EA042821B1 - COMPOSITIONS AND METHODS FOR DIAGNOSTICS OF LUNG CANCER - Google Patents

Description

The invention relates to in vitro diagnostics of cancer, more specifically, it relates to in vitro diagnostic methods for lung cancer. The compositions of the invention comprise a progastrin binding molecule, in particular an anti-hPG (human progastrin) antibody, while the methods of the invention comprise the use of a progastrin binding molecule, and in particular an anti-hPG antibody.

Lung cancer remains the most lethal malignant tumor in the world. Despite improvements in surgical treatment, systemic therapy, and radiotherapy, the 5-year survival rate for all patients diagnosed with lung cancer remains 15 to 20%.

Lung cancer includes two main types of tumors, namely small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). SCLC represents 15-18% of all lung cancers, while NSCLC accounts for approximately 80% to 85% of lung cancers. Other types of lung cancer, such as adenocystic carcinomas, lymphomas, and sarcomas, and benign lung tumors, such as hamartomas, are rare.

Small cell and non-small cell lung cancer are treated differently. In particular, SCLC is more responsive to chemotherapy and radiation therapy than other cell types of lung cancer. However, it is difficult to achieve a cure, as SCLC tends to be more widely disseminated by the time of diagnosis. To date, there are no molecular biomarkers that have been translated into widespread clinical practice in lung cancer. Treatment options depend on the development of the cancer and usually include surgery for small, localized tumors or chemotherapy, possibly in combination with radiation therapy.

Therefore, there is still a need for methods that provide rapid, reliable and cost-effective diagnosis of lung cancer.

This is the purpose of the invention.

Description of the invention

The present invention now provides methods for diagnosing lung cancer in vitro, wherein said methods include detecting progastrin in a biological sample from a subject. Preferably, the amount of progastrin in said sample is determined, thus providing a quantitative measurement of progastrin.

Human pre-progastrin, a 101 amino acid peptide (reference amino acid sequence: AAB19304.1), is the primary translation product of the gastrin gene. Progastrin is formed by cleavage of the first 21 amino acids (signal peptide) from preprogastrin. The 80 amino acid chain of progastrin is further processed by cleaving and modifying enzymes to several biologically active hormonal forms of gastrin: gastrin 34 (G34) and gastrin 34 with glycine extension (G34-Gly), containing amino acids 38-71 of progastrin, gastrin 17 (G17) and gastrin 17 with glycine extension (G17-Gly) containing progastrin amino acids 55-71.

Monoclonal antibodies against human progastrin (anti-hPG antibodies) and their use in diagnosis and therapy have been described in the following documents: WO 2011/083088 for colorectal cancer, WO 2011/083090 for breast cancer, WO 2011/083091 for pancreatic cancer, WO 2011/116954 for colorectal and gastrointestinal cancers, and WO 2012/013 609 and WO 2011/083089 for liver pathologies.

The present invention will become clearer from the detailed description provided herein and from the accompanying drawings, which are provided by way of illustration only and do not limit the intended scope of the present invention.

In a first aspect, the present invention relates to a method for assessing the in vitro risk of the presence of lung cancer, which method includes the step of detecting progastrin in a biological sample from a subject. The presence of progastrin in the sample indicates that there is a risk of lung cancer being present.

Thus, in a first embodiment, this invention relates to a method for assessing the in vitro risk of lung cancer in a subject, said method comprising the following steps:

a) contacting a biological sample from said subject with at least one progastrin-binding molecule, and

b) detecting the binding of said progastrin-binding molecule to progastrin in said sample, wherein said binding is indicative of a risk for the presence of lung cancer.

Binding of the progastrin binding molecule can be detected by various assays available to the skilled artisan. While any suitable assay methods are included in the present invention, mention may be made in particular of FACS (fluorescent cell sorting), ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), Western blotting, and IHC (immunohistochemistry).

In a preferred embodiment, the method of the invention for assessing the in vitro risk of having lung cancer in a subject comprises the following steps:

a) contacting said biological sample from said subject with at least one progastrin-binding molecule,

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b) determining the concentration of progastrin in the specified biological sample, where the concentration of progastrin at least 10 pM in the specified biological sample indicates the risk of the presence of lung cancer.

Once the concentration of progastrin present in the sample is determined, this result can be compared with the results of the control sample(s), which are obtained in a similar way to the experimental samples, but from the individual(s) for whom it is known that that he (they) does not suffer from lung cancer. If the concentration of progastrin is significantly higher in the test sample, it can be concluded that there is an increased likelihood that the subject from which it was obtained has lung cancer.

Thus, in a more preferred embodiment, the method of the invention comprises the following additional steps:

c) determination of the control concentration of progastrin in the control sample,

d) comparing the concentration of progastrin in the specified biological sample with the specified control concentration of progastrin,

e) assessment from stage comparison d) risk of lung cancer presence.

In another aspect, the invention relates to a method for in vitro diagnosis of lung cancer in a subject, said method comprising the steps of:

a) contacting a biological sample from said subject with at least one progastrin-binding molecule, and

b) detecting the binding of said progastrin-binding molecule to progastrin in said sample, wherein said binding indicates the presence of lung cancer in said subject.

In a preferred embodiment, the present invention relates to a method for in vitro diagnosis of lung cancer in a subject, comprising the following steps:

a) contacting said biological sample from said subject with at least one progastrin-binding molecule,

b) determining the concentration of progastrin in said biological sample, wherein a progastrin concentration of at least 10 pM in said biological sample indicates the presence of lung cancer in said subject.

In a more specific embodiment of the method of the invention, a progastrin concentration of at least 10 pM, at least 20 pM, at least 30 pM in said biological sample indicates the presence of lung cancer in said subject.

In a more preferred embodiment, the method of the invention comprises the following additional steps:

a) determination of the control concentration of progastrin in the control sample,

b) comparing the concentration of progastrin in the specified biological sample with the specified control level or concentration of progastrin,

c) making a diagnosis from a comparison by stage d) the presence of lung cancer.

In another aspect, the invention relates to a method for performing an in vitro diagnosis of metastatic lung cancer in a subject, said method comprising the steps of:

a) contacting a biological sample from said subject with at least one progastrin-binding molecule, and

b) detecting the binding of said progastrin-binding molecule to progastrin in said sample, wherein said binding indicates the presence of metastatic lung cancer in said subject.

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

a) bringing said biological sample into contact with at least one progastrin-binding molecule,

b) determining by biochemical analysis a level or concentration of progastrin in said biological sample, wherein a progastrin concentration of at least 10 pM or higher in said biological sample indicates the presence of metastatic lung cancer in said subject.

In a more specific embodiment of the method of 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 said biological sample indicates the presence of metastatic lung cancer in said subject.

In a more preferred embodiment, the method of the invention comprises the following additional steps:

a) determination of the control concentration of progastrin in the control sample,

b) comparing the concentration of progastrin in the specified biological sample with the specified control concentration of progastrin,

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c) making a diagnosis from the comparison by stage d) the presence of metastatic lung cancer.

In a specific embodiment, the present invention relates to a method for diagnosing lung cancer in vitro in a subject, comprising determining the concentration of progastrin in a biological sample and comparing this obtained value with the concentration of progastrin in a control sample.

In a more specific embodiment, in the method for diagnosing lung cancer according to the present invention, a biological sample of said subject is brought into contact with at least one progastrin-binding molecule, wherein said progastrin-binding molecule is an antibody or an antigen-binding fragment thereof.

