EA043033B1 - METHOD FOR CANCER RISK ASSESSMENT, CORRESPONDING SET, DATA STORAGE UNIT AND PROCESSING SYSTEM - Google Patents

Description

Introduction

Cancer is a multifactorial disease in which a group of cells exhibits uncontrolled growth, invasion that invades and destroys adjacent tissues, and in some cases metastases or spreads to other locations in the body through the lymph or blood. These three detrimental properties of cancer distinguish it from benign tumors, which do not invade or metastasize.

Currently, there are several treatments for each type of cancer, including surgery, radiotherapy, chemotherapy, and targeted therapy. Successful cancer therapy targets the primary tumor and any metastases, whether clinical or microscopic.

Choosing the right treatment is critical for the patient. It is essential to know at what point an intensive and aggressive treatment protocol should be used immediately in order to prevent the spread of aggressive cancer. Conversely, performing intensive and aggressive treatment of a patient's tumor unnecessarily is also disadvantageous for the patient. Moreover, intensive and aggressive treatment always leads to severe intoxications, which can significantly impair the patient's quality of life. For example, such treatments are for the most part mutagenic and thus tend to induce secondary tumors. In addition, such intensive and aggressive treatment is usually very expensive and should only be performed when needed.

Currently, the choice of treatment for solid tumors is based on the assessment of the stage of tumor spread, which is usually performed using the American Joint Committee on Cancer (AJCC) tumor/node/metastasis (TNM) classification test system for malignant tumors. ). The TNM system assigns a number based on three categories. T indicates the size of the tumor, N is the degree of involvement of the lymph nodes, and M is the degree of metastasis. The pronounced stage of cancer is usually given a score by the number I, II, III, IV, derived from the TNM value, grouped by prognosis; a higher number indicates a later stage of the cancer and more likely a poor treatment outcome.

It is generally accepted that although this test and staging system offer some valuable information about the stage at which solid cancer was diagnosed in a patient, they are inaccurate and insufficient. In particular, they are limited to solid tumors. Fluid tissue tumors, on the other hand, are mostly characterized by the detection of cellular alterations.

Most importantly, the TNM test does not detect the earliest stages of tumor progression. These early stages provide the most promising therapeutic window. Detection of cancer at the very beginning of its development allows for targeted, effective therapy with reduced side effects. Therefore, it is important to identify patients at the earliest possible stage as part of screening of the entire population. Cancer can thus be detected early in the community, allowing for earlier intervention and treatment strategies to reduce mortality and suffering from the disease. There is a real need for better cancer predictive tests, not only to improve global patient survival, but also to improve their quality of life, and to keep intensive and expensive chemotherapy for patients who will actually benefit from it. In particular, there is a need for a test that assesses a subject's risk of developing cancer.

Description

The present invention provides a simple and effective method for predicting the occurrence of cancer in subjects who have never been diagnosed with cancer before. The present inventors have shown that the presence of progastrin in a subject's sample is a good and reliable indicator of whether the subject will develop cancer. This relationship does not depend on age or any other parameter. Progastrin thus offers a simple and effective tool for determining a subject's cancer risk. Progastrin is thus a marker for the earliest stages of cancer.

Prognostic tests based on progastrin levels have been described previously. However, such tests are limited to predicting the risk of colonic neoplasia for a patient with a hyperplastic polyp after resection of said hyperplastic polyp (WO 2012/164035, Do et al., Cancer Prev. Res., 5(4): 675-684, 2012). Thus, such tests have a narrow application as they are limited to the prognosis of known cancers. They cannot be used to assess risk in a subject who does not have evidence of cancer when developing the disease.

In a first aspect, the invention relates to a method for assessing the risk of cancer in a subject who has not previously been diagnosed with cancer, said method comprising the steps of:

a) determine the level of progastrin in the sample of the specified subject;

b) determine the risk that the specified subject will develop cancer, based on the level obtained in stage (a).

Analysis of clinical data from the ROC-curve (Receiver Operating Characteristic) showed that the presence of progastrin is a highly specific and sensitive marker. Progastrin is in most cases not detectable in samples of subjects who do not develop cancer. On the other hand, a subject who has never been diagnosed with cancer will have a substantial risk of developing cancer in the future if progastrin is detected in his/her sample.

The method according to the invention makes it possible to easily and reliably assess the risk of developing cancer in a subject who has not previously been diagnosed with cancer. In other words, this method also makes it possible to identify subjects who will develop cancer even if they are not currently symptomatic. These patients already have cancer, even if they do not recognize any symptoms.

The term cancer risk assessment for a subject means determining the relative likelihood of a given subject experiencing cancer symptoms in the future. The method according to the invention is a tool for assessing said risk in combination with other methods or indicators such as clinical examination, biopsy and determination of the level of a known cancer biomarker such as, for example, CA125 and/or OVA1.

The subject to be subjected to the procedure described herein may be any mammal, including humans, dogs, cats, cattle, goats, pigs, pigs, sheep, and monkeys. The person being tested is called the patient. Preferably, the subject is a non-cancer mammal in which cancer is not suspected or previously diagnosed. Cancer test subject as used herein refers to a mammal suffering from cancer that is exhibiting symptoms of cancer or has been diagnosed with cancer. A subject was diagnosed with cancer when a medical test performed by a medical practitioner revealed the presence of cancer.

As used herein, a symptom is any subjective evidence of a disease, such as cancer. A symptom is understood to mean a deviation from normal function or feeling that is noted by the patient, reflecting the presence of an unusual condition or disease, such as cancer. A disease is considered asymptomatic if the patient is a carrier of the specified disease, but does not show symptoms. Asymptomatic conditions may not be detected until the patient is subjected to medical tests, such as measuring progastrin levels, for example.

The present method is also particularly useful in that it can detect cancer in a subject even when the subject has never been diagnosed with cancer and/or does not show any symptoms of cancer. Progastrin is a highly specific and sensitive cancer marker. The detection of progastrin in a subject indicates that there is a high probability that said subject will develop cancer. Thus, progastrin is, in particular, an important biomarker for identifying test subjects who will develop cancer even if they do not yet show any symptoms. The prospect of the invention, in particular, lies in the fact that it allows screening of the population by the appearance of healthy subjects, i.e. those who have never been diagnosed with cancer and/or who have not shown any symptoms, and to identify those who develop cancer. Screening refers here to a method used to detect within a population the possible presence of an as yet undiagnosed disease in individuals with no signs or symptoms. This may include individuals with pre-symptomatic or unrecognized symptomatic disease. One of skill in the art will appreciate that screening tests are unique in that they are performed on people who appear to be healthy. The immediate goal of cancer screening is to detect early-stage cancer or precancerous lesions before a person develops symptoms, and at a point in the disease trajectory where treatment is more likely to be curative.

In yet another aspect, the invention provides a method for predicting cancer in a subject who has not previously been diagnosed with cancer, said method comprising the steps of:

a) determine the level of progastrin in the sample of the specified subject;

b) predict cancer based on the level of stage (a).

The term prognosis as used herein means the likelihood of recovery after a disease, or a prediction of the possible development or outcome of a disease. For example, if a subject's sample for the presence of progastrin is negative, then the prognosis for that subject is better than if the sample for progastrin is positive.

By progastrin, a mammalian progastrin peptide is referred to herein. Progastrin is formed by cleavage of the first 21 amino acids (signal peptide) from preprogastrin, a 101 amino acid peptide (amino acid sequence reference: AAB19304.1), which is the primary translation product of the gastrin gene. The 80-amino acid chain of progastrin is further processed by cleavage and modification of enzymes into several biologically active forms of the hormone gastrin: gastrin-34 (G34) and glycine-extended gastrin-34 (G34-Gly), containing progastrin amino acids 38 to 71, gastrin-17 ( G17) and glycine-extended gastrin-17 (G17-Gly) containing progastrin amino acids 55 to 71.

In a preferred embodiment, the progastrin peptide of the invention is human progastrin. More preferably, the expression human progastrin refers to human PG (progastrin, from English - progastrin) with the sequence of SEQ ID NO: 1. It is noteworthy that human progastrin contains an N-terminal and C-terminal domains that are absent in biologically active forms of the hormone gastrin, mentioned above. Preferably, the sequence of said N-terminal domain is represented by SEQ ID NO: 2. In another preferred embodiment, the sequence of said C-terminal domain is represented by SEQ ID NO: 3.

The present invention provides methods for detecting progastrin in samples, especially biological samples such as body fluids and cells, tissues, biopsy samples and organ sites, and the like.

By biological sample here refers to any sample that can be taken from the subject. Such a sample should be able to determine the level of expression of progastrin. Progastrin is known to be a secreted protein. Preferred biological samples for determining progastrin protein levels thus include body fluids. As used herein, a biological fluid means any fluid that includes material of biological origin. Preferred body fluids for use in the present invention include the body fluids of an animal, such as a mammal, preferably a human subject. The physiological fluid can be any physiological fluid, including, but not limited to, blood, plasma, serum, lymph, cerebrospinal fluid (CSF), saliva, sweat, and urine. Preferably, said preferred liquid biological samples include samples such as a blood sample, a plasma sample, or a lymph sample. More preferably, the biological sample is a blood sample. Moreover, such a blood sample can be obtained by completely safe blood sampling from a patient and thus allows a non-invasive assessment of the risks of developing a tumor in the subject.

The biological sample used herein also includes a solid cancer sample of the patient being tested in case the cancer is a solid cancer. Such a solid cancer sample makes it possible for a person skilled in the art to perform any type of biomarker level measurement according to the invention. In some instances, the methods of the invention may further comprise the preliminary step of collecting a solid cancer sample from a patient. A sample of solid cancer is referred to here as a sample of tumor tissue. Even in a patient suffering from cancer, the tissue in which the tumor is localized still contains non-tumor healthy tissue. Thus, the cancer sample must be limited to tumor tissue taken from the patient. Said cancer sample may be a biopsy sample or a sample taken during surgical resection therapy.

