Compositions and methods for detecting and treating esophageal cancer

Abstract

This invention relates to compositions and methods for detecting and treating esophageal cancer, including methods for in vitro diagnosis of esophageal cancer, and to compositions and methods for preventing or treating esophageal cancer, wherein the compositions comprise antibodies that bind progastrin and the methods comprise using antibodies that bind progastrin.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201780013902.0, filed on January 2, 2017, entitled "Composition and Method for Detecting and Treating Esophageal Cancer". Technical Field

[0002] This invention relates to the in vitro diagnosis, prevention, and treatment of cancer, and more specifically to methods for the in vitro diagnosis of esophageal cancer, and to methods and compositions for the prevention and treatment of esophageal cancer. Compositions according to the invention comprise progastrin-binding molecules, particularly anti-hPG antibodies, while methods according to the invention involve the use of progastrin-binding molecules, and particularly anti-hPG antibodies. Background Technology

[0003] Esophageal cancer originates from esophageal cells in the region between the throat and stomach, and has been described as the eighth most common cancer, affecting more men than women, with significant differences in proportions across countries.

[0004] The two most common types of esophageal cancer are squamous cell carcinoma and adenocarcinoma. Many rarer subtypes are also known. Squamous cell carcinoma originates from the epithelial cells of the esophagus, while adenocarcinoma originates from glandular cells located in the lower part of the esophagus.

[0005] Clinical diagnosis is based on endoscopic biopsy. Adverse outcomes for this disease are particularly due to late diagnosis, especially the lack of early signs and symptoms. To date, no molecular biomarkers have been converted into widely used clinical markers for esophageal cancer (Kaz et al., Cancer Letters, 2014). Treatment depends on the progression of the cancer and typically involves surgery or chemotherapy for small, localized tumors, possibly in combination with radiation therapy.

[0006] Therefore, there remains a need for methods that allow for rapid, reliable, and cost-effective diagnosis of esophageal cancer, as well as for new compositions and methods for the prevention and treatment of esophageal cancer.

[0007] This is the purpose of the present invention. Summary of the Invention

[0008] The present invention now provides a method for the in vitro diagnosis of esophageal cancer, wherein the method includes detecting progastrin in a biological sample from a subject. Preferably, the amount of progastrin in the sample is determined, thus allowing for the quantification of progastrin. The present invention also provides a composition for the prevention or treatment of esophageal cancer, wherein the composition comprises an antibody that binds to progastrin, and a method for the prevention or treatment of esophageal cancer, the method comprising using the composition comprising an antibody that binds to progastrin, alone or in combination with any other known preventive or therapeutic method for esophageal cancer.

[0009] Human progastrinogen, a 101-amino acid peptide (amino acid sequence reference number: AAB19304.1), is the primary translation product of the gastrin gene. Progastrin is formed by cleaving the first 21 amino acids (signal peptide) from progastrinogen. The 80-amino acid chain of progastrin is further processed by cleaving and modifying enzymes into several biologically active gastrin hormone forms: gastrin 34 (G34) containing amino acids 38-71 of progastrin and glycine-extended gastrin 34 (G34-Gly), and gastrin 17 (G17) containing amino acids 55-71 of progastrin and glycine-extended gastrin 17 (G17-Gly).

[0010] Anti-human progastrin (anti-hPG) monoclonal antibodies and their use for diagnosis or treatment have been described in the following documents: WO 2011 / 083 088 for colorectal cancer, WO 2011 / 083 090 for breast cancer, WO 2011 / 083 091 for pancreatic cancer, WO 2011 / 116 954 for colorectal and gastrointestinal cancers, and WO2012 / 013 609 and WO 2011 / 083089 for liver pathology.

[0011] The invention will be more fully understood from the detailed description and accompanying drawings provided herein, which are given by way of example only and do not limit the scope of the invention.

[0012] In a first aspect, the present invention relates to a method for in vitro assessment of the risk of esophageal cancer, wherein the method includes the step of detecting progastrin in a biological sample from a subject. The presence of progastrin in the sample indicates a risk of esophageal cancer.

[0013] Therefore, in a first embodiment, the present invention relates to an in vitro method for evaluating the risk of esophageal cancer in a subject, the method comprising the following steps:

[0014] a) Contact a biological sample from the subject with at least one progastrin-binding molecule, and

[0015] b) Detect the binding of the progastrin-binding molecule to progastrin in the sample, wherein the binding indicates a risk of esophageal cancer.

[0016] The binding of progastrin-binding molecules can be detected by a variety of assays available to those skilled in the art. While any suitable method for performing such an assay is included in this invention, FACS, ELISA, RIA, Western blot, and IHC may be specifically mentioned.

[0017] In a preferred embodiment, the method according to the present invention for in vitro evaluation of the risk of esophageal cancer in a subject includes the following steps:

[0018] a) Contact the biological sample with at least one progastrin-binding molecule.

[0019] b) Determine the concentration of progastrin in the biological sample, wherein a progastrin concentration of at least 10 pM in the biological sample indicates a risk of esophageal cancer.

[0020] Once the concentration of progastrin in the sample is determined, this result is compared with the progastrin concentration in a control sample obtained in a similar manner to the test sample but from an individual known to have esophageal cancer. If the concentration of progastrin in the test sample is significantly higher, it can be concluded that the subject from whom the sample was derived has an increased likelihood of having esophageal cancer.

[0021] Therefore, in a more preferred embodiment, the method of the present invention further includes the following additional steps:

[0022] c) Determine the reference concentration of rumenin in the reference sample.

[0023] d) Compare the concentration of progastrin in the biological sample with the reference concentration of progastrin.

[0024] e) Evaluate the risk of esophageal cancer based on the comparison in step d).

[0025] According to another aspect, the present invention relates to an in vitro method for diagnosing esophageal cancer in a subject, the method comprising the following steps:

[0026] a) Contact a biological sample from the subject with at least one progastrin-binding molecule, and

[0027] b) Detect the binding of the progastrin-binding molecule to progastrin in the sample, wherein the binding indicates the presence of esophageal cancer in the subject.

[0028] In a preferred embodiment, the present invention relates to an in vitro diagnostic method for esophageal cancer in a subject, comprising the following steps:

[0029] a) Contact the biological sample with at least one progastrin-binding molecule, and

[0030] b) Determine the level or concentration of progastrin in the biological sample, wherein a progastrin concentration of at least 10 pM in the biological sample indicates the presence of esophageal cancer in the subject.

[0031] In a more specific embodiment of a method according to the invention, a progastrin concentration of at least 10 pM, at least 20 pM, or at least 30 pM in the biological sample indicates the presence of esophageal cancer in the subject.

[0032] In a more preferred embodiment, the method of the present invention includes the following additional steps:

[0033] c) Determine the reference concentration of progastrin in the reference sample.

[0034] d) Compare the concentration of progastrin in the biological sample with the reference level or concentration of progastrin.

[0035] e) Diagnose the presence of esophageal cancer based on the comparison in step d).

[0036] According to another aspect, the present invention relates to an in vitro method for diagnosing metastatic esophageal cancer in a subject, the method comprising the following steps:

[0037] a) Contact a biological sample from the subject with at least one progastrin-binding molecule, and

[0038] b) Detect the binding of the progastrin-binding molecule to progastrin in the sample, wherein the binding indicates the presence of metastatic esophageal cancer in the subject.

[0039] In a preferred embodiment, the present invention relates to a method for in vitro diagnosis of metastatic esophageal cancer from a biological sample of a subject, comprising the following steps:

[0040] a) Contact the biological sample with at least one progastrin-binding molecule, and

[0041] b) Determine the level or concentration of progastrin in the biological sample by biochemical assay, wherein a progastrin concentration of at least 10 pM or higher in the biological sample indicates the presence of metastatic esophageal cancer in the subject.

[0042] In a more specific embodiment of a method according to the invention, a progastrin concentration of at least 10 pM, at least 20 pM, at least 30 pM, at least 40 pM, or at least 50 pM in the biological sample indicates the presence of metastatic esophageal cancer in the subject.

[0043] In a more preferred embodiment, the method of the present invention includes the following additional steps:

[0044] c) Determine the reference concentration of progastrin in the reference sample.

[0045] d) Compare the level or concentration of progastrin in the biological sample with the reference concentration of progastrin.

[0046] e) Diagnose metastatic esophageal cancer based on the comparison in step d).

[0047] In one specific embodiment, the present invention relates to a method for the in vitro diagnosis of esophageal cancer in a subject, comprising determining the concentration of rumenin in a biological sample and comparing the obtained value with the concentration of rumenin in a reference sample.

[0048] In a more specific embodiment, in a method for diagnosing esophageal cancer according to the invention, a biological sample of the subject is contacted with at least one progastrin-binding molecule, wherein the progastrin-binding molecule is an antibody or an antigen-binding fragment thereof.

[0049] The statement "assessing the risk of esophageal cancer in a subject" means determining the relative probability of a given subject having esophageal cancer when compared to a reference subject or reference value. The method according to the invention represents a tool for assessing said risk, which is combined with other methods or indicators such as clinical examination, biopsy, and the determination of the levels of known biomarkers for esophageal cancer.

[0050] According to a specific embodiment, the present invention relates to an in vitro diagnostic method for esophageal cancer, comprising determining the concentration of progastrin in a biological sample from a subject, wherein the subject exhibits at least one clinical symptom of esophageal cancer. Clinical symptoms of esophageal cancer include weight loss, pain or dysphagia, cough, indigestion, and heartburn.

[0051] According to another specific embodiment, the present invention relates to an in vitro diagnostic method for esophageal cancer, which includes determining the concentration of progastrin in a biological sample from a subject, wherein the subject exhibits at least one clinical symptom of cancer and / or metastatic cancer.

[0052] Therefore, a method for in vitro diagnosis of esophageal cancer according to the present invention can be considered as a tool in the diagnostic process.

[0053] In a more specific embodiment, the present invention relates to a method for in vitro diagnosis of esophageal cancer in a subject, comprising determining the concentration of progastrin in the biological sample and identifying known biomarkers of esophageal cancer.

