon chain of a fatty acid can be of any length; however, it is preferable that the carbon chain length be at least 2, 3, 4, Petition 870250097700, dated 10 / 24 / 2025, page 47 / 145 43 / 118 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms, and any derivable interval therefrom. In certain embodiments, the carbon chain length varies from 4 to 18 carbon atoms in the fatty acid chain portion. In certain embodiments, the fatty acid carbon chain may comprise an odd number of carbon atoms; however, an even number of carbon atoms in the chain may be preferable in certain embodiments. A fatty acid comprising only single bonds in its carbon chain is termed saturated, while a fatty acid comprising at least one double bond in its chain is termed unsaturated. The fatty acid may be branched, although in preferred embodiments of the present invention, it is not branched.Specific fatty acids include, among others, linoleic acid, oleic acid, palmitic acid, linolenic acid, stearic acid, lauric acid, myristic acid, arachidic acid, palmitoleic acid, and arachidonic acid.

[00106] In a preferred embodiment, the chemical moiety that facilitates cellular uptake of the polypeptide comprising the sequence SEQ ID NO: 1 or a functional equivalent variant thereof is a cell-penetrating peptide sequence, in which case the conjugate would comprise a fusion protein comprising the polypeptide comprising the SEQ ID NO: 1 or a functional equivalent variant thereof and the cell-penetrating peptide sequence.

[00107] The term fusion protein refers to proteins generated by genetic technology that consist of two or more functional domains derived from different proteins. A fusion protein can be obtained by means Petition 870250097700, dated 10 / 24 / 2025, page 48 / 145 44 / 118 conventional methods, for example, through gene expression of the nucleotide sequence encoding said fusion protein in a suitable cell. It is understood that the cell-penetrating peptide refers to a cell-penetrating peptide different from the cell-penetrating peptide that is part of the polypeptide comprising SEQ ID NO: 1 or the functional equivalent variant of SEQ ID NO: 1.

[00108] The term “cell-penetrating peptide sequence” is used in this specification interchangeably with “CPP,” protein transducer domain, or PTD. It refers to a peptide chain of variable length that directs the transport of a protein into the cell. The delivery process into the cell usually occurs by endocytosis, but the peptide can also be internalized into the cell by direct translocation across the membrane. CPPs typically have an amino acid composition that contains a high relative abundance of positively charged amino acids, such as lysine or arginine, or sequences that contain an alternating pattern of polar / charged amino acids and nonpolar hydrophobic amino acids.

[00109] Examples of CPPs that can be used in the present invention include, without limitation, the CPP found in the DNA-binding protein VP22 of Drosophila CPP of Bac7 (RRIRPRPPRLPRPRPRPLPFPRPG; SEQ ID NO: 15), the CPPs of the HIV-1 TAT protein consisting of amino acids 49-57 (RKKRRQRRR, SEQ ID NO: 16), amino acids 48-60 (GRKKRRQRRRTPQ, SEQ ID NO: 17), amino acids 47-57 (YGRKKRRQRRR; SEQ ID NO: 18); the CPP peptide of S413-PV (ALWKTLLKKVLKAPKKKRKV; SEQ ID NO: 19), the penetratin CPP Petition 870250097700, dated 10 / 24 / 2025, p. 49 / 145 45 / 118 (RQIKWFQNRRMKWKK; SEQ ID NO: 20), o CPP de SynBl (RGGRLSYSRRRFSTSTGR; SEQ ID NO: 21), o CPP de SynB3 (RRLSYSRRRF; SEQ ID NO: 22), o CPP de PTD-4 (PIRRRKKLRRLK; SEQ ID NO: 23), o CPP de PTD-5 (RRQRRTSKLMKR; SEQ ID NO: 24), o CPP do FHV Coat-(35-49) (RRRRRNRTRRNRRRVR; SEQ ID NO: 25), o CPP de BMV Gag-(7-25) (KMTRAQRRAAARRNRWTAR; SEQ ID NO: 26), o CPP de HTLV-II Rex-(4-16) (TRRQRTRRARRNR; SEQ ID NO: 27), o CPP of D-Tat (GRKKRRQRRRPPQ; SEQ ID NO: 28), o CPP of R9-Tat (GRRRRRRRRRPPQ; SEQ ID NO: 29), o CPP of MAP (KLALKLALKLALALKLA; SEQ ID NO: 30), o CPP of SBP (MGLGLHLLVLAAALQGAWSQPKKKRKV; SEQ ID NO: 28). 31), o CPP of FBP (GALFLGWLGAAGSTMGAWSQPKKKRKV; SEQ ID NO: 32), o CPP of MPG (ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cya; SEQ ID NO: 33), o CPP of MPG(ENLS) (ac-GALFLGFLGAAGSTMGAWSQPKSKRKV-cya; SEQ ID NO: 31). 34), o CPP de Pep-1 (ac-KETWWETWWTEWSQPKKKRKV-cya; SEQ ID NO: 35), o CPP de Pep-2 (ac-KETWFETWFTEWSQPKKKRKV-cya; SEQ ID NO: 36), some sequence of poliarginina tending to an estrutura RN (where N is between 4 and 17), the sequence GRKKRRQRRR (SEQ ID NO: 37), the sequence RRRRRRLR (SEQ ID NO: 38), the sequence RRQRRTS KLMKR (SEQ ID NO: 39); Transport GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 40); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 41); RQIKIWFQNRRMKWKK (SEQ ID NO: 42), the sequence YGRKKRRQRRR (SEQ ID NO: 43); the sequence RKKRRQRR (SEQ ID NO: 44); the sequence YARAAARQARA (SEQ ID NO: 45); the sequence THRLPRRRRRR (SEQ ID NO: 46); the sequence GGRRARRRRRR (SEQ ID NO: 47).

[00110] In a preferred embodiment, the aforementioned cell-penetrating peptide is not the endogenous one contained in SEQ ID NO: 1. Petition 870250097700, dated 10 / 24 / 2025, p. 50 / 145 46 / 118

[00111] In a preferred embodiment, the CPP is the CPP of the HIV-1 TAT protein, consisting of amino acids 4, 9-57 (RKKRRQRRR, SEQ ID NO: 16). In another preferred embodiment, the CPP is the sequence GRKKRRQRRR (SEQ ID NO: 37) or RRRRRRLR (SEQ ID NO: 38). In yet another embodiment, the CPP is the sequence GRKKRRQRRR (SEQ ID NO: 37) or RRRRRRRR (SEQ ID NO: 65).

[00112] In some modalities, a CPP is like a CPP as described in WO2019 / 018898, the content of which is incorporated herein by reference in its entirety.

[00113] In one embodiment, the cell-penetrating peptide sequence is fused to the N-terminal end of the polypeptide of the invention or to a functionally equivalent variant of said polypeptide. In another embodiment, the cell-penetrating peptide is fused to the C-terminal end of the polypeptide of the invention or to a functionally equivalent variant of said polypeptide.

[00114] In preferred embodiments, the conjugates or fusion proteins of the combination according to the invention comprise, in addition to the cell-penetrating peptide itself found in the polypeptide of SEQ ID NO: 1 or the functional equivalent variant of said polypeptide, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more additional cell-penetrating peptides.

[00115] Suitable fusion proteins of the invention include the Omomyc*TAT and Omomyc*LZArg polypeptides as defined below: TABLE 2: Petition 870250097700, dated 10 / 24 / 2025, page 51 / 145 47 / 118 Name SEQ ID NO: Sequence Omomyc*TAT 11 MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVI LKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCAGRK KRRQRRR Omomyc*LZArg 12 MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVI LKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCARRR RRRLR

[00116] Thus, in a preferred embodiment, the fusion protein is the polypeptide selected from SEQ ID NO: 11 and 12.

[00117] Suitable assays to determine whether a conjugate preserves the ability of Omomyc to translocate across the cell membrane include, without limitation, assays that measure the conjugate's ability to transduce cells in culture. This assay is based on contact of the conjugate with cultured cells and detection of the presence of the conjugate at an intracellular location.

[00118] In another preferred embodiment, the conjugate of the invention further comprises an additional nuclear localization signal.

[00119] The term nuclear localization signal (NLS), as used herein, refers to an amino acid sequence approximately 4 to 20 amino acid residues in length that serves to direct a protein to the nucleus. Typically, the nuclear localization sequence is 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 the NLS of the large T antigen SV40 (PKKKRKV, SEQ Petition 870250097700, dated 10 / 24 / 2025, p. 52 / 145 48 / 118 ID NO: 48); the nucleoplasmin NLS (KRPAATKKAGQAKKKK, SEQ ID NO: 49); the CBP8 0 NLS (RRRHSDENDGGQPHKRRK, SEQ ID NO: 50); the HIV-I Rev protein NLS (RQARRNRRRWE, SEQ ID NO: 51); the HTLV-I Rex NLS (MPKTRRRPRRSQRKRPPT, SEQ ID NO: 52); the hnRNP A NLS (NQSSNFGPMKGGNFGGRSSGPYGGGGQYFKPRNQGGY, SEQ ID NO: 53); the rpL23a NLS (VHSHKKKKIRTSPTFTTPKTLRLRRQPKYPRKSAPRRNKLDHY, SEQ ID NO: 54). In one embodiment of the invention, the nuclear localization signal comprises the K (K / R) X (K / R) motif.

[00120] In an even more preferred embodiment, the nuclear locator 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).

[00121] In another preferred embodiment, the NLS may be N-terminal or C-terminal to the conjugate or to the fusion protein comprising the SEQ ID NO: 1 polypeptide or a functionally equivalent variant thereof.

[00122] A person skilled in the art will understand that it may be desirable for the conjugate of the invention to further comprise one or more flexible peptides connecting the polypeptide comprising SEQ ID NO: 1 or a functional equivalent variant thereof, to the cell-penetrating peptide sequence and / or the NLS. Thus, in one particular embodiment, the polypeptide comprising SEQ ID NO: 1 or a functional equivalent variant thereof is directly connected to the cell-penetrating peptide sequence. In another particular embodiment, the polypeptide comprising SEQ ID NO: 1 or a functional equivalent variant thereof is connected to the cell-penetrating peptide sequence by means of a flexible peptide. In one embodiment, the polypeptide comprising SEQ ID NO: 1 or a variant Petition 870250097700, dated 10 / 24 / 2025, page 53 / 145 49 / 118 functional equivalent of this is directly connected to the NLS. In another embodiment, the polypeptide comprising SEQ ID NO: 1 or a functional equivalent variant thereof is connected to the NLS via a flexible peptide.

[00123] In a particular embodiment, the conjugate polypeptide according to the invention is directly connected to the cell-penetrating peptide sequence and to the NLS.

[00124] In one embodiment, the NLS is one of the NLS that appear endogenously in the Myc sequence, such as the M1 peptide (PAAKRVKLD, SEQ ID NO: 56) or the M2 peptide (RQRRNELKRSF, SEQ ID NO: 57).

[00125] In another embodiment, the additional NLS refers to an NLS different from the endogenous NLS found in a polypeptide comprising SEQ ID NO: 1 or in the functional equivalent variant of SEQ ID NO: 1.

[00126] In preferred embodiments, the conjugates or fusion proteins according to the invention comprise, in addition to the endogenous NLS found in the polypeptide of the invention or in the functional equivalent variant thereof, 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 NLS.

[00127] In another particular embodiment, the conjugate polypeptide according to the invention is connected to the cell-penetrating peptide sequence by means of a first flexible peptide linker and to the NLS by means of a second flexible peptide linker.

[00128] As used in this document, the term flexible peptide, spacer peptide or peptide Petition 870250097700, dated 10 / 24 / 2025, page 54 / 145 50 / 118 ligand refers to a peptide that covalently binds to two proteins or fragments, but is not part of either polypeptide, allowing movement of one relative to the other without causing a substantial detrimental effect on the function of the protein or fragment. Thus, the flexible ligand does not affect the tumor tracking activity of the polypeptide sequence, the cell penetration activity of the cell-penetrating peptide, or the nuclear localization capability of the NLS.

[00129] The flexible peptide comprises 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 10 amino acids, at least 12 amino acids, at least 14 amino acids, at least 16 amino acids, at least 18 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, or about 100 amino acids. In some embodiments, the flexible peptide will allow movement of one protein relative to another in order to increase protein solubility and / or improve its activity. Suitable binding regions include a polyglycine region, the GPRRRR sequence (SEQ ID NO: 58) of combinations of glycine, proline, and alanine residues.