In one aspect, the sample taken from the patient is a cancer cell or tissue.

This sample can be taken and, if necessary, prepared according to methods known to the person skilled in the art. In particular, it is well known in the art that the sample should be taken from the subject on an empty stomach.

The cancer cell or cancer tissue in the present invention is not particularly limited.

As used herein, the term cancer refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell proliferation. The terms cancer and cancerous as used herein are intended to cover all stages of the disease. As used herein, cancer is any malignant neoplasm caused by unwanted growth, invasion, and under certain conditions, metastasis of damaged cells in the body. The cells that give rise to cancer are genetically damaged and usually lose their ability to control cell division, cell migration dynamics, differentiation status and/or cell death mechanism. Most cancers form a tumor, with the exception of some hematopoietic cancers such as leukemia.

Thus, the cancer used here may include both benign and malignant cancers. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specifically, the type of cancer according to the present invention may be selected from the group consisting of squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung, oropharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, peritoneal cancer, esophageal cancer, hepatocellular cancer, gastric or gastric cancer, including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, brain cancer, nervous system cancer, cervical cancer, ovarian cancer, liver cancer , bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, gallbladder cancer, vulvar cancer , testicular cancer, thyroid cancer, Kaposi's sarcoma, liver carcinoma, anal carcinoma, penile carcinoma, non-melanoma skin cancer, melanoma,

- 3 043033 melanoma of the skin, superficial spreading melanoma, precancerous melanosis of Dubrey, acral lentiginous melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma (including Hodgkin's lymphoma; non-Hodgkin's lymphoma, such as, for example, follicular non-Hodgkin's lymphoma (NHL) of low malignancy ; small cell lymphocytic (SL) NHL; moderate follicular NHL; diffuse moderate NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small cell NHL with undivided nuclei; massive NHL lymphadenopathy; mantle cell lymphoma ; AIDS-associated lymphoma; and Waldenström's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia (CML); acute myeloid leukemia (AML) and post-transplant lymphoproliferative disorder (PTLL), as well as abnormal vascular proliferation associated with phakomatoses, edema (eg, associated with brain tumors), Meig's syndrome, brain, and cancer of the head and neck, including the lip and oral cavity, and associated metastases.

In a preferred embodiment, said cancer is lung cancer, lip and mouth cancer, oropharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, prostate cancer, esophageal cancer, gallbladder cancer, liver cancer, hepatocellular cancer, gastric cancer, or gastric cancer, including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, Kaposi's sarcoma, kidney cancer, bladder cancer, colon cancer, rectal cancer, colorectal cancer, hepatoma, liver carcinoma, anal carcinoma, thyroid cancer, non-melanoma skin cancer, skin melanoma, brain cancer, nervous system cancer, testicular cancer, cervical cancer, uterine cancer, endometrial cancer, ovarian cancer, or breast cancer.

In a more preferred embodiment, said cancer is esophageal cancer, liver cancer, hepatocellular cancer, gastric cancer, or gastric cancer, including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, Hodgkin's lymphoma, colon cancer, rectal cancer, colorectal cancer, hepatoma , liver carcinoma, anal carcinoma, non-melanoma skin cancer, skin melanoma, cervical cancer, uterine cancer, endometrial cancer, ovarian cancer, or breast cancer.

The cancer risk of the subject is preferably determined in step (b) by comparing the level of step (a) with a reference level.

The term reference level, as used herein, refers to the level of expression of the cancer marker in question, i.e. progastrin, in the reference sample. As used herein, a reference sample means a sample obtained from known healthy subjects, preferably two or more subjects, or alternatively from the general population. Suitable reference levels of expression of a cancer marker can be determined by measuring the expression levels of said cancer marker in a number of suitable subjects, such reference levels may be adjusted for a particular population of subjects. The reference value or reference level may be an absolute value; relative value; a value having an upper or lower limit; range of values; average value; median, mean, or value compared to a particular control or baseline. The reference value may be based on the value of a single sample, for example, obtained from the test subject's sample, but at an earlier point in time. The reference value may be based on a large number of samples, such as a population of test subjects from a chronological age matched group, or based on a pool of samples including or excluding the test sample.

Preferably, the reference level is a predetermined progastrin level obtained from a biological sample of a subject with a known cancer private status. In particular embodiments, the reference level used for comparison with the test sample in step (b) can be obtained from a biological sample of a healthy test subject, or from a biological sample of a test subject suffering from cancer; it is contemplated that the reference expression profile can also be obtained from a pool of biological samples from healthy subjects or from a pool of samples from subjects suffering from cancer. The authors of the present invention showed that the level of progastrin in healthy subjects on an empty stomach is 0 pM. In a preferred embodiment, the reference level is 0 pM.

The level of progastrin can be measured by any method known to the person skilled in the art.

Preferably, determining the level of progastrin in a sample comprises contacting said sample with a progastrin-binding molecule, and measuring the binding of said progastrin-binding molecule to progastrin.

Measurement of the level of expression at the protein level can in particular be carried out using specific progastrin-binding molecules, such as for example antibodies, in particular using well-known techniques such as cell membrane staining using biotinylation, or other equivalent methods, followed by immunoprecipitation. spec

- 4 043033 phytic antibodies, Western blotting, enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunosorbent assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry (IHC), immunofluorescence (IF), microchips antibodies or tissue microarrays coupled with immunohistochemistry. Other suitable methods include fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET), single cell microscopic or histochemical methods using single or multiple excitation wavelengths, and the use of any of the adapted optical methods such as electrochemical methods (methods voltammetry and amperometry), atomic force microscopy, and radio frequency techniques, such as multipolar resonance spectroscopy, confocal and non-confocal, detection of fluorescence, luminescence, chemiluminescence, absorption, reflection, transmission, and birefringence or refraction (e.g., surface plasmon resonance, ellipsometry, resonant mirrors, waveguide grating beam splitter method or interferometry), cellular ELISA (ELISA), flow cytometry, radioisotope, magnetic resonance imaging, analysis by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS-PAGE electrophoresis, English. - SDS-PAGE); HPLC-mass spectroscopy; liquid chromatography/mass spectrometry/mass spectrometry LC-MS/MS (LC-MS/MS)). All of these methods are well known in the art and need not be further detailed. These various methods can be used to measure progastrin levels.

The progastrin binding molecules of the present invention, especially anti-progastrin antibodies, are particularly useful in immunoassays. The immunochemical assay can be an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), an immunodiffusion assay, or an immunoassay such as a surface plasmon resonance assay (eg, Biacore® assay), ELISPOT assay, slot blot, or Western blot. For a general guide to such methods, see, for example, Ausubel et al. (ed.) (1987) in Current Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y.

Antibodies are key reagents in numerous assays used in medical, veterinary, and other areas of immunoassay. Such tests include many routinely used immunoassay methods such as, for example, enzyme immunoassay (ELISA), RIA (RIA), IHC (IHC) and IF (IF). The level of progastrin is preferably analyzed by any method known to the person skilled in the art, using antibodies directed against the specified protein. Preferably, the level of progastrin is determined using an enzyme immunoassay, preferably based on methods selected between RIA (RIA) and ELISA (ELISA) with at least one progastrin-binding molecule. Most preferably, this level is determined by ELISA, using at least one progastrin-binding molecule. More preferably, the progastrin level is measured with a single progastrin-binding molecule using an enzyme-linked immunosorbent assay, most preferably an ELISA assay.

In one particularly useful embodiment, the method for assessing the risk of cancer in accordance with the invention comprises determining the level of progastrin in a biological sample of the subject using an enzyme immunoassay, preferably based on methods selected between RIA (RIA) and ELISA (ELISA), using progastrin binding molecule.

These methods are particularly useful as they allow the person skilled in the art to analyze the presence of progastrin with a simple, reproducible and reliable test. The prior art method was based on a semi-quantitative test, i. IHC (IHC) staining of epithelial cells throughout the polyp. This method is rather unreliable. In particular, due to the degree of subjectivity associated with analysis, it is difficult to compare results obtained by different pathologists. On the contrary, the method according to the present invention is completely quantitative, objective and highly sensitive.

In another particularly useful embodiment, the method according to the invention comprises determining the level of progastrin in a biological sample of the subject using an enzyme immunoassay, preferably based on methods selected between RIA (RIA) and ELISA (ELISA), using a progastrin binding molecule.

Thus, the level of progastrin is determined in step (a) of the present method by determining the amount of progastrin that is bound by a progastrin-binding molecule, preferably a progastrin-recognizing antibody.

A progastrin-binding molecule refers here to any molecule that binds progastrin but does not bind gastrin-17 (G17), gastrin-34 (G34), glycine-extended gastrin-17 (G17-Gly) or glycine-extended gastrin-34 ( G34-Gly). The progastrin-binding molecule of the present invention may be any progastrin-binding molecule, such as, for example, an antibody molecule or a receptor molecule. Preferably, the progastrin-binding molecule is an anti-progastrin antibody, or an antigen-binding fragment thereof.

- 5 043033

Under the terms connecting, binds, etc. here it is understood that the antibody or its antigennegative fragment forms a complex with an antigen, which under physiological conditions is relatively stable. Methods for determining the binding of two molecules to each other are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. In a particular embodiment, said antibody or antigen-binding fragment thereof binds progastrin with an affinity that is at least twice its binding affinity for a non-specific molecule such as BSA bovine serum albumin (BSA) or casein. In a more particular embodiment, said antibody, or antigen-binding fragment thereof, binds only to progastrin.

In a particular embodiment, the biological sample of the test subject is brought into contact with at least one progastrin-binding molecule, wherein the binding affinity of said agent for progastrin is at least 100 nM, at least 90 nM, at least nM, at least 70 nM, at least 60 nM, at least 50 nM, at least nM, at least 30 nM, at least 20 nM, at least 10 nM, at least nM, at least 1 nM, at least 100 pM, at least 10 pM or at least pM, as determined by the method described above.