[0054] The term "progastrin" refers to mammalian progastrin peptides, particularly human progastrin. For the avoidance of ambiguity, without further explanation, the expression "human progastrin" refers to human PG as shown in sequence 1. Human progastrin clearly includes N-terminal and C-terminal domains that are absent in the biologically active gastrin hormone forms mentioned above. Preferably, the sequence of the N-terminal domain is represented by sequence 2. In another preferred embodiment, the sequence of the C-terminal domain is represented by sequence 3.

[0055] In the method according to the invention, the concentration of progastrin is determined by any method known to a person skilled in the art of biochemistry.

[0056] Preferably, determining the concentration of progastrin in the sample includes contacting the sample with a progastrin-binding molecule and measuring the binding of the progastrin-binding molecule to progastrin.

[0057] When determining expression levels at the protein level, specific progastrin-binding molecules, such as antibodies, can be used, specifically with known techniques, such as biotinylated cell membrane staining or other equivalent techniques, followed by specific antibody immunoprecipitation, Western blotting, ELISA or ELISPOT, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry (IHC), immunofluorescence (IF), antibody microarrays or tissue microarrays combined with immunohistochemistry. Other suitable techniques include FRET or BRET, which use single-cell microscopy or histochemical methods with one or more excitation wavelengths and apply any suitable optical methods, such as electrochemical methods (voltammetry and current analysis techniques), atomic force microscopy, and radio frequency methods, e.g., multipolar resonance spectroscopy, confocal and non-confocal methods, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, elliptic polarization, resonant mirror method, grating coupler waveguide method, or interferometry), cell ELISA, flow cytometry, radioisotopes, magnetic resonance imaging, analysis by polyacrylamide gel electrophoresis (SDS-PAGE); HPLC-mass spectrometry; liquid chromatography / mass spectrometry / mass spectrometry (LC-MS / MS). All of these techniques are well known in the art and do not require further detail here. These different techniques can be used to determine progastrin levels.

[0058] In particular, the method may be selected from: immunoassay-based methods, Western blotting-based methods, mass spectrometry-based methods, chromatography-based methods, and flow cytometry-based methods. While any suitable method for performing the assay is included in this invention, methods such as FACS, ELISA, RIA, Western blotting, and IHC are particularly useful for performing the assay.

[0059] In a more specific embodiment, the method for in vitro diagnosis of esophageal cancer according to the invention includes using an immunoenzyme assay, preferably based on a technology selected from RIA and ELISA, to contact a biological sample from a subject with a progastrin-binding molecule.

[0060] As used herein, a “biological sample” is a sample of biological tissue or fluid containing nucleic acids or polypeptides (e.g., nucleic acids or polypeptides of esophageal cancer proteins), polynucleotides, or transcripts. Such a sample must allow for the determination of progastrin expression levels. Progastrin is known to be a secretory protein. Therefore, preferred biological samples for determining progastrin protein levels include biological fluids. As used herein, “biological fluid” means any liquid containing biologically derived substances. Preferred biological fluids for use in this invention include bodily fluids of animals (e.g., mammals, preferably human subjects). Bodily fluids can be any bodily fluid, including but not limited to blood, plasma, serum, lymph, cerebrospinal fluid (CSF), saliva, sweat, and urine. Preferably, the preferred liquid biological sample includes samples such as blood samples, plasma samples, or serum samples. More preferably, the biological sample is a blood sample. In practice, such blood samples can be obtained through completely harmless blood collection from a patient, and thus allow for non-invasive assessment of the subject’s risk of developing a tumor.

[0061] When the cancer is a solid cancer, the term "biological sample" as used herein also includes a solid cancer sample from the patient to be tested. Such solid cancer samples allow those skilled in the art to perform any type of determination of the levels of the biomarkers of the present invention. In some cases, the method according to the invention may further include a preparatory step of obtaining a solid cancer sample from a patient. "Solid cancer sample" refers to a tumor tissue sample. Even in cancerous patients, the tissue at the tumor site still contains non-tumor healthy tissue. Therefore, "cancer sample" should be limited to tumor tissue obtained from a patient. The "cancer sample" may be a biopsy sample or a sample obtained from surgical resection treatment.

[0062] Biological samples are typically obtained from eukaryotic organisms, most preferably mammals, birds, reptiles, or fish. In fact, a “subject” who can undergo the methods described herein can be any mammal, including humans, dogs, cats, cattle, goats, pigs, swine, sheep, and monkeys; or birds; reptiles; or fish. Preferably, the subject is a human; a human subject may be referred to as a “patient.”

[0063] In this document, “obtaining a biological sample” means obtaining a biological sample used in the methods described in this invention. Most often, this is done by removing a cell sample from an animal, but it can also be done by using previously isolated cells (e.g., isolated by another person, at another time and / or for another purpose) or by performing the methods of this invention in vivo. Archives with a history of treatment or outcomes can be particularly useful.

[0064] The sample can be obtained and prepared according to methods known to those skilled in the art, and if necessary, according to methods known to those skilled in the art. In particular, it is known in the art that the sample should be extracted from a fasting subject.

[0065] Determining the concentration of progastrin involves determining the amount of progastrin in a known volume of sample. The progastrin concentration can be expressed relative to a reference sample, for example, as a ratio or percentage. The concentration can also be expressed as the intensity or location of a signal, depending on the method used to determine the concentration. Preferably, the concentration of a single compound in the sample is expressed after standardizing the total concentration of relevant compounds in the sample; for example, the level or concentration of a protein is expressed after standardizing the total concentration of proteins in the sample.

[0066] Preferably, the risk of the subject having esophageal cancer is determined by comparing the level of progastrin measured in the biological sample with a reference level.

[0067] As used herein, the term "reference level" refers to the expression level of a cancer biomarker (i.e., progastrin) considered in a reference sample. As used herein, "reference sample" means a sample obtained from subjects known not to have the disease, preferably two or more subjects, or optionally from the general population. An appropriate reference expression level for progastrin can be determined by measuring the expression level of the biomarker in a suitable group of subjects, and such a reference level can be adjusted to a specific subject population. A reference value or reference level can be an absolute value; a relative value; a value with an upper or lower limit; a range of values; an average value; a median, a mean value, or a value compared to a specific control or baseline value. Reference values ​​can be based on individual sample values, such as values ​​obtained, for example, from samples from subjects being tested but at an earlier time point. Reference values ​​can be based on a large number of samples, such as a population of subjects from a chronologically age-matched group, or on a pool of samples that includes or excludes samples to be tested.

[0068] Advantageously, the “reference level” is a predetermined progastrin level obtained from a biological sample from a subject with a known specific state of cancer. In a specific implementation, the reference level used for comparison with the test sample in step (b) may be obtained from a biological sample from a healthy subject or from a biological sample from a subject with cancer; it should be understood that a reference expression profile may also be obtained from a pool of biological samples from healthy subjects or from a pool of samples from subjects with cancer.

[0069] In one specific embodiment of the method of the present invention, a reference sample is collected from a subject exempt from any cancer, and preferably from a subject exempt from any pathology. It will be understood that, depending on the nature of the biological sample collected from the patient, the reference sample will be a biological sample having the same properties as the biological sample.

[0070] In the method of the present invention, the level of progastrin is determined by identifying the amount of progastrin bound by progastrin-binding molecules, preferably by antibodies that recognize progastrin.

[0071] The term "progastrin-binding molecule" herein refers to any molecule that binds progastrin but not 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 can be any progastrin-binding molecule, such as, for example, an antibody molecule or a receptor molecule. Preferably, the progastrin-binding molecule is an antigastrin antibody or its antigen-binding fragment.

[0072] According to one specific embodiment, the present invention relates to an in vitro diagnostic method for esophageal cancer, the method comprising determining the concentration of progastrin in a biological sample from a subject, wherein the subject exhibits at least one clinical symptom of esophageal cancer.

[0073] According to another specific embodiment, the present invention relates to an in vitro diagnostic method for esophageal cancer, the method comprising determining the concentration of progastrin in a biological sample from a subject, wherein the subject exhibits at least one clinical symptom of cancer and / or metastasis.

[0074] The terms "binding" and similar terms refer to an antibody or its antigen-binding fragment forming a complex with an antigen that is relatively stable under physiological conditions. Methods for determining whether two molecules are bound are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. In one specific embodiment, the antibody or its antigen-binding fragment binds to progastrin with an affinity at least twice that of its binding to nonspecific molecules such as BSA or casein. In a more specific embodiment, the antibody or its antigen-binding fragment binds only to progastrin.

[0075] In one specific embodiment, in a method for diagnosing esophageal cancer according to the invention, a biological sample from a subject is contacted with at least one progastrin-binding molecule, wherein the affinity of said molecule for progastrin is at least 100 nM, at least 90 nM, at least 80 nM, at least 70 nM, at least 60 nM, at least 50 nM, at least 40 nM, at least 30 nM, at least 20 nM, at least 10 nM, at least 5 nM, at least 1 nM, at least 100 pM, at least 10 pM, or at least 1 pM, as determined by methods such as those described above.

[0076] In one specific embodiment, the present invention relates to a method for diagnosing esophageal cancer, the method comprising detecting the concentration of progastrin in a biological sample from a subject, wherein the biological sample is contacted with an anti-hPG antibody or an antigen-binding fragment thereof.

[0077] As used herein, the term "antibody" is intended to include both polyclonal and monoclonal antibodies. An antibody (or "immunoglobulin") is composed of a glycoprotein that comprises at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain contains a heavy chain variable region (or domain) (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into highly variable regions called "complementarity-determining regions" (CDRs) or "hypervariable regions," which are primarily responsible for binding epitopes of the antigen and are interspersed with more conserved regions called framework regions (FRs). Methods for identifying CDRs in the light and heavy chains of antibodies and for determining their sequences are well known to those skilled in the art. To avoid ambiguity, and in the absence of any indication to the contrary in the context, the expression CDR refers to the hypervariable region of the heavy and light chains of the antibody as defined by the IMGT, wherein the unique IMGT number provides a standardized definition of the framework region and complementarity-determining region, CDR1-IMGT: 27 to 38, CDR2.