[00130] In a particular embodiment, the Petition 870250097700, dated 10 / 24 / 2025, page 55 / 145 51 / 118 Conjugates according to the invention comprise a tag attached to the conjugate or C-terminal or N-terminal domain of said polypeptide or fusion protein or variant thereof. Said tag is generally a peptide or amino acid sequence that can be used in the isolation or purification of said fusion protein. Thus, said tag is capable of binding to one or more ligands, for example, one or more ligands of an affinity matrix, such as a chromatographic support or sphere with high affinity. An example of said tag is a histidine tag (His-tag or HT), such as a tag comprising 6 histidine residues (His6 or H6), which can bind to a nickel (Ni2+) or cobalt (Co2+) column with high affinity. Histag has the desirable characteristic of being able to bind to its ligands under conditions that are denaturing to most proteins and disruptive to most protein-protein interactions.Thus, it can be used to remove the protein from the H6-labeled bait after the interruption of the protein-protein interactions in which the bait participated.

[00131] Additional, non-limiting, illustrative examples of useful markers for isolating or purifying the conjugate or polypeptide comprising SEQ ID NO: 1 or a variant thereof or a fusion protein include Arg-tag, FLAG-tag (DYKDDDDK; SEQ ID NO:59), Strep-tag (WSHPQFEK, SEQ ID NO:60), an antibody-recognized epitope such as c-myc-tag (recognized by an 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, domain Petition 870250097700, dated 10 / 24 / 2025, page 56 / 145 52 / 118 chitin-binding protein, glutathione S-transferase-tag, maltose-binding protein, NusA, TrxA, DsbA, Avi-tag, etc. Microbiol. Biotechnol. 2003, 60:523-525), an amino acid sequence such as AHGHRP (SEQ ID NO:63) or PIHDHDHPHLVIHSGMTCXXC (SEQ ID NO:64), β-galactosidase and the like.

[00132] The label can be used, if desired, for the isolation or purification of said fusion protein.

[00133] In another preferred embodiment, the anticancer treatment is a polynucleotide encoding the polypeptide or fusion protein disclosed above. In a preferred embodiment, the anticancer treatment is a polynucleotide encoding a polypeptide comprising the sequence SEQ ID NO: 1 or a functional equivalent variant thereof. In another embodiment, the anticancer treatment is a polynucleotide encoding a conjugate comprising the polypeptide comprising the sequence SEQ ID NO: 1 or a functional equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or the functional equivalent variant thereof; more preferably, it is a polynucleotide encoding a fusion protein between the polypeptide comprising the sequence SEQ ID NO: 1 or a functional equivalent variant thereof and a cell-penetrating peptide sequence.

[00134] 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 Petition 870250097700, dated 10 / 24 / 2025, page 57 / 145 53 / 118 and its analogues. Nucleotides can have any three-dimensional structure and can perform any function, known or unknown. The term polynucleotide includes, for example, single-stranded, double-stranded and triple-helix molecules, a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA isolated from any sequence, RNA isolated from any sequence, nucleic acid probes and primers. In addition to a native nucleic acid molecule, a nucleic acid molecule of the present invention may also comprise modified nucleic acid molecules. As used herein, mRNA refers to RNA that can be translated in a cell.

[00135] In a preferred embodiment, the polynucleotide of the invention is an mRNA.

[00136] The mRNA can be chemically synthesized, obtained through in vitro transcription, or synthesized in vivo in the target cell. The nucleotide sequences that form the polynucleotide encoding the conjugated or fusion protein of the invention are in the same correct reading frame for its expression.

[00137] In a preferred embodiment, the anticancer treatment is an mRNA encoding a polypeptide consisting of the sequence SEQ ID NO: 1 or a polypeptide consisting of a functionally equivalent variant of SEQ ID NO: 1 or a polypeptide consisting of SEQ ID NO: 4.

[00138] In another embodiment, the anticancer treatment is a vector comprising a polynucleotide of the invention. Petition 870250097700, dated 10 / 24 / 2025, page 58 / 145 54 / 118

[00139] The term vector, as used herein, refers to a nucleic acid sequence comprising the sequences necessary for a polypeptide encoded by the polynucleotide of the invention to be generated after transcription and translation of said sequences in a cell. Said sequence is operatively linked to additional segments that ensure its autonomous replication in a host cell of interest. Preferably, the vector is an expression vector, which is defined as a vector that, in addition to the autonomous replication regions in a host cell, contains regions operatively linked to the nucleic acid of the invention and that are capable of increasing the expression of the nucleic acid products according to the invention. The vectors of the invention can be obtained by means of techniques widely known in the art.

[00140] Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensation agents, DNA or RNA expression vectors encapsulated in liposomes and certain eukaryotic cells, such as producer cells. Suitable vectors comprising a polynucleotide of the invention are vectors derived from expression vectors in prokaryotes, such as pUC18, pUC19, pBluescript and their derivatives, mp18, mp19, pBR322, pMB9, ColEl, pCRl, RP4, phages and “shuttle” vectors such as pSA3 and pAT28, expression vectors in yeast such as 2-micron plasmid-type vectors, integration plasmids, YEP vectors, centromeric plasmids and Petition 870250097700, dated 10 / 24 / 2025, page 59 / 145 55 / 118 similar vectors, expression vectors in insect cells such as the pAC and pVL series vectors, expression vectors in plants such as the pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE and similar series vectors, and expression vectors in higher eukaryotic cells based on viral vectors (adenoviruses, adenovirus-associated viruses, as well as retroviruses and, in particular, lentiviruses), as well as non-viral vectors such as 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. In a preferred embodiment, the polynucleotide of the invention is contained in a vector selected from the group consisting of pEGFP or pBabe retroviral vectors and pTRIPZ or pSLIK lentiviral vectors.

[00141] The vector of the invention can be used to transform, transfect, or infect cells that can be transformed, transfected, or infected by said vector. Said cells can be prokaryotic or eukaryotic.

[00142] The vector preferably comprises the polynucleotide of the invention operationally linked to sequences that regulate the expression of the polynucleotide of the invention. The regulatory sequences used in the present invention may be nuclear promoters or, alternatively, enhancer sequences and / or other regulatory sequences that increase the expression of the heterologous nucleic acid sequence. In principle, any promoter may be used in the present invention, provided it is compatible with the cells in which the polynucleotide is used. Petition 870250097700, dated 10 / 24 / 2025, p. 60 / 145 56 / 118 will be expressed. Thus, suitable promoters for carrying out the present invention include, but are not necessarily limited to, constitutive promoters, such as those derived from eukaryotic virus genomes, such as polyomavirus, adenovirus, SV40, CMV, avian sarcoma virus, hepatitis B virus, the metallothionein gene promoter, the thymidine kinase gene promoter of herpes simplex virus, retrovirus LTR regions, the immunoglobulin gene promoter, the actin gene promoter, the EF-1alpha gene promoter, as well as inducible promoters in which protein expression depends on the addition of an exogenous molecule or signal, such as tetracycline systems, the NFkB / UV light system, the Cre / Lox system and the heat shock gene promoter, the reducible RNA polymerase II promoters described in WO / 2006 / 135436 and tissue-specific promoters.

[00143] In another embodiment, the anticancer treatment consists of a cell capable of secreting into the medium the polypeptide of the invention or the conjugate of the invention, preferably the polypeptide of the invention or the fusion protein of the invention.

[00144] Suitable cells capable of secreting a polypeptide of the invention include, among others, cardiomyocytes, adipocytes, endothelial cells, epithelial cells, lymphocytes (B and T cells), mast cells, eosinophils, vascular intima cells, primary cell cultures isolated from different organs, preferably isolated from islets of Langerhans, hepatocytes, leukocytes, including mononuclear, mesenchymal, umbilical cord or adult leukocytes (skin, lung, kidney and liver), osteoclasts, chondrocytes and others. Petition 870250097700, dated 10 / 24 / 2025, page 61 / 145 57 / 118 connective tissue cells. Cells from established cell lines, such as Jurkat T cells, NIH-3T3, CHO, Cos, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 cells, C2C12 myoblasts, and W138 cells, are also suitable. Skilled individuals will recognize that cells capable of secreting a polypeptide of the invention into the medium can be found forming microparticles or microcapsules, so that the cells have a longer lifespan in patients. Suitable materials for forming the microparticles of the invention include any biocompatible polymeric material that allows continuous secretion of the therapeutic products and acts as a support for the cells. Thus, said biocompatible polymeric material can be, for example, thermoplastic polymers or hydrogen polymers.Among the thermoplastic polymers we have acrylic acid, acrylamide, 2-aminoethyl methacrylate, poly(tetrafluoroethylenecohexafluoropropylene), methacrylic acid-(7-coumaroxy) ethyl ester, N-isopropyl acrylamide, polyacrylic acid, polyacrylamide, polyamidoamine, poly(amino)-p-xylylene, poly(chloroethylvinyl ether), polycaprolactone, poly(caprolactone-co-trimethylene carbonate), poly(urea carbonate) urethane, poly(carbonate) urethane, polyethylene, polyethylene-acrylamide copolymer, polyethylene glycol, polyethylene glycol methacrylate, poly(ethylene terephthalate), poly(4-hydroxybutyl acrylate), poly(hydroxyethyl methacrylate), poly(N-2-hydroxypropyl methacrylate), poly(lactic glycolic acid), poly(L-lactic acid). poly(gamma-methyl, L-glutamate), poly(methyl methacrylate), poly(propylene fumarate), poly(propylene oxide), polypyrrole, polystyrene. Petition 870250097700, dated 10 / 24 / 2025, page 62 / 145 58 / 118 poly(tetrafluoroethylene), polyurethane, polyvinyl alcohol, ultra-high molecular weight polyethylene, 6-(p-vinylbenzamide)-hexanoic acid N-p-vinylbenzyl-D-maltonamide and copolymers containing more than one of these polymers. Among the hydrogel-type polymers we have natural materials such as alginate, agarose, collagen, starch, hyaluronic acid, bovine serum albumin, cellulose and its derivatives, pectin, chondroitin sulfate, fibrin and fibroin, as well as synthetic hydrogels such as Sepharose® and Sephadex®.

[00145] The method for evaluating or monitoring the clinical response of the invention is performed on a post-treatment sample, that is, on a patient sample obtained after the patient has received the treatment whose response is being evaluated.

[00146] Thus, the first step of the first method of the invention comprises determining the level of at least one biomarker selected from the group consisting of IL-17A, IFN-γ and CD62E in a sample from an individual with cancer obtained after administration of treatment.

[00147] The expression "obtained after treatment administration" means that the individual's sample was obtained at any time after the start of treatment, i.e., it is a post-treatment sample.

[00148] The sample can be obtained immediately after the end of the treatment dose administration (i.e., after the end of the intravenous infusion), preferably at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours after the end of the treatment dose administration. In a Petition 870250097700, dated 10 / 24 / 2025, page 63 / 145 59 / 118 preferred method, the sample is obtained within 7 days, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, within 24 hours, within 23 hours, within 22 hours, within 21 hours, within 20 hours, within 19 hours, within 18 hours, within 17 hours, within 16 hours, within 15 hours, within 14 hours, within 13 hours, within 12 hours, within 11 hours, within 10 hours, within 9 hours, within 8 hours, within 7 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours, within 1 hour after the end of treatment administration.In a preferred embodiment, the sample is obtained at a time between 15 minutes and 7 days after the end of the treatment dose administration, preferably between 15 minutes and 6 days, preferably between 15 minutes and 5 days, preferably between 15 minutes and 4 days, preferably between 15 minutes and 3 days, preferably between 15 minutes and 2 days, preferably between 15 minutes and 24 hours, more preferably between 15 minutes and 20 hours, even more preferably between 15 minutes and 18 hours, preferably between 15 minutes and 15 hours, preferably between 15 minutes and 12 hours, preferably between 15 minutes and 10 hours, most preferably between 15 minutes and 6 hours. In another embodiment, the sample is obtained at a time between 1 hour and 8 hours, preferably between 1 hour and 6 hours, preferably between 1 hour and 4 hours, preferably between 1 hour and 2 hours after the end of the administration of the treatment dose.In one preferred embodiment, the sample is obtained 1 hour after the end of the treatment dose administration. In another preferred embodiment, the sample is obtained 2 hours later. Petition 870250097700, dated 10 / 24 / 2025, page 64 / 145 60 / 118 hours after the end of the treatment dose administration. In another preferred embodiment, the sample is obtained 6 hours after the end of the treatment dose administration.

[00149] The time frame after the start of therapy at which the response is assessed is not particularly limited. Thus, the response to therapy can be determined 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 therapy. The response to therapy can be determined at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks after the start of the first therapy. Preferably, the response to therapy can be determined at least 9 weeks after the start of the first therapy. The response to therapy can be determined at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months after the start of the first therapy, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years after the start of the first therapy.In one particular embodiment, the response to therapy can be assessed when the individual has received at least two, preferably at least three, more preferably at least four, even more preferably at least five, and even more preferably at least six prior cycles of anticancer treatment. In a preferred embodiment, the response to therapy can be assessed when the individual has received at least three prior cycles of anticancer treatment. In another preferred embodiment, the response to therapy can be assessed when the individual has received at least six prior cycles of anticancer treatment.