In a particular embodiment, the present invention relates to a method for assessing the risk of cancer in a test subject who has not previously been diagnosed with cancer, the method comprising detecting the concentration of progastrin in a biological sample of a test subject who has not been diagnosed with cancer, wherein said biological sample is brought into contact with an antibody against human progastrin (anti-hPG antibody) or an antigen-binding fragment thereof.

In a particular embodiment, the present invention relates to a method for predicting cancer, comprising detecting the concentration of progastrin in a biological sample of a subject who has not been diagnosed with cancer, and said biological sample is brought into contact with an anti-hPG antibody or antigen-binding fragment.

As used herein, the term antibody includes polyclonal and monoclonal antibodies. An antibody (or immunoglobulin) consists of a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain contains a heavy chain variable region (or domain) (abbreviated here as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, C _H 2 and C _H 3. Each light chain contains a light chain variable region (abbreviated here as LCVR or VL) and a light chain constant region. The light chain constant region contains one C _L domain. The V _H and V _L regions can be further subdivided into regions of hypervariability, referred to as complementarity determining regions (CDRs) or hypervariable regions, which are primarily responsible for antigen epitope binding and which are interspersed with regions that are more conserved, referred to as framework regions (FRs) . Each V _H and VL consists of three CDRs and four FRs, arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. Antibody constant regions can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. The antibodies may be of different isotypes (ie IgA, IgD, IgE, IgG or IgM).

In a more particular embodiment, said biological sample is contacted with a progastrin-binding antibody, or an antigen-binding fragment thereof, selected from the group consisting of: polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, camel antibody, IgA1 antibodies, IgA2 antibodies, IgD antibodies, IgE antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, and IgM antibodies.

In addition, one skilled in the art of antibody synthesis will easily select and implement a method for synthesizing polyclonal and/or monoclonal antibodies to a given antigen. Also, a person skilled in the art has a method for determining the CDR in the light and heavy chains of an antibody.

A polyclonal antibody is an antibody produced between or in the presence of one or more other, non-identical antibodies. In general, polyclonal antibodies are produced from B lymphocytes in the presence of several other B lymphocytes producing non-identical antibodies. Typically, polyclonal antibodies are obtained directly from the immunized animal.

The term monoclonal antibody means an antibody derived from an approximately homogeneous population of antibodies, the population containing identical antibodies except for a small number of possible natural mutations that can be found in minimal proportions. A monoclonal antibody is obtained by growing a single cell clone, such as a hybridoma, and is characterized by one class or subclass of heavy chains and one type of light chains. Monoclonal antibodies to human progastrin (anti-hPG) and their use for diagnosis or therapy are already known in the art.

- 6 043033 prior art, see for example WO 2011/083088 for colorectal cancer, WO 2011/083090 for breast cancer, WO 2011/083091 for pancreatic cancer, WO 2011/116954 for colorectal and gastrointestinal cancer and WO 2012/ 013609 and WO 2011/083089 for liver pathologies.

An antigen-binding fragment of an antibody refers to any peptide, polypeptide, or enzyme that retains the ability to bind to the target (also commonly referred to as antigen) of said antibody, generally of the same epitope, and contains an amino acid sequence of at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues or at least 200 consecutive amino acid residues of the antibody amino acid sequence.

In a particular embodiment, said antigen-binding fragment contains at least one CDR of the antibody from which it is derived. However, in a preferred embodiment, said antigen-binding fragment contains 2, 3, 4 or 5 CDRs, more preferably 6 CDRs of the antibody of which it is derived.

Antigen binding fragments can be selected, without limitation, from the group consisting of Fv, scFv (sc for single chain), Fab, F(ab') ₂ , Fab', scFv-Fc fragments or dimers, or fusion proteins with random peptides such as XTEN (extended recombinant polypeptide) or PAS (Per-ARNT-Sim) motifs, or any fragment whose half-life can be increased by chemical modification, such as the addition of a poly(alkylene) glycol such as poly(ethylene) glycol (PEG) ( pegylation) (pegylated fragments are referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab') ₂ -PEG or Fab'-PEG), or incorporation into a liposome, said fragments having at least one characteristic CDR of an antibody according to invention. Preferably, said antigen-binding fragments consist of or contain a partial sequence of the heavy or light variable chain of the antibody from which they are derived, said partial sequence being sufficient to retain the same binding specificity as that of the antibody from which it is derived, and having sufficient affinity for the target. , preferably at least 1/100, more preferably at least 1/10, of the affinity of the antibody from which it is derived.

In another particular embodiment, the biological sample of the test subject is brought into contact with at least one antibody that binds progastrin, and the specified antibody is obtained by an immunization method known to a person skilled in the art, while a peptide is used as an immunogen, the amino acid sequence of which contains the full set or part of the amino acid sequence of progastrin. Said antibody may be polyclonal or monoclonal. When more than one antibody is used (eg 2), the method of the invention is performed either on antibodies of the same type only (eg two monoclonal antibodies) or on antibodies belonging to different types (eg one monoclonal and one polyclonal).

In another particular embodiment, the specified biological sample is brought into contact with one such antibody. More preferably, said immunogen contains a peptide selected from:

a peptide, the amino acid sequence of which contains or consists of the amino acid sequence of full length progastrin, in particular full length human progastrin SEQ ID NO: 1;

peptide, the amino acid sequence corresponds to part of the amino acid sequence of progastrin, in particular full-length human progastrin SEQ ID NO: 1;

a peptide whose amino acid sequence corresponds to the entire amino acid sequence of the N-terminal portion of progastrin or a portion thereof, and in particular a peptide comprising or consisting of the amino acid sequence SWKPRSQQPDAPLG (SEQ ID NO: 2);

a peptide whose amino acid sequence corresponds to the entire amino acid sequence of the C-terminal portion of progastrin or a portion thereof, and in particular a peptide comprising or consisting of the amino acid sequence: QGPWLEEEEEAYGWMDFGRRSAEDEN (SEQ ID NO: 3);

a peptide whose amino acid sequence corresponds to a portion of the amino acid sequence of the C-terminal portion of progastrin, and in particular a peptide comprising the amino acid sequence FGRRSAEDEN (SEQ ID NO: 40) corresponding to amino acids 71-80

- 7 043033 progastrin.

One of skill in the art will readily appreciate that such immunization can be used to synthesize both polyclonal and monoclonal antibodies, if desired. Methods for producing each of these types of antibodies are well known in the art.

Examples of monoclonal antibodies that have been synthesized using an immunogen containing the amino acid sequence SWKPRSQQPDAPLG corresponding to amino acid sequence 1-14 of human progastrin (N-terminal end) include, but are not limited to, the monoclonal antibodies designated mAb3, mAb4, mAb16, mAb19 and mAb20, as described in the following table. 1-4. Other antibodies have also been described, but it is not clear whether these antibodies bind progastrin (WO 2006/032980). Experimental epitope mapping results show that mAb3, mAb4, mAb16, mAb19 and mAb20 specifically bind an epitope at the specified hPG N-terminal amino acid sequence.

Polyclonal antibodies specifically recognizing the epitope at the N-terminus of progastrin represented by SEQ ID NO: 2 have been described in the art (see, for example, WO 2011/083088).

Table 1

Depository number of hybridoma mAb Amino acid subsequence SEQID NO 6V5V11S10 tal3 VH CDR 1 GYIFTSYW SEQID NO 4 VH CDR 2 FYPGNSDS SEQID NO 5 VH CDR 3 TRRDSPQY SEQ ID NO 6 VL CDR 1 QSIVHSNGNTY SEQ ID NO 7 VL CDR 2 KVS SEQID NO 8 VL CDR 3 FQGSHVPFT SEQ ID NO 9

table 2

Depository number of hybridoma mAb Amino acid subsequence SEQID NO 20D2C3G2 rnAb4 VH CDR 1 GYTFSSW SEQID NO 10 VH CDR 2 FLPGSGST SEQ ID NO 11 VH CDR 3 ATDGNYDWFAY SEQ ID NO 12 VL CDR 1 QSLVHSSGVTY SEQ ID NO 13 VL CDR 2 KVS SEQ ID NO 14 VL CDR 3 SQSTHVPPT SEQ ID NO 15

Table 3

Depository number of hybridoma mAb Amino acid subsequence SEQID NO 1E9D9B6 mAb16 VH CDR 1 GYTFTSYY SEQ ID NO 16 VH CDR 2 INPSNGGT SEQ ID NO 17 VH CDR 3 TRGGYYPFDY SEQ ID NO 18 VL CDR 1 QSLLDSDGKTY SEQ ID NO 19 VL CDR 2 LVS SEQID NO 20 VL CDR 3 WQGTHSPYT SEQ ID NO 21

- 8 043033

Table 4

Depository number of the hybridoma mAb Amino acid subsequence SEQID NO 1B3B4F11 taL19 VH CDR 1 GYSITSDYA SEQ ID NO 22 VH CDR2 ISFSGYT SEQ ID NO 23 VH CDR 3 AREVNYGDSYHFDY SEQ ID NO 24 VLCDR 1 SQHRTYT SEQ ID NO 25 VLCDR2 VKKDGSH SEQ ID NO 26 VLCDR3 GVGDAIKGQSVFV SEQ ID NO 27

Examples of monoclonal antibodies that can be synthesized using an immunogen containing the amino acid sequence QGPWLEEEEEAYGWMDFGRRSAEDEN (progastrin C-terminal) corresponding to amino acid sequence 55-80 of human progastrin include, but are not limited to, the antibodies designated as mAb8 and mAb13 in the following tables. . 5 and 6. Experimental epitope mapping results showed that mAb13 specifically binds an epitope at the indicated C-terminal amino acid sequence of hPG.