[0078] The IMGT unique number has been defined as a comparative variable domain, regardless of antigen receptor, chain type, or species [Lefranc M.-P., Immunology Today 18, 509 (1997) / Lefranc M.-P., TheImmunologist, 7, 132-136 (1999) / Lefranc, M.-P., Pommié, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. In the IMGT unique number, conserved amino acids have always had the same site, for example, cysteine ​​23 (1 st -CYS), tryptophan 41 (conserved TRP), hydrophobic amino acid 89, cysteine ​​104 (2 nd-CYS), phenylalanine, or tryptophan 118 (J-PHE or J-TRP). The unique IMGT number provides a standardized definition of the frame region (FR1-IMGT: positions 1-26, FR2-IMGT: positions 39-55, FR3-IMGT: positions 66-104, and FR4-IMGT: positions 118-128) and the complementarity-determining region (CDR1-IMGT: positions 27-38, CDR2-IMGT: positions 56-65, and CDR3-IMGT: positions 105-117). Since vacancy indicates an unoccupied position, the CDR-IMGT length (shown between parentheses and separated by dots, e.g., [8.8.13]) becomes important information. The unique IMGT identifier is used in the two-dimensional illustration and is named IMGTColliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883(2002) / Kaas, Q. and Lefranc, M.-P., Current Bioinformatics, 2, 21-30(2007)], and is also used in the three-dimensional structure in IMGT / 3Dstructure-DB [Kaas, Q., Ruiz, M.and Lefranc, M.-P., T cell receptor and MHC structural data. Nucl. Acids.Res., 32, D208-D210 (2004)].

[0079] Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the typical complement system. Antibodies can be different isotypes (i.e., IgA, IgD, IgE, IgG, or IgM).

[0080] In one specific embodiment, the progastrin-binding antibody or its antigen-binding fragment is selected from the group consisting of: polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, camelified antibodies, IgA1 antibodies, IgA2 antibodies, IgD antibodies, IgE antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, and IgM antibodies.

[0081] A polyclonal antibody is an antibody produced in the presence of one or more other distinct antibodies. Generally, polyclonal antibodies are produced from a single B lymphocyte in the presence of several other B lymphocytes that produce distinct antibodies. Typically, polyclonal antibodies are obtained directly from immunized animals.

[0082] The term "monoclonal antibody" refers to antibodies produced from a nearly homogeneous population of antibodies, which contain identical antibodies except for a very small number of potentially naturally occurring mutations that can be found in a minimal proportion. Monoclonal antibodies are produced from the growth of single-cell clones (such as hybridomas) and are characterized by a class and a subclass of heavy chains and a type of light chain.

[0083] The term "antigen-binding fragment" describing an antibody is intended to represent any peptide, polypeptide, or protein that retains the ability to bind to the target (also generally referred to as an antigen) of the antibody (typically the same epitope) and comprises 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 amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 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.

[0084] In one specific embodiment, the antigen-binding fragment comprises at least one CDR of the antibody from which it is derived. Still in a preferred embodiment, the antigen-binding fragment comprises 2, 3, 4, or 5 CDRs of the antibody from which it is derived, more preferably 6 CDRs.

[0085] The “antigen-binding fragment” may be selected from, but is not limited to, the following: Fv, scFv (sc refers to single chain), Fab, F(ab')2, Fab', scFv-Fc fragments or biantibodies, or fusion proteins having a disordered peptide such as XTEN (extended recombinant polypeptide) or PAS motif, or may be chemically modified to add a poly(alkylene) alcohol such as polyethylene glycol (“PEGylation”) (PEGylated fragments are referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG) (“PEG” stands for poly(alkylene) alcohol), or any fragment whose half-life is increased by incorporation into liposomes, said fragment having at least one characteristic CDR of the antibody according to the invention. Preferably, the “antigen-binding fragment” is constructed or contains a partial sequence of the heavy or light variable chain of the antibody from which it is derived, the partial sequence being sufficient to maintain the same binding specificity and sufficient affinity as the antibody from which it is derived, the affinity being preferably at least 1 / 100 of the affinity of the antibody from which it is derived, and more preferably at least 1 / 10, relative to the target.

[0086] In another specific embodiment, in a method for diagnosing esophageal cancer according to the invention, a biological sample from a subject is contacted with an antibody that binds to progastrin, wherein the antibody has been obtained by an immunoassay known to those skilled in the art, wherein the immunoassay uses a peptide whose amino acid sequence comprises all or part of the amino acid sequence of progastrin as an immunogen. More specifically, the immunogen comprises a peptide selected from:

[0087] - A peptide whose amino acid sequence contains or is composed of the full-length progastrin and, in particular, the amino acid sequence of the full-length human progastrin of sequence 1.

[0088] - A peptide whose amino acid sequence corresponds to that of progastrin and, in particular, a portion of the full-length amino acid sequence of human progastrin, sequence 1.

[0089] - Peptides whose amino acid sequences correspond to a portion of the N-terminal portion of foregutrin or the full amino acid sequence corresponding to the N-terminal portion of foregutrin, and particularly to peptides containing the amino acid sequence SWKPRSQQPDAPLG (Sequence 2) or composed thereof, and

[0090] - A peptide whose amino acid sequence corresponds to a portion of or the entire amino acid sequence corresponding to the C-terminal portion of progastrin, and particularly to a peptide containing the amino acid sequence QGPWLEEEEEAYGWMDFGRRSAEDEN (Sequence 3) or composed thereof.

[0091] - Peptides whose amino acid sequence corresponds to a portion of the amino acid sequence of the C-terminal portion of progastrin, and in particular to peptides containing the amino acid sequence FGRRSAEDEN (Sequence 40) corresponding to amino acids 71-80 of progastrin.

[0092] Those skilled in the art will recognize that such immunization can be used to generate polyclonal or monoclonal antibodies, as desired. Methods for obtaining each of these types of antibodies are well known in the art. Therefore, those skilled in the art will readily select and perform methods for generating polyclonal and / or monoclonal antibodies against any given antigen.

[0093] Examples of monoclonal antibodies generated using an immunogen containing the amino acid sequence “SWKPRSQQPDAPLG”, which corresponds to amino acid sequences 1-14 (N-terminus) of human progastrin, include, but are not limited to, monoclonal antibodies named as follows: mAb3, mAb4, mAb16, and mAb19 and mAb20, as described in Tables 1 through 4 below. Other monoclonal antibodies have been described, but it is unclear whether these antibodies actually bind to progastrin (WO 2006 / 032980). Epitope mapping results show that mAb3, mAb4, mAb16, and mAb19 and mAb20 do indeed specifically bind to the epitope within the N-terminal amino acid sequence of the hPG. Polyclonal antibodies that specifically recognize the epitope within the N-terminus of progastrin represented by sequence 2 have been described in the art (see, for example, WO 2011 / 083088).

[0094] Table 1

[0095]

[0096] Table 2

[0097]

[0098] Table 3

[0099]

[0100] Table 4

[0101]

[0102] Examples of monoclonal antibodies generated using an immunogen containing the amino acid sequence “QGPWLEEEEEAYGWMDFGRRSAEDEN” (the C-terminal portion of progastrin) corresponding to amino acid sequences 55-80 of human progastrin include, but are not limited to, antibodies named mAb8 and mAb13 as shown in Tables 5 and 6 below. Epitope mapping results show that mAb13 does indeed specifically bind to the epitope in the C-terminal amino acid sequence of the hPG.

[0103] Table 5

[0104]

[0105] Table 6

[0106]

[0107] Other examples include anti-hPG monoclonal and / or polyclonal antibodies generated using an immunogen containing an amino acid sequence of sequence 40.

[0108] In a more specific embodiment, in the method according to the invention, the biological sample is contacted with an anti-hPG antibody or an antigen-binding fragment thereof, wherein the anti-hPG antibody is selected from N-terminal anti-hPG antibodies and C-terminal anti-hPG antibodies.

[0109] The terms "N-terminal anti-hPG antibody" and "C-terminal anti-hPG antibody" refer to antibodies that bind to an epitope containing an amino acid located at the N-terminal portion of hPG, or to an epitope containing an amino acid located at the C-terminal portion of hPG, respectively. Preferably, the term "N-terminal anti-hPG antibody" refers to an antibody that binds to an epitope located in the domain of progastrin, represented by sequence 2. In another preferred embodiment, the term "C-terminal anti-hPG antibody" refers to an antibody that binds to an epitope located in the domain of progastrin, represented by sequence 3.

[0110] The term "epitope" is an antigenic region to which an antibody binds. An epitope can be defined as structural or functional. Functional epitopes are typically a subset of structural epitopes and contain amino acids that directly contribute to the interaction. Epitopes can also be conformational. In some embodiments, an epitope may include determinant clusters, i.e., chemically active surface groups of a molecule (such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups), and in some embodiments, may have specific three-dimensional structural features and / or specific charge features. Epitopes bound by antibodies can be determined using epitope mapping techniques known to those skilled in the art. An epitope may comprise different amino acids that are sequentially located in the amino acid sequence of a protein. An epitope may also comprise amino acids that are not sequentially located in the amino acid sequence of a protein.

[0111] In one specific implementation, the antibody is a monoclonal antibody selected from the group consisting of:

[0112] - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, and preferably three, sequences of amino acid sequences CDR-H1, CDR-H2, and CDR-H3, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 4, 5, and 6, respectively; and the light chain comprises at least one, preferably at least two, and preferably three, sequences of amino acid sequences CDR-L1, CDR-L2, and CDR-L3, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 7, 8, and 9, respectively.

[0113] - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, and preferably three of the following sequences: CDR-H1, CDR-H2, and CDR-H3 of amino acid sequences 10, 11, and 12, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 10, 11, and 12, respectively; and the light chain comprises at least one, preferably at least two, and preferably three of the following sequences: CDR-L1, CDR-L2, and CDR-L3 of amino acid sequences 13, 14, and 15, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 13, 14, and 15, respectively.

[0114] - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, and preferably three, sequences of amino acid sequences CDR-H1, CDR-H2, and CDR-H3, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 16, 17, and 18, respectively; and the light chain comprises at least one, preferably at least two, and preferably three, sequences of amino acid sequences CDR-L1, CDR-L2, and CDR-L3, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 19, 20, and 21, respectively.

[0115] - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, and preferably three of the following sequences: CDR-H1, CDR-H2, and CDR-H3 of amino acid sequences 22, 23, and 24, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 22, 23, and 24, respectively; and the light chain comprises at least one, preferably at least two, and preferably three of the following sequences: CDR-L1, CDR-L2, and CDR-L3 of amino acid sequences 25, 26, and 27, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 25, 26, and 27, respectively.