[00150] The levels of these biomarkers can be detected in any type of sample. The term sample, as used herein, refers to any sample that Petition 870250097700, dated 10 / 24 / 2025, page 65 / 145 61 / 118 can be obtained from the individual and contains any biological material suitable for detecting RNA or protein levels. In one particular embodiment, the sample contains genetic material, for example, DNA, genomic DNA (gDNA), complementary DNA (cDNA), RNA, heterogeneous nuclear RNA (hnRNA), mRNA, etc., from the individual under study. In another particular embodiment, the sample contains proteins. The sample may comprise cellular and / or non-cellular material from the individual. The present method can be applied to any type of biological sample from an individual, such as a biopsy sample, tissue, cell or biological fluid (blood, plasma, serum, saliva, urine, semen, sputum, cerebrospinal fluid (CSF), tears, mucus, sweat, milk), feces, brain extracts, bone marrow, nipple aspirate, samples obtained by bronchial lavage, bronchoscopy, fine needle aspiration biopsy (FNAB), solid tumor biopsy sample, buccal or pharyngeal swab and the like.The sample in question can be obtained by conventional methods, for example, biopsy, surgical excision, or aspiration, using methods well known to those with common knowledge in related medical techniques. Methods for obtaining the biopsy sample include macroscopic distribution of a mass, microdissection, or other cell separation methods known in the art. Tumor cells can also be obtained by fine-needle aspiration cytology. In a preferred embodiment, samples are obtained by fine-needle aspiration biopsy (FNAB). To simplify sample preservation and handling, samples can be formalin-fixed and paraffin-embedded first, or first. Petition 870250097700, dated 10 / 24 / 2025, page 66 / 145 62 / 118 frozen and then embedded in a cryosolidifiable medium, such as OCT-Compound, by immersion in a highly cryogenic medium that allows rapid freezing. Samples can also be a cell suspension, cell bead, cell slide, or frozen solid tumor biopsy.

[00151] In a particular embodiment, the sample is a tumor tissue sample, preferably a biopsy of a tumor tissue sample, fresh or frozen.

[00152] In a particular embodiment, the sample comprises cancer cells, preferably breast cancer, ovarian cancer, prostate cancer, gastric cancer, pancreatic cancer, lung cancer, colorectal cancer, stomach / gastric cancer, endometrial / uterine / cervical cancer, bladder cancer, head and neck cancer, leukemia, sarcoma, cholangiocarcinoma, glioblastoma, multiple myeloma, and lymphoma cells. In a preferred embodiment, the sample comprises cells selected from the group consisting of pancreatic cancer cells, lung cancer cells, colorectal cancer cells, salivary gland cells, sarcoma cells, and mesothelioma cells; more preferably, it comprises a cell selected from the group consisting of pancreatic cancer cells, lung cancer cells, colorectal cancer cells, salivary gland cells, and sarcoma cells.In a preferred embodiment, it is a sample of tumor tissue or part thereof. Preferably, said sample of tumor tissue is a breast tumor, ovarian tumor, prostate tumor, gastric tumor, pancreatic tumor, lung tumor, or other tumor. Petition 870250097700, dated 10 / 24 / 2025, p. 67 / 145 63 / 118 colorectal, stomach / 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, a sarcoma tumor sample, or a mesothelioma tumor sample; more preferably, it is a pancreatic tumor sample, a lung tumor sample, a colorectal tumor sample, a salivary gland tumor sample, or a sarcoma tumor sample.

[00153] In another particular embodiment, the individual's sample, according to the methods of the invention, is a biofluid, preferably a biofluid from affected organs. A biofluid, biological fluid sample, or body fluid sample, as used herein, refers to any biological secretion or fluid, whether physiological or pathological, produced in an individual's body. Such biofluids include, without limitation, blood, plasma, serum, bronchoalveolar lavage fluid, urine, nasal secretion, ear secretion, urethral secretion, cerebrospinal fluid, pleural fluid, synovial fluid, peritoneal fluid, ascitic fluid, pericardial fluid, amniotic fluid, gastric juice, lymphatic fluid, interstitial fluid, saliva, sputum, fluid deposition, tears, mucus, sweat, milk, semen, vaginal secretions, fluid from ulcers, blisters, abscesses, and other superficial eruptions. Said samples may be Petition 870250097700, dated 10 / 24 / 2025, page 68 / 145 64 / 118 obtained by conventional methods, using processes known in the state of the art by those skilled in the art, such as blood extraction, instillation and aspiration of fluid during bronchofibroscopy, cisternal, ventricular or lumbar puncture, pleural puncture or thoracentesis, percutaneous articular or synovial puncture, abdominal puncture, amniocentesis, expectoration, percutaneous peritoneal puncture, percutaneous pericardial puncture, etc., or by simple collection.

[00154] In a preferred embodiment, the sample is selected from the group consisting of blood, serum, plasma, saliva, cerebrospinal fluid (CSF), and a tumor biopsy sample; more preferably, it is selected from the group consisting of blood, plasma, and serum; even more preferably, it is serum.

[00155] The blood sample is typically obtained by puncturing an artery or vein, usually a vein on the inside of the elbow or the back of the hand, and the blood sample is collected in a sealed vial or syringe. A capillary puncture, usually in the heel or distal phalanges of the fingers, can be performed for analysis using a micromethod. Serum can be obtained from the whole blood sample, in the absence of anticoagulant, by letting the sample stand for 10 minutes to coagulate and then centrifuging it at 1,500 rpm for 10 minutes to separate the cells (precipitate) from the serum (supernatant). In turn, to obtain the plasma sample, whole blood is contacted with an anticoagulant and centrifuged at 3,000 rpm for 20 minutes. The precipitate from this centrifugation corresponds to the formed elements, and the supernatant corresponds to the plasma. Petition 870250097700, dated 10 / 24 / 2025, page 69 / 145 65 / 118 The serum or plasma obtained can be transferred to a storage tube for sample analysis using the method of the invention. The term biomarker, as used herein, refers to a gene product of a gene of interest, understood as the transcriptional product or the translational product of a gene of interest (i.e., the mRNA transcribed from said gene or the protein encoded by said gene), the quantity of which reflects a specific situation, for example, a condition such as the response of an individual suffering from cancer to an anticancer treatment.

[00156] The biomarkers of the invention are primarily (i.e., the primary biomarkers of the invention are) IL-17A, IFN-γ, and CD62E.

[00157] The term IL-17A, IL17A, IL-17, IL17, CTLA-8 or CTLA8 refers to interleukin-17A or cytotoxic T lymphocyte-associated antigen 8. As used in this document, this term refers to the protein or gene encoding the protein with UniProt accession number Q16552 (version 193 of the entry on February 22, 2023). The term interleukin-17A includes any of the isoforms described for this protein.

[00158] The term IFN-γ, IFN-gamma, IFN-g or IFNG refers to interferon gamma. As used in this document, this term refers to the protein or gene encoding the protein with UniProt accession number P01579 (version 238 of the entry on October 12, 2022). The term interferon gamma includes any of the isoforms described for this protein.

[00159] The term CD62E refers to the E member of the CD62 antigen-like family and is also known Petition 870250097700, dated 10 / 24 / 2025, p. 70 / 145 66 / 118 as SELE, ELAM1, ELAM-1, LECAM2, E-selectin, “leukocyte-endothelial adhesion molecule 1 or leukocyte-endothelial cell adhesion molecule 2.” As used in this document, this term refers to the protein or gene encoding the protein with UniProt accession number P16581 (version 235 of the entry on October 12, 2022). The term CD62 antigen-like family member E includes any of the isoforms described for this protein.

[00160] The expression "at least one biomarker selected from the group consisting of IL-17A, IFN-γ and CD62E, as used herein" means that, according to the present invention, the expression level of one, two or three of said biomarkers can be determined. In a particular embodiment, the method comprises determining the expression level of one of said biomarkers. In a more preferred embodiment, the biomarker determined is IL-17A. In another preferred embodiment, the biomarker determined is IFN-γ. In another preferred embodiment, the biomarker determined is CD62E. In another particular embodiment, the method of the invention comprises determining the expression level of two of said biomarkers. In a preferred embodiment, the biomarkers determined are IL-17A and IFN-γ. In another preferred embodiment, the biomarkers determined are IL-17A and CD62E. In another embodiment, the biomarkers determined are IFNγ and CD62E.In another particular embodiment, the method of the invention comprises determining the expression level of the three biomarkers IL-17A, IFN-γ, and CD62E.

[00161] Any combination of biomarkers Petition 870250097700, dated 10 / 24 / 2025, p. 71 / 145 67 / 118 primary is covered by the methods of the invention.

[00162] The method of the invention may further comprise the determination of other biomarkers besides IL-17A, IFN-γ and CD62E.

[00163] Therefore, in a preferred embodiment, the method further comprises determining the expression level of additional biomarkers other than the primary biomarkers of the invention (i.e., secondary biomarkers). Preferably, the method further comprises determining the level of at least one additional biomarker selected from the group consisting of MCP-1, IL-1α, MIP-1β, TNFα, MIP-1α, CD62P, IL-10 and combinations thereof.

[00164] The term MCP-1 or MCP1 refers to monocyte chemotactic protein 1 or monocyte chemotactic protein 1, and is also known as CCL2 or chemokine ligand 2 (CC motif) or CC motif chemokine 2 or small inducible cytokine A2 or SCYA2. As used in this document, this term refers to the protein or gene encoding the protein with UniProt accession number P13500 (version 246 of the entry on October 12, 2022). The term monocyte chemotactic protein 1 includes any of the isoforms described for this protein.

[00165] The terms IL-Ια, IL-1a, IL-1 alpha, IL1A or IL1F1 refer to interleukin-1 alpha, also called hematopoietin-1. As used herein, this term refers to the protein or gene encoding the protein with UniProt accession number P01583 (version 224 of the entry on October 12, 2022). The term interleukin-1 alpha includes any of the isoforms described for this Petition 870250097700, dated 10 / 24 / 2025, p. 72 / 145 68 / 118 protein.

[00166] The term MIP-1β, MIP-1b, MIP1B, or MIP-1beta refers to macrophage inflammatory protein-1beta and is now officially named CCL4 or chemokine ligand 4 (CC motif) or CC motif chemokine 4. Older names for human CCL4 are also AT 744, Act-2, LAG-1, HC21, and G-26. As used herein, this term refers to the protein or gene encoding the protein with UniProt accession number P13236 (version 216 of the entry on October 12, 2022). The term CC motif chemokine 4 includes any of the isoforms described for this protein.

[00167] The terms TNF-α, TNF-alpha, TNF-a, TNFA, TNFSF2, DIF, TNLG1F, or TNF refer to tumor necrosis factor or tumor necrosis factor alpha, also called cachectin, cachexin, or tumor necrosis factor ligand superfamily member 2. As used herein, this term refers to the protein or gene encoding the protein with UniProt accession number P01375 (version 267 of the entry on February 22, 2023). The term “tumor necrosis factor” includes any of the isoforms that have been described for this protein.

[00168] The term “MIP-1”, “MIP-1-alpha” or “MIP1A”, also referred to as “CCL3”, “LD78-alpha”, “G0S19-1”, “PAT 464.1”, “SIS-beta” or “SCYA3” refers to “macrophage inflammatory protein 1-alpha”, also known as “CC motif chemokine 3”, “G0 / G1 exchange regulatory protein 19-1”, “small cell-inducible cytokine A3” or “tonsillar lymphocyte LD78 alpha protein”. According Petition 870250097700, dated 10 / 24 / 2025, p. 73 / 145 69 / 118 used in this document, this term refers to the protein or gene encoding the protein with UniProt accession number P10147 (version 217 of the entry on February 22, 2023). The term “macrophage inflammatory protein 1-alpha” includes any of the isoforms described for this protein.

[00169] The term “CD62P”, “SELP”, “GMRP”, “GRMP”, “GMP-140”, “GMP140”, “LECAM3”, “PADGEM”, or “PSEL”, also referred to as “P-selectin” or “P-selectin”, refers to “CD62P antigen-like family member”, “granular membrane protein 140”, “leukocyte-endothelial cell adhesion molecule 3”, or “platelet activation-dependent granule outer membrane protein”. As used in this document, this term refers to the protein or gene encoding the protein with UniProt accession number P16109 (version 240 of the entry on February 22, 2023). The term “CD62P antigen-like family member” includes any of the isoforms described for this protein.

[00170] The term “IL-10”, “IL10” or “IL10A”, also referred to as “CSIF”, refers to “interleukin-10” or “cytokine synthesis inhibitory factor”. As used in this document, this term refers to the protein or gene encoding the protein with UniProt accession number P22301 (version 216 of the entry on February 22, 2023). The term “interleukin-10” includes any of the isoforms that have been described for this protein.