Table 5

Depository number of the hybridoma mAb Amino acid subsequence SEQID NO 1C10D3B9 mAb8 VH CDR 1 GF IFI I YA SEQ ID NO 28 VH CDR 2 ISSGGTYT SEQ ID NO 29 VH CDR 3 ATQGNYSLDF SEQID NO 30 VLCDR 1 KSLRHTKGITF SEQ ID NO 31 VLCDR2 QMS SEQ ID NO 32 VLCDR3 AQNLELPLT SEQ ID NO 33

Table 6

Depository number of the hybridoma mAb Amino acid subsequence SEQID NO 2S6SZS7 TAYZ VH CDR 1 GFIFSSYG SEQ ID NO 34 VH CDR 2 INTFGDRT SEQ ID NO 35 VH CDR 3 ARGTGTY SEQID NO36 VLCDR 1 QSLLDSDGKTY SEQ ID NO 37 VLCDR2 LVS SEQ ID NO 38 VLCDR3 WQGTHFPQT SEQ ID NO 39

Other examples include anti-hPG monoclonal and/or polyclonal antibodies synthesized using an immunogen containing the amino acid sequence of SEQ ID NO: 40.

In a more particular embodiment of the method according to the invention, said biological sample is contacted with at least one anti-hPG antibody or antigen-binding fragment thereof, preferably with one anti-hPG antibody or antigen-binding fragment, said anti-hPG antibody being selected from N -terminal and C-terminal anti-hPG antibodies.

The terms N-terminal anti-hPG antibodies and C-terminal anti-hPG antibodies refer to antibodies

- 9 043033 la binding an epitope containing amino acids located in the N-terminal part of hPG, or an epitope containing amino acids located in the C-terminal part of hPG, respectively. Preferably, the term N-terminal anti-hPG antibodies refers to antibodies that bind an epitope located in the progastrin domain with the sequence shown in SEQ ID NO: 2. In yet another preferred embodiment, the term C-terminal anti-hPG antibodies refers to antibodies that bind an epitope located in the domain of progastrin with the sequence shown in SEQ ID NO: 3.

The term epitope refers to the region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a type of structural epitopes and contain amino acids that directly determine the affinity of the interaction. Epitopes can also be conformational. In specific embodiments, epitopes may include determinants that are reactive surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in specific embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics. Determination of an epitope bound by an antibody can be performed by any epitope mapping method known to those skilled in the art. An epitope may contain various amino acids arranged sequentially within the amino acid sequence of the enzyme. An epitope may also contain amino acids that are not in sequence within the amino acid sequence of the enzyme.

In a particular embodiment of the method according to the invention, said antibody is a monoclonal antibody selected from the group consisting of:

a monoclonal antibody comprising a heavy chain containing at least one, preferably at least two, preferably three CDR-H1, CDR-H2 and CDR-H3 regions of the amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively, or sequences with identity after optimal alignment of at least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 4, 5 and 6, respectively; and a light chain containing at least one, preferably at least two, preferably three CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 7, 8 and 9, respectively, or sequences with an identity after optimal alignment of at least least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 7, 8 and 9, respectively;

a monoclonal antibody comprising a heavy chain containing at least one, preferably at least two, preferably three CDR-H1, CDR-H2 and CDR-H3 regions of the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively, or sequences with identity after optimal alignment of at least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 10, 11 and 12, respectively; and a light chain containing at least one, preferably at least two, preferably three CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 13, 14 and 15, respectively, or sequences with an identity after optimal alignment of at least least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 13, 14 and 15, respectively;

a monoclonal antibody containing a heavy chain containing at least one, preferably at least two, preferably three CDR-H1, CDR-H2 and CDR-H3 regions of the amino acid sequences of SEQ ID NOs: 16, 17 and 18, respectively, or sequences with identity after optimal alignment of at least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 16, 17 and 18, respectively; and a light chain containing at least one, preferably at least two, preferably three CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 19, 20 and 21, respectively, or sequences with an identity after optimal alignment of at least least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 19, 20 and 21, respectively;

a monoclonal antibody containing a heavy chain containing at least one, preferably at least two, preferably three CDR-H1, CDR-H2 and CDR-H3 regions of the amino acid sequences of SEQ ID NOs: 22, 23 and 24, respectively, or sequences with identity after optimal alignment of at least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 22, 23 and 24, respectively; and a light chain containing at least one, preferably at least two, preferably three CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 25, 26 and 27, respectively, or sequences with an identity after optimal alignment of at least least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 25, 26 and 27, respectively;

a monoclonal antibody comprising a heavy chain containing at least one, preferably at least two, preferably three CDR-H1, CDR-H2 and CDR-H3 regions of the amino acid sequences of SEQ ID NOs: 28, 29 and 30, respectively, or sequences with identity after optimal alignment of at least 80, preferably 85, 90, 95 and

- 10 043033

98% with sequences of SEQ ID NO: 28, 29 and 30, respectively; and a light chain containing at least one, preferably at least two, preferably three CDR-L1, CDR-L2 and

CDR-L3 amino acid sequences of SEQ ID NOs: 31, 32 and 33, respectively, or sequences with an identity after optimal alignment of at least 80, preferably

85, 90, 95 and 98% with the sequences of SEQ ID NO: 31, 32 and 33, respectively;

a monoclonal antibody containing a heavy chain containing at least one, preferably at least two, preferably three CDR-H1, CDR-H2 and CDR-H3 regions of the amino acid sequences of SEQ ID NOs: 34, 35 and 36, respectively, or sequences with identity after optimal alignment of at least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 34, 35 and 36, respectively; and a light chain containing at least one, preferably at least two, preferably three CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 37, 38 and 39, respectively, or sequences with an identity after optimal alignment of at least least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 37, 38 and 39, respectively; and a monoclonal antibody produced by a hybridoma deposited in the National Collection of Cultures of Microorganisms NKCM (CNCM), Pasteur Institute, 25-28 st. Docteur Roux, 75724 Paris CEDEX 15, France, December 27, 2016, reference I-5158.

As used herein, the percentage identity or % identity between two nucleic acid or amino acid sequences refers to the percentage of identical nucleotides or amino acid residues between two sequences when compared, obtained after optimal alignment and is statistical, and the differences between the two sequences are randomly distributed throughout their length. Comparison of two nucleic acid or amino acid sequences is traditionally done by comparing the sequences after they have been optimally aligned, the comparison being possible by segment or using an alignment window. Optimal alignment of sequences for comparison can be done manually and by methods known to the person skilled in the art.

Preferred examples of an amino acid sequence having at least 80%, preferably 85%, 90%, 95%, and 98% identity with the reference amino acid sequence include sequences containing the reference sequence as well as specific modifications, such as deletion, addition, or substitution of at least one amino acid. , truncate or lengthen. In the case of substitution of one or more consecutive or non-consecutive amino acids, substitution variants are preferred in which the substituted amino acids are replaced with equivalent amino acids. Here, the expression equivalent amino acids refers to any amino acids, the possible replacement of which with one of the structural amino acids does not lead to a modification of the biological activity of the corresponding antibodies, as well as specific examples defined below.

Equivalent amino acids can be determined either on the basis of their structural homology with their replacement amino acids, or on the basis of the results of comparative tests for biological activity between different likely synthesized antibodies.

In yet another particular embodiment, the antibody used in the method of the invention is a humanized antibody.

As used herein, a humanized antibody refers to an antibody that contains CDR regions derived from antibodies of non-human origin, while other parts of the antibody molecule are derived from one or more human antibodies. In addition, certain skeletal segment residues (referred to as scaffold FRs) can be modified, if necessary, to maintain binding affinity using methods well known to those skilled in the art (Jones et al., Nature, 321:522-525, 1986). The goal of humanization is to reduce the immunogenicity of a foreign antibody, such as a mouse antibody, for administration to a human while maintaining full antigen binding affinity and antibody specificity.

Antibodies can be humanized using a variety of techniques, including antigen binding region (CDR) transplantation (EP 0239400; WO 91/09967; U.S. Patent Nos. 5530101 and 5585089), veneering or resurfacing (EP 0592106; EP 0519596; Padlan B.A., 1991 , Molecular Immunology 28(4/5):489-498 Studnicka G.M. et al., 1994, Protein Engineering, 7(6):805-814 Roguska M.A. et al., 1994, Proc. Natl. Acad. ScL U.S.A. , 91:969-973), and strand permutation (U.S. Patent No. 5565332). Human antibodies can be generated by a variety of methods known in the art, including phage display methods. See also U.S. patents No. 4444887, 4716111, 5545806 and 5814318 and international patent applications with publication numbers WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741 .