The expression risk score for the presence of lung cancer in a subject means a determination of the relative likelihood for a given subject of suffering from lung cancer compared to a control subject or value. According to the method according to the invention, an agent is provided in the assessment of said risk, in combination with other methods or indicators, such as clinical examination, biopsy and determination of the level of a known lung cancer biomarker.

The expression in vitro diagnostics means determining whether a subject is suffering from a particular disease. It is known that the diagnosis of lung cancer involves at least clinical observation of the symptoms of said subject, such as, for example, a low-dose helical computed tomography (CT) scan. Although some biomarkers have been identified in the discovery phase, their transfer to clinical practice is still an important challenge, mainly due to the lack of a method for systematic evaluation (Li et al, Neoplasma. 2012, 59(5): 500-507).

Therefore, the in vitro diagnostic method for lung cancer according to the present invention can be considered as a tool within the diagnostic process.

The expression lung cancer refers to cancer that occurs in lung tissue, usually in the cells that line the airways. The term lung cancer as used herein includes, in particular, small cell lung cancer (SCLC), including small cell carcinoma and combined small cell carcinoma, and non-small cell lung cancers (NSCLC), including squamous cell carcinoma, large cell carcinoma and adenocarcinoma. Other types of lung cancers, such as adenocystic carcinomas, lymphomas, and sarcomas, as well as benign lung tumors, such as hamartomas, are also included in lung cancers as the term is used herein.

The term progastrin refers to the peptide of mammalian progastrin and in particular human progastrin. For the avoidance of doubt, without any precise definition, the expression human progastrin refers to the human PG (progastrin) sequence of SEQ ID NO: 1. Namely, human progastrin contains N-terminal and C-terminal domains that are not present in the biologically mentioned above. active forms of the hormone gastrin. Preferably, the sequence of said N-terminal domain is represented by SEQ ID NO: 2. In another preferred embodiment, the sequence of said C-terminal domain is represented by SEQ ID NO: 3.

Determination of the concentration of progastrin in the method according to the invention is carried out by any method known to a person skilled in the field of biochemistry.

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

When measuring expression levels at the protein level, this can in particular be carried out using specific progastrin-binding molecules such as, for example, antibodies, in particular using well known techniques such as cell membrane staining using biotinylation or other equivalent techniques. , followed by immunoprecipitation with specific antibodies, Western blotting, ELISA or ELISPOT (enzymatic immunostain method), enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunohistochemistry (IHC), immunofluorescence (IF), antibody bioarrays or tissue microarrays in combination with immunohistochemistry. Other suitable techniques include FRET (fluorescence resonant energy transfer) or BRET (bioluminescence resonant energy transfer), microscopic or histochemical methods on single cells using single or multiple excitation wavelengths, and the use of any of the adapted optical methods such as electrochemical methods (voltametry techniques). and amperometry), atomic force microscopy, and radio frequency techniques, such as multipolar resonance spectroscopy, confocal and non-confocal microscopy, detection of fluorescence, luminescence, chemiluminescence, absorption, reflectivity, transmittance and birefringence or refractive index (for example, surface plasmon resonance, ellipsometry, resonating mirror method, grating lead light guide interferometry method), cellular ELISA, flow cytometry, radioisotope, magnetic resonance imaging, analysis by polyacrylamide gel electrophoresis (SDS-PAGE - sodium dodecyl sulfate polyacrylamide gel electrophoresis); HPLC (high performance liquid chromatography)-mass spectrometry; liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS). All of these techniques are well known in the art and need not be described in further detail here. These different techniques can be used to measure progastrin levels.

Said method may in particular be selected from among the following: immunodetection method, Western blot method, mass spectrometry method, chromatography method and flow cytometry method. While any suitable methods for performing assays are included within the scope of this invention, methods such as FACS, ELISA, RIA, Western blot, and IHC are particularly useful for carrying out the method of the invention.

In a more specific embodiment, the in vitro diagnostic method for lung cancer of the invention comprises contacting a biological sample from a subject with a progastrin binding molecule using an enzyme immunoassay, preferably based on techniques selected from RIA and ELISA.

A biological sample, as used herein, is a biological tissue or fluid sample that contains nucleic acids or polypeptides, such as a protein, polynucleotide, or lung cancer transcript. Such a sample should provide for the determination of progastrin expression levels. It is known that progastrin is a secreted protein. Preferred biological samples for determining progastrin protein levels thus include body fluids. The term biological fluid as used herein means any fluid that includes a substance of biological origin. Preferred biological fluids for use in the present invention include body fluids of an animal, eg a mammal, preferably a human subject. The body fluid can be any body fluid, including but not limited to blood, plasma, serum, lymph, cerebrospinal fluid (CSF), saliva, sweat, and urine. Preferably, said preferred liquid biological samples include samples such as a blood sample, a plasma sample, or a serum sample. More preferably, the biological sample is a blood sample. Indeed, such a blood sample can be obtained by completely harmless blood sampling from a patient and thus provides a non-invasive assessment of the risks that the subject will develop a tumor.

The term biological sample, as used herein, also includes the patient's solid cancer sample to be analyzed when the cancer is a solid cancer. Such a sample of solid cancer allows a specialist to perform any type of measurement of the level of the biomarker according to the invention. In some instances, the methods of the invention may further include the preliminary step of collecting a solid cancer sample from the patient. A sample of solid cancer is a sample of tumor tissue. Even in a cancer patient, the tissue that is the site of the tumor still contains non-tumor healthy tissue. The cancer sample should therefore be limited to tumor tissue taken from the patient. Said cancer sample may be a biopsy sample or a sample taken as a result of surgical resection therapy.

The biological sample is typically obtained from a eukaryotic organism, most preferably a mammal or bird, reptile or fish. Indeed, the subject that can be subjected to the method described herein can be any mammalian animal, including human, dog, cat, cattle, goat, pig, boar, sheep and monkey; or a bird; reptile; or fish. Preferably the subject is a human; the human subject may be known as a patient.

By obtaining a biological sample in this document means obtaining a biological sample for use in the methods described in this invention. This will most often be done by removing a sample of cells from the animal, but can also be done by using previously isolated cells (eg, isolated by another person, at a different time and/or for a different purpose) or by performing the methods of the invention in vivo. Archival tissues with a history of treatment or outcome will be especially useful.

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

Progastrin concentration determination refers to the determination of the amount of progastrin in a known volume of a sample. The concentration of progastrin can be expressed relative to the control sample, for example, as a ratio or percentage. A given concentration can also be expressed as signal intensity or localization, depending on the method used to determine said concentration. Preferably, the concentration of a compound in a sample is expressed after normalizing the total concentration of related compounds in said sample, for example, the level or concentration of a protein is expressed after normalizing the total concentration of proteins in the sample.

Preferably, the risk that said subject suffers from lung cancer is determined by comparing the level of progastrin measured in said biological sample with a control level.

The term reference level, as used herein, refers to the level of expression of the lung cancer marker in question, i.e. progastrin, in the control sample. Control sample, as the term is used herein, means a sample obtained from subjects, preferably two or more than two subjects, who are known not to have the disease or, alternatively, come from the general population. . Suitable control levels of progastrin expression can be determined by measuring the levels of expression of the specified marker in several suitable subjects, and such control levels can be adjusted in relation to specific populations of subjects. The reference value or reference level may be an absolute value; relative value; a value that has an upper or lower bound; range of values; average value; median value; an average value, or a value that is compared to a particular control or baseline value. The control value may be based on the value of an individual sample, such as, for example, the value obtained from a sample from an analyzed subject, but at an earlier point in time. The reference value may be based on a large number of samples, such as from a population of subjects in a chronologically age-matched group, or based on a pool of samples that includes or excludes the sample to be analyzed.