[0116] - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, preferably three, sequences of the following: CDR-H1, CDR-H2, and CDR-H3, amino acid sequences 28, 29, and 30, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 28, 29, and 30, respectively; and the light chain comprises at least one, preferably at least two, preferably three, sequences of the following: CDR-L1, CDR-L2, and CDR-L3, amino acid sequences 31, 32, and 33, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 31, 32, and 33, respectively.

[0117] - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, and preferably three, sequences of amino acid sequences CDR-H1, CDR-H2, and CDR-H3, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 34, 35, and 36, respectively; and the light chain comprises at least one, preferably at least two, and preferably three, sequences of amino acid sequences CDR-L1, CDR-L2, and CDR-L3, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 37, 38, and 39, respectively.

[0118] As used herein, “percentage identity” or “% identity” between two nucleic acid sequences or two amino acid sequences refers to the percentage of identical nucleotide or amino acid residues between the two sequences to be compared after optimal alignment. This percentage is entirely statistical, and the differences between the two sequences are randomly distributed along their lengths. Comparison of two nucleic acid or amino acid sequences is conventionally performed by comparing the sequences after they have been aligned, which can be done by segment or by using an “alignment window.” In addition to manual comparison, optimal alignment of sequences for comparison can be performed using methods known to those skilled in the art.

[0119] For amino acid sequences exhibiting at least 80%, preferably 85%, 90%, 95%, and 98% identity with the reference amino acid sequence, preferred examples include those containing the reference sequence, certain modifications, particularly the deletion, addition, or substitution of at least one amino acid, truncation, or extension. In the case of substitution of one or more continuous or discontinuous amino acids, such substitution is preferred, wherein the substituted amino acid is replaced by an "equivalent" amino acid. Here, the term "equivalent amino acid" means any amino acid that can be substituted with a structural amino acid without modifying the biological activity of the corresponding antibody, as well as any amino acid in the specific examples described below.

[0120] Equivalent amino acids can be determined by their structural similarity (homology) to the amino acids they replace or by a comparative test of the biological activities of the various antibodies that may be generated.

[0121] In another specific embodiment, the antibody used in the method of the present invention is a humanized antibody.

[0122] As used herein, "humanized antibody" refers to an antibody containing a CDR region derived from a non-human antibody, with other portions of the antibody molecule derived from one or more human antibodies. Additionally, some backbone residues (called framework FRs) may be modified to maintain binding affinity using techniques known to those skilled in the art (Jones et al., Nature, 321:522-525, 1986). The purpose of humanization is to reduce the immunogenicity of xenobiotic antibodies (such as mouse antibodies) for introduction into humans while maintaining the overall antigen-binding affinity and specificity of the antibody.

[0123] The humanized antibodies or fragments thereof of the present invention can be prepared by techniques known to those skilled in the art (such as, for example, those described in the document Singer et al., J. Immun., 150:2844-2857, 1992). Such humanized antibodies are preferred for use in methods involving in vitro diagnostics or in vivo prophylactic and / or therapeutic treatments. Other humanization techniques are also known to those skilled in the art. In fact, various techniques can be used to humanize antibodies, including CDR transplantation (EP 0 451 261; EP 0 682 040; EP 0 939 127; EP 0 566647; US 5,530,101; US ​​6,180,370; US 5,585,089; US 5,693,761; US ​​5,639,641; US6,054,297; US 5,886,152; and US 5,877,293), veneering, or resurfacing (EP 0 592 106; EP 0 519 596; Padlan EA, 1991, Molecular Immunology 28(4 / 5): 489-498;). Studnicka GM et al., 1994, ProteinEngineering 7(6): 805-814; Roguska MA et al., 1994, Proc. Natl. Acad. ScLU.SA, 91:969-973), and chain shuffling (US Patent No. 5,565,332). Human antibodies can be prepared by a variety of methods known in the art, including phage display methods. See also US Patent Nos. 4,444,887, 4,716,111, 5,545,806 and 5,814,318; and International Patent Application Publications Nos. WO 98 / 46645, WO 98 / 50433, WO98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735 and WO 91 / 10741.

[0124] In a more specific embodiment, the antibody is a humanized antibody selected from the group consisting of:

[0125] - A humanized antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, preferably three, sequences of the following amino acid sequences: CDR-H1, CDR-H2, and CDR-H3, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 4, 5, and 6, respectively; and the light chain comprises at least one, preferably at least two, preferably three, sequences of the following amino acid sequences: CDR-L1, CDR-L2, and CDR-L3, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 7, 8, and 9, respectively.

[0126] - A humanized antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, preferably three of the following sequences: CDR-H1, CDR-H2, and CDR-H3 of amino acid sequences 10, 11, and 12, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 10, 11, and 12, respectively; and the light chain comprises at least one, preferably at least two, preferably three of the following sequences: CDR-L1, CDR-L2, and CDR-L3 of sequences 13, 14, and 15, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 13, 14, and 15, respectively.

[0127] - A humanized antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, preferably three, sequences of the following: CDR-H1, CDR-H2, and CDR-H3, amino acid sequences 16, 17, and 18, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 16, 17, and 18, respectively; and the light chain comprises at least one, preferably at least two, preferably three, sequences of the following: CDR-L1, CDR-L2, and CDR-L3, amino acid sequences 19, 20, and 21, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 19, 20, and 21, respectively.

[0128] - A humanized antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, preferably three, sequences of the following amino acid sequences: CDR-H1, CDR-H2, and CDR-H3, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 22, 23, and 24, respectively; and the light chain comprises at least one, preferably at least two, preferably three, sequences of the following amino acid sequences: CDR-L1, CDR-L2, and CDR-L3, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 25, 26, and 27, respectively.

[0129] - A humanized antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3 of amino acid sequences 28, 29, and 30, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 28, 29, and 30, respectively; and the light chain comprises at least one, preferably at least two, preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3 of amino acid sequences 31, 32, and 33, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 31, 32, and 33, respectively.

[0130] - A humanized antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, preferably three, sequences of the following: CDR-H1, CDR-H2, and CDR-H3, amino acid sequences 34, 35, and 36, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 34, 35, and 36, respectively; and the light chain comprises at least one, preferably at least two, preferably three, sequences of the following: CDR-L1, CDR-L2, and CDR-L3, amino acid sequences 37, 38, and 39, respectively, or sequences having at least 80%, preferably 85%, 90%, 95%, and 98% identity after optimal alignment with sequences 37, 38, and 39, respectively.

[0131] The antibody also includes constant regions of the light and heavy chains derived from human antibodies.

[0132] In a first embodiment, the method according to the invention includes contacting a biological sample with an anti-hPG antibody that binds to an epitope of hPG, wherein the epitope is located within the C-terminal portion of hPG, or contacting a biological sample with an anti-hPG antibody that binds to an epitope located within the N-terminal portion of hPG.

[0133] In a more specific embodiment, the method according to the invention includes contacting a biological sample with an anti-hPG antibody that binds to an epitope of hPG, wherein the epitope comprises an amino acid sequence corresponding to an amino acid sequence of the N-terminal portion of progastrin, the amino acid sequence corresponding to the N-terminal portion of progastrin being selected from amino acid sequences corresponding to amino acids 10 to 14, 9 to 14, 4 to 10, 2 to 10, and 2 to 14 of hPG, wherein the amino acid sequence of hPG is Sequence 1.

[0134] In a more specific embodiment, the method according to the invention includes contacting a biological sample with an anti-hPG antibody that binds to an epitope of hPG, wherein the epitope comprises an amino acid sequence corresponding to an amino acid sequence of the C-terminal portion of progastrin, the amino acid sequence corresponding to the C-terminal portion of progastrin being selected from amino acid sequences corresponding to amino acids 71 ​​to 74, 69 to 73, 71 to 80, 76 to 80, and 67 to 74 of hPG, wherein the amino acid sequence of hPG is Sequence 1.

[0135] In a first embodiment, the composition according to the invention comprises an antibody that recognizes an epitope, said epitope comprising an amino acid sequence corresponding to the amino acid sequence of progastrin.

[0136] In a more specific embodiment, the composition according to the invention comprises an antibody that recognizes an epitope of progastrin, wherein the epitope comprises an amino acid sequence corresponding to an amino acid sequence of the N-terminal portion of progastrin, wherein the amino acid sequence may comprise residues 10 to 14, 9 to 14, 4 to 10, 2 to 10, or 2 to 14 of hPG, wherein the amino acid sequence of hPG is Sequence 1.

[0137] In a more specific embodiment, the composition according to the invention comprises an antibody that recognizes an epitope of progastrin, wherein the epitope comprises an amino acid sequence corresponding to an amino acid sequence of the C-terminal portion of progastrin, wherein the amino acid sequence may comprise residues 71 to 74 of hPG, residues 69 to 73 of hPG, residues 71 to 80 of hPG (Sequence 40), residues 76 to 80 of hPG, or residues 67 to 74 of hPG, wherein the amino acid sequence of hPG is Sequence 1.

[0138] In one specific embodiment of the method for in vitro diagnosis of esophageal cancer according to the present invention, the method includes contacting a biological sample from a subject with a first molecule of a first portion of progastrin and a second molecule of a second portion of progastrin. In a more specific embodiment, wherein the progastrin-binding molecule is an antibody, and the biological sample from the subject is contacted with an antibody binding to a first epitope of progastrin and a second antibody binding to a second epitope of progastrin.

[0139] In a preferred embodiment, the method of the present invention for diagnosing esophageal cancer includes detecting progastrin in a biological sample from a human subject.

[0140] In a more preferred embodiment, the method of the present invention for diagnosing esophageal cancer includes determining the concentration of progastrin in a biological sample from a human subject.

[0141] In another specific embodiment, the method of the present invention for diagnosing esophageal cancer includes detecting the concentration of progastrin in a biological sample from a human subject, wherein the biological sample is selected from blood, serum, and plasma.

[0142] In another preferred embodiment, the method of the present invention includes contacting a sample from the subject with the anti-hPG antibody described above, wherein the binding of the anti-hPG antibody in the sample indicates the presence of esophageal cancer in the subject.