In a more particular embodiment, the antibody used in the method of the invention is a humanized antibody selected from the group consisting of:

a humanized antibody containing a heavy chain containing at least one,

- 11 043033 preferably at least two, preferably three regions of CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively, or sequences with identity after optimal alignment of at least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 4, 5 and 6, respectively, and a light chain containing at least one, preferably at least two, preferably three CDR-L1, CDR-L2 and CDR-L3 amino acid sequences SEQ ID NOs: 7, 8, and 9, respectively, or sequences with an identity after optimal alignment of at least 80, preferably 85, 90, 95, and 98% with sequences of SEQ ID NOs: 7, 8, and 9, respectively;

a humanized antibody comprising a heavy chain comprising at least one, preferably at least two, preferably three CDR-H1, CDR-H2 and CDR-H3 regions of the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively, or sequences with identity after optimal alignment of at least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 10, 11 and 12, respectively, and a light chain containing at least one, preferably at least two, preferably three, CDR- L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NO: 13, 14 and 15, respectively, or sequences with an identity after optimal alignment of at least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 13, 14 and 15 respectively;

a humanized antibody comprising a heavy chain comprising at least one, preferably at least two, preferably three CDR-H1, CDR-H2, and CDR-H3 regions of the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively, or sequences with identity after optimal alignment of at least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 16, 17 and 18, respectively, and a light chain containing at least one, preferably at least two, preferably three, CDR- L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NO: 19, 20 and 21, respectively, or sequences with an identity after optimal alignment of at least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 19, 20 and 21 respectively;

a humanized antibody comprising a heavy chain containing at least one, preferably at least two, preferably three, CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 22, 23 and 24, respectively, or sequences with identity after optimal alignment of at least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 22, 23 and 24, respectively, and a light chain containing at least one, preferably at least two, preferably three, CDR- L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NO: 25, 26 and 27, respectively, or sequences with an identity after optimal alignment of at least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 25, 26 and 27 respectively;

a humanized antibody comprising a heavy chain comprising at least one, preferably at least two, preferably three CDR-H1, CDR-H2, and CDR-H3 regions of the amino acid sequences of SEQ ID NOs: 28, 29, and 30, respectively, or sequences with identity after optimal alignment of at least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 28, 29 and 30, respectively, and a light chain containing at least one, preferably at least two, preferably three, CDR- L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NO: 31, 32 and 33, respectively, or sequences with an identity after optimal alignment of at least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 31, 32 and 33 respectively; and a humanized antibody comprising a heavy chain containing at least one, preferably at least two, preferably three, CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 34, 35 and 36, respectively, or sequences with identity after optimal alignment of at least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 34, 35 and 36, respectively, and a light chain containing at least one, preferably at least two, preferably three, CDR -L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NO: 37, 38 and 39, respectively, or sequences with an identity after optimal alignment of at least 80, preferably 85, 90, 95 and 98% with the sequences of SEQ ID NO: 37 , 38 and 39, respectively, wherein said antibody also contains light chain and heavy chain constant regions derived from a human antibody.

In a first embodiment, the method of the invention comprises contacting a biological sample with at least one anti-hPG antibody that binds an hPG epitope, preferably one anti-hPG antibody that binds an hPG epitope, said epitope being located at the C-terminal portion of the hPG. Alternatively, the method of the invention comprises contacting a biological sample with at least one anti-hPG antibody that binds an hPG epitope, preferably one anti-hPG antibody that binds an hPG epitope, said epitope being located at the N-terminal portion of the hPG.

- 12 043033

In a more specific embodiment, the method of the invention comprises contacting a biological sample with at least one anti-hPG antibody that binds an hPG epitope, preferably one anti-hPG antibody that binds an hPG epitope, said epitope comprising an amino acid sequence corresponding to the amino acid sequence An N-terminal portion of progastrin selected from the amino acid sequence corresponding to hPG amino acids 10-14, hPG amino acids 9-14, hPG amino acids 4-10, hPG amino acids 2-10, and hPG amino acids 2-14, wherein the hPG amino acid sequence is characterized by SEQ ID NO: 1.

In a more specific embodiment, the method of the invention comprises contacting a biological sample with at least one anti-hPG antibody that binds an hPG epitope, preferably one anti-hPG antibody that binds an hPG epitope, said epitope comprising an amino acid sequence corresponding to the amino acid sequence a C-terminal portion of progastrin selected from the amino acid sequence corresponding to hPG amino acids 71-74, hPG amino acids 69-73, hPG amino acids 71-80 (SEQ ID NO: 40), hPG amino acids 76-80, and hPG amino acids 67-74, wherein the amino acid sequence of hPG is characterized by SEQ ID NO: 1.

In a particular embodiment, the method according to the invention comprises the step of contacting the biological sample of the subject with a first agent that binds the first part of progastrin and with a second agent that binds the second part of progastrin. In a more particular embodiment, wherein said progastrin-binding molecule is an antibody, the subject's biological sample is contacted with an antibody that binds a first progastrin epitope and a second antibody that binds a second progastrin epitope.

In a preferred embodiment, said first antibody is associated with an insoluble or partially soluble carrier. Binding of progastrin by said first antibody results in capture of progastrin from said biological sample. Preferably, said first antibody is an antibody that binds an hPG epitope, said epitope comprising an amino acid sequence corresponding to the amino acid sequence of the C-terminal portion of progastrin as described above. More preferably, said first antibody is a Mab14 monoclonal antibody produced by the 2H9F4B7 hybridoma described in WO 2011/083088. Hybridoma 2H9F4B7 was deposited under the Budapest Treaty with the CNCM (CNCM), Pasteur Institute, 25-28 st. Docteur Roux, 75724 Paris CEDEX 15, France, December 27, 2016, reference I-5158.

In yet another preferred embodiment, said second antibody is labeled with a detectable moiety as described below. Binding of progastrin by the second antibody allows the detection of progastrin molecules present in the biological sample. Additionally, binding of progastrin to the second antibody allows for the quantification of progastrin molecules present in the biological sample. Preferably, said second antibody is an antibody that binds an hPG epitope, said epitope comprising an amino acid sequence corresponding to the amino acid sequence of the N-terminal portion of progastrin as described above. More preferably, said N-terminal antibody is a polyclonal antibody as described above. Alternatively, a monoclonal antibody that binds an epitope at the N-terminus of progastrin can be used, such as, for example, an N-terminal monoclonal antibody as described above, namely a heavy chain containing monoclonal antibody containing CDR-H1, CDR-H2 and CDR -H3 amino acid sequences of SEQ ID NOS: 16, 17, and 18, respectively, and a light chain containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequences of SEQ ID NOS: 19, 20, and 21.

In a particular preferred embodiment, the first antibody is linked to an insoluble or partially soluble carrier and the second antibody is labeled with a detectable molecular moiety.

In a particular embodiment, the method of the present invention comprises determining the concentration of progastrin in a biological sample from a human test subject who has not previously been diagnosed with cancer.

In yet another particular embodiment, the method of the present invention comprises determining the concentration of progastrin in a biological sample of a human subject who has not previously been diagnosed with cancer, said biological sample being selected from blood, serum and plasma.

In a more particular embodiment, the method of the present invention comprises contacting a plasma sample of said subject with at least one anti-hPG antibody, namely one anti-HPG antibody, and determining the concentration of progastrin in said sample, wherein the progastrin concentration is greater than 10 pM in said plasma is an indicator of cancer risk in said subject. In other words, a progastrin concentration greater than 10 pM in the indicated plasma is an indicator of poor prognosis for the indicated test.

- 13 043033 Tumuy.

However, more preferably, the method of the present invention comprises contacting a plasma sample of said subject with at least one anti-hPG antibody, namely one anti-hPG antibody, and determining the concentration of progastrin in said sample, wherein the progastrin concentration, greater than 10 pM, preferably 20 pM, more preferably 30 pM, even more preferably 40 pM and even more preferably 50 pM in said plasma sample, is an indicator of cancer risk in said subject.

In yet another aspect, the present invention relates to a method for treating cancer in a patient who has never been diagnosed with cancer, said method comprising the steps of:

a) assessing the risk of developing cancer in said patient by any of the methods described above; And

b) treat said cancer if a risk is detected in stage (a).

The method according to the invention is particularly useful in that it makes it possible to detect cancer at a very early stage. It is known from the prior art that the earlier cancer is detected, the higher the chances of remission. In addition, the patient is treated with anti-cancer drugs that are not too aggressive, thereby reducing the chances of side effects while maintaining therapeutic efficacy.

In a particular embodiment, the method according to the invention comprises comparing the concentration of progastrin in a biological sample obtained from a patient with a predetermined value for the concentration of progastrin in the sample, in a more particular embodiment, the specified, predetermined value is selected from: the mathematical expectation or average value for the sample, based on determining the mathematical expectation or mean value of the concentration of progastrin in a healthy population, and the value of the concentration of progastrin is obtained at the time when it was known that the patient is healthy.

In yet another aspect, the invention also provides a composition for use in the methods described above. According to an aspect of the invention, a composition is provided for assessing the risk of cancer in a subject who has never previously been diagnosed with cancer, said composition comprising at least one progastrin-binding antibody or antigen-binding fragment thereof.

In a first embodiment, the composition of the invention comprises an antibody that recognizes an epitope comprising an amino acid sequence corresponding to the amino acid sequence of progastrin.

In a more specific embodiment, the composition of the invention comprises an antibody that recognizes a progastrin epitope, said epitope comprising an amino acid sequence corresponding to the amino acid sequence of the N-terminal portion of progastrin, said amino acid sequence may include residues 10-14 from hPG, residues 9-14 from hPG, residues 4-10 from hPG, residues 2-10 from hPG, or residues 2-14 from hPG, wherein the hPG amino acid sequence is characterized by SEQ ID NO: 1.

In a more particular embodiment, the composition of the invention comprises an antibody that recognizes a progastrin epitope, said epitope comprising an amino acid sequence corresponding to the amino acid sequence of the C-terminal portion of progastrin, said amino acid sequence may include residues 71-74 from hPG, residues 69-73 from hPG, residues 71-80 from hPG (SEQ ID NO: 40), residues 76-80 from hPG, residues 67-74 from hPG, the amino acid sequence being characterized by SEQ ID NO: 1.

In yet another aspect, the present invention provides a kit for use in the methods described above, said kit containing any of the antibodies of the invention. Also within the scope of the invention are packaged materials containing a combination of reagents in predetermined amounts with instructions for performing the methods described above, such as kits. Preferably said kit contains at least one antibody according to the invention, more preferably two antibodies according to the invention.