Preferably, the control level is a target level of progastrin obtained from a biological sample from a subject with known specific cancer status. In specific embodiments, the control level used for comparison with the test sample in step (b) may have been obtained from a biological sample from a healthy subject or from a biological sample from a subject suffering from cancer; it is clear that the control expression profile can also be obtained from the pool of biological samples of healthy subjects or from the pool of biological samples of healthy subjects, or from the pool of samples from subjects with cancer.

In a specific embodiment of the method of the invention, a control sample is taken from subjects free of any cancer and preferably no pathology. It should be understood that, according to the nature of the biological sample taken from the patient, the control sample will be a biological sample of the same nature as the specified biological sample.

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

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

According to a specific embodiment, the present invention relates to a method for diagnosing lung cancer in vitro, comprising determining the concentration of progastrin in a biological sample from a subject, where the specified subject shows at least one clinical symptom of lung cancer.

According to another specific embodiment, the present invention relates to a method for diagnosing lung cancer in vitro, including determining the concentration of progastrin in a biological sample from a subject, where the specified subject shows at least one clinical symptom of cancer and/or metastasis.

By binding, binding, or the like, is meant that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining whether two molecules bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. In a specific embodiment, said antibody, or antigen-binding fragment thereof, binds to progastrin with an affinity that is at least two times greater than its binding affinity for a non-specific molecule such as BSA (bovine serum albumin) or casein. In a more specific embodiment, said antibody, or antigen-binding fragment thereof, binds only to progastrin.

In a specific embodiment, in the lung cancer diagnostic method of the invention, a biological sample from a subject is contacted with at least one progastrin-binding molecule, wherein said molecule has an affinity for progastrin of 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, according to

- 5 042821 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 when determined in a manner such as the method described above.

In a specific embodiment, the present invention relates to a method for diagnosing lung cancer, comprising detecting the concentration of progastrin in a biological sample from a subject, where the specified biological sample is brought into contact with an anti-hPG antibody or antigen-binding fragment.

The term antibody, as used herein, is intended to include polyclonal and monoclonal antibodies. An antibody (or immunoglobulin) consists of a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain contains a heavy chain variable region (or domain) (abbreviated 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 regions of hypervariability, referred to as complementarity determining regions (CDRs) or hypervariable regions, which are primarily responsible for binding to an antigen epitope, and interspersed with regions that are more conserved, referred to as framework regions. (FR). A method for identifying CDRs within the light and heavy chains of an antibody and determining their sequence is well known to those skilled in the art. For the avoidance of doubt, in the absence of any indication to the contrary in the text, the expression CDR means the hypervariable regions of the heavy and light chains of an antibody, as defined by IMGT, where the unique numbering of the IMGT provides a standardized delimitation of framework regions and complementarity determining regions, CDR1-IMGT: 27-38 , CDR2.

A unique IMGT numbering has been defined to compare variable domains, whatever the antigen receptor, chain type, or species [Lefranc M.-P., Immunology Today 18, 509 (1997)/ Lefranc M.-P., The Immunologist, 7, 132-136 (1999)/ Lefranc, M.-P., Pommie, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, Dev . Associate Immunol., 27, 55-77 (2003)]. In the unique IMGT numbering, conserved amino acids always have the same position, e.g. cysteine 23 (1st CYS), tryptophan 41 (CONSERVATIVE TRP), hydrophobic amino acid 89, cysteine 104 (2nd CYS), phenylalanine or tryptophan 118 (J -PHE or J-TRP). The unique IMGT numbering provides a standardized delimitation of the framework regions (FR1-IMGT: positions 1-26, FR2-IMGT: 39-55, FR3IMGT: 66-104 and FR4-IMGT: 118-128) and complementarity-determining regions: CDR1-IMGT: 27-38, CDR2-IMGT: 56-65 and CDR3-IMGT: 105-117. Since spaces represent unoccupied positions, the CDR-IMGT lengths (shown between brackets and separated by dots, eg [8.8.13]) become key information. The unique IMGT numbering is used in 2D (two-dimensional) graphical representations, designated as IMGT Colliers 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 in 3D structures 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)].

Each VH and VL consists of three CDRs and four FRs, organized from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. Antibody constant regions can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. Antibodies can be of different isotypes (namely IgA, IgD, IgE, IgG or IgM).

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

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

The term monoclonal antibody refers to an antibody arising from a nearly homogeneous population of antibodies, where this population contains identical antibodies, except for a few possible naturally occurring mutations, which may be in minimal proportions. A monoclonal antibody arises from the growth of a single cell clone, such as a hybridoma, and is distinguished by one class and subclass of heavy chains and one type of light chains.

The expression antigen-binding fragment of an antibody is intended to indicate any

- 6 042821 peptide, polypeptide or protein that retains the ability to bind to the target (also commonly referred to as an antigen) of the specified antibody, usually with the same epitope, and contains an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues , at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues or at least 200 contiguous amino acid residues of an antibody amino acid sequence.

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

Antigen binding fragments may be selected, without limitation, from the group consisting of Fv, scFv (sc stands for single chain), Fab, F(ab') ₂ , Fab', scFv-Fc, or diabodies, or fusion proteins with disordered peptides such as as XTEN (extended recombinant polypeptide) or PAS motifs, or any fragment whose half-life would be increased by chemical modification such as the addition of a poly(alkylene) glycol such as poly(ethylene) glycol (PEGylation) (pegylated fragments are called Fv-PEG, scFv-PEG, Fab-PEG, F(ab') ₂ -PEG or Fab'-PEG) (PEG is polyethylene glycol), or by incorporation into a liposome, said fragments having at least one of the characteristic CDRs of an antibody of the invention. Preferably, said antigen-binding fragments will be or will consist of a partial sequence of the heavy or light chain variable region of the antibody from which they are derived, said partial sequence being sufficient to retain the same binding specificity as the antibody from which it is derived and sufficient affinity, preferably at least equal to 1/100, more preferably at least - 1/10 of the affinity of the antibody from which it is derived, in relation to the target.

In another specific embodiment, in the method for diagnosing lung cancer according to the invention, a biological sample from a subject is brought into contact with an antibody that binds to progastrin, where the specified antibody was obtained by an immunization method known to a person skilled in the art, where a peptide is used as an immunogen, the amino acid sequence of which contains all or part of the progastrin amino acid sequence. More specifically, said immunogen comprises a peptide selected from a peptide whose amino acid sequence contains or consists of the amino acid sequence of full length progastrin and in particular full length human progastrin of SEQ ID NO: 1, a peptide whose amino acid sequence corresponds to a portion of the amino acid sequence of progastrin and, in in particular full-length human progastrin SEQ ID NO: 1, a peptide whose amino acid sequence corresponds to part or all of the amino acid sequence of the N-terminal part of progastrin, and in particular peptides containing or consisting of the amino acid sequence: SWKPRSQQPDAPLG (SEQ ID NO: 2), and a peptide whose amino acid sequence corresponds to part or all of the amino acid sequence of the C-terminal portion of progastrin and, in particular, peptides containing or consisting of the amino acid sequence: QGPWLEEEEEAYGWMDFGRRSAEDEN (SEQ ID NO: 3), a peptide whose amino acid sequence corresponds to part of the amino acid sequence C -terminal part of progastrin and, in particular, peptides containing the amino acid sequence FGRRSAEDEN (SEQ ID NO: 40) corresponding to amino acids 71-80 of progastrin.