[0143] In a more specific embodiment, the method of the present invention includes contacting a sample from the subject with the anti-hPG antibody described above, wherein a concentration of progastrin in the sample greater than 10 pM indicates the presence of esophageal cancer in the subject.

[0144] More preferably, the method of the present invention includes contacting a sample from the subject with the anti-hPG antibody described above, wherein a concentration of foregutrin in the sample greater than 10 pM, 20 pM, 30 pM or 40 pM indicates the presence of esophageal cancer in the subject.

[0145] More preferably, the method of the present invention includes contacting a sample from the subject with the anti-hPG antibody described above, wherein a concentration of progastrin in the plasma greater than 10 pM, 20 pM, 30 pM, or 40 pM indicates the presence of metastatic esophageal cancer in the subject.

[0146] The present invention also relates to a method for monitoring the efficacy of esophageal cancer treatment in patients, such as chemotherapy, biotherapy, immunotherapy, or antibody therapy, wherein the method involves determining the concentration of progastrin in a first sample (such as bodily fluids or a biopsy of esophageal cancer) obtained from the patient prior to treatment for esophageal cancer, and then comparing the concentration of progastrin in the first sample with the concentration of progastrin in a second sample obtained from the same patient after treatment, wherein a decrease in the concentration of progastrin in the second sample compared to the first sample indicates that the treatment is effective.

[0147] In one specific embodiment, the method according to the invention includes comparing the concentration of progastrin in a biological sample obtained from a patient with a predetermined value of the progastrin concentration in the sample. In one specific embodiment, the predetermined value is selected from: the mean or average of sample values ​​determined based on the mean or average value in a population without esophageal cancer, where the progastrin concentration value is obtained when the patient is known to be without esophageal cancer.

[0148] In one specific embodiment, the method for in vitro diagnosis of esophageal cancer according to the present invention includes determining the concentration of progastrin in a sample from the patient and a second esophageal cancer diagnostic test. In a more specific embodiment, the method for in vitro diagnosis of esophageal cancer according to the present invention includes determining the concentration of progastrin in a sample from the patient and a second diagnostic test for esophageal cancer.

[0149] In one specific embodiment of the invention, the method according to the invention includes determining the level of progastrin in samples from patients who have received or are receiving treatment for esophageal cancer over time.

[0150] In another aspect, the subject matter of the present invention relates to a composition for the prevention or treatment of esophageal cancer, wherein the composition comprises a progastrin-binding antibody or an antigen-binding fragment thereof.

[0151] The antibody compositions used in the methods of the present invention can be prepared into various formulations, including but not limited to aqueous suspensions for administration via a variety of routes, including but not limited to parenteral, intrathecal, subcutaneous, intravenous, intramuscular, intraperitoneal, infusion, or pellet administration. In some embodiments, the compositions are prepared for parenteral administration, and in some specific embodiments, for intravenous infusion.

[0152] In one specific embodiment, the composition according to the invention for the prevention or treatment of esophageal cancer comprises an effective dose of the anti-progastrin antibody of the invention ranging from 0.001 mg / kg to about 250 mg / kg, which may be administered in a single administration or in multiple intermittent administrations.

[0153] In one specific embodiment, the composition according to the invention for the prevention or treatment of esophageal cancer comprises a progastrin-binding antibody or an antigen-binding fragment thereof selected from: polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, camelified antibodies, IgA1 antibodies, IgA2 antibodies, IgD antibodies, IgE antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, and IgM antibodies. Preferably, the antibody is one of those described above. More preferably, the antibody is a humanized antibody.

[0154] In a more specific embodiment, the composition according to the invention for the prevention or treatment of esophageal cancer comprises a progastrin-binding antibody or an antigen-binding fragment thereof having an affinity for progastrin of at least 5000 nM, at least 500 nM, 100 nM, 80 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 7 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 50 pM, 10 pM, 5 pM, 1 pM, or at least 0.1 pM, as determined by methods such as those described above.

[0155] In one or even more specific embodiments, the composition for the prevention or treatment of esophageal cancer comprises a progastrin-binding antibody, wherein the progastrin-binding molecule or its antigen-binding fragment is a neutralizing antibody.

[0156] The term "neutralizing anti-PG antibody" refers to an antibody that binds to PG and blocks PG-dependent signaling, thereby inhibiting the PG-induced response in tumor cells (especially gastric tumor cells). Inhibition of the PG-induced response in esophageal cancer cells can be mediated by inhibiting cell differentiation, inhibiting cell death, and / or stimulating cell proliferation.

[0157] In another specific embodiment, the composition for the prevention or treatment of esophageal cancer comprises a progastrin-binding antibody, wherein the progastrin-binding molecule or its antigen-binding fragment is a humanized antibody.

[0158] In one specific embodiment, the composition for the prevention or treatment of esophageal cancer comprises a progastrin-binding antibody, wherein the progastrin-binding molecule or its antigen-binding fragment is conjugated to a cytotoxic molecule.

[0159] In another specific embodiment, the composition for the prevention or treatment of esophageal cancer in a patient comprises a progastrin-binding antibody, wherein the patient has been diagnosed with esophageal cancer by the method according to the invention, and wherein the concentration of progastrin in a biological sample from the patient is higher than that in a reference sample.

[0160] In a more specific aspect, the present invention relates to compositions for the prevention or treatment of esophageal cancer according to the present invention, wherein the progastrin-binding antibody or its antigen-binding fragment is selected from N-terminal antigastrin antibodies and C-terminal antigastrin antibodies.

[0161] In another aspect, the present invention relates to a pharmaceutical composition comprising a composition according to the invention for the prevention or treatment of esophageal cancer and a pharmaceutically acceptable carrier. More specifically, the pharmaceutical composition according to the invention for the prevention or treatment of esophageal cancer comprises the antibody described above and a pharmaceutically acceptable carrier.

[0162] In a more specific aspect, the present invention relates to a pharmaceutical composition comprising a composition according to the invention for the prevention or treatment of esophageal cancer and a pharmaceutically acceptable carrier, wherein the anti-progastrin antibody is administered at a dose of 0.001 mg / kg to 250 mg / kg, and preferably at a dose of at least 0.005 mg / kg, at least 0.01 mg / kg, at least 0.05 mg / kg, at least 0.1 mg / kg, at least 0.5 mg / kg, at least 1 mg / kg, at least 5 mg / kg, at least 10 mg / kg, at least 50 mg / kg, or at least 100 mg / kg. In another aspect, the present invention relates to a kit comprising a composition according to the invention for the prevention or treatment of esophageal cancer and an anticancer therapeutic molecule.

[0163] In fact, treatment using anti-PG monoclonal antibodies as described in this article can be combined with or used as adjunct to other therapies. Non-limiting examples of other therapies include chemotherapy, radiation therapy, surgical resection, and antibody therapy.

[0164] In another aspect, the present invention relates to a kit comprising a composition according to the invention for the prevention or treatment of esophageal cancer and an anticancer therapeutic molecule selected from chemotherapy molecules and targeted therapy molecules.

[0165] In one specific embodiment, the present invention relates to a kit comprising a composition and chemotherapy molecule according to the invention for the treatment of esophageal cancer, for simultaneous, sequential, or individual administration. Useful chemotherapy molecules for this purpose include, but are not limited to, folic acid antagonists, purine antagonists, pyrimidine antagonists, DNA alkylating molecules, DNA cross-linking agents, antibiotics, platinum complexes, proteasome inhibitors, mitotic spindle toxins, topoisomerase inhibitors, tyrosine kinase inhibitors, and others.

[0166] In another specific embodiment, the present invention relates to a kit comprising a composition according to the invention for simultaneous, sequential, or individual administration, and a composition comprising another targeted therapeutic molecule. Such targeted therapeutic molecules include, but are not limited to, antibodies targeting EGFR such as cetuximab or panitumumab, antibodies targeting VEGF such as bevacizumab, antibodies targeting HER2 such as trastuzumab or pertuzumab, antibodies targeting PD-1 and PDL-1 such as pembrolizumab, antibodies targeting CTLA-4 such as ipilimumab, small molecule drugs targeting EGFR such as erlotinib, small molecule drugs targeting BRAF such as vemurafenib or dabrafenib, and recombinant fusion proteins targeting VEGF such as aflibercept.

[0167] In another specific aspect, the present invention relates to the use of progastrin-binding antibodies or antigen-binding fragments thereof for the diagnosis of esophageal cancer.

[0168] In another specific aspect, the present invention relates to the use of progastrin-binding antibodies or antigen-binding fragments thereof for the prevention or treatment of esophageal cancer.

[0169] In a more specific aspect, the present invention relates to the use of a progastrin-binding antibody or an antigen-binding fragment thereof for the prevention or treatment of esophageal cancer in a patient, wherein the concentration of progastrin in a biological sample of said patient has been determined and is higher than the concentration of progastrin in a reference biological sample.

[0170] In a more specific aspect, the present invention relates to the use of progastrin-binding antibodies or antigen-binding fragments thereof for the prevention or treatment of esophageal cancer in patients exhibiting metastasis.

[0171] In one or more specific aspects, the present invention relates to the use of a progastrin-binding antibody or an antigen-binding fragment thereof for the prevention or treatment of esophageal cancer in a patient exhibiting metastasis, and wherein the concentration of progastrin in a biological sample of the patient has been determined to be higher than the concentration of progastrin in a reference biological sample.

[0172] The components of the combination can be administered simultaneously, individually, or sequentially to achieve the maximum efficacy of the combination; for each component, the administration may be varied during the period from rapid administration to continuous infusion.

[0173] As used herein, "simultaneous administration" means administration of two compounds in a composition according to the invention in a single and unique pharmaceutical form. As used herein, "single administration" means simultaneous administration of two compounds in a composition according to the invention in different pharmaceutical forms. As used herein, "sequential administration" means sequential administration of two compounds in a composition according to the invention, each in a different pharmaceutical form.

[0174] As used in this article, “therapeutic effective amount” refers to the minimum concentration or amount of one or more compounds that effectively prevent, alleviate, reduce or improve the symptoms of a disease or prolong the survival of a patient undergoing treatment.