[00171] In a preferred embodiment, the method of the invention further comprises determining the expression level of MCP-1. In another embodiment, the method comprises Petition 870250097700, dated 10 / 24 / 2025, p. 74 / 145 70 / 118 further the determination of the expression level of IL-Iα. In another embodiment, the method further comprises the determination of the expression level of MIP-Iβ. In another embodiment, the method further comprises the determination of the expression level of TNF-α. In another embodiment, the method further comprises the determination of the expression level of MIP-Iα. In another embodiment, the method further comprises the determination of the expression level of CD62P. In another embodiment, the method further comprises the determination of the expression level of IL10. In another embodiment, the method of the invention further comprises the determination of the expression level of a combination of MCP-1, IL-1α, MIP-Iβ, TNF-α, MIP-Iα, CD62P and IL-10.

[00172] Any combination of secondary biomarkers is covered by the methods of the invention.

[00173] Any combination of a primary biomarker with a secondary biomarker is also covered by the methods of the invention.

[00174] In a preferred embodiment, the method of the invention comprises determining the levels of a combination of biomarkers selected from the group consisting of: a) a combination of biomarkers comprising IL-17A and MCP-1; b) a combination of biomarkers comprising IL-17A and IL-1α; c) a combination of biomarkers comprising IL-17A and MIP-1α; d) a combination of biomarkers comprising IL-17A and TNF-α; e) a combination of biomarkers comprising IL-17A and MIP-1α; f) a combination of biomarkers comprising IL-17A and CD62P; g) a combination of biomarkers comprising IL-17A and IL-10; and a combination of biomarkers comprising IL-17A and CD62E. More Petition 870250097700, dated 10 / 24 / 2025, page 75 / 145 71 / 118 Preferably, the combination is selected from: a) a biomarker combination comprising IL-17A and MCP-1; b) a biomarker combination comprising IL-17A and IL-1α; c) a biomarker combination comprising IL-17A and CD62P; d) a biomarker combination comprising IL-17A and IL-10; and e) IL-17A and CD62E. Even more preferably, the combination is selected from: a) a biomarker combination comprising IL-17A and MCP-1; and b) a biomarker combination comprising IL-17A and IL-1α. In a preferred embodiment, the biomarker combination consists of the combinations defined above.

[00175] In a preferred embodiment, the method of the first aspect of the invention further comprises: i) determine the level of at least one additional biomarker selected from the group consisting of MCP-1, IL-1β, MIP-10, TNF-α, MIP-1β, CD62P, and IL-10 in a sample from the individual obtained after treatment administration; and ii) compare the level of said at least one biomarker with a reference value, where: - an increased level of at least one biomarker compared to the reference value is indicative of a good clinical response of the individual to cancer treatment, or - A level equal to or lower than the reference value for at least one biomarker is indicative of an unsatisfactory clinical response to cancer treatment.

[00176] Any combination of primary and secondary biomarkers is covered by the methods of Petition 870250097700, dated 10 / 24 / 2025, p. 76 / 145 72 / 118 invention.

[00177] The term level is also understood as “expression level” or “expression level” and, as used in this document, refers to the expression level of a gene product, more specifically to a measurable quantity of a gene product produced by a specific gene in a specific sample from the individual. The term gene product, as used in this document, refers to a transcriptional or translational product and therefore corresponds to the mRNA transcribed from said gene or the protein encoded by a specific gene. When referring to the expression level of a biomarker, it is understood that the gene product is said biomarker, which, as indicated below, may be an mRNA or a protein transcribed or encoded by the selected genes of the invention. As understood by those skilled in the art, the gene expression level can be quantified by measuring the messenger RNA levels of said gene or the protein encoded by said gene.In the context of the present invention, the expression level of the gene encoding IL-17A, IFN-γ, CD62E, MCP-1, IL-1a, MIP-Iβ, TNF-α, MIP-Iα, CD62P or IL-10 can be determined by measuring the levels of mRNA encoded by said genes, or by measuring the levels of the protein encoded by said genes, i.e., IL-17A, IFN-γ, CD62E, MCP1, IL-1a, MIP-Iβ, TNF-α, MIP-Iα, CD62P or IL-10 protein or variants thereof. The protein variants IL-17A, IFN-γ, CD62E, MCP-1, IL-1a, MIP-Iβ, TNF-α, MIP-Iα, CD62P, and IL-10 include all forms of physiologically relevant post-translational chemical modifications of the protein, for example, glycosylation, phosphorylation, acetylation, etc. Petition 870250097700, dated 10 / 24 / 2025, p. 77 / 145 73 / 118 that the functionality of the protein is maintained. The term encompasses the proteins IL-17A, IFN-γ, CD62E, MCP-1, IL-1a, MIP-Iβ, TNF-α, MIP-Iα, CD62P, and IL-10 from any mammalian species, including, but not limited to, domestic and farm animals (cows, horses, pigs, sheep, goats, dogs, cats, or rodents), primates, and humans. Preferably, the proteins IL-17A, IFN-γ, CD62E, MCP-1, IL-1a, MIP-Iβ, TNFα, MIP-Iα, CD62P, and IL-10 are human proteins.

[00178] As the expert in the technique understands, the expression levels of said biomarkers can be measured by determining the mRNA expression levels of the genes that encode them or by determining the protein levels encoded by said genes.

[00179] In a preferred embodiment, the levels of at least one biomarker that are determined are the mRNA levels.

[00180] To measure mRNA levels of the aforementioned biomarkers, the biological sample can be treated to physically, mechanically, or chemically disrupt tissues or cellular structures, to release intracellular components into an aqueous or organic solution and prepare nucleic acids for further analysis. Nucleic acids are extracted from the sample by procedures known to the expert, using commercially available reagents. RNA is then extracted from frozen or fresh samples by any of the typical methods of the technique, for example, Sambrook, J., et al., 2001. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, NY, Vol. 1-3. Preferably, care should be taken to avoid RNA degradation during the process. Petition 870250097700, dated 10 / 24 / 2025, p. 78 / 145 74 / 118 extraction process.

[00181] The expression level can be determined using mRNA obtained from a formalin-fixed, paraffin-embedded tissue sample. The mRNA can be isolated from an archival pathological sample or a biopsy sample that is first deparaffinized. An exemplary deparaffinization method involves washing the paraffinized sample with an organic solvent, such as xylene. The deparaffinized samples can be rehydrated with an aqueous solution of a lower alcohol. Suitable lower alcohols, for example, include methanol, ethanol, propanols, and butanols. The deparaffinized samples can be rehydrated with successive washes with decreasing concentration lower alcohol solutions, for example. Alternatively, the sample is simultaneously deparaffinized and rehydrated. The sample is then lysed and RNA is extracted from the sample. Samples can also be obtained from fresh tumor tissue, such as a resected tumor.In one particular modality, samples can be obtained from fresh tumor tissue or from frozen tissue embedded in OCT.

[00182] Suitable methods for determining gene expression levels at the mRNA level include, without limitation, standard assays for determining mRNA expression levels such as qPCR, RT-PCR, RNA shielding analysis, Northern blot, RNA dot blot, TaqMan®, marker-based methods such as serial gene expression analysis (SAGE), including variants such as LongSAGE and SuperSAGE, microarrays, nucleic acid sequence-based amplification (NASBA), fluorescence in situ hybridization, Petition 870250097700, dated 10 / 24 / 2025, page 79 / 145 75 / 118 including variants such as Flow-FISH, qFiSH and double fusion fish (D-FISH), and similar.

[00183] In one particular embodiment, the expression levels of the biomarkers are determined by a microarray. In another particular embodiment, the expression levels of the biomarkers are determined by quantitative PCR, preferably real-time PCR.

[00184] To normalize mRNA expression values ​​between different samples, it is possible to compare the expression levels of the mRNA of interest in the test samples with the expression of a control RNA. A control RNA, as used herein, refers to an RNA whose expression levels do not change or change only in limited amounts in tumor cells compared to non-tumorigenic cells. Preferably, the control RNA is an mRNA derived from maintenance genes that encode proteins that are constitutively expressed and perform essential cellular functions. Examples of maintenance genes for use in the present invention include β2-microglobulin, ubiquitin, ribosomal protein 18-S, cyclophilin, GAPDH, PSMB4, tubulin, and β-actin.

[00185] In one embodiment, the quantification of relative gene expression is calculated according to the comparative threshold cycle (Ct) method, using GAPDH, β-actin or PSMB4 as endogenous controls and commercial RNA controls as calibrators. The final results are determined according to the formula 2-(ACt sample-ACt calibrator), where the ACT values ​​of the calibrator and the sample are determined by subtracting the CT value of the target gene from the value of the control gene. Petition 870250097700, dated 10 / 24 / 2025, p. 80 / 145 76 / 118

[00186] In a preferred embodiment, the levels of at least one biomarker determined are the protein levels.

[00187] The expression levels of said biomarkers can also be measured by determining the levels of proteins or variants thereof encoded by the genes. Virtually any conventional method can be used in the context of the present invention to quantify the levels of said proteins. Suitable methods for determining gene expression levels at the protein level include, without limitation, conventional methods such as the use of antibodies capable of specifically binding to the proteins encoded by said genes (or to fragments thereof containing antigenic determinants) and the subsequent quantification of the resulting antibody-antigen complexes.In a particular embodiment, the protein levels of the biomarkers of the invention can be quantified using standard assays to determine protein expression levels, such as Western blot or Western transfer, immunoassays such as ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (antibody-double sandwich ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of biochips or protein microarrays that include specific antibodies, or assays based on colloidal precipitation in formats such as dipsticks or immunoassays based on Luminex technology.

[00188] The antibodies to be used in these assays may be, for example, polyclonal sera, Petition 870250097700, dated 10 / 24 / 2025, page 81 / 145 77 / 118 hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab' and F(ab')2, ScFv, diabodies, triabodies, tetrabodies and humanized antibodies. At the same time, antibodies may be labeled or unlabeled. Illustrative, but not exclusive, examples of markers that can be used include radioactive isotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes, etc.There is a wide variety of well-known assays that can be used in the present invention, employing unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies); these techniques include Western blot or Western transfer, ELISA, RIA, competitive EIA, DAS-ELISA, immunocytochemical and immunohistochemical techniques, techniques based on the use of biochips or protein microarrays, including specific antibodies or assays based on colloidal precipitation in formats such as dipsticks. Other ways to detect and quantify the levels of the protein of interest include affinity chromatography techniques, ligand-binding assays, etc.

[00189] On the other hand, the determination of biomarker protein levels can be performed by constructing a tissue microarray (TMA) containing the collected samples and by determining the expression levels of the corresponding protein using immunohistochemical techniques. The intensity of the immunostaining can be evaluated by two or more different pathologists and scored using uniform and clear cutoff criteria in order to maintain the reproducibility of the method. Discrepancies may be Petition 870250097700, dated 10 / 24 / 2025, page 82 / 145 78 / 118 resolved by simultaneous reassessment. In summary, the immunostaining result can be recorded as negative expression (0) versus positive expression, and low expression (1+) versus moderate (2+) and high (3+) expression, taking into account the expression in tumor cells and the specific cutoff point for each marker. As a general criterion, cutoff points are selected to facilitate reproducibility and, when possible, to translate biological events. Alternatively, the intensity of immunostaining can be evaluated using imaging techniques and automated methods, such as those reported in Rojo, MG et al. (Folia Histochem. Cytobiol. 2009; 47: 349-54) or Mulrane, L. et al. (Expert Rev. Mol. Diagn. 2008; 8: 70725).

[00190] Alternatively, in another particular embodiment, the levels of biomarker proteins are determined by Western blot. Western blot is based on the detection of proteins previously resolved by gel electrophoresis under denaturing conditions and immobilized on a membrane, usually nitrocellulose, by incubation with a specific antibody and a development system (e.g., chemiluminescent).

[00191] In one particular embodiment, expression levels are determined as protein levels. In a more specific embodiment, protein levels are determined by ELISA, Western blot, or an immunoassay.

[00192] In a preferred embodiment, levels are determined by an immunoassay. The term immunoassay, as used in this document, refers to a biochemical test that measures the presence or concentration of a Petition 870250097700, dated 10 / 24 / 2025, p. 83 / 145 79 / 118 molecule in a solution by means of the use of an antibody or an antigen-binding fragment thereof, capable of specifically recognizing said molecule. In a preferred embodiment, the immunoassay is an antibody-based immunoassay bound to Luminex beads, particularly an immunoassay performed using Luminex technology, more specifically using a ProcartaPlex™ kit.

[00193] As mentioned previously, variants of said proteins can be used to measure the expression levels of IL-17A, IFN-γ, CD62E, MCP1, IL-1a, MIP-Iβ, TNF-α, MIP-Iα, CD62P and IL-10, in order to put the method of the invention into practice.