The specialist will understand that such immunization can be used to obtain either polyclonal or monoclonal antibodies, depending on what is desired. Methods for obtaining each of these types of antibodies are well known in this field. Thus, one skilled in the art will readily select and use a method for producing polyclonal and/or monoclonal antibodies against any given antigen.

Examples of monoclonal antibodies that have been generated using an immunogen containing the amino acid sequence SWKPRSQQPDAPLG corresponding to amino acid sequence 1-14 of human progastrin (N-terminus) include the monoclonal antibodies designated mAb3, mAb4, mAb16, mAb19, and mAb20 as described in next

- 7 042821 tab. 1-4, but are not limited to them. Other monoclonal antibodies have been described, although it is not clear whether these antibodies actually bind to progastrin (WO 2006/032980). Experimental epitope mapping results show that mAb3, mAb4, mAb16, mAb19, and mAb20 indeed bind specifically to an epitope within the indicated N-terminal amino acid sequence of hPG. Polyclonal antibodies specifically recognizing an epitope within the N-terminus of progastrin represented by SEQ ID NO: 2 have been described in the art (see, for example, WO 2011/083088).

Table 1

Deposition of hybridoma mAb Amino acid sequences SEQID NO 6V5V11S10 tal3 CDR 1 VH GYIFTSYW SEQID NO 4 CDR 2 VH FYPGNSDS SEQID NO 5 CDR3VH TRRDSPQY SEQ ID NO 6 CDR 1 VL QSIVHSNGNTY SEQ ID NO 7 CDR2 VL KVS SEQID NO 8 CDR3VL FQGSHVPFT SEQ ID NO 9 mVH 3 EVQLQQSGTVLARPGASVKMS CKASGYIFTSYWVHWVKQRPG QGLEWIGGFYPGNSDSRYNQ KFKGKATLTAVTSASTAYMDLS SLTNEDSAVYFCTRRDSPQYW GQGTTLTVSS SEQ ID NO 41 mvl 3 DVLMTQTPLSLPVSLGDQASIS CRSSQSIVHSNGNTYLEWYLQ KPGQSPKLLIYKVSNRFSGVPD RFSGSGSGTDFTLKISRLEAED LGVYYCFQGSHVPFTFGGGTK SEQ ID NO 42 huVH 3 QVQLVQSGAEVKKPGASVKVS CKASGYI FTSYWVHWVRQAPG QRLEWMGGFYPGNSDSRYSQ KFQGRVTITRDTSASTAYMELS SLRSEDTAVYYCTRRDSPQYW GQGTLVTVSS SEQ ID NO 53 huVL 3 DWMTQSPLSLPVTLGQPASIS CRSSQSIVHSNGNTYLEWFQQ RPGQSPRRLIYKVSNRFSGVP DRFSGSGSGTDFTLKISRVEAE DVGVYYCFQGSHVPFTFGGGT KVEIK SEQ ID NO 54

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table 2

20D2C3G2 taL4 CDR 1 VH GYTFSSW SEQID NO 10 CDR 2 VH FLPGSGST SEQ ID NO 11 CDR3VH ATDGNYDWFAY SEQ ID NO 12 CDR 1 VL QSLVHSSGVTY SEQ ID NO 13 CDR2 VL KVS SEQ ID NO 14 CDR 3 VL SQSTHVPPT SEQ ID NO 15 mvh 4 QVQLQQSGAELMKPGASVKIS CKATGYTFSSSWIEWLKQRPG HGLEWIGEFLPGSGSTDYNEK FKGKATFTADTSSDTAYMLLSS LTSEDSAVYYCATDGNYDWFA YWGQGTLVTVSA SEQ ID NO 43 mvl 4 DLVMTQTPLSLPVSLGDQASIS CRSSQSLVHSSGVTYLHWYLQ KPGQSPKLLIYKVSNRFSGVPD RFSGSGSGTDFTLKISRVEAED LGVYFCSQSTHVPPTFGSGTK LEIK SEQ ID NO 44 huVH 4 QVQLVQSGAEVKKPGASVKVS CKASGYTFSSSWMHWVRQAP GQGLEWMGIFLPGSGSTDYAQ KFQGRVTMTRDTSTSTVYMEL SSLRSEDTAVYYCATDGNYDW FAYWGQGTLVTVSS SEQ ID NO 55 huVL4 DIVMTQTPLSLSVTPGQPASIS CKSSQSLVHSSGVTYLYWYLQ KPGQSPQLLIYKVSNRFSGVP DRFSGSGSGTDFTLKISRVEAE DVGVYYCSQSTHVPPTFGQGT KLEIK SEQ ID NO 56

Table 3

Deposition of hybridoma mAb Amino acid sequences SEQID NO 1E9D9B6 taL16 CDR 1 VH GYTFTSYY SEQ ID NO 16 CDR 2 VH INPSNGGT SEQ ID NO 17 CDR 3 VH TRGGYYPFDY SEQ ID NO 18 CDR 1 VL QSLLDSDGKTY SEQ ID NO 19 CDR2 VL LVS SEQID NO 20 CDR3VL WQGTHSPYT SEQ ID NO 21 mvh 16 QVQLQQSGAELVKPGASVKLS CKASGYTFTSYYMYWVKQRP GQGLEWIGEINPSNGGTNFNE KFKSKATLTVDKSSSTAYMQLS SLTSEDSAVYYCTRGGYYPFD YWGQGTTLTVSS SEQ ID NO 45 mvl 16 DWMTQTPLTLSVTIGRPASIS CKSSQSLLDSDGKTYLYWLLQ RPGQSPKRLIYLVSELDSGVPD RITGSGSGTDFTLKISRVEAED LGVYYCWQGTHSPYTFGGGT KLEIK SEQ ID NO 46 huVH 16a QVQLVQSGAEVKKPGASVKVS CKASGYTFTSYYMYWVRQAP GQGLEWMGIINPSNGGTSYAQ KFQGRVTMTRDTSTSTVYMEL SSLRSEDTAVYYCTRGGYYPF DYWGQGTTVTVSS SEQ ID NO 57 huVH 16b QVQLVQSGAEVKKPGASVKVS CKASGYTFTSYYMHWVRQAP GQGLEWMGIINPSNGGTSYAQ KFQGRVTMTRDTSTSTVYMEL SSLRSEDTAVYYCTRGGYYPF SEQ ID NO 58

- 9 042821

DYWGQGTTVTVSS huVH 16s QVQLVQSGAEVKKPGASVKVS CKASGYTFTSYYMYWVRQAP GQGLEWMGEINPSNGGTNYA QKFQGRVTMTRDTSTSTVYME LSSLRSEDTAVYYCTRGGYYP FDYWGQGTTVTVSS SEQ ID NO 59 huVL 16a DWMTQSPLSLPVTLGQPASIS CRSSQSLLDSDGKTYLYWFQQ RPGQSPRRLIYLVSNRDSGVP DRFSGSGSGTDFTLKISRVEAE DVGVYYCWQGTHSPYTFGQG TKLEIK SEQ ID NO 60 huVL 16b DWMTQSPLSLPVTLGQPASIS CRSSQSLLDSDGKTYLNWFQQ RPGQSPRRLIYLVSNRDSGVP DRFSGSGSGTDFTLKISRVEAE DVGVYYCWQGTHSPYTFGQG TKLEIK SEQ ID NO 61 huVL 16c DWMTQSPLSLPVTLGQPASIS CRSSQSLLDSDGKTYLYWFQQ RPGQSPRRLIYLVSERDSGVP DRFSGSGSGTDFTLKISRVEAE DVGVYYCWQGTHSPYTFGQG TKLEIK SEQ ID NO 62