[0175] The features of embodiments of the present invention will become more apparent from the following detailed description of the examples. Attached Figure Description

[0176] Figure 1 Median plasma concentrations of progastrin in esophageal cancer patients (n=12) and control patients (n=103) — two-tailed Mann-Whitney test, ***p<0.001.

[0177] Figure 2 The number of OE33 spheres formed after treatment with control (CT Hz) or anti-PG humanized antibody (PG Hz) under ultra-low adsorption conditions — two-tailed t-test, * p < 0.05. Detailed Implementation

[0178] Example

[0179] Example 1: Detection of plasma progastrin concentration using polyclonal antibodies

[0180] Plasma progastrin levels were quantified using ELISA by using two specific anti-progastrin antibodies: a capture antibody was coated onto the wells of a plate, while a revelation antibody was used to detect progastrin and mediate the revelation of the signal.

[0181] In this embodiment, quantification is based on the ELISA method, which allows for the allocation of values ​​by using the substrate that emits light in response, and these values ​​are proportional to the amount of light emitted by the antibody that binds to the antigen held by the capture antibody.

[0182] Material

[0183] The reagents and instruments are listed in Table 7:

[0184] Table 7

[0185]

[0186] According to the standard protocol, polyclonal antibodies are obtained by immunizing rabbits with N-terminal progastrin (sequence 2) or C-terminal progastrin corresponding to amino acids 71 ​​to 80 of hPG and having the sequence FGRRSAEDEN (sequence 40).

[0187] The polyclonal antibodies against progastrin used in this assay have the following binding characteristics: they do not bind to G34-Gly, G34, G17-Gly, or G17, but bind to full-length progastrin.

[0188] A 50 mM carbonate-bicarbonate solution at pH 9.6 was prepared to coat a 96-well plate by dissolving the contents of one capsule in 100 ml of MilliQ water. A capture antibody (3 µg / ml) corresponding to a polyclonal antibody obtained using the C-terminal FGRRSAEDEN (Sequence 40) of progastrin was prepared in carbonate buffer. 100 μL of the antibody solution was added to each well and incubated at 4°C for 16 hours (overnight). The plate was then blocked by removing the antibody solution and washing three times with 300 μL of 1X PBS / 0.1% Tween-20. 200 μL of blocking buffer (1X PBS / 0.1% Tween-20 / 0.1% BSA) was added to each well, and the plate was incubated at 22°C for 2 hours to block the plate. The blocking buffer was then removed, and the wells were washed three times with 300 μL of 1X PBS / 0.1% Tween-20.

[0189] Plasma dilutions were performed as follows: pure plasma was used, diluted to 1 / 2, 1 / 5, and 1 / 10. Diluents were prepared from pure plasma in 1X PBS / 0.1% Tween 20 / 0.1% BSA.

[0190] For the control test, in the presence of known concentrations of progastrin, the progastrin dilution was prepared as follows: Stored recombinant PG (full-length human progastrin produced in *E. coli* and purified via glutathione agarose / tag removal (Tev) / IMAC Counter / dialysis affinity purification, from the Institut Pasteur, Paris, France) at a concentration of 0.45 mg / ml (45 microM), in triplicate. The range of progastrin concentrations was prepared as follows:

[0191] Solution A: Pre-diluted 1 / 10, 2 μl of stock solution + 18 μl of buffer

[0192] Solution B: Pre-diluted 1 / 100, 10 μl of A + 90 μl of buffer.

[0193] Solution C: Pre-diluted 1 / 1000, 10 μl of B + 90 μl of buffer.

[0194] Solution D: 500 pM, 5.55 μl of C+, 494.5 μl of diluent

[0195] Solution E: 250 pM, 250 μl of D + 250 μl of diluent

[0196] Solution F: 100 pM, 200 μl of E + 300 μl of diluent

[0197] Solution G: 50 pM, 250 μl of F + 250 μl of diluent

[0198] Solution H: 25 pM, 200 μl of G + 200 μl of diluent

[0199] Solution I: 10 pM, 100 μl H+, 150 μl diluent

[0200] The range of recombinant PG is linear and therefore can be broadly wide depending on the antibody used.

[0201] For test sample preparation, reserve approximately 500 μl of each sample and store it until analysis (and confirmation, if necessary) of the results. Measure each point in the range of 100 μl and / or plasma (pure, diluted to 1 / 2, 1 / 5, and 1 / 10) and incubate on a plate at 22°C for 2 hours.

[0202] For the test, the plates were washed three times with 300 μl of 1X PBS / 0.1% Tween-20. A solution of a 0.5 μg / ml biotin-bound polyclonal rabbit anti-progastrin antibody, obtained by dilution in 1X PBS / 0.1% Tween-20 / 0.1% BSA, was prepared by dilution of the N-terminal portion of progastrin, which was obtained using the immunogen. 100 μl of this solution was added to each well. Incubation was performed at 22°C for 1 hour. Disclosure using streptavidin-HRP was performed by the following steps: removing the detection antibody and washing three times with 300 μl of 1X PBS / 0.1% Tween-20, then preparing a 20 ng / ml solution of streptavidin-HRP diluted in 1X PBS / 0.1% Tween-20 / 0.1% BSA, adding 100 μl of this solution to each well, and then incubating at 22°C for 1 hour.

[0203] The assay consists of the following steps: Streptavidin-HRP is removed, and the sample is washed three times with 300 μl of 1X PBS / 0.1% Tween-20. Then, 100 μl of chemiluminescent substrate solution is added to each well. The substrate solution is prepared 30 minutes before use by mixing equal volumes of the two solutions (20 ml + 20 ml) in the SuperSignal ELISA Femto kit and storing in the dark at room temperature. After incubation in the dark at room temperature for 5 minutes, the luminescence is read.

[0204] For each condition, tests were performed in triplicate, and the results for that range were presented as a graph showing the changes in luminescence depending on the progastrin concentration. For each plasma dilution, the progastrin concentration was determined using an equation with a linear regression line for the corresponding range (1 / 10 for samples diluted to 1 / 10).

[0205] Methods and Results

[0206] The median plasma progastrin concentration was 42.3 pM in patients with esophageal cancer (n=12), while the median plasma progastrin concentration was 0 pM in control patients (n=103). Figure 1 These data demonstrate that patients with esophageal cancer have higher concentrations of progastrin in their plasma compared to healthy controls.

[0207] These data demonstrate that patients with esophageal cancer have higher levels of progastrin in their plasma compared to healthy controls.

[0208] Example 2: Detection of progastrin concentration using monoclonal anti-progastrin antibody

[0209] The wells of the Nunc MaxiSORP 96-well plate were coated with a first-stage progastrin-specific antibody as follows: An anti-progastrin monoclonal antibody specific for the C-terminal region of progastrin was diluted to a concentration of 3 μg / ml in 50 mM sodium carbonate / bicarbonate buffer at pH 9.6 in MilliQ water.

[0210] A total of 100 μl of the antibody solution was then added to each well of a 96-well plate and incubated overnight at 4°C. After binding, the antibody solution was removed from the wells, and the wells were washed three times with 100 μl of wash buffer (IX PBS / 0.1% Tween-20). A total of 100 μl of blocking buffer (IX PBS / 0.1% Tween-20 / 0.1% BSA) was then added to each well and incubated at 22°C for 2 hours. The blocking buffer was then removed, and the wells were washed three times with wash buffer. Plasma or serum samples isolated from the patient were then added to the wells in 100 μl volumes, using typical dilution series of 1:1, 1:2, 1:5, and 1:10, and incubated at 22°C for 2 hours. Plasma or serum samples were analyzed in duplicate.

[0211] The assay also includes two standard curves. The first standard curve was prepared using recombinant progastrin at dilutions of 1 ng, 0.5 ng, 0.25 ng, 0.1 ng, 0.05 ng, 0.01 ng, and 0 ng per well. The second standard curve, serving as a negative control, was prepared from progastrin-negative human serum diluted in blocking buffer at the same dilutions as the test samples (i.e., 1:1, 1:2, 1:5, and 1:10). Alternatively, when plasma samples were assayed, the second standard curve, serving as a negative control, was prepared from progastrin-negative human plasma diluted in blocking buffer at the same dilutions as the test samples (i.e., 1:1, 1:2, 1:5, and 1:10).

[0212] After incubation with plasma or serum samples, the contents of the wells are removed, and the wells are washed three times with washing buffer at 100 μl / well. Then, the progastrin bound to the first antibody is detected using a second antibody that is specific for progastrin, as described below.

[0213] A biotin-conjugated anti-progastrin monoclonal antibody, specific for the N-terminal region of progastrin, was diluted to a concentration of 0.1–10 μl g / ml in blocking buffer, depending on the antibody. A total of 100 μl of antibody solution was then added to each well and incubated at 22 °C for 1 hour.

[0214] After the second antibody binding is complete, the plate is washed three times with 100 μl / well of wash buffer, and then 100 μl of streptavidin-HRP (25 ng / ml in blocking buffer) is added to each well and incubated at 22°C for 1 hour. After incubation with streptavidin-HRP solution, the plate is washed three times with 100 μl / well of wash buffer. Then, 100 μl of chemiluminescent substrate prepared using the Pierce SuperSignal ELISA Femto Maximum Sensitivity Chemiluminescent Substrate Kit is added to each well, and the plate is incubated in the dark at room temperature for 5 minutes before reading on a photometer.

[0215] Based on photometer readings, linear regression analysis was used to obtain a linear equation corresponding to the standard curve data. This equation was then used to calculate the progastrin concentrations in different patient samples.

[0216] Median plasma progastrin concentrations were calculated in patients with esophageal cancer and compared with median plasma progastrin concentrations in control patients. These data demonstrate that patients with esophageal cancer have elevated progastrin levels in their plasma compared with healthy controls.

[0217] Example 3: Neutralizing activity of anti-hPG in cancer cell lines

[0218] 3.1 Neutralizing activity of anti-hPG monoclonal antibody

[0219] TE-1, TE-4, TE-6, KYSE30, FLO-1, OE-19, and OE-33 are commonly used cell lines for esophageal cancer research, producing and secreting progastrin. The ability of monoclonal antibodies against PG to inhibit proliferation in these different cell lines was tested. Cell viability from various TE-1, TE-4, TE-6, KYSE30, FLO-1, OE-19, and OE-33 cell lines was tested using different anti-hPG monoclonal antibodies.