For example, in a first embodiment, said kit contains a first antibody coupled to an insoluble or partially soluble carrier. Preferably, said first antibody is an antibody that binds an hPG epitope, said epitope comprising an amino acid sequence corresponding to the amino acid sequence at the C-terminal portion of progastrin, as described above. More preferably, said first antibody is a Mab14 monoclonal antibody produced by the 2H9F4B7 hybridoma described in WO 2011/083088. Hybridoma 2H9F4B7 was deposited under the Budapest Treaty with the CNCM (CNCM), Pasteur Institute, 25-28 st. Docteur Roux, 75724 Paris CEDEX 15, France, December 27, 2016, reference I-5158. In yet another embodiment, polyclonal or monoclonal antibodies, or antigen-binding fragments or derivatives thereof, as detailed herein, are labeled as detectable molecular fragments so that they can be packaged and used, for example, in kits to detect cells having the above antigen, both before secretion and associated with the progastrin receptor. Non-limiting iso

- 14 043033 shaving examples of such labels include fluorophores, such as fluorescent isothiocyanate; chromophores, radionuclides, biotin or enzymes. Such labeled antibodies or binding fragments can be used, for example, for histological localization of the antigen, ELISA, cell sorting, and other immunological methods to detect or quantify progastrin and cells bearing such an antigen. Preferably, said labeled antibody is an antibody that binds an hPG epitope, said epitope comprising an amino acid sequence corresponding to the amino acid sequence at the N-terminal portion of progastrin, as described above. More preferably, said N-terminal antibody is a polyclonal antibody as described above. Alternatively, you can also use a monoclonal antibody that binds an epitope at the N-terminus of progastrin, such as, for example, the N-terminal monoclonal antibodies described above, in particular a heavy chain containing monoclonal antibody containing CDR-H1, CDR-H2 and CDR -H3 amino acid sequences of SEQ ID NOS: 16, 17, and 18, respectively, and a light chain containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequences of SEQ ID NOS: 19, 20, and 21.

Thus, in the most preferred embodiment, the kit according to the invention contains:

the first antibody to progastrin, while the specified antibody is the first antibody to progastrin and is a monoclonal antibody produced by a hybridoma deposited in the NKCM (CNCM), Pasteur Institute, 25-28 st. Docteur Roux, 75724 Paris CEDEX 15, France, December 27, 2016, reference I-5158; and a second antibody to progastrin, wherein said second antibody to progastrin is a polyclonal antibody that binds an epitope at the N-terminus of progastrin, or a monoclonal antibody containing a heavy chain containing the following three CDR regions: CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NO: 16, 17 and 18, respectively, and a light chain containing the following three CDRs: CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NO: 19, 20 and 21, respectively.

The invention includes kits in which the antibody or its antigen-binding fragment or derivative are labeled.

The reagents may be dry powders, usually lyophilized, including excipients which, when dissolved, provide a reagent solution having a suitable concentration.

The kit contains antibodies for in vitro detection and quantification of progastrin, for example by ELISA or Western blotting. The antibody of the present invention can be provided in a kit for the detection and quantification of progastrin in vitro, for example by ELISA or Western blotting. When an enzyme label is attached to an antibody, the kit includes the substrates and cofactors required by the enzyme (eg, a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives such as stabilizers, buffers (eg blocking buffer or lysis buffer) and the like may be included. Such a kit may include a compartmentable reservoir to hold one or more containers such as vials, test tubes, and the like, such containers being used to store individual elements of the invention. For example, one container may contain the first antibody associated with an insoluble or partially soluble carrier. The second container may contain a soluble, labeled second antibody in lyophilized form or in solution. The container may also contain a third container for storing the labeled third antibody in lyophilized form or in solution. A kit of this kind can be used in a sandwich assay according to the invention. A description of the composition, as well as instructions for the proposed in vitro or diagnostic use, may be provided on the label or package insert.

Also provided are kits for use as a positive control for purification or immunoprecipitation of progastrin from cells. To isolate and purify progastrin, the kit may contain an antibody as described herein, or an antigen-binding fragment or bead-bound derivative thereof (eg, Sepharose beads). Kits may be provided that contain antibodies for in vitro or ex vivo detection or quantification of progastrin, for example by ELISA or Western blotting. Such a kit contains a container and a label or leaflet on the package attached to the container or on the container. The container stores a composition containing at least one antibody or binding fragment or derivative according to the invention. Additional containers may be included which contain, for example, diluents and buffers, control antibodies. The label or package insert provides a description of the composition as well as instructions for intended in vitro or diagnostic use.

The invention also relates to a product/computer program containing a set of instructions specific to carrying out the method according to the invention.

The invention also relates to a processing system including a computing unit and

- 15 043033 input interface, characterized in that said system includes a means for implementing a method for determining the risk of developing cancer in accordance with the invention disclosed in this document.

In the footnote to FIG. 13, a device 1 according to a particular embodiment of the present invention includes a computing unit 10 configured to follow computer instructions as well as to process data. One such computing unit 10 preferably includes a microprocessor 110 of any type known in the art. Computing unit 10 also has a data storage unit 100 configured to receive a computer program including a set of instructions specific to implementing the method and also configured to store data.

The device 1 also includes an input interface 12 connected to a computing unit 10 enabling the operator O of the device 1 to enter the data to be processed. One such input interface 12 includes any element capable of inputting such data addressed to the computing unit 10, such as a keyboard element, optionally connected to a pointing device element.

Preferably, the computing unit 10 further includes an output interface 14, such as a screen, which, on the one hand, allows the user to check the correctness of the entered data, and on the other hand, allows the computing unit 10 to interact with the operator O.

The device 1 can be integrated into a single system, such as a computer, smart phone or any other system known in the art, enabling the implementation of the method according to the invention. Operator O may be of any skill level and thus may or may not have a medical qualification.

A particular embodiment of the present invention specifically provides that the data entered by the operator O is sent via a network (for example, the Internet), preferably with secure access, to a remote server containing a computing unit capable of implementing the method according to the invention, and thus process the data received by the server. Optionally, after said processing, the server returns the result of the analysis to the user via the same network or via another network. Optionally, the server records the data and/or the result of the analysis on a recording means. Undoubtedly, a means may be provided to ensure the anonymity of the physiological/clinical characteristics of the donor and recipient.

Thus, one such device 1 makes it possible to carry out the method according to the invention, i.e. it enables the following steps to be carried out:

entering physiological/clinical characteristics using the input interface 12 into the computing unit 10, stage 22, while these characteristics include the level of progastrin in the sample 10, stage 23;

optionally, normalization of said progastrin level via data processing by computing unit 10, step 24; and analyzing said risk score to determine the risk of developing stage 25 cancer.

Thus, the method according to the invention can be carried out not only by clinical or hospital staff, but also by all persons involved in clinical research (pharmaceutical industry, scientists, doctors, etc.), or even by the general public.

The following examples are presented here solely to illustrate the scope of the invention and the content of the disclosure. A person skilled in the art can design and construct numerous modifications of the examples listed below without departing from the scope of this invention.

Description of figures

Fig. 1: ROC curve for colorectal cancer (upper panel) with area under the ROC curve and statistical analysis (lower panel).

Fig. 2: Median plasma progastrin concentration in a patient with colorectal cancer (n=148) and in controls (n=103), using a combination of N-terminal polyclonal antibody and C-terminal polyclonal antibody - two-sample Mann-Whitney test, ** *p<0.0001.

Fig. 3: ROC curve for hepatocellular carcinoma (top panel) with area under the ROC curve and statistical analysis (bottom panel).

Fig. 4: Median plasma progastrin concentration in a patient with colorectal cancer (n=47) and in controls (n=103), using a combination of N-terminal polyclonal antibody and C-terminal polyclonal antibody - two-sample Mann-Whitney test, ** *p<0.0001.

Fig. 5: ROC curve for esophageal cancer (top panel) with area under the ROC curve and statistical analysis (bottom panel).

Fig. 6: Median plasma progastrin concentration in patients (n=12) with esophageal cancer and controls (n=103), using a combination of N-terminal polyclonal antibody and C-terminal polyclonal antibody - two-sample Mann-Whitney test, ** *p<0.0001.

- 16 043033

Fig. 7: ROC curve for gastric cancer (top panel) with area under the ROC curve and statistical analysis (bottom panel).

Fig. 8: Median plasma progastrin concentration in patients (n=15) with gastric cancer and controls (n=103), using a combination of N-terminal polyclonal antibody and C-terminal polyclonal antibody - two-sample Mann-Whitney test, ** p<0.0001.

Fig. 9: ROC curve for pancreatic cancer (top panel) with area under the ROC curve and statistical analysis (bottom panel).

Fig. 10: Median plasma progastrin concentration in patients (n=44) with pancreatic cancer and controls (n=103), using a combination of N-terminal polyclonal antibody and C-terminal polyclonal antibody - two-sample Mann-Whitney test, * **p<0.0001.

Fig. 11: ROC curve for ovarian cancer (upper panel) with ROC area under the curve and statistical analysis (lower panel).

Fig. 12: Median plasma progastrin concentration in patients (n=8) with ovarian cancer and controls (n=103) using a combination of N-terminal polyclonal antibody and C-terminal polyclonal antibody.

Fig. 13: Schematic representation of a processing system according to a particular embodiment of the present invention.

Fig. 14: Functional graph representing the method according to a particular embodiment of the present invention.

Fig. 15: Median plasma progastrin concentration in patients (n=231) with various types of cancer and in controls (n=322), using a combination of polyclonal and monoclonal antibodies.

Fig. 16: Median plasma progastrin concentration in patients (n=10) with different types of cancer, using a combination of polyclonal antibody and monoclonal antibody (mAb-pAb) or a combination of monoclonal antibodies (mAb-mAb) - two-sample Mann-Whitney test, NS p >0.05.

Examples

Example 1 Detection of Plasma Progastrin Concentration Using Polyclonal Antibodies.

Plasma progastrin levels were quantified by ELISA using two specific anti-progastrin antibodies: plate walls were coated with landing (immobilized) antibodies, and detection antibodies were used to detect progastrin and mediate signal detection.

In the present example, the quantitation is based on the ELISA method, which allows, through the use of a substrate, upon reaction with which light is emitted, to assign a value proportional to the amount of luminescence of antibodies bound to the antigen retained by the immobilized antibodies.

Material.

Reagents and equipment are listed in Table. 7.