Table 4

Deposition of hybridoma mAb Amino acid sequences SEQID NO 1B3B4F11 mAb19 CDR 1 VH GYSITSDYA SEQ ID NO 22 CDR 2 VH ISFSGYT SEQ ID NO 23 CDR3VH AREVNYGDSYHFDY SEQ ID NO 24 CDR 1 VL SQHRTYT SEQ ID NO 25 CDR2 VL VKKDGSH SEQ ID NO 26 CDR3VL GVGDAIKGQSVFV SEQ ID NO 27 mvh 19 DVQLQESGPGLVKPSQSLSLT CTVTGYSITSDYAWNWIRQFP GNKLEWMGYISFSGYTSYNPS LKSRISVTRDTSRNQFFLQLTS VTTEDTATYYCAREVNYGDSY HFDYWGQGTIVTVSS SEQ ID NO 47 mvl 19 QLALTQSSSASFSLGASAKLTC TLSSQHRTYTIEWYQQQSLKP PKYVMEVKKDGSHSTGHGIPD RFSGSSSGADRYLSISNIQPED EAIYICGVGDAIKGQSVFVFGG GTKVTVL SEQ ID NO 48 huVH 19a QVQLQESGPGLVKPSQTLSLT CTVSGYSITSDYAWNWIRQHP GKGLEWIGYISFSGYTYYNPSL KSRVTISVDTSKNQFSLKLSSV TAADTAVYYCAREVNYGDSYH FDYWGQGTLVTVSS SEQ ID NO 63 huVH 19b QVQLQESGPGLVKPSQTLSLT CTVSGYSITSDYAWSWIRQHP GKGLEWIGYISFSGYTYYNPSL KSRVTISVDTSKNQFSLKLSSV TAADTAVYYCAREVNYGDSYH FDYWGQGTLVTVSS SEQ ID NO 64 huVH 19c QVQLQESGPGLVKPSQTLSLT CTVSGYSITSDYAWNWIRQHP GKGLEWIGYISFSGYTSYNPSL KSRVTISVDTSKNQFSLKLSSV TAADTAVYYCAREVNYGDSYH FDYWGQGTLVTVSS SEQ ID NO 65 huVL 19a QLVLTQSPSASASLGASVKLTC TLSSQHRTYTIEWHQQQPEKG PRYLMKVKKDGSHSKGDGIPD SEQ ID NO 66

- 10 042821

RFSGSSSGAERYLTISSLQSED EADYYCGVGDAIKGQSVFVFG GGTKVEIK huVL 19b QLVLTQSPSASASLGASVKLTC TLSSQHRTYTIAWHQQQPEKG PRYLMKVKKDGSHSKGDGIPD RFSGSSSGAERYLTISSLQSED EADYYCGVGDAIKGQSVFVFG GGTKVEIK SEQ ID NO 67 huVL 19s QLVLTQSPSASASLGASVKLTC TLSSQHRTYTIEWHQQQPEKG PRYLMEVKKDGSHSKGDGIPD RFSGSSSGAERYLTISSLQSED EADYYCGVGDAIKGQSVFVFG GGTKVEIK SEQ ID NO 68

Examples of monoclonal antibodies that can be obtained using an immunogen containing the amino acid sequence QGPWLEEEEEAYGWMDFGRRSAEDEN (C-terminal part of progastrin) corresponding to the amino acid sequence 55-80 of human progastrin include the antibodies indicated in the following table. 5 and 6 as but not limited to mAb8 and mAb13. The experimental results of epitope mapping show that mAb13 indeed specifically binds to an epitope within the indicated C-terminal amino acid sequence of hPG. Another example of a monoclinal antibody that can be obtained in this way is the mAb14 antibody produced by the 2H9F4B7 hybridoma described in WO 2011/083088. Hybridoma 2H9F4B7 was deposited under the Budapest Agreement at CNCM, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris CEDEX 15, France on December 27, 2016, according to reference I-5158 (see WO 2017/114973).

Table 5

Deposition of hybridoma mAb Amino acid sequences SEQID NO validity 1C10D3B9 mAb8 CDR 1 VH GFTFTTYA SEQ ID NO 28 CDR 2 VH ISSGGTYT SEQ ID NO 29 CDR3VH ATQGNYSLDF SEQID NO 30 CDR 1 VL KSLRHTKGITF SEQ ID NO 31 CDR2 VL QMS SEQ ID NO 32 CDR3VL AQNLELPLT SEQ ID NO 33 mVH 8 EVQLVESGGGLVKPGGSLRL SCAASGFTFTTYAMSWVRQA PGKGLEWVATISSGGTYTYY ADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCATQGN YSLDFWGQGTTVTVSS SEQ ID NO 49 mvl 8 DIVMTQSPLSLPVTPGEPASI SCRSSKSLRHTKGITFLYWYL QKPGQSPQLLIYQMSNLASG VPDRFSSSGSGTDFTLKISRV EAEDVGVYYCAQNLELPLTF GGGTKVEIK SEQ ID NO 50 VH hZ8CV1 EVQLVESGGGLVKPGGSLRL SCAASGFTFTTYAMSWVRQA PGKGLEWVSSISSGGTYTYY ADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCATQGN YSLDFWGQGTTVTVSS SEQ ID NO 69 VL hZ8CV1 DIVMTQSPLSLPVTPGEPASI SCRSSKSLRHTKGITFLYWYL QKPGQSPQLLIYQMSNRASG VPDRFSGSGSGTDFTLKISRV EAEDVGVYYCAQNLELPLTF GGGTKVEIK SEQ ID NO 70 VH hZ8CV2 EVQLVESGGGLVKPGGSLRL SCAASGFTFTTYAMSWVRQA PGKGLEWVATISSGGTYTYY ADSVKGRFTISRDNAKNSLYL SEQ ID NO 71

- 11 042821

QMNSLRAEDTAVYYCATQGN YSLDFWGQGTTVTVSS VL hZ8CV2 DIVMTQSPLSLPVTPGEPASI SCRSSKSLRHTKGITFLYWYL QKPGQSPQLLIYQMSNLASG VPDRFSSSGSGTDFTLKISRV EAEDVGVYYCAQNLELPLTF GGGTKVEIK SEQ ID NO 72 CH hZ8CV2 EVQLVESGGGLVKPGGSLRL SCAASGFTFTTYAMSWVRQA PGKGLEWVATISSGGTYTYY ADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCATQGN YSLDFWGQGTTVTVSSASTK GPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCWVD VSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWS VLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSP GK SEQ ID NO 73 CL hZ8CV2 DIVMTQSPLSLPVTPGEPASI SCRSSKSLRHTKGITFLYWYL QKPGQSPQLLIYQMSNLASG VPDRFSSSGSGTDFTLKISRV SEQ ID NO 74 EAEDVGVYYCAQNLELPLTF GGGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASWCLLNNF YPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC

Table 6

Deposition of hybridoma mAb Amino acid sequences SEQID NO 2C6C3C7 TAYZ CDR 1 VH GFIFSSYG SEQ ID NO 34 CDR 2 VH INTFGDRT SEQ ID NO 35 CDR 3 VH ARGTGTY SEQ ID NO 36 CDR 1 VL QSLLDSDGKTY SEQ ID NO 37 CDR2 VL LVS SEQ ID NO 38 CDR3VL WQGTHFPQT SEQ ID NO 39 mvh 13 EVQLVESGGGLVQPGGSLKL SCAASGFIFSSYGMSWVRQS PDRRLELVASINTFGDRTYYP DSVKGRFTISRDNAKNTLYLQ MTSLKSEDTAIYYCARGTGTY WGQGTTLTVSS SEQ ID NO 51 mvl 13 DVVLTQTPLTLSVTIGQPASIS CKSSQSLLDSDGKTYLNWLL QRPGQSPKRLIYLVSKLDSGV PDRFTGSGSGTDFTLKISRVE AEDLGVYYCWQGTHFPQTF GGGTKLEIK SEQ ID NO 52 huVH 13a EVQLVESGGGLVQPGGSLRL SCAASGFIFSSYGMSWVRQA PGKGLEWVANINTFGDRTYY VDSVKGRFTISRDNAKNSLYL SEQ ID NO 75

- 12042821

QMNSLRAEDTAVYYCARGTG TYWGQGTLVTVSS huVH 13b EVQLVESGGGLVQPGGSLRL SCAASGFIFSSYGMSWVRQA PGKGLEWVASINTFGDRTYY VDSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARGTG TYWGQGTLVTVSS SEQ ID NO 76 huVL 13a DVVMTQSPLSLPVTLGQPASI SCRSSQSLLDSDGKTYLNWF QQRPGQSPRRLIYLVSNRDS GVPDRFSGSGSGTDFTLKIS RVEAEDVGVYYCWQGTHFP QTFGGGTKVEIK SEQ ID NO 77 huVL 13b DVVMTQSPLSLPVTLGQPASI SCRSSQSLLDSDGKTYLNWF QQRPGQSPRRLIYLVSKRDS GVPDRFSGSGSGTDFTLKIS RVEAEDVGVYYCWQGTHFP QTFGGGTKVEIK SEQ ID NO 78

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

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

The terms anti-hPG N-terminal antibody and anti-hPG C-terminal antibody refer to antibodies that bind to an amino acid-containing epitope located at the N-terminal portion of hPG or an amino acid-containing epitope located at the C-terminal portion of hPG, respectively. Preferably, the term antibodies against the N-terminus of hPG refers to antibodies that bind to an epitope located in the progastrin domain, the sequence of which is shown by SEQ ID NO: 2. In another preferred embodiment, the term antibodies against the C-terminus of hPG refers to antibodies that bind to an epitope located in the progastrin domain, the sequence of which is presented by SEQ ID NO: 3.

The term epitope refers to the region of an antigen to which an antibody binds. Epitopes can be defined as structural or functional. Functional epitopes are usually a subset of structural epitopes, and they contain those amino acids that directly contribute to the affinity of the interaction. Epitopes can also be conformational. In some embodiments, epitopes may include determinants that are reactive surface groupings of molecules, such as amino acids, side sugar chains, phosphoryl groups, or sulfonyl groups, and in some embodiments they may have specific three-dimensional structural characteristics and/or specific charge characteristics. Determination of an epitope bound by an antibody can be performed by any epitope mapping technique known to one of skill in the art. An epitope may contain different amino acids that are consecutive within the amino acid sequence of a protein. An epitope may also contain amino acids that are not sequential within the amino acid sequence of the protein.

In a specific embodiment, said antibody is a monoclonal antibody selected from the group consisting of the following:

a monoclonal antibody containing a heavy chain containing at least one, preferably at least two, preferably three of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively, or sequences with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NO: 4, 5 and 6, respectively, and a light chain, containing at least one, preferably at least two, preferably three of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NO: 7, 8 and 9, respectively, or sequences with at least 80% , preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NO: 7, 8 and 9, respectively, a monoclonal antibody containing a heavy chain containing at least one, preferably at least two, preferably three of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively, or sequences with at least 80%, preferably 85% -th, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NO: 10, 11 and 12, respectively, and easy

- 13 042821 chain containing at least one, preferably at least two, preferably three of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NO: 13, 14 and 15, respectively, or sequences with at least at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NO:

13, 14 and 15, respectively, a monoclonal antibody containing a heavy chain containing at least one, preferably at least two, preferably three of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOS: 16, 17 and 18, respectively, or sequences with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NOS: 16, 17 and 18, respectively, and a light chain containing at least one, preferably at least two, preferably three of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOS: 19, 20 and 21, respectively, or sequences with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NO: 19, 20 and 21, respectively, a monoclonal antibody containing a heavy chain containing at least one, preferably at least two, preferably three of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 22, 23 and 24, respectively, or sequences with at least 80 %, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NO: 22, 23 and 24, respectively, and a light chain containing at least one, preferably at least two, preferably three of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 25, 26 and 27, respectively, or sequences with at least 80%, preferably 85% 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NO: 25, 26 and 27, respectively, a monoclonal antibody containing a heavy chain containing at least one, preferably at least at least two, preferably at least three of the CDR-H1, CDR-H2 and CDRN3 amino acid sequences of SEQ ID NOs: 28, 29 and 30, respectively, or sequences with at least 80%, preferably 85%, 90 %, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NOS: 28, 29 and 30, respectively, and a light chain containing at least one, preferably at least two, preferably three of CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 31, 32 and 33, respectively, or sequences with at least 80%, preferably 85%, 90%, 95% identity and 98% identity after optimal alignment with the sequences of SEQ ID NOS: 31, 32 and 33, respectively, and a monoclonal antibody containing a heavy chain containing at least one, preferably at least two, preferably three of CDR-H1 , CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 34, 35 and 36, respectively, or sequences with at least 80%, preferably 85%, 90%, 95% and 98 % identity after optimal alignment with SEQ ID NOs: 34, 35 and 36, respectively, and a light chain containing at least one, preferably at least two, preferably three of CDR-L1, CDR-L2 and CDR- L3 amino acid sequences of SEQ ID NOs: 37, 38 and 39, respectively, or sequences with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with sequences of SEQ ID NO: 37, 38 and 39, respectively.

In another embodiment, the antibody is a monoclonal antibody produced by a hybridoma deposited with CNCM, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris CEDEX 15, France, December 27, 2016, according to reference I-5158 (see WO 2017 /114973).

In the meaning of the present invention, percentage identity or % identity between two nucleic acid or amino acid sequences means the percentage of identical nucleotides or amino acid residues between two sequences to be compared, obtained after optimal alignment, this percentage being purely statistical and the difference between the two data sequences are randomly distributed along their length. The comparison of two nucleic acid or amino acid sequences is traditionally carried out by comparing the sequences after their optimal alignment, and this comparison can be carried out in segments or using an alignment window. Optimal alignment of sequences for comparison can be carried out, in addition to manual comparison, by methods known to the person skilled in the art.

For an amino acid sequence showing at least 80%, preferably 85%, 90%, 95% and 98% identity with the control amino acid sequence, preferred examples include sequences containing a reference sequence, certain modifications , namely: deletion, addition or replacement by

- 14 042821 at least one amino acid, truncation or extension. In the case of substitution of one or more consecutive or non-sequential amino acids, substitutions are preferred in which the replaced amino acids are replaced with equivalent amino acids. It is understood here that the expression equivalent amino acids indicates any amino acids that are likely to be replaced by one of the structural amino acids, however, without modifying the biological activities of the respective antibodies, and these specific examples are defined below.

Equivalent amino acids can be determined either by their structural homology with the amino acids they replace, or by comparisons of biological activity between different antibodies that are likely to be generated.