[00194] Thus, variants of the proteins IL-17A, IFN-γ, CD62E, MCP-1, IL-1a, MIP-Iβ, TNF-α, MIP-Iα, CD62P and IL-10 may be: (i) one in which one or more amino acid residues are replaced by a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be a residue encoded by the genetic code; (ii) one in which there are one or more modified amino acid residues, for example, residues that are modified by the attachment of substituent groups; (iii) one in which the protein is an isoform or an alternative splicing variant of the proteins of the present invention and / or; (iv) fragments of the proteins. The fragments include proteins generated by proteolytic cleavage (including multisite proteolysis) of an original sequence. The variations are considered within the scope of those versed in the technique, based on the teachings presented here.

[00195] The variants, according to the present Petition 870250097700, dated 10 / 24 / 2025, p. 84 / 145 80 / 118 invention, include amino acid sequences that are at least 60%, 70%, 80%, 90%, 95%, or 96% similar or identical to the original amino acid sequence. As is known in the art, the similarity between two proteins is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one protein with the sequence of a second protein. The degree of identity between two proteins is determined using algorithms and computational methods widely known to those skilled in the art. The identity between two amino acid sequences is preferably determined using the BLASTP algorithm [BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S. et al., J. Mol. Biol. 215: 403-410 (1990)].

[00196] Proteins can be modified by post-translation. For example, post-translational modifications that fall within the scope of the present invention include cleavage of signaling peptides, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding, and proteolytic processing, etc. In addition, proteins can include non-natural amino acids formed by post-translational modification or by the introduction of non-natural amino acids during translation.

[00197] In a particular embodiment, the variant in question is a mammalian variant, preferably a human variant, more preferably with at least 60%, 70%, 80%, 90%, 95% or 96% similarity or identity to the original amino acid sequence.

[00198] The second step of the first method of the invention involves comparing the level of said at least Petition 870250097700, dated 10 / 24 / 2025, page 85 / 145 81 / 118 a biomarker obtained in step (i) with a reference value.

[00199] The term reference value, as used herein, refers to a laboratory value used as a reference for values / data obtained from samples collected from individuals. The 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 value, a median value, an average value, or a value compared to a specific control or baseline value. A reference value may be based on an individual sample value, such as a value obtained from a sample of the individual to be tested, but at an earlier time.The reference value can be based on a large number of samples, for example, on a group of patients considered representative, such as the values ​​obtained from a population of individuals of a corresponding chronological age group that coincides with that of the patient under study, or based on a set of samples including or excluding the sample to be tested.

[00200] In a preferred embodiment, the reference value is the expression level of IL-17A, IFN-γ, CD62E, MCP-1, IL-1a, MIP-Iβ, TNF-α, MIP-Iα, CD62P, and IL-10 determined in a sample from an individual with cancer prior to the administration of said treatment, i.e., before the start of the first administration of treatment, in a pre-treatment sample. More preferably, the reference value is the expression level of IL-17A, IFN-γ, CD62E, MCP-1, IL-1a, MIP-Iβ, TNF-α, MIP-Iα, CD62P, and IL-10 determined in a sample from the same individual that will be Petition 870250097700, dated 10 / 24 / 2025, page 86 / 145 82 / 118 tested before treatment administration.

[00201] In another embodiment, the reference value is the expression level of IL-17A, IFN-γ, CD62E, MCP-1, IL-1a, MIP-Iβ, TNF-α, MIP-Iα, CD62P and IL-10 determined in a set of samples from individuals with cancer before the administration of said treatment, i.e., before the start of the first administration of treatment, in a pre-treatment sample.

[00202] In a preferred embodiment, the reference value is obtained from a pretreatment sample.

[00203] In the context of the present invention, a pre-treatment sample is considered to be a sample obtained before the individual receives the first administration of an anticancer treatment selected from the group consisting of items (a) to (e) of the first aspect of the invention.

[00204] In a preferred embodiment, when the reference value is determined on a pretreatment sample, preferably the individual from whom the pretreatment sample is obtained has not received any anticancer treatment at any time, preferably in the last 168 hours, more preferably preferably preferably preferably sample. In received a preferably in the last in the last in the last in the last 24 hours modality other anticancer treatment in the last hours, hours, hours, prior to the preferred, the hours hours, more even more even more even more individual isolation not prior to sample isolation, but preferably in the last 23 hours, preferably in the last 22 hours, more Petition 870250097700, dated 10 / 24 / 2025, page 87 / 145 83 / 118 preferably in the last 21 hours, even more preferably in the last 20 hours, even more preferably in the last 15 hours, even more preferably in the last 10 hours, even more preferably in the last 5 hours, and even more preferably in the last 2 hours. Preferably, the sample was taken within the last hour prior to isolation. In a further preferred embodiment, the individual from whom the sample was isolated had not received any anticancer treatment at any time prior to isolation, i.e., the individual had never received any anticancer treatment. In another preferred embodiment, the individual had not received any anticancer treatment in the 24 hours prior to isolation.

[00205] In another particular embodiment, when the reference value is determined on a pretreatment sample, preferably the individual from whom the pretreatment sample is obtained has not received systemic anticancer treatment for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 50 weeks prior to sample isolation. In a more preferred embodiment, the individual has not received systemic anticancer treatment for at least 4 weeks prior to sample isolation.

[00206] In another particular modality, when the Petition 870250097700, dated 10 / 24 / 2025, page 88 / 145 The reference value 84 / 118 is determined on a pretreatment sample, preferably the individual from whom the pretreatment sample was obtained received anticancer treatment at any time prior to sample isolation, preferably within the last 168 hours, more preferably within the last 72 hours, even more preferably within the last 48 hours, even more preferably within the last 36 hours, even more preferably within the last 24 hours before sample isolation. If administered 24 hours before sample isolation, preferably within the last 24 hours before sample isolation, said anticancer treatment is different from an anticancer treatment selected from the group consisting of items (a) to (e) of the first aspect of the invention.In a preferred embodiment, the individual from whom the sample was isolated received anticancer treatment prior to the isolation of the sample, and wherein the anticancer treatment is different from a treatment selected from the group consisting of items (a) to (e) of the first aspect of the invention. Preferably, said anticancer treatment is selected from surgery, chemotherapy, radiotherapy, hormone therapy and targeted therapy, including immunotherapy, and combinations thereof. In a preferred embodiment, said anticancer therapy is selected from the group consisting of surgery and radiotherapy. In a more preferred embodiment, said anticancer therapy is radiotherapy.

[00207] In a preferred embodiment, the reference value is the expression level of IL-17A, IFN-γ, CD62E, MCP-1, IL-1a, MIP-Iβ, TNF-α, MIP-Iα, CD62P and IL-10 Petition 870250097700, dated 10 / 24 / 2025, page 89 / 145 85 / 118 determined in a sample from an individual with cancer at a time prior to treatment administration, i.e., after the start of treatment administration, in a post-treatment sample obtained at an earlier time. More preferably, the reference value is the expression level of IL-17A, IFN-γ, CD62E, MCP-1, IL-1α, MIP-1β, TNF-α, MIP1α, CD62P, and IL-10 determined in a sample from the same individual to be tested at a time prior to treatment administration, i.e., after the start of treatment administration, in a post-treatment sample obtained at an earlier time.

[00208] In another embodiment, the reference value is the expression level of IL-17A, IFN-γ, CD62E, MCP-1, IL-1a, MIP-Iβ, TNF-α, MIP-Iα, CD62P and IL-10 determined in a set of samples from individuals with cancer at a time prior to treatment administration, i.e., after the start of treatment administration, in a post-treatment sample obtained at an earlier time.

[00209] According to the present invention, a post-treatment sample obtained at an earlier time is a sample obtained after the first administration of the selected anticancer treatment from the group consisting of items (a) to (e) of the first aspect of the invention and before receiving a new dose of said anticancer treatment. This new dose of the anticancer treatment may be the second dose, the third dose, the fourth dose, the fifth dose, the sixth dose, or any subsequent dose received by the patient during the course of treatment. In a preferred embodiment, the post-treatment sample obtained at an earlier time is a sample obtained immediately before receiving a new dose. Petition 870250097700, dated 10 / 24 / 2025, pp. 90 / 145 86 / 118 of the anticancer treatment, that is, a pre-dose sample.In a preferred embodiment, the post-treatment sample obtained at a prior time point is obtained within 7 days, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, within 24 hours, within 23 hours, within 22 hours, within 21 hours, within 20 hours, within 19 hours, within 18 hours, within 17 hours, within 16 hours, within 15 hours, within 14 hours, within 13 hours, within 12 hours, within 11 hours, within 10 hours, within 9 hours, within 8 hours, within 7 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours, within 1 hour, within 50 minutes, within 45 minutes, within 40 minutes, within 35 minutes, within 30 minutes, within 25 minutes, within 20 minutes, within 15 minutes, within 10 minutes, or within 5 minutes before receiving a new dose of cancer treatment.In a preferred embodiment, the post-treatment sample obtained at an earlier time point is obtained within 15 minutes before receiving a new dose of the anticancer treatment; more preferably within 10 minutes before receiving a new dose of the anticancer treatment; even more preferably, it is obtained 10 minutes before receiving a new dose of the anticancer treatment.

[00210] If the method of the invention aims to determine the effect of a therapy on a patient, the reference sample is preferably a sample obtained from said patient before receiving said treatment or at different time periods during a course of treatment.

[00211] In a preferred embodiment, the value of Petition 870250097700, dated 10 / 24 / 2025, pp. 91 / 145 87 / 118 reference is a value obtained from a sample from the same individual before treatment administration, preferably before the first dose of treatment.

[00212] In a preferred embodiment, the reference value is a value obtained from a sample of the same individual at a time prior to the administration of the treatment.

[00213] Thus, in a particular embodiment, the method of the invention involves determining the expression level of biomarkers in a sample from an individual obtained at a first time point (first individual sample) and determining the expression level of biomarkers in a sample from an individual obtained at a second time point (second individual sample), which are compared, allowing the effectiveness of the response to therapy in said individual with cancer to be evaluated or monitored. The second individual sample can be collected from the same individual with cancer from whom the first measurement is derived, at a second time point, i.e., at any time after the first time point, for example, one day, one week, one month, two months, three months, 1 year, 2 years or more after the first individual sample.In one particular embodiment, the individual's first sample is collected before the individual receives treatment, and the individual's second sample is collected after treatment. In another particular embodiment, the individual's first sample is collected after the individual has started / received treatment, and the individual's second sample is collected subsequently, at different time points during a [session / period]. Petition 870250097700, dated 10 / 24 / 2025, page 92 / 145 88 / 118 course of treatment whose effectiveness must be evaluated or monitored.

[00214] The reference value may also be determined in a sample of one or more individuals with cancer who have never been treated with an anticancer treatment selected from the group consisting of items (a) to (e) of the first aspect of the invention. The reference value may also be determined in a sample of one or more individuals with cancer who have shown an unsatisfactory clinical response to an anticancer treatment selected from the group consisting of items (a) to (e) of the first aspect of the invention.

[00215] In one embodiment, the reference value can be obtained by determining the median value of the expression levels of each of the biomarkers measured in a collection of samples from individuals with cancer who do not show an increase in said biomarkers or from normal tissue.

[00216] Alternatively, the use of a reference value to determine whether the expression level of a biomarker is increased or decreased could correspond to the median value of the expression levels of each biomarker measured in an RNA sample obtained by combining equal amounts of RNA from each of the samples obtained from individuals with cancer who do not show increased levels of the biomarkers of the invention, preferably from individuals with the same type of cancer.

[00217] The sample collection from which the reference level is derived will preferably consist of individuals with the same type of cancer or a mixture of Petition 870250097700, dated 10 / 24 / 2025, page 93 / 145 89 / 118 tissues from normal individuals not affected by cancer. More preferably, the sample is from the same individual who will be tested.

[00218] In another modality, the reference value is the expression level of biomarkers in a healthy individual, that is, an individual who has not been diagnosed with the type of cancer for which the response to therapy is being evaluated.

[00219] In the present invention, the reference value can be an arbitrary cutoff point, established according to the ROC methodology. Once this cutoff point is established, the level of this marker expressed in biofluids or tumor tissues of the individual can be compared with this cutoff point and thus be assigned a low expression level if it is below this cutoff point or a high expression level if it is above this cutoff point. Therefore, in a preferred embodiment, the expression levels of each of the biomarkers in a combination are compared with predetermined cutoff values ​​for each of the genes, wherein the predetermined cutoff values ​​for each gene preferably correspond to the gene expression level that correlates with the highest specificity at the desired sensitivity in an ROC curve calculated based on the expression levels of the biomarkers.

[00220] Once this reference value is established, the level of the biomarker expressed in the sample can be compared with the said reference value and thus be assigned an increased, decreased, or equal level of expression. For example, an increase in levels Petition 870250097700, dated 10 / 24 / 2025, pp. 94 / 145 90 / 118 expression levels above the reference value 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 even more compared to the reference value is considered an increased expression level. Conversely, a decrease in expression levels 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 even less compared to the reference value is considered a decreased expression level.