[0220] For each experiment, 50,000 cells were seeded into 6-well plates in medium containing fetal bovine serum and incubated for 8 hours. The cells were starved of serum overnight, and then, starting 24 hours post-seeding (time "T0"), cells were treated in six replicates every 12 hours for 48 hours as follows: in the absence of fetal bovine serum, a monoclonal control antibody (anti-purulentinum monoclonal antibody) (CT mAb) of 1–20 µg / ml was used, or an anti-hPG mAb of 1–20 µg / ml was used, wherein the mAb was a C-terminal or N-terminal anti-hPG monoclonal antibody.

[0221] The mAb is selected from the following C-terminal anti-hPG antibodies:

[0222] -An antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3 of amino acid sequences 28, 29, and 30, and the light chain comprises CDR-L1, CDR-L2, and CDR-L3 of amino acid sequences 31, 32, and 33.

[0223] - An antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3 of amino acid sequences 34, 35, and 36, and the light chain comprises CDR-L1, CDR-L2, and CDR-L3 of amino acid sequences 37, 38, and 39.

[0224] Alternatively, select from the following N-terminal anti-hPG antibodies:

[0225] - An antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3 with amino acid sequences 4, 5, and 6, respectively, and the light chain comprises CDR-L1, CDR-L2, and CDR-L3 with amino acid sequences 7, 8, and 9.

[0226] - An antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3 with amino acid sequences 10, 11, and 12, respectively, and the light chain comprises CDR-L1, CDR-L2, and CDR-L3 with amino acid sequences 13, 14, and 15, respectively.

[0227] - An antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3 with amino acid sequences 16, 17, and 18, respectively, and the light chain comprises CDR-L1, CDR-L2, and CDR-L3 with amino acid sequences 19, 20, and 21, respectively.

[0228] -An antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises CDR-H1, CDR-H2 and CDR-H3 with amino acid sequences of sequences 22, 23 and 24, respectively, and the light chain comprises CDR-L1, CDR-L2 and CDR-L3 with amino acid sequences of sequences 25, 26 and 27, respectively.

[0229] For each experiment, the number of T0 cells in the control wells was counted.

[0230] Specifically, at 48 hours, the number of viable cells in both the control and anti-hPG mAb-treated wells was counted, and the difference between each cell count and the cell count determined at T0 was calculated. The resulting number of anti-hPG mAb-treated cells was then expressed as a percentage of the number of cells treated with the control mAb.

[0231] Treatment with anti-hPG monoclonal antibody reduced cell number compared to treatment with control antibody. Statistical significance was determined using Tukey's post-hoc test in a one-way ANOVA: * = p < 0.05, ** = p < 0.01, and *** = p < 0.001. In each cell line, anti-hPG antibody reduced cell survival.

[0232] 3.2 Neutralizing activity of anti-hPG humanized antibodies in cell survival

[0233] The ability of humanized anti-hPG antibodies to inhibit the proliferation of TE-1, TE-4, TE-6, KYSE30, FLO-1, OE-19, and OE-33 cell lines was tested. Cell viability from each of the TE-1, TE-4, TE-6, KYSE30, FLO-1, OE-19, and OE-33 cell lines was tested using different humanized anti-hPG antibodies.

[0234] For each experiment, 50,000 cells were seeded into 6-well plates containing fetal bovine serum and incubated for 8 hours. Cells were starved of serum overnight, and then, starting 24 hours post-seeding (time "T0"), cells were treated in six replicates every 12 hours for 48 hours as follows: in the absence of fetal bovine serum, using a 1–20 µg / ml humanized control antibody (anti-human FcG1, from BioXCell) (CT Hz), or using a 1–20 µg / ml anti-hPG Hz, where the Hz is a C-terminal or N-terminal anti-hPG humanized antibody. For each experiment, the number of cells at T0 in the control wells was counted.

[0235] Specifically, at 48 hours, the number of viable cells in both the control and anti-hPG Hz-treated wells was counted, and the difference between each cell count and the cell count determined at T0 was calculated. The resulting number of anti-hPG mAb-treated cells was then expressed as a percentage of the number of cells in the control Hz-treated wells.

[0236] Treatment with anti-hPG Hz antibody reduced cell number compared to treatment with control antibody. Statistical significance was determined using a one-way ANOVA with the Tuki post-hoc test: * = p < 0.05, ** = p < 0.01, and *** = p < 0.001. In each cell line, anti-hPG antibody reduced cell survival.

[0237] 3.3. Neutralizing activity of anti-hPG monoclonal antibodies in cancer stem cell frequency

[0238] The ability of anti-PG monoclonal antibodies to reduce the frequency of cancer stem cells (CSCs) in TE-1, TE-4, TE-6, KYSE30, FLO-1, OE-19, and OE-33 cell lines was tested using the Extreme Limiting Dilution Assay (ELDA). The frequency of CSCs from each of the TE-1, TE-4, TE-6, KYSE30, FLO-1, OE-19, and OE-33 cell lines was tested using different anti-hPG monoclonal antibodies.

[0239] For each experiment, cells were seeded at a fixed per-well concentration in ultra-low adsorption (ULA) P96 (96-well plate) using a FACS Aria flow cytometer, ranging from 1 to 500 cells / well. Cells were cultured in ULA plates containing M11 medium (Macari et al., Oncogene, 2015) for up to 11 days and treated every 3 or 4 days with 1–20 µg / ml of a monoclonal control antibody (anti-purulentinum monoclonal antibody) (CT mAb) or with 1–20 µg / ml of an anti-hPG mAb (wherein the mAb is a C-terminal or N-terminal anti-hPG monoclonal antibody, as disclosed in Example 3.1).

[0240] Specifically, at the end of the incubation phase, the number of positive wells for each cell concentration was assessed using a phase-contrast microscopy plate. Finally, the CSC frequency for each treatment group and any statistical differences between the test groups (modified chi-square test) were calculated using the ELDA site tool (http: / / www.bioinf.wehi.edu.au / software / elda / ).

[0241] Treatment with anti-hPG monoclonal antibody reduced the CSC frequency compared to treatment with control antibody.

[0242] 3.4 Neutralizing activity of anti-hPG humanized antibodies in cancer stem cell frequency

[0243] Sphere formation determination

[0244] The ability of humanized anti-PG antibodies to reduce the frequency of cancer stem cells (CSCs) in FLO-1, OE19, and OE33 cell lines was tested using a spheroidization assay.

[0245] For each experiment, 200 cells were seeded in 24-well ultralow adsorption (ULA) plates. Cells were cultured in ULA plates containing M11 medium (Macari et al, Oncogene, 2015) for up to 10 days and treated every 3 or 4 days with either 20 µg / ml of humanized control antibody (anti-human FcG1, from BioXCell) (CT Hz) or 20 µg / ml of anti-hPG Hz (PG Hz) (wherein the Hz is a C-terminal or N-terminal anti-hPG humanized antibody).

[0246] Specifically, at the end of the incubation phase, the holes were photographed using a bright-field microscope, the photographs were analyzed, and spheres with an average diameter greater than 25 µm were counted.

[0247] Treatment with anti-hPG humanized antibodies reduced the CSC frequency compared to treatment with control antibodies.

[0248] Extremely limiting dilution assay

[0249] The ability of humanized anti-PG antibodies to reduce the frequency of cancer stem cells (CSCs) in TE-1, TE-4, TE-6, KYSE30, FLO-1, OE19, and OE33 cell lines was tested using an extremely limiting dilution assay (ELDA). The frequency of CSCs from each of the TE-1, TE-4, TE-6, KYSE30, FLO-1, OE19, and OE33 cell lines was tested using different humanized anti-hPG antibodies.

[0250] For each experiment, cells were seeded at a fixed per-well concentration in ultra-low adsorption (ULA) P96 (96-well plate) using a FACS Aria flow cytometer, ranging from 1 to 500 cells / well. Cells were cultured in ULA plates containing M11 medium (Macari et al, Oncogene, 2015) for up to 11 days and treated every 3 or 4 days with 1–20 µg / ml of humanized control antibody (anti-human FcG1, from BioXCell) (CT Hz) or with 1–20 µg / ml of anti-hPG Hz (wherein the Hz is a C-terminal or N-terminal anti-hPG humanized antibody).

[0251] Specifically, at the end of the incubation phase, the number of positive wells for each cell concentration was assessed using a phase-contrast microscopy plate. Finally, the CSC frequency for each treatment group and any statistical differences between the test groups (modified chi-square test) were calculated using the ELDA site tool (http: / / www.bioinf.wehi.edu.au / software / elda / ).

[0252] Treatment with anti-hPG humanized antibodies reduced the CSC frequency compared to treatment with control antibodies.

[0253] 3.5 Neutralizing activity of anti-hPG monoclonal antibody in the WNT / β-linkin pathway

[0254] TE-1, TE-4, TE-6, KYSE30, FLO-1, OE19, and OE33 are commonly used cell lines for esophageal cancer research, producing and secreting progastrin. The ability of monoclonal antibodies against hPG to inhibit the WNT / β-linkin pathway in these different cell lines was tested, using the expression of the protein survival protein (a known target gene of the WNT / β-linkin pathway) as a readout. Survival protein expression from each of the TE-1, TE-4, TE-6, KYSE30, FLO-1, OE19, and OE33 cell lines was tested using different anti-hPG monoclonal antibodies.

[0255] For each experiment, 50,000 cells were seeded into 6-well plates containing fetal bovine serum and incubated for 8 hours. Cells were starved of serum overnight, and then, starting 24 hours post-seeding, treated in quadruplicate every 12 hours for 72 hours, as follows: in the absence of fetal bovine serum, a monoclonal control antibody (anti-purulentinum monoclonal antibody) (CT mAb) of 1–20 µg / ml was used, or an anti-hPG mAb of 1–20 µg / ml was used, wherein the mAb was a C-terminal or N-terminal anti-hPG monoclonal antibody.

[0256] Specifically, after 72 hours of treatment, cells were harvested and total protein was extracted using RIPA buffer. Equal amounts of protein from cells treated with either CT mAb or anti-hPGmAb were then Western blotted using an anti-survival protein antibody (monoclonal antibody, #2802 from Cell Signaling) and an anti-actin antibody (monoclonal antibody, #A4700 from SIGMA) as loading controls. Quantification was performed using the GBOX chemistry system from Syngene.