In a more specific embodiment, said antibody is a monoclonal antibody selected from the group consisting of the following:

a monoclonal antibody containing the heavy chain of the amino acid sequence of SEQ ID NO: 41 and the light chain of the amino acid sequence of SEQ ID NO: 42;

a monoclonal antibody containing the heavy chain of the amino acid sequence of SEQ ID NO: 43 and the light chain of the amino acid sequence of SEQ ID NO: 44;

a monoclonal antibody containing the heavy chain of the amino acid sequence of SEQ ID NO: 45 and the light chain of the amino acid sequence of SEQ ID NO: 46;

a monoclonal antibody containing the heavy chain of the amino acid sequence of SEQ ID NO: 47 and the light chain of the amino acid sequence of SEQ ID NO: 48;

a monoclonal antibody containing the heavy chain of the amino acid sequence of SEQ ID NO: 49 and the light chain of the amino acid sequence of SEQ ID NO: 50; and a monoclonal antibody comprising the heavy chain of the amino acid sequence of SEQ ID NO: 51 and the light chain of the amino acid sequence of SEQ ID NO: 52.

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

The expression humanized antibody, as used herein, means an antibody that contains CDR regions derived from a non-human antibody, with other portions of that antibody molecule derived from one or more human antibodies. In addition, some of the residues of the skeletal segments (referred to as FR, which stands for frame) can be modified to maintain binding affinity according to techniques known to the person skilled in the art (Jones et al, Nature, 321:522-525, 1986). The goal of humanization is to reduce the immunogenicity of a xenogeneic antibody, such as a mouse antibody, for administration to a human while maintaining the full antigen binding affinity and specificity of the antibody.

Humanized antibodies of the invention, or fragments thereof, can be prepared by techniques known to those skilled in the art (such as, for example, those described in Singer et al., J. Immun., 150:2844-2857, 1992). Such humanized antibodies are preferred for use in methods involving in vitro diagnostics or prophylactic and/or therapeutic treatment in vivo. Other humanization techniques are also known to the person skilled in the art. Indeed, antibodies can be humanized using a variety of techniques, including CDR grafting (EP 0451261; EP 0682040; EP 0939127; EP 0566647; US 5530101; US 6180370; US 5585089; US 5693761; US 5639641; US 6054297; and US 5877293), masking of surface residues (venation) or surface modification (EP 0592106; EP 0519596; Padlan E. A., 1991, Molecular Immunology 28(4/5): 489-498; Studnicka G. M. et al., 1994, Protein Engineering 7(6): 805814; Roguska M.A. et al., 1994, Proc. Natl. Acad. ScL U.S.A., 91:969-973) and strand shuffling (US Pat. No. 5,565,332). Human antibodies can be generated by a variety of methods known in the art, including phage display methods. See also US patent No. 4444887, 4716111, 5545806 and 5814318; and international patent applications with publication numbers WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741.

In a more specific embodiment, said antibody is a humanized antibody selected from the group consisting of the following:

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

- 15 042821 respectfully at least two, preferably three of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively, or sequences with at least 80%, preferably 85 %, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NOS: 10, 11 and 12, respectively, and a light chain containing at least one, preferably at least two, preferably three of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 13, 14 and 15, respectively, or sequences with at least 80%, preferably 85%, 90% 95% and 98% identity after optimal alignment with the sequences of SEQ ID NOS: 13, 14 and 15, respectively, a humanized antibody containing a heavy chain containing at least one, preferably at least two, preferably three of CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 16, 17 and 18, respectively, or sequences with at least 80%, preferably 85%, 90%, 95% identical and 98% identity after optimal alignment with the sequences of SEQ ID NOS: 16, 17 and 18, respectively, and a light chain containing at least one, preferably at least two, preferably three of CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 19, 20 and 21, respectively, or sequences with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NOS: 19, 20 and 21, respectively, a humanized antibody containing a heavy chain containing at least one, preferably at least two, preferably three of CDR-H1, CDR-H2 and CDR-H3 amino acids sequences of SEQ ID NO: 22, 23 and 24, respectively, or sequences with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with sequences SEQ ID NOs: 22, 23 and 24, respectively, and a light chain containing at least one, preferably at least two, preferably three of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NO: 25, 26 and 27, respectively, or sequences with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NOS: 25, 26 and 27, respectively, a humanized heavy chain antibody comprising at least one, preferably at least two, preferably three of the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of SEQ ID NOS: 28, 29, and 30, respectively. , or sequences with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NOS: 28, 29 and 30, respectively, and a light chain containing at least one, preferably at least two, preferably three of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 31, 32 and 33, respectively, or sequences with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NOS: 31, 32 and 33, respectively, and a humanized antibody containing a heavy chain, containing at least one, preferably at least two, preferably three of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 34, 35 and 36, respectively, or sequences with at least 80% , preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NO: 34, 35 and 36, respectively, and a light chain containing at least one, preferably at least two, preferably three of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 37, 38 and 39, respectively, or sequences with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NOS: 37, 38 and 39, respectively, where the specified antibody also contains constant regions of the light chain and heavy chain derived from a human antibody.

In another more specific embodiment, said antibody is a humanized antibody selected from the group consisting of the following:

a humanized antibody comprising the heavy chain variable region of the amino acid sequence of SEQ ID NO: 53 and the light chain variable region of the amino acid sequence of SEQ ID NO: 54;

a humanized antibody comprising the heavy chain variable region of the amino acid sequence of SEQ ID NO: 55 and the light chain variable region of the amino acid sequence of SEQ ID NO: 56;

a humanized antibody comprising a heavy chain variable region of an amino acid sequence selected between SEQ ID NOs: 57, 58 and 59 and a light chain variable region of an amino acid sequence selected between SEQ ID NOs: 60, 61 and 62;

a humanized antibody comprising a heavy chain variable region of an amino acid sequence selected between SEQ ID NOs: 63, 64 and 65 and a light chain variable region of an amino acid sequence selected between SEQ ID NOs: 66, 67 and 68;

a humanized antibody comprising a heavy chain variable region of an amino acid sequence selected between SEQ ID NOs: 69 and 71 and a light chain variable region of an amino acid sequence selected between SEQ ID NOs: 70 and 72; and a humanized antibody comprising a heavy chain variable region of an amino acid sequence selected between SEQ ID NOs: 75 and 76 and a light chain variable region of an amino acid sequence selected between SEQ ID NOs: 77 and 78;

wherein said antibody also contains light chain and heavy chain constant regions derived from a human antibody.

In a first embodiment, the method of the invention comprises contacting a biological sample with an anti-hPG antibody that binds to an hPG epitope, where said epitope is located within the C-terminal portion of hPG, or an epitope located within the N-terminal portion of hPG.

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

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

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

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

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

In a specific embodiment of the method for diagnosing lung cancer in vitro according to the invention, said method comprises the step of contacting a biological sample from a subject with a first molecule that binds to a first portion of progastrin and a second molecule that binds to a second portion of progastrin. In a more specific embodiment, wherein said progastrin-binding molecule is an antibody, a biological sample from the subject is contacted with an antibody that binds to a first progastrin epitope and a second antibody that binds to a second progastrin epitope.

In a specific embodiment of the method of the invention, said method includes the step of contacting a biological sample from a subject with a first agent that binds to a first portion of progastrin and a second agent that binds to a second portion of progastrin. In a more specific embodiment, wherein said progastrin-binding molecule is an antibody, a biological sample from the subject is contacted with an antibody that binds to a first progastrin epitope and a second antibody that binds to a second progastrin epitope.