[00221] This comparison allows the detection of a decrease in the expression of one or more of the aforementioned biomarkers in relation to the reference value. The expression “decreased levels” of one or more biomarkers selected from the group consisting of IL-17A, IFN-γ, CD62E, MCP1, IL-1a, MIP-Iβ, TNF-α, MIP-Iα, CD62P, and IL-10 means a decrease in the expression of a given biomarker relative to the reference value 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.

[00222] The expression “increased levels” of one or more biomarkers selected from the group consisting of IL-17A, IFN-γ, CD62E, MCP-1, IL-1α, MIP-1β, TNFα, MIP-1α, CD62P and IL-10 means an increase in the expression of a given biomarker relative to the value of Petition 870250097700, dated 10 / 24 / 2025, pp. 95 / 145 91 / 118 reference 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.

[00223] Expression levels may be considered equal to the reference value if the levels differ 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.

[00224] When a comparison is made between the level of one or more selected biomarkers from the group consisting of IL-17A, IFN-γ and CD62E and the reference value, the method of the invention allows determining whether the individual has a good clinical response to an anticancer treatment according to items (a) to (e) of the first aspect of the invention or an unsatisfactory clinical response. Thus, individuals who have an increased level of at least one biomarker relative to the reference value have a good clinical response to anticancer treatment.

[00225] The expression “good clinical response”, as used herein, means that the individual presents a favorable response to anticancer treatment in accordance with items (a) to (e) of the first aspect of the invention, which is understood by the expert as a complete response, Petition 870250097700, dated 10 / 24 / 2025, pp. 96 / 145 92 / 118 a partial response or stabilization of the disease.

[00226] In a preferred modality, a good clinical response is stabilization of the disease.

[00227] Alternatively, patients with a level equal to or reduced from the reference value of at least one biomarker have an unsatisfactory clinical response to cancer treatment. Preferably, patients with an unsatisfactory clinical response have a level equal to or reduced from the reference value of at least one biomarker.

[00228] The term unsatisfactory clinical response, as used in this document, means that the individual's response is not as favorable as indicated. Unsatisfactory clinical response includes a prediction of disease progression. It may also include a prediction of relapse, mortality, or the need to change or administer a new medical treatment.

[00229] In a preferred modality, the unsatisfactory clinical response is disease progression.

[00230] As will be understood by those versed in the art, such a probability assessment, although preferred, may generally not be correct for 100% of the individuals to be analyzed. The term, however, requires that a statistically significant portion of individuals can be identified as having a predisposition to respond to chemotherapy treatment. Whether a portion is statistically significant can be determined without major complications by the subject matter expert using various well-known statistical assessment tools, for example, determination of intervals of Petition 870250097700, dated 10 / 24 / 2025, page 97 / 145 93 / 118 confidence, determination of the p-value, Student's t-test, Mann-Whitney test, etc. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 95%. The p-values ​​are preferably 0.1, 0.05, 0.01, 0.005, or 0.0001. More preferably, at least 60%, at least 70%, at least 80%, or at least 90% of the individuals in a population can be adequately identified by the method of the present invention.

[00231] This method allows the evaluation of a specific treatment for a selected individual previously diagnosed with cancer. Consequently, patients with a good clinical response can continue receiving the same anticancer treatment. Otherwise, patients with an unsatisfactory clinical response should discontinue the anticancer treatment of the invention and be treated with a different therapy. The course of the new treatment can be easily followed according to this method. THERAPEUTIC METHODS OF THE INVENTION

[00232] The results obtained in the present invention demonstrate that a high level of IL-17A, IFN-γ and CD62E in a sample from a cancer patient, collected after the start of treatment, indicates a high probability of therapeutic success with an anticancer treatment, as defined in items (a) to (f) of the first aspect of the invention. This indicates that this may be the best treatment available for such patients and that they should continue to receive said treatment. Petition 870250097700, dated 10 / 24 / 2025, pp. 98 / 145 94 / 118

[00233] In a second aspect, the invention relates to an agent selected from the group consisting of: a) a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof; (b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) a polynucleotide that codes for the polypeptide in a) or the conjugate of b); d) a vector comprising the polynucleotide according to c); and (e) a cell capable of secreting into the environment the polypeptide according to a) or the conjugate according to b); for use in the treatment of cancer in an individual, wherein the individual has been identified as a good responder to said agent by a method in accordance with the first aspect of the invention.

[00234] Alternatively, the invention relates to the use of an agent selected from the group consisting of: a) a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a variant Petition 870250097700, dated 10 / 24 / 2025, pp. 99 / 145 95 / 118 functional equivalent thereof and a chemical fraction that facilitates cellular uptake of the polypeptide or its functional equivalent variant; c) a polynucleotide that codes for the polypeptide in a) or the conjugate of b); d) a vector comprising the polynucleotide according to c); and (e) a cell capable of secreting into the environment the polypeptide according to a) or the conjugate according to b); for the manufacture of a medicament for the treatment of an individual suffering from cancer, wherein the individual has been identified as a good responder to said agent by a method in accordance with the first aspect of the invention.

[00235] Alternatively, the invention relates to a method for treating cancer in an individual, comprising administering to said individual an agent selected from the group consisting of: a) a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) a polynucleotide that codes for the polypeptide in a) or the conjugate of b); d) a vector comprising the polynucleotide of Petition 870250097700, dated 10 / 24 / 2025, pages 100 / 145 96 / 118 in accordance with c); and (e) a cell capable of secreting into the environment the polypeptide according to a) or the conjugate according to b); whereby the individual was identified as a good responder to said agent by a method in accordance with the first aspect of the invention.

[00236] The agent preferably forms part of a medicament, that is, a composition comprising a therapeutically effective amount of the agent, comprising at least one pharmaceutically acceptable excipient or carrier. The medicament may comprise more than one agent, either more than one agent defined in items (a) to (e) of the first aspect of the invention, or additional agents useful for the treatment of cancer.

[00237] The term therapeutically effective amount, as used in this document, refers to the amount of the agent sufficient to produce the desired effect and will generally be determined, among other factors, by the characteristics of the compound itself and the therapeutic effect to be achieved. It will also depend on the individual being treated, the severity of the oncological disease suffered by said individual, the pharmaceutical form chosen, the route of administration, etc.

[00238] The term pharmaceutically acceptable excipient or pharmaceutically acceptable carrier, as used in this document, refers to any compound or combination of compounds that is essentially non-toxic to the individual at the dosage and concentration used and is compatible with the others Petition 870250097700, dated 10 / 24 / 2025, pp. 101 / 145 97 / 118 components of a medicine. Thus, an excipient is an inactive substance formulated along with the active ingredient of a medicine for the purpose of increasing the volume of compositions containing said active ingredients. Excipients may also serve various therapeutic enhancement purposes, such as facilitating the absorption or solubility of the active ingredient, or other pharmacokinetic considerations. Excipients may also be useful in the manufacturing process, to aid in the handling of the active substance in question, such as facilitating powder flowability or anti-adherent properties, as well as aiding in in vitro stability, such as preventing denaturation over the expected shelf life.

[00239] In a preferred embodiment, the anticancer treatment is administered intravenously; preferably as an intravenous infusion over 30 to 45 minutes. In another embodiment, the anticancer therapy is administered once a week.

[00240] In a preferred embodiment, the agent is a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant; more preferably, it is a polypeptide consisting of SEQ ID NO: 4.

[00241] All embodiments of the first aspect of the invention are also applicable to the second aspect of the invention.

[00242] All terms have been defined previously in the context of the first aspect of the invention and are used with the same meaning in the context of the second aspect of the invention. KITS OF THE INVENTION AND THEIR USES Petition 870250097700, dated 10 / 24 / 2025, pp. 102 / 145 98 / 118

[00243] The inventors of the present invention have discovered that the levels of IL-17A, IFN-γ and CD62E can be determined by an immunoassay to evaluate or monitor the response of an individual with cancer to an anticancer treatment with OMO-103.

[00244] In another aspect, the 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 IL-17A, IFN-γ and CD62E.

[00245] In another aspect, the invention relates to the use of a kit comprising a specific reagent to determine the expression level of at least one biomarker selected from the group consisting of IL-17A, IFN-γ and CD62E, or to the use of a specific reagent to determine the expression level of at least one biomarker selected from the group consisting of IL-17A, IFN-γ, CD62E, MCP1, IL-1α, MIP-Iβ, TNF-α, MIP-Iα, CD62P and IL-10, preferably selected from the group consisting of IL-17A, MCP-1 and IL-Iα, in a sample from an individual with cancer to evaluate or monitor the clinical response of said individual to treatment with any of the agents defined in items (a) to (e) of the first aspect of the invention. In a preferred embodiment, the kit or reagent is used in a method according to the first aspect of the invention.

[00246] In the context of the present invention, a kit is understood to be a product containing the different reagents necessary for carrying out the different uses of the invention, packaged in a way that allows for its transport and storage. The materials suitable for packaging the Petition 870250097700, dated 10 / 24 / 2025, pp. 103 / 145 99 / 118 components of the kit include crystal, plastic (polyethylene, polypropylene, polycarbonate and the like), bottles, jars, paper, envelopes and the like. Additionally, the kits used in the invention may contain instructions for the simultaneous, sequential or separate use of the different components that comprise it. Such instructions may be in the form of printed material or in the form of an electronic medium capable of storing instructions that can be read or understood, such as, for example, electronic storage media (e.g., magnetic disks, tapes), or optical media (e.g., CD-ROM, DVD), or audio materials. Alternatively, the media may contain internet addresses that provide said instructions.

[00247] In a preferred embodiment, the kit further comprises specific reagents to determine the expression level of at least one additional biomarker selected from the group consisting of MCP-1, IL-Iα, MIP-Iβ, TNF-α, MIP-Iα, CD62P, IL-10 and combinations thereof.

[00248] In a more preferred embodiment, the kit comprises a specific reagent for determining the expression level of IL-17A and at least one additional specific reagent for determining the expression level of a biomarker selected from the group consisting of IFN-γ, CD62E, MCP-1, IL-Iα, MIP-Iβ, TNF-α, MIP-Iα, CD62P and IL-10.

[00249] The term reagent, as used in this document, refers to any compound or composition that can be used to detect the level of any of the biomarkers of the present invention, for example, the level of Petition 870250097700, dated 10 / 24 / 2025, pp. 104 / 145 100 / 118 any of the biomarkers IL-17A, IFN-γ, and CD62E, or the level of any of the biomarkers MCP-1, IL-1α, MIP-Iβ, TNF-α, MIP-Iα, CD62P, and IL-10. In a preferred embodiment, the reagent is a reagent for detecting the level of IL-17A. In another embodiment, the reagent is a reagent for detecting the level of IFN-γ. 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 MCP-1. In another embodiment, the reagent is a reagent for detecting the level of IL-10. In another embodiment, the reagent is a reagent for detecting the level of MIP-13. In another embodiment, the reagent is a reagent for detecting the level of TNF-α. In another embodiment, the reagent is a reagent for detecting the level of MIP-1α. In another embodiment, the reagent is a reagent for detecting the level of CD62P. In another embodiment, the reagent is a reagent for detecting the level of IL-10.

[00250] In another embodiment, the reagent is a reagent for detecting the levels of one or more biomarkers selected from the group consisting of IL-17A, IFNγ, CD62E, MCP-1, IL-γα, MIP-10, TNF-α, MIP-γα, CD62P and IL10, i.e., for detecting their genes, proteins or variants. The reagent may optionally include reagents for detecting one or more maintenance genes or the protein encoded by said maintenance gene(s).

[00251] The expression “specific reagent for determining the expression level” of a biomarker, as used herein, refers to any compound or set of compounds that allows the specific determination of the expression level of a gene or protein by detection methods well known to those skilled in the art. In particular, the term Petition 870250097700, dated 10 / 24 / 2025, pp. 105 / 145 101 / 118 “specific reagent”, as used herein, refers to any reagent that can be used for the specific quantification of one of the biomarkers of the invention.

[00252] In one embodiment, the specific reagent for determining the expression level of a biomarker is a nucleic acid capable of specifically hybridizing with any of the genes IL-17A, IFN-γ, CD62E, MCP-1, IL-1β, MIP-1P, TNF-α, MIP-1β, CD62P, and IL-10. In a preferred embodiment, said nucleic acid is an oligonucleotide that specifically hybridizes with the biomarker.