Table 7

Name Provider Link 96-well flat bottom plates Dutscher #055221

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(MaxiSORP, Nunc) Sodium carbonate/hydrocarbonate Sigma #21851 Phosphate-saline buffer Dulbecco single (1HFSB) (DPBS 1X) Lonza # P04-36500 Polysorbate Twin-20 Biosolve #20452335 Bovine whey albumin BSA (BSA) Euromedex # 04-100-810-C Streptavidin horseradish peroxidase (HRP) Pierce (Thermo) #21130 Substrate chemiluminescent SuperSignal for ELISA, Femto Maximum Sensitivity substrate) Pierce (Thermo) #37074 polyclonal antibody to progastrin Eurogentec /

Polyclonal antibodies were generated by immunizing a rabbit with N-terminal progastrin (SEQ ID NO: 2) or C-terminal progastrin corresponding to hPG amino acids 71-80 and having the sequence FGRRSAEDEN (SEQ ID NO: 40) according to standard protocols.

The binding characteristics of the anti-progastrin polyclonal antibodies used in this assay are as follows: no binding to G34-Gly, G34, G17-Gly, G17, binding to full length progastrin.

96-well plates were coated as follows: a solution of sodium carbonate-hydrocarbonate, 50 mM, pH 9.6 was prepared by dissolving the contents of one capsule in 100 ml of MilliQ purified water. A solution of immobilized antibody (3 μg/ml) corresponding to polyclonal antibodies prepared using the C-terminal progastrin FGRRSAEDEN (SEQ ID NO: 40) was prepared in carbonate buffer. 100 μl of antibody solution was added to each well and incubated at 4°C for 16 hours (1 night). The plates were then blocked by removing the antibody solution and washing 3 times with 300 µl of 1X PBS (1X PBS)/0.1% Tween-20 1X phosphate-buffered saline solution, then adding 200 µl of blocking buffer (1X PBS (1X PBS)/0 .1% Tween-20/0.1% BSA) per well and incubated for 2 hours at 22°C. Then the blocking buffer was removed, the wells were washed 3 times with a solution of 300 μl of 1X PBS (1X PBS)/0.1% Tween-20.

Plasma dilution was performed as follows: Plasma was used neat, diluted 1/2, 1/5 and 1/10. A dilution was prepared from pure plasma in 1X PBS (1X PBS)/0.1% Tween-20/0.1% BSA.

For a control ELISA test in the presence of a known concentration of progastrin, a progastrin dilution was prepared as follows: stock solution of recombinant PG (full-length human progastrin prepared in E. coli and affinity purified with glutathione garose/affinity tag removal (tobacco etch virus (Tev))/affinity immobilized metal ion chromatography (IMAC counter purification)/dialysis, from the Institut Pasteur, Paris, France) was prepared at a concentration of 0.45 mg/ml (45 μM) in three solutions. A series of progastrin concentrations was prepared as follows:

Solution A: pre-diluted 1/10, 2 µl of stock solution plus 18 µl of buffer.

Solution B: pre-diluted 1/100, 10 µl of solution A plus 90 µl of buffer.

Solution C: pre-diluted 1/1000, 10 µl of solution B plus 90 µl of buffer.

Solution D: 500 pM, 5.55 µl of solution C plus 494.5 µl of diluent.

Solution E: 250 pM, 250 µl of solution D plus 250 µl of diluent.

Solution F: 100 pM, 200 µl of solution E plus 300 µl of diluent.

Solution G: 50 pM, 250 µl of solution F plus 250 µl of diluent.

Solution H: 25 pM, 200 µl of solution G plus 200 µl of diluent.

Solution I: 10 pM, 100 µl of solution H plus 150 µl of diluent.

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The region of recombinant progastrin is linear and therefore may be more or less wide for the antibody used.

For the preparation of test samples, approximately 500 µl of each sample was retained until analysis (and confirmation, if necessary) of the results. 100 μl of each point from the series of concentrations and/or plasma were analyzed in pure form, as well as diluted in the ratio of 1/2, 1/5 and 1/10, and incubated for 2 hours at 22°C on the plates.

To detect the test, the plates were washed 3 times with 300 μl of a solution of 1X PBS (1X PBS) / 0.1% Tween-20. A solution of polyclonal rabbit antibodies to progastrin, coupled with biotin to 0.5 μg/ml, and these antibodies were obtained using the N-terminal part of progastrin as an immunogen, was prepared by dilution in 1X PBS (1X PBS) / 0.1% Tween-20 /0.1% BSA (BSA). 100 μl of this solution was added to each well. Spent incubation for 1 h at 22°C. Detection of the test with streptavidin-peroxidase (HRP) conjugate was performed as follows: the detection antibody was removed and a 3-fold wash was performed with 300 μl of a solution of 1X PBS (1X PBS)/0.1% Tween-20, then a streptavidin-peroxidase solution was prepared with a concentration of 20 ng/ml diluted in 1X PBS (1X PBS)/0.1% Tween-20/0.1% BSA (BSA), while 100 additions of 100 µl of this solution were added to each well before incubation for 1 hour at 22°C.

Detection was performed as follows: streptavidin peroxidase was removed and washes were performed 3 times with 300 µl of 1X PBS (1X PBS)/0.1% Tween-20 solution, then 100 µl of chemiluminescent substrate solution was added per well. Substrate solution was prepared by mixing equal volumes of two solutions of SuperSignal ELISA Femto Kit (SuperSignal Substrate ELISA Kit), 20 ml plus 20 ml, stored at room temperature in the dark 30 min before use. Luminescence was read after 5 min of incubation at room temperature in the dark.

For each condition, the test was performed three times, the results for each series are presented as a graph showing the change in luminescence depending on the concentration of progastrin. For each plasma dilution, the progastrin concentration was determined using a linear regression equation for the respective series (1/10 series for sample diluted to 1/10).

Methods and results.

The level of progastrin was determined in plasma samples of the subjects, who, as it turned out, were later diagnosed with cancer. Progastrin was captured by polyclonal antibodies specific to the C-terminus. Detection was performed with labeled polyclonal antibodies specific for the N-terminus.

It is important to note that at the time of sampling, these subjects had never been diagnosed with cancer and had never shown any symptoms of cancer. The control consisted in the collection of plasma samples from the general population.

The results are shown in FIG. 1-12. The median plasma progastrin concentration was 17 pM in patients who later developed colorectal cancer (n=148), 100 pM in patients who later developed hepatocellular carcinoma (n=47), 42.3 pM in patients who who were observed to develop esophageal cancer (n=12), 17.90 pM in patients who were observed to develop gastric cancer (n=15), 16.6 pM in patients who were observed to develop pancreatic cancer (n=44) and 8.45 in patients who developed ovarian cancer (n=8). For comparison, the median plasma concentration of progastrin in control subjects was 0 pM (n=103).

These data demonstrate that patients who will develop cancer have detectable levels of progastrin in their plasma, while healthy controls do not. Progastrin can be detected even before any type of cancer can be diagnosed, thus making progastrin a useful biomarker for cancer occurrence. Analysis of the ROC curve confirmed the predictive nature of progastrin for each of the types of cancer described above.

These data demonstrate that patients at risk of developing cancer have a higher concentration of progastrin in their plasma compared to healthy controls.

Example 2 Detection of Plasma Progastrin Concentration Using a Combination of Polyclonal Antibodies and Monoclonal Antibodies.

In the present example, plasma progastrin levels were quantified by ELISA using an antibody specific for human progastrin (hPG) previously coated on a 96-well plate. Standards and samples were added to the wells and any hPG present was bound by the immobilized antibody. The wells were washed and horseradish peroxidase (HRP) anti-hPG detection antibody conjugates were added, producing an antibody-antigen-antibody sandwich. After the second wash, a TMB (tetramethylbenzidine) substrate solution was added, which produces a blue color in direct proportion to the amount of hPG present in the original sample. After the color changed from blue to yellow with Stop Reagent, the wells were read at 450 nm with a microplate reader.

Polyclonal antibodies were obtained by immunizing a rabbit with N-terminal progastrin (SEQ ID NO: 2) or C-terminal progastrin corresponding to hPG amino acids 71-80 and having the sequence FGRRSAEDEN (SEQ ID NO: 40) according to standard protocols.

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Monoclonal antibodies were obtained using hybridomas producing antibodies to

N-terminal progastrin (SEQ ID NO: 2) or C-terminal progastrin corresponding to hPG amino acids 71-80 and having the sequence FGRRSAEDEN (SEQ ID NO: 40), according to standard protocols.

The binding characteristics of the polyclonal and monoclonal antibodies to progastrin used in this assay are as follows: no binding to G34-Gly, G34, G17-Gly, G17, binding to full length progastrin.

For a control ELISA test (ELISA) in the presence of a known concentration of progastrin, a dilution of progastrin was prepared as follows: stock solution of recombinant PG (full length human progastrin prepared in E. coli and affinity purified by glutathione garose/affinity tag removal (Tev tobacco engraving virus)/affinity chromatography on immobilized metal ions (IMAC counterpurification)/and dialysis, from the Pasteur Institute, Paris, France) were prepared at a concentration of 0.45 mg/ml (45 μM) in three solutions. Progastrin concentration series were prepared as follows:

Solution A: pre-diluted 1/10, 2 µl of stock solution plus 18 µl of buffer.

Solution B: pre-diluted 1/100, 10 µl of solution A plus 90 µl of buffer.

Solution C: pre-diluted 1/1000, 10 µl of solution B plus 90 µl of buffer.

Solution D: 500 pM, 5.55 µl of solution C plus 494.5 µl of diluent.

Solution E: 250 pM, 250 µl of solution D plus 250 µl of diluent.

Solution F: 100 pM, 200 µl of solution E plus 300 µl of diluent.

Solution G: 50 pM, 250 µl of solution F plus 250 µl of diluent.

Solution H: 25 pM, 200 µl of solution G plus 200 µl of diluent.

Solution I: 10 pM, 100 µl of solution H plus 150 µl of diluent.

The recombinant PG region is linear and therefore may be more or less wide for the antibody used.

Methods and results.