[0257] Treatment with anti-hPG monoclonal antibody reduced the expression of surviving proteins compared with treatment with control antibody. Statistical significance was determined using an unpaired Student's t-test: * = p < 0.05, ** = p < 0.01 and *** = p < 0.001.

[0258] 3.6 Neutralizing activity of anti-hPG humanized antibody in the WNT / β-linkin pathway

[0259] The ability of humanized anti-hPG antibodies to inhibit the WNT / β-linkin pathway in TE-1, TE-4, TE-6, KYSE30, FLO-1, OE19, and OE33 cell lines was tested, using the expression of the protein survival protein (a known target gene of the WNT / β-linkin pathway) as readout. The expression of the survival protein from each of the TE-1, TE-4, TE-6, KYSE30, FLO-1, OE19, and OE33 cell lines was tested using different anti-hPG humanized antibodies.

[0260] For each experiment, 50,000 cells were seeded into 6-well plates in medium containing fetal bovine serum and incubated for 8 hours. The cells were starved of serum overnight, and then, starting 24 hours post-seeding, the cells were treated in quadruplicate every 12 hours for 72 hours as follows: in the absence of fetal bovine serum, a humanized control antibody (anti-human FcG1, from BioXCell) (CT Hz) of 1–20 µg / ml was used, or an anti-hPG Hz of 1–20 µg / ml was used, wherein the Hz was a C-terminal or N-terminal anti-hPG humanized antibody.

[0261] Specifically, after 72 hours of treatment, cells were harvested and total protein was extracted using RIPA buffer. Equal amounts of protein from cells treated with either CT Hz or anti-hPG Hz were then Western blotted using an anti-survival protein antibody (monoclonal antibody, #2802 from Cell Signaling) and an anti-actin antibody (monoclonal antibody, #A4700 from SIGMA) as loading controls. Quantification was performed using the GBOX chemistry system from Syngene.

[0262] Treatment with the anti-hPG humanized antibody reduced the expression of the survival protein compared with treatment with the control antibody. Statistical significance was determined using an unpaired Student's t-test: * = p < 0.05, ** = p < 0.01 and *** = p < 0.001.

[0263] This disclosure relates to the following implementation plan.

[0264] 1. A method for in vitro diagnosis of esophageal cancer in a subject, comprising the following steps:

[0265] a) Contact a biological sample from the subject with at least one progastrin-binding molecule.

[0266] b) Detect the binding of the progastrin-binding molecule to progastrin in the sample, wherein the binding indicates the presence of esophageal cancer in the subject.

[0267] 2. The method of implementation 1, wherein step b) further includes determining the concentration of progastrin, and wherein a progastrin concentration of at least 10 pM in the biological sample indicates the presence of esophageal cancer in the subject.

[0268] 3. The method of implementation scheme 2, which includes the following additional steps:

[0269] c) Determine the reference concentration of rumenin in the reference sample.

[0270] d) Compare the concentration of progastrin in the biological sample with the reference concentration of progastrin.

[0271] e) Determine the presence of esophageal cancer from the comparison in step d).

[0272] 4. The method of any one of embodiments 1-3, wherein the progastrin-binding molecule is an antibody or an antigen-binding fragment thereof.

[0273] 5. The method of any one of embodiments 1-4, wherein the antibody or its antigen-binding molecule is selected from N-terminal anti-progastrin monoclonal antibody and C-terminal anti-progastrin monoclonal antibody.

[0274] 6. The method of any one of embodiments 1-5, wherein the antibody binding to progastrin is a monoclonal antibody selected from the group consisting of:

[0275] - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3, with amino acid sequences 4, 5, and 6 respectively; and the light chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3, with amino acid sequences 7, 8, and 9 respectively.

[0276] - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3, with amino acid sequences 10, 11, and 12 respectively; and the light chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3, with amino acid sequences 13, 14, and 15 respectively.

[0277] - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3, with amino acid sequences 16, 17, and 18, respectively; and the light chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3, with amino acid sequences 19, 20, and 21, respectively.

[0278] - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3, with amino acid sequences 22, 23, and 24, respectively; and the light chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3, with amino acid sequences 25, 26, and 27, respectively.

[0279] - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3, with amino acid sequences 28, 29, and 30 respectively; and the light chain comprises at least one, preferably at least two, preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3, with amino acid sequences 31, 32, and 33 respectively.

[0280] - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3, with amino acid sequences 34, 35, and 36 respectively; and the light chain comprises at least one, preferably at least two, preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3, with amino acid sequences 37, 38, and 39 respectively.

[0281] 7. The method of any one of embodiments 1-6, wherein the biological sample is contacted with a first molecule of the first portion of the progastrin and a second molecule of the second portion of the progastrin.

[0282] 8. The method of any one of implementation schemes 1-7, wherein the biological sample is selected from: blood, serum and plasma.

[0283] 9. The method of any one of embodiments 1-8, wherein the biological sample is plasma, and wherein a pregastrin concentration of at least 10 pM indicates the presence of esophageal cancer in the subject.

[0284] 10. A composition for use in the prevention or treatment of esophageal cancer, wherein the composition comprises a progastrin-binding antibody or an antigen-binding fragment thereof.

[0285] 11. The composition of embodiment 10, wherein the progastrin-binding antibody or its antigen-binding fragment is selected from humanized antibodies, single-chain antibodies, camelified antibodies, IgA1 antibodies, IgA2 antibodies, IgD antibodies, IgE antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, and IgM antibodies.

[0286] 12. The composition of any one of embodiments 10 or 11, wherein the progastrin-binding antibody or its antigen-binding fragment is selected from N-terminal antigastrin antibody and C-terminal antigastrin antibody.

[0287] 13. The composition of any one of embodiments 10-12, wherein the progastrin-binding molecule or its antigen-binding fragment is a neutralizing antibody.

[0288] 14. The composition of any one of embodiments 10-13, wherein the progastrin-binding molecule is a humanized antibody selected from:

[0289] - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3, with amino acid sequences 4, 5, and 6 respectively; and the light chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3, with amino acid sequences 7, 8, and 9 respectively.

[0290] - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3, with amino acid sequences 10, 11, and 12 respectively; and the light chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3, with amino acid sequences 13, 14, and 15 respectively.

[0291] - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3, with amino acid sequences 16, 17, and 18, respectively; and the light chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3, with amino acid sequences 19, 20, and 21, respectively.

[0292] - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3, with amino acid sequences 22, 23, and 24, respectively; and the light chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3, with amino acid sequences 25, 26, and 27, respectively.

[0293] - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3, with amino acid sequences 28, 29, and 30 respectively; and the light chain comprises at least one, preferably at least two, preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3, with amino acid sequences 31, 32, and 33 respectively.

[0294] - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3, with amino acid sequences 34, 35, and 36 respectively; and the light chain comprises at least one, preferably at least two, preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3, with amino acid sequences 37, 38, and 39 respectively.

[0295] 15. The use of the progastrin-binding antibody or its antigen-binding fragment as described in any one of embodiments 10-14 for the in vitro diagnosis of esophageal cancer.

[0296] References

[0297] Kaz et al, Cancer Letters, 2014, Jan 28;342(2):193-9. “Epigenetic biomarkers in esophageal cancer.”.

Claims

1. A method for in vitro diagnosis of esophageal cancer in a subject, comprising the following steps: a) Contact a biological sample from the subject with at least one progastrin-binding molecule. b) Detect the binding of the progastrin-binding molecule to progastrin in the sample, wherein the binding indicates the presence of esophageal cancer in the subject.

2. The method of claim 1, wherein step b) further comprises determining a progastrin concentration, and wherein a progastrin concentration of at least 10 pM in the biological sample indicates the presence of esophageal cancer in the subject.

3. The method of claim 2, further comprising the following additional steps: c) Determine the reference concentration of rumenin in the reference sample. d) Compare the concentration of progastrin in the biological sample with the reference concentration of progastrin. e) Determine the presence of esophageal cancer from the comparison in step d).

4. The method of any one of claims 1-3, wherein the progastrin-binding molecule is an antibody or an antigen-binding fragment thereof.

5. The method of any one of claims 1-4, wherein the antibody or its antigen-binding molecule is selected from N-terminal anti-progastrin monoclonal antibody and C-terminal anti-progastrin monoclonal antibody.

6. The method of any one of claims 1-5, wherein the antibody binding to progastrin is a monoclonal antibody selected from the group consisting of: - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3, with amino acid sequences 4, 5, and 6 respectively; and the light chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3, with amino acid sequences 7, 8, and 9 respectively. - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3, with amino acid sequences 10, 11, and 12 respectively; and the light chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3, with amino acid sequences 13, 14, and 15 respectively. - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3, with amino acid sequences 16, 17, and 18, respectively; and the light chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3, with amino acid sequences 19, 20, and 21, respectively. - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3, with amino acid sequences 22, 23, and 24, respectively; and the light chain comprises at least one, preferably at least two, and preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3, with amino acid sequences 25, 26, and 27, respectively. - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3, with amino acid sequences 28, 29, and 30 respectively; and the light chain comprises at least one, preferably at least two, preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3, with amino acid sequences 31, 32, and 33 respectively. - A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one, preferably at least two, preferably three, of the following sequences: CDR-H1, CDR-H2, and CDR-H3, with amino acid sequences 34, 35, and 36 respectively; and the light chain comprises at least one, preferably at least two, preferably three, of the following sequences: CDR-L1, CDR-L2, and CDR-L3, with amino acid sequences 37, 38, and 39 respectively.

7. The method of any one of claims 1-6, wherein the biological sample is contacted with a first molecule of the first portion of the progastrin and a second molecule of the second portion of the progastrin.

8. The method of any one of claims 1-7, wherein the biological sample is selected from blood, serum, and plasma.

9. The method of any one of claims 1-8, wherein the biological sample is plasma, and wherein a pregastrin concentration of at least 10 pM indicates the presence of esophageal cancer in the subject.

10. A composition for use in the prevention or treatment of esophageal cancer, wherein the composition comprises a progastrin-binding antibody or an antigen-binding fragment thereof.

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