[00253] The term oligonucleotide, as used in this document, refers to a nucleic acid with preferably at least 10 nucleotides, preferably at least 15 nucleotides, preferably at least 20 nucleotides, preferably at least 25 nucleotides and preferably no more than 100 nucleotides, including polyribonucleotides, polydeoxyribonucleotides and combinations thereof. The term oligonucleotide also refers to molecules formed from conventional nucleotides linked by conventional phosphodiester bonds, as well as variants thereof, including modifications in purines or pyrimidines or modifications in riboses or deoxyriboses designed to increase the stability of the oligonucleotide. Alternatively or additionally, oligonucleotides may contain modified linkages, such as phosphotriester, phosphorothioate, methylphosphonate, etc., or they may be peptide nucleic acids (PNA).The methods for synthesizing oligonucleotides are well known to experts in the field. Petition 870250097700, dated 10 / 24 / 2025, pp. 106 / 145 102 / 118 area. The oligonucleotide can be a primer or a probe. In one embodiment, the oligonucleotide is a probe. In another embodiment, the oligonucleotide is a primer, preferably a pair of primers. In one embodiment, the present invention provides a set of one or more pairs of oligonucleotide primers designed to specifically amplify the biomarker of the methods of the invention.

[00254] Nucleic acids capable of hybridizing specifically with the genes IL-17A, IFN-γ, CD62E, MCP-1, IL-1a, MIP-Iβ, TNF-α, MIP-Iα, CD62P or IL-10 are, for example, one or more pairs of oligonucleotide primers for the specific amplification of fragments of mRNA (or its corresponding cDNA) of the said gene.

[00255] As one skilled in the art understands, the primers and oligonucleotide probes of the invention kit can be used in all gene expression profiling techniques (RT-PCR, SAGE, TaqMan, Real-Time PCR, FISH, NASBA, etc.).

[00256] In another embodiment, the specific reagent for determining the expression level of a biomarker is a compound that binds specifically to the biomarker protein, being particularly selected from the group consisting of antibodies, aptamers and fragments thereof.

[00257] In a preferred embodiment, the specific reagent for determining the expression level of a biomarker is an antibody or fragment thereof capable of specifically binding to (i.e., specifically recognizing) the proteins IL-17A, IFN-γ, CD62E, MCP-1, IL-1α, MIP-Iβ, TNF-α, MIP-Iα, CD62P, or IL-10, or variants thereof. Petition 870250097700, dated 10 / 24 / 2025, pp. 107 / 145 103 / 118 (including fragments thereof containing antigenic determinants).

[00258] Antibodies, or a fragment thereof, capable of detecting an antigen, capable of specifically binding to a protein or its variants are, for example, monoclonal and polyclonal antibodies, antibody fragments, Fv, Fab, Fab' and F(ab')2, ScFv, diabodies, triabodies, tetrabodies and humanized antibodies. The antibodies of the invention kit can be used in conventional methods for detecting protein expression levels, such as Western blot or Western transfer, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of biochips, protein microarrays including specific antibodies or assays based on colloidal precipitation in formats such as dipsticks, etc. In a preferred embodiment, said antibodies are used in an immunoassay.Ideally, the immunoassay is based on Luminex technology, more preferably a ProcartaPlex™ kit.

[00259] The said reagents, specifically the probes and antibodies, can be fixed to a solid support, such as a membrane, plastic or glass, optionally treated to facilitate the fixation of said probes or antibodies to the support.

[00260] The kits of the invention optionally comprise additional reagents for detecting a maintenance gene, a polypeptide encoded by a maintenance gene, or the mRNA encoded by said gene. Petition 870250097700, dated 10 / 24 / 2025, pp. 108 / 145 104 / 118 Maintenance. The availability of said additional reagent allows for the normalization of measurements performed on different samples (e.g., the test sample and the control sample) to rule out that differences in biomarker expression are due to a different amount of total protein in the sample, rather than actual differences in relative expression levels. Maintenance genes, as used herein, refer to genes that encode proteins that are constitutively expressed and perform essential cellular functions. Preferred maintenance genes for use in the present invention include β-2-microglobulin, ubiquitin, ribosomal protein 18-S, cyclophilin, PSMB4, GAPDH, tubulin, and β-actin.

[00261] In a preferred embodiment, the kit referred to in any of the above uses further comprises a specific reagent for detecting or determining the expression level of a gene or protein whose expression determines whether a cell in a sample is proliferating. Preferably, the kit comprises an antibody capable of specifically recognizing a protein whose expression determines whether a cell in a sample is proliferating, preferably selected from the group consisting of geminin, KI-67, proliferating cell nuclear antigen, and cyclin A2.

[00262] The kits of the invention also contain other reagents that allow the determination of the expression level of a biomarker, but which are not specific to said biomarker, for example, reagents for the extraction of RNA material, etc., for example, primers for the synthesis of the corresponding cDNA by RT, reagents for the Petition 870250097700, dated 10 / 24 / 2025, pp. 109 / 145 105 / 118 DNA amplification, such as DNA polymerases, dNTPs, buffers, etc.

[00263] In another preferred embodiment, any of the reagents mentioned in this section, particularly any of the oligonucleotides or antibodies mentioned in this section, more preferably any of the antibodies, each comprises 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 quantity of reagents that make up the kits of the invention, preferably of the total quantity of reagents specific for determining the expression level of one or more biomarkers that make up the kit.

[00264] In a more preferred embodiment, the specific reagent for determining the expression level of at least one biomarker comprises at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of the total quantity of reagents that make up the kit. Preferably, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of the specific reagents for biomarker determination contained in the kit are specific reagents for determining the expression level of at least one biomarker selected from the group consisting of IL-17A, IFN-γ, CD62E, MCP-1, IL-1α, MIP-Iβ, TNF-α, MIP-Iα, CD62P, and IL-10.

[00265] All particular modalities Petition 870250097700, dated 10 / 24 / 2025, pp. 110 / 145 106 / 118 disclosed for the methods of the present invention are applicable to the kit of the invention and the uses thereof.

[00266] All terms have been defined previously in the context of the first and second aspects of the invention and are used with the same meaning in the context of the invention kits and their uses.

[00267] The invention also relates to the following aspects:

[00268] 1. An in vitro method for evaluating or monitoring the clinical response of an individual suffering from cancer to an anticancer treatment selected from the group consisting of: a) a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof; (b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) a polynucleotide that codes for the polypeptide in a) or the conjugate of b); d) a vector comprising the polynucleotide of c); and (e) a cell capable of secreting into the environment the polypeptide of a) or the conjugate of b); the aforementioned method comprising: I. determine the level of at least one selected biomarker from the group consisting of IL-17A, IFN-γ, and CD62E in a sample of said biomarker. Petition 870250097700, dated 10 / 24 / 2025, pp. 111 / 145 107 / 118 individual obtained after administration of treatment, and ii. compare the level of said biomarker with a reference value, where: - an increased level of at least one biomarker compared to the reference value is indicative of a good clinical response of the individual to cancer treatment, or - A level equal to or lower than the reference value for at least one biomarker is indicative of a poor clinical response to cancer treatment.

[00269] 2. The method according to aspect 1, in which a good clinical response is the stabilization of the disease. [0027 0] 3. The method according to either aspect 1 or 2, where a poor clinical response is disease progression.

[00271] 4. The method, according to any one of claims 1 to 3, wherein the reference value is a value obtained from a sample of the same individual before administration of the treatment, preferably before administration of the first dose of the treatment.

[00272] 5. The method, according to any one of claims 1 to 3, wherein the reference value is a value obtained from a sample of the same individual at a time prior to administration of the treatment.

[00273] 6. The method, according to any of aspects 1 to 5, in which the sample is blood, serum or plasma; most preferably serum. Petition 870250097700, dated 10 / 24 / 2025, pp. 112 / 145 108 / 118

[00274] 7. The method, according to any of aspects 1 to 6, in which the sample from step (i) was obtained within 8 hours after the end of the treatment administration, preferably within 6 hours, more preferably between 2 and 6 hours.

[00275] 8. The method, according to any of aspects 1 to 7, in which the levels of at least one biomarker determined are the mRNA levels. [0027 6] 9. The method according to any of aspects 1 to 7, wherein the levels of at least one biomarker determined are protein levels.

[00277] 10. The method according to aspect 9, in which the level of at least one biomarker protein is determined by an immunoassay.

[00278] 11. The method according to any of aspects 1 to 10, wherein the anticancer treatment comprises the use of a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof.

[00279] 12. The method according to aspect 11, wherein the anticancer treatment comprises the use of a polypeptide consisting of the sequence SEQ ID NO: 4.

[00280] 13. The method according to any of aspects 1 to 12, where the cancer is a solid tumor.

[00281] 14. The method according to any of aspects 1 to 13, further comprising the determination of the level of at least one additional biomarker selected from the group consisting of MCP-1, IL-1a, MIP-Iβ, TNFα, MIP-Iα, CD62P and IL-10 and combinations thereof.

[00282] 15. The method according to any Petition 870250097700, dated 10 / 24 / 2025, pp. 113 / 145 109 / 118 of aspects 1 to 14, where the method comprises the determination of the levels of a combination of biomarkers selected from the group consisting of: a) a combination of biomarkers comprising IL-17A and MCP-1; b) a combination of biomarkers comprising IL-17A and IL-1a; c) a combination of biomarkers comprising IL-17A and MIP-Iβ; d) a combination of biomarkers comprising IL-17A and TNF-α; e) a combination of biomarkers comprising IL-17A and MIP-1a; f) a combination of biomarkers comprising IL-17A and CD62P; g) a combination of biomarkers comprising IL-17A and IL-10; and a combination of biomarkers comprising IL-17A and CD62E.

[00283] 16. The method according to aspect 15, wherein the method comprises determining the levels of a combination of biomarkers selected from the group consisting of: a) a combination of biomarkers comprising IL-17A and MCP-1; and b) a combination of biomarkers comprising IL-17A and IL-1a.

[00284] 17. An agent selected from the group consisting of: a) a polypeptide comprising the sequence SEQ Petition 870250097700, dated 10 / 24 / 2025, pp. 114 / 145 110 / 118 ID NO: 1 or a functionally equivalent variant thereof; (b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) a polynucleotide that codes for the polypeptide in a) or the conjugate of b); d) a vector comprising the polynucleotide of c); and (e) a cell capable of secreting into the environment the polypeptide of a) or the conjugate of b); for use in the treatment of cancer in an individual, wherein the individual has been identified as a good responder to said agent by a method as defined in any of aspects 1 to 16.

[00285] 18. The agent for use according to aspect 17, wherein the agent is a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof.

[00286] 19. The agent for use according to aspect 18, wherein the agent is a polypeptide consisting of SEQ ID NO: 4.

[00287] 20. A kit comprising a specific reagent for determining the expression level of at least one biomarker selected from the group consisting of IL-17A, IFN-γ and CD62E.

[00288] 21. The kit according to aspect 20, also comprising specific reagents to determine the Petition 870250097700, dated 10 / 24 / 2025, pages 115 / 145 111 / 118 expression level of at least one additional biomarker selected from the group consisting of MCP-1, IL-Iα, MIP-Iβ, TNF-α, MIP-Iα, CD62P, IL-10 and combinations thereof.

[00289] 22. The kit according to aspect 21, wherein the kit comprises a specific reagent for determining the expression level of IL-17A and at least one additional specific reagent for determining the expression level of a biomarker selected from the group consisting of IFN-γ, CD62E, MCP-1, IL-Iα, MIP-Iβ, TNF-α, MIP-Iα, CD62P and IL-10.

[00290] 23. The kit conforms to any of aspects 20 to 22, wherein the reagent is an antibody capable of specifically recognizing at least one of the biomarkers.

[00291] 24. The kit conforms to any of aspects 20 to 23, wherein the specific reagent for determining the expression level of at least one biomarker comprises at least 10% of the total quantity of reagents comprising the kit.

[00292] 25. Use of a kit in accordance with any of aspects 20 to 24 or of a specific reagent to determine the expression level of at least one biomarker selected from the group consisting of IL-17A, IFNγ, CD62E, MCP-1, IL-Iα, MIP-Iβ, TNF-α, MIP-Iα, CD62P and IL10 in the method as defined in any of aspects 1 to 16.

[00293] The invention will be described by means of the following examples which should be considered merely illustrative and not limiting the scope of the invention. EXAMPLES Experimental details Petition 870250097700, dated 10 / 24 / 2025, pp. 116 / 145 112 / 118

[00294] Blood samples were collected pre-treatment (immediately before patients received the first OMO-103 infusion and before each subsequent infusion) and 1h, 2h, and 6h post-infusion. OMO-103 (SEQ ID NO: 4) is the administered product, corresponding to the Omomyc peptide sequence, also including a methionine at the N-terminal end. OMO-103 is the final product, composed of the active pharmaceutical ingredient (API) and a mixture of excipients, to be used clinically for intravenous infusion. Serum was then isolated using standard procedures and cryopreserved at -80°C until use. Cytokine and chemokine levels were measured using the Luminex technique with the Inflammation 20-Plex Human ProcartaPlex™ kit (Invitrogen). Cytokine and chemokine levels were extrapolated from standard curves using ProcartaPlex Analyst software.