Progastrin levels were determined in plasma samples from subjects who were later found to have cancer. Progastrin was captured by the C-terminal mAb14 monoclonal antibody produced by the 2H9F4B7 hybridoma described in WO 2011/083088 (the 2H9F4B7 hybridoma was deposited under the Treaty of Budapest with the CNCM, Institut Pasteur, 25-28 rue Docteur Roux, 75724, Paris, CEDEX 15, France, December 27, 2016, ref. I-5158.). Detection was performed with labeled polyclonal antibodies specific for the N-terminus.

It is important to note that at the time of sampling, these subjects were not diagnosed with cancer and did not show symptoms related to cancer. The control consisted in the collection of plasma samples from the general population.

The results are shown in FIG. 15. The median plasma progastrin concentration was distributed between 2.750 and 21.5 pM in patients depending on the type of cancer (n=231). For comparison, the median plasma concentration of progastrin in control subjects (n=322) was 0 pM.

These data demonstrate that patients have a detectable plasma progastrin level if they develop cancer, while healthy controls do not. Progastrin can be detected even before cancer can be diagnosed, thus making progastrin a useful biomarker for cancer occurrence.

These data demonstrate that patients at risk of developing cancer have a higher concentration of progastrin in their plasma compared to healthy controls.

Example 3 Detection of Plasma Progastrin Concentration Using a Combination of Monoclonal Antibodies.

In the present example, plasma progastrin levels were quantified by ELISA using an antibody specific for human progastrin (hPG) previously coated on a 96-well plate. Standards and samples were added to the wells and any hPG present was bound to the immobilized seeding antibody. The wells were washed and horseradish peroxidase (HRP) anti-hPG detection antibody conjugates were added to form an antibody-antigen sandwich. After the second wash, a TMB substrate solution was added producing a blue color in direct proportion to the amount of hPG present in the original sample. After the color changed from blue to yellow with Stop Reagent, the wells were read at 450 nm with a microplate reader.

Polyclonal antibodies were obtained by immunizing a rabbit with N-terminal progastrin (SEQ ID NO: 2) or C-terminal progastrin corresponding to hPG amino acids 71-80 and having the sequence FGRRSAEDEN (SEQ ID NO: 40) according to standard protocols.

Monoclonal antibodies were obtained using hybridomas producing antibodies to

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N-terminal progastrin (SEQ ID NO: 2) or C-terminal progastrin corresponding to hPG amino acids 71-80 and having the sequence FGRRSAEDEN (SEQ ID NO: 40), according to standard protocols.

The binding characteristics of the polyclonal and monoclonal antibodies to progastrin used in this assay are as follows: no binding to G34-Gly, G34, G17-Gly, G17, binding to full length progastrin.

For a control ELISA test (ELISA) in the presence of a known concentration of progastrin, a dilution of progastrin was prepared as follows: stock solution of recombinant PG (full length human progastrin prepared in E. coli and affinity purified by glutathione garose/affinity tag removal (Tev tobacco engraving virus)/affinity chromatography on immobilized metal ions (IMAC counterpurification)/dialysis, from the Institut Pasteur, Paris, France) were prepared at a concentration of 0.45 mg/ml (45 µmol/ml) in three solutions. Progastrin concentration series were prepared as follows:

Solution A: pre-diluted 1/10, 2 µl of stock solution plus 18 µl of buffer.

Solution B: pre-diluted 1/100, 10 µl of solution A plus 90 µl of buffer.

Solution C: pre-diluted 1/1000, 10 µl of solution B plus 90 µl of buffer.

Solution D: 500 pM, 5.55 µl of solution C plus 494.5 µl of diluent.

Solution E: 250 pM, 250 µl of solution D plus 250 µl of diluent.

Solution F: 100 pM, 200 µl of solution E plus 300 µl of diluent.

Solution G: 50 pM, 250 µl of solution F plus 250 µl of diluent.

Solution H: 25 pM, 200 µl of solution G plus 200 µl of diluent.

Solution I: 10 pM, 100 µl of solution H plus 150 µl of diluent.

The recombinant PG region is linear and therefore may be more or less wide for the antibody used.

Methods and results.

The level of progastrin was determined in plasma samples of the subjects, who, as it turned out, were later diagnosed with cancer. Progastin was captured by the C-Contain Monoclonal Antitle MAB14, produced by 2H9F4B7 hybridim, described in Wo 2011/083088 (hybridum 2H9F4B7 was deposited under the Budapest Agreement at the NKKM (CNCM) Institute of Pasteur, 25-28 Ul. Docteur Rounx, 75724 , Paris, Sedex 15, France , Dec. 27, 2016, ref. I-5158.). Detection was performed with labeled mAb16 monoclinal antibodies described in WO 2011/083088 specific for the N-terminus.

It is important to note that at the time of sample collection, these subjects were not diagnosed with cancer and did not show symptoms related to cancer. Control consisted of collecting plasma samples from the general population.

The results are shown in FIG. 16. The median plasma progastrin concentration obtained using mAb-pAb and mAb-mAb was similar for median (8.2 vs. 6.6 pM, respectively) and mean (18.99 vs. 16.79 pM ) respectively (n=10).

These data demonstrate that both mAb-pAb and mAb-mAb ELISA sandwich assays can detect progastrin in patients' plasma. It is important to note that there was no significant difference in the results of the two tests. In particular, the sensitivity of mAb-pAb and mAb-mAb sandwiches was equally high. Therefore, the mAb-mAb ELISA sandwich can be reliably used to detect progastrin in a patient's plasma even before cancer is diagnosed, making progastrin a useful biomarker for the onset of cancer.

These data demonstrate that patients at risk of developing cancer have a higher concentration of progastrin in their plasma compared to healthy controls.

Claims (13)

1. A method for assessing the risk of cancer in a test person who has not previously been diagnosed with cancer, including the steps: a) determination of the level of progastrin in the sample of the specified subject, including: contacting said sample with at least a hybridoma-produced Mab14 monoclonal antibody deposited with CNCM reference I-5158 and measuring the binding of said monoclonal antibody to progastrin; And b) determining the risk that said subject will develop cancer based on the stage (a) level by comparing the stage (a) level with a reference level, where said reference level is 0 pM. 2. The method according to claim 1, where stage (a) of determining includes bringing into contact the specified sample - 21 043033 additionally with at least one other progastrin-binding molecule, wherein said progastrin-binding molecule binds to a portion of the progastrin that is not bound by Mab14. 3. The method of claim 2, wherein said other progastrin-binding molecule is an anti-progastrin antibody or an antigen-binding fragment thereof. 4. The method of claim 3, wherein said antibody binds the N-terminus of progastrin. 5. The method of claim 3 or 4, wherein said antibody is a monoclonal antibody or a polyclonal antibody. 6. The method according to any one of claims 3-5, wherein said monoclonal antibody is selected from the group consisting of: mAb3 monoclonal antibody containing a heavy chain containing the following three complementarity determining regions (CDRs) CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively, and a light chain containing the following three CDR regions -L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 7, 8 and 9, respectively; monoclonal antibody mAb4 containing a heavy chain containing the following three regions CDR-H1 CDR-H2 and CDR-H3 of the amino acid sequences of SEQ ID NO: 10, 11 and 12, respectively, and a light chain containing the following three regions: CDR-L1, CDR- L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 13, 14 and 15, respectively; mAb16 monoclonal antibody containing a heavy chain containing the following three regions CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequences of SEQ ID NO: 16, 17 and 18, respectively, and a light chain containing the following three regions: CDR-L1, CDR -L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 19, 20 and 21, respectively; and a mAb19 monoclonal antibody containing a heavy chain containing the following three regions CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequences of SEQ ID NOs: 22, 23 and 24, respectively, and a light chain containing the following three regions CDR-L1, CDR -L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 25, 26 and 27, respectively. 7. The method according to any one of claims 3 to 6, wherein the Mab14 monoclonal antibody is linked to an insoluble or partially soluble carrier, and a detectable molecular moiety is labeled to the second antibody. 8. The method according to any one of claims 3 to 7, wherein the progastrin level is determined in step (a) using an enzyme-linked immunosorbent assay (ELISA). 9. The method according to any one of claims 1 to 8, wherein the biological sample is selected from blood, blood serum and plasma. 10. A kit for assessing the risk of cancer, characterized in that it contains the monoclonal antibody Mab14 produced by hybridoma, deposited in CNCM under the reference number I-5158. 11. The kit according to claim 10, containing: a first anti-progastrin antibody, said first anti-progastrin antibody being a Mab14 monoclonal antibody produced by a hybridoma deposited with CNCM reference number I-5158; and additionally a second anti-progastrin antibody, wherein said second anti-progastrin antibody is: a polyclonal antibody that binds the N-terminus of progastrin, or a monoclonal antibody selected from the group consisting of: mAb3 monoclonal antibody containing a heavy chain containing the following three regions CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequences of SEQ ID NO: 4, 5 and 6, respectively, and a light chain containing the following three regions CDR-L1, CDR- L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 7, 8 and 9, respectively; monoclonal antibody mAb4 containing a heavy chain containing the following three regions CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequences of SEQ ID NO: 10, 11 and 12, respectively, and a light chain containing the following three regions CDR-L1, CDR- L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 13, 14 and 15, respectively; monoclonal antibody mAb16 containing a heavy chain containing the following three regions CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequences of SEQ ID NO: 16, 17 and 18, respectively, and a light chain containing the following three regions CDR-L1, CDR- L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 19, 20 and 21, respectively; and a mAb19 monoclonal antibody containing a heavy chain containing the following three regions CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequences of SEQ ID NOs: 22, 23 and 24, respectively, and a light chain containing the following three regions CDR-L1, CDR -L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 25, 26 and 27, respectively. 12. A data storage unit, including a computer program containing a set of instructions specific to the implementation of the method according to any one of claims 1-9. 13. Processing system, including a computing unit and an input interface, where indicated - 22 043033 said system includes means for carrying out the method according to any one of claims 1 to 9.