[00295] Patients had not received experimental systemic anticancer treatment in the 4 weeks prior to isolation of the pre-treatment sample and had not received any anticancer treatment in the 24 hours prior to isolation of the sample.

[00296] The patients presented with different types of solid tumors, including pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), salivary gland carcinoma, sarcoma, and pleural mesothelioma.

[00297] OMO-103 was administered as an intravenous infusion over 30 to 45 minutes, at doses of 2.88 mg / kg, 4.32 mg / kg, 6.48 mg / kg and 9.72 mg / kg.

[00298] The patients' response to OMO-103 was Petition 870250097700, dated 10 / 24 / 2025, pp. 117 / 145 113 / 118 measured after three treatment cycles (9 weeks) using computed tomography scans based on RECIST 1.1 criteria (Eisenhauer, EA et al. (2009). New criteria for assessing response in solid tumors: RECIST guideline revised (version 1.1), Eur J Cancer 45: 228-247). Depending on the RECIST results, patients were then classified as Progressive Disease (PD) or Stable Disease (SD). Only patients evaluable for response were considered. In total, 11 patients were evaluable for soluble biomarker analysis, 4 were classified as PD and 6 as SD. One evaluable patient with SD was excluded from the analysis due to a different biomarker profile at C3 compared to the other SD patients. However, it presented the same biomarker profile in C6.

[00299] To determine whether the increase in serum levels differed between SD and PD patients at the start of C3, their pre-dose levels for this specific cycle were calculated. A total of 11 patients were considered in this analysis (7 PD and 4 PD). The p-value was used to reject the null hypothesis. A two-tailed Welch t-test was used to determine whether the levels of cytokines, chemokines, and other soluble factors between the two groups were significantly different. Bonferroni correction was used to adjust for multiple comparisons. Among the differentially expressed soluble factors, only those that increased in all PD patients were selected (except for patient 102001, who showed an increase at C6) (Figure 1).

[00300] Individual biomarker models Petition 870250097700, dated 10 / 24 / 2025, pp. 118 / 145 114 / 118 selected solubles that can correctly stratify patients between PD and SD were found using QLattice (Abzu) modeling technology (René, K et al. (2021). An approach to symbolic regression using Feyn. arXiv:2014.05417 [cs.LG]; Wilsup, C. et al. (2021). Symbolic regression outperforms other models for small datasets. arXiv:2013.15147 [cs.LG]; Christensen, N. et al. (2022) Identifying interactions in omics data for clinical biomarker discovery using symbolic regression, Bioinformatics, Volume 38, Issue 15: 3749-3758) using only one time point per patient (the maximum peak) or all measured time points. QLattice is a symbolic regression algorithm that searches for combinations of input variables and mathematical functions that provide prediction for the output variable. It employs an evolutionary approach to conduct the search and selects the best models by minimizing a calculated error metric between the prediction and the actual result. QLattice was executed within a Leave-one-out cross-validation loop, meaning the algorithm was run 11 times, excluding a single patient in each iteration.The selected models appeared in each iteration. The diagnostic accuracy of individual models for identifying patients with SD was estimated using the area under the receiver operator characteristic curve (ROC-AUC). As a global measure to compare the predictive power of each individual biomarker, ROC-AUC was used. In general, an AUC of 0.5 suggests a lack of discrimination, 0.7 to 0.8 is considered acceptable, 0.8 to 0.9 is considered excellent, and more than 0.9 is considered exceptional, knowing that a model is a perfect classifier when the AUC is 1 (DW). Petition 870250097700, dated 10 / 24 / 2025, pp. 119 / 145 115 / 118 Hosmer, S Lemeshow (2000). Applied Logistic Regression, 2nd Ed. Chapter 5, John Wiley and Sons, New York, NY (2000), pp. 160-164). The analysis was performed by fitting a logistic regression model to each solvable factor individually, with RECIST (C3) as the binary output variable. The Python package Scikit-learn (Pedragosa F. et al. (2011). Scikitlearn: Machine Learning in Python. Journal of Machine Learning Research, 12, pp. 2825-2830) was used to fit the models. The confidence interval ranges of the models were estimated using the parameter values ​​of all the models found (Figure 2).

[00301] Combination models of IL-17A with two other soluble biomarkers that can correctly stratify patients between PD and SD were found using QLattice modeling technology and using all time points measured per patient. Two models appeared in 9 iterations: IL-17A + MCP-1 and IL-17A + IL-1a, models that appeared in 8: IL-17A + MIP-Iβ, IL-17A + TNFa, IL-17A + MIP-Iα and IL-17A + CD62P, 1 model that appeared in 7: IL-17A + IL-10; and 1 model that appeared in 6: IL-17A + CD62E. The AUC-ROC was also calculated for all combination models to determine their accuracy and predictive power (Figure 3). The confidence interval ranges of the models were estimated using the parameter values ​​of all models found. Results

[00302] Patients who responded to treatment with OMO-103 showing stabilization of the disease in cycle 3 had increased levels of three soluble factors. Petition 870250097700, dated 10 / 24 / 2025, pp. 120 / 145 116 / 118 after OMO-103 infusion, starting from C3. This signature is already detected ~2-3 weeks before the predefined 9-week CT scan assessment and is observed only in patients who then presented with SD, while absent in patients with PD. In fact, all patients who presented with SD after 9 weeks of treatment showed significantly increased levels of Interferon-γ (IEN-γ), CD62E, and Interleukin-17A (IL-17A) at various time points after OMO-103 infusion. In contrast, in patients who presented with PD, the levels of these markers remained completely stable (Figure 1). It is worth noting that this transient increase in soluble factor levels was observed at the beginning of cycle 3 in all patients who presented with SD, except for patient 102-001, who showed the increase in cycle 6 (data not shown). It is worth noting that this signature was independent of oncological indication or previous treatments (Table 3). Patient ID Cohort Dose (mg / kg) Tumor Type Time Point Response RECIST 1 Pharmaceutical Signature 102-001 3 2.88 Salivary Gland 06* SD YES 101-002 3 2.88 PDAC 03 SD YES 103-003 4 4.32 Sarcoma 03 SD YES 101-005 5 6.48 NSCLC 03 SD YES 101-007 5 6.48 ORC 03 SD YES 101-008 6 6.48 CRC 03 SD YES 101-009 5 9.72 CRC 03 SD YES 103-002 3 2.88 Mesothelioma 03 PD NO Petition 870250097700, dated 10 / 24 / 2025, pp. 121 / 145 117 / 118 Table 3: Patients with SD present a signature. 101-004 4 4.32 CRC C3 PD NO 102-003 4 4.32 PDAC C3 PD NO 101-006 5 6.48 CRC C3 PD NO Specific pharmacodynamics. Patient characteristics used for the pharmacodynamic signature are shown. 102-001 presented the pharmacodynamic signature at C6 instead of C3. PDAC: pancreatic ductal adenocarcinoma; NSCLC: non-small cell lung cancer; CRC: colorectal cancer.

[00303] Further analysis of these data using QLattice technology generated an independent model for each of the selected soluble factors, capable of efficiently distinguishing between patients with SD and PD. The same models were identified whether only one time point per patient was used (the point where peak levels were detected) (Figure 2A) or whether all time points after infusion were considered (Figure 2B). In order to investigate the predictive power of the identified individual models, the inventors performed Receiver Operating Characteristic (ROC) curve analysis. For the single-time point models, the ROC AUCs of the identified models are 0.96 for IFN-γ, 0.96 for CD62E, and 1 for IL-17A. For the all-time point models, the ROC AUCs are 0.96 for IFN-γ, 0.98 for CD62E, and 0.94 for IL-17A.All values ​​are close to 1, which indicates that they represent excellent predictors of SD outcomes and have very strong prognostic power.

[00304] Again, using technology Petition 870250097700, dated 10 / 24 / 2025, pp. 122 / 145 118 / 118 Using the levels of selected soluble factors from all time points measured after OMO-103 infusion, the inventors identified combination models capable of stratifying patients between SD and PD. These models are all combinations of the cytokine IL-17A with other soluble factors such as MCP-1, IL-1α, MIP-Iβ, TNFα, MIP-Iα, CD62P, IL-10, and CD62E (Figure 3). Again, the predictive power of the identified cytokine / chemokine combinations was investigated through curve analysis. ROC. The results show that the ROC curves of all models have an AUC very close to 1 or even 1, which means that all models are excellent predictors of the SD outcome. The ROC-AUC values ​​of the combined models are 1 for IL-17A + MCP-1, 1 for IL-17A + IL-1a, 0.96 for IL-17A + MIP-Ιβ, 0.95 for IL-17A + TNFa, 0.96 for IL-17A + MIP-1a, 1 for IL-17A + CD62P, 1 for IL-17A + IL-10 and 1 for IL-17A + CD62E.

[00305] It is worth noting that none of these patients presented anti-drug antibodies (ADAs) at any time during treatment. Petition 870250097700, dated 10 / 24 / 2025, pp. 123 / 145

Claims

1 / 4 CLAIMS 1. An in vitro method for evaluating or monitoring the clinical response of an individual with cancer to an anticancer treatment selected from the group consisting of: a) a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant; b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant and a chemical moiety that facilitates cellular uptake of the polypeptide or the functionally equivalent variant; 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 into the medium the polypeptide of a) or the conjugate of b);the aforementioned method is characterized by the fact that it comprises: (i) determining the level of at least one biomarker selected from the group consisting of IL-17A, IFN-γ, and CD62E in a sample from the individual obtained after administration of the treatment, and (ii) comparing the level of said at least one biomarker with a reference value, wherein: - an increased level of said at least one biomarker relative to the reference value is indicative of a good clinical response of the individual to the anticancer treatment, or Petition 870250093984, dated 10 / 14 / 2025, p. 8 / 118 2 / 4 - an equal or decreased level of said at least one biomarker relative to the reference value is indicative of an unsatisfactory clinical response of the individual to the anticancer treatment.

2. A method, according to claim 1, characterized in that a good clinical response is stabilization of the disease and an unsatisfactory clinical response is disease progression.

3. A method, according to either claim 1 or 2, characterized in that the reference value is a value obtained from a sample of the same individual before administration of the first dose of treatment.

4. A method, according to either of claims 1 or 2, characterized in that the reference value is a value obtained from a sample of the same individual at a time prior to the administration of the treatment.

5. A method, according to any one of claims 1 to 4, characterized in that the sample is blood, serum or plasma.

6. A method, according to any one of claims 1 to 5, characterized in that the levels of at least one biomarker determined are either mRNA levels or protein levels.

7. Method according to claim 6, characterized in that the level of at least one biomarker protein is determined by an immunoassay.

8. Method, according to any one of claims 1 to 7, characterized in that Petition 870250093984, dated 10 / 14 / 2025, p. 9 / 118 3 / 4 anticancer treatment comprises the use of a polypeptide consisting of SEQ ID NO:

4.

9. A method, according to any one of claims 1 to 8, characterized in that it further comprises determining the level of at least one additional biomarker selected from the group consisting of MCP-1, IL-1a, MIP-10, TNF-α, MIP-1a, CD62P, IL-10 and combinations thereof.

10. Agent, characterized by being selected from the group consisting of: a) a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof; b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO: 1 or a functionally equivalent variant thereof and a chemical moiety that facilitates cellular uptake of the polypeptide or the functionally equivalent variant thereof; c) a polynucleotide encoding the polypeptide of a) or the conjugate of b); d) a vector comprising the polynucleotide according to c); and e) a cell capable of secreting into the medium the polypeptide according to a) or the conjugate according to b); for use in the treatment of cancer in an individual, wherein the individual has been identified as a good responder to said agent by a method as defined in any one of claims 1 to 9.

11. Kit, characterized in that it comprises a specific reagent for determining the expression level of at least one biomarker selected from the group consisting of IL-17A, IFN-γ and CD62E.

12. Kit, according to claim 11, characterized in that it further comprises specific reagents for determining the expression level of at least one additional biomarker selected from the group consisting of MCP-1, IL-1a, MIP-1p, TNF-α, MIP-1a, CD62P, IL10 and combinations thereof.

13. Kit, according to any one of claims 11 or 12, characterized in that the reagent is an antibody capable of specifically recognizing at least one of the biomarkers.

14. Kit, according to any one of claims 11 to 13, characterized in that the specific reagent for determining the expression level of at least one biomarker comprises at least 10% of the total quantity of reagents that make up the kit.

15. Use of a kit, according to any one of claims 11 to 14, or of a specific reagent, characterized in that it is for determining the expression level of at least one biomarker selected from the group consisting of IL-17A, IFN-γ, CD62E, MCP-1, IL-1a, MIP-1e, TNF-α, MIP-1a, CD62P and IL-10 in the method as defined in any one of claims 1 to 9. Petition 870250093984, dated 10 / 14 / 2025, p. 11 / 118

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