According to a preferred embodiment, said first antibody is bound to an insoluble or partially soluble carrier. Binding of progastrin by said first antibody results in capture of progastrin from said biological sample. Preferably, said first antibody is an antibody that binds to an hPG epitope, wherein said epitope comprises an amino acid sequence corresponding to the amino acid sequence of the C-terminal portion of progastrin, as described above. More preferably said first antibody

- 17 042821 is a mAb14 monoclonal antibody produced by 2H9F4B7 hybridoma described in WO 2011/083088. Hybridoma 2H9F4B7 was deposited under the Budapest Agreement in

CNCM, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris CEDEX 15, France, December 27

2016 according to reference I-5158 (see WO 2017/114973).

In another preferred embodiment, said second antibody is labeled with a detectable moiety as described below. Binding of progastrin by the second antibody allows detection of progastrin molecules that were present in the biological sample. In addition, the binding of progastrin by the second antibody provides a quantitative measurement of the progastrin molecules that were present in the biological sample. Preferably, said second antibody is an antibody that binds to an hPG epitope, wherein said epitope comprises an amino acid sequence corresponding to the amino acid sequence of the N-terminal portion of progastrin, as described above. More preferably, said N-terminal antibody is a polyclonal antibody as described above. Alternatively, it is also possible to use a monoclonal antibody that binds to an epitope within the N-terminus of progastrin, such as, for example, the N-terminal monoclonal antibodies described above, namely: a monoclonal antibody containing a heavy chain containing CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequences of SEQ ID NOS: 16, 17 and 18, respectively, and a light chain containing CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequences of SEQ ID NOS: 19, 20 and 21.

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

In a preferred embodiment, the method of the present invention for diagnosing lung cancer comprises detecting progastrin in a biological sample from a human subject.

In a more preferred embodiment, the method of the present invention for diagnosing lung cancer comprises determining the concentration of progastrin in a biological sample from a human subject.

In another specific embodiment, the method of the present invention for diagnosing lung cancer includes determining the concentration of progastrin in a biological sample from a human subject, where the specified biological sample is selected from blood, serum and plasma.

In another preferred embodiment, the method of the present invention comprises contacting a sample from said subject with an anti-hPG antibody as described above, wherein binding of said anti-hPG antibody in the sample is indicative of said subject having lung cancer.

In a more specific embodiment, the method of the present invention comprises contacting a sample from said subject with an anti-hPG antibody as described above, wherein said plasma concentration of progastrin greater than 10 pM is indicative of said subject having lung cancer.

More preferably, the method of the present invention comprises contacting a sample from said subject with an anti-hPG antibody as described above, wherein a progastrin concentration greater than 10 pM, 20 pM, 30 pM, or 40 pM in said sample is indicative of the presence of lung cancer in said subject.

Even more preferably, the method of the present invention comprises contacting a sample from said subject with an anti-hPG antibody as described above, wherein the progastrin concentration is greater than 10 pM, preferably greater than 20 pM, more preferably greater than 30 pM, even more preferably greater than 40 pM, even more preferably, greater than 50 pM in said sample indicates the presence of metastatic lung cancer in said subject.

The present invention also relates to methods for monitoring the effectiveness of a lung cancer treatment in a patient, such as chemotherapy, biological therapy, immunotherapy, or antibody therapy, by determining the concentration of progastrin in a first sample, such as a body fluid or a lung cancer biopsy obtained from the patient prior to lung cancer treatment. , and then comparing the concentration of progastrin in the first sample with the concentration in the second sample obtained from the same patient after treatment, where the decrease in the concentration of progastrin in said second sample compared to said first sample indicates that the treatment was effective.

In a specific embodiment, the method of the invention comprises comparing the concentration of progastrin in a biological sample obtained from a patient with a target value for the concentration of progastrin in the sample, in a more specific embodiment, said target value is selected from: the average or average of the sample based on the average or average determination of the value in the population without lung cancer, progastrin concentration values obtained when the patient was known to be free of lung cancer.

In a specific embodiment, the method of the invention for in vitro diagnosis of lung cancer comprises determining the concentration of progastrin in a sample from said patient and a second diagnostic assay for lung cancer. In a more specific embodiment, the method of the invention for in vitro diagnosis of lung cancer comprises determining the concentration of progastrin in a sample from said patient and a second lung cancer diagnostic assay, wherein said second diagnostic assay comprises detecting a specific biomarker selected from the following: carcinoembryonic antigen (CEA),

- 18 042821 neuron specific enolase (NSE), cytokeratin 19 (CYFRA-21-1), alpha-fetoprotein, carbohydrate antigen-125 (CA-125), carbohydrate antigen-19.9 (CA-19.9) and ferritin, alone or in combination ( Li et al, 2012).

In a specific embodiment of the invention, the method of the present invention includes determining the level of progastrin over time in samples from a patient who has been treated or is being treated for lung cancer.

The characteristics of the embodiments of the present invention will become clearer from the following detailed description of the examples below.

Graphics Legends

In Figure 1, progastrin concentration was measured in 40 plasma samples from lung cancer patients and 119 plasma samples from healthy donors using the DECODE Lab ELISA kit (capture antibody: mAb14, detection antibody: polyclonal against hPG).

Examples

Example 1 Determination of plasma concentration of progastrin using polyclonal antibodies.

Plasma progastrin levels were quantified by ELISA using two specific anti-progastrin antibodies: capture antibodies coat the wells of the plate, while detection antibodies are used to detect progastrin and mediate signal detection.

In the present example, the quantitative measurement is based on the ELISA method, which provides, through the use of a substrate whose reaction emits light, attributing a value proportional to luminescence to the amount of antibodies bound to the antigen retained by the capture antibodies.

Material.

Reagents and instrument are listed in Table. 7.

Designation Salesman Link MaxiSORP Nunc white plates, 96 wells Dutscher #055221 Sodium carbonate/bicarbonate Sigma #21851 DPBS 1x Lonza # P04-36500 Tween-20 Biosolve #20452335 BSA Euromedex #04-100-810-C Streptavidin-HPP (horseradish peroxidase) Pierce (Thermo) #21130 Substrate for maximum sensitivity SuperSignal ELISA Femto Pierce (Thermo) #37074 Polyclonal antibody against progastrin Eurogentec /

Table 7

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

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

96-well plates are coated with a 50 mM sodium carbonate-bicarbonate pH 9.6 solution by dissolving the contents of one capsule in 100 ml MilliQ water. A solution of a capture antibody (3 μg/ml) corresponding to polyclonal antibodies prepared using the C-terminus of progastrin FGRRSAEDEN (SEQ ID NO: 40) is prepared in carbonate buffer. 100 μl of antibody solution is added to each well and incubated at 4° C. for 16 h (1 night). The plates are then blocked by removing the antibody solution and washing 3 times with 300 µl 1x PBS/0.1% Tween-20, then adding 200 µl blocking buffer (1x PBS/0.1% Tween-20/0, 1% BSA) per well and incubated for 2 hours at 22°C. The blocking buffer is then discarded, the wells are washed 3 times with 300 μl 1x PBS/0.1% Tween-20.

Plasma dilution is carried out as follows: plasma is used in its pure form, diluted 1/2, 1/5 and 1/10. Dilutions are made from pure plasma in 1x PBS/0.1% Tween-20/0.1% BSA.

For control analysis, ELISA is performed in the presence of a known concentration of progastrin, progastrin dilution is prepared as follows: uterine recombinant PG (full-length human progastrin produced in E. coli and affinity purified using glutathione-agarose/tag removal (Tev)/purification on the IMAC Counter/ dialysis, from the Institut Pasteur, Paris, France) are prepared at a concentration of 0.45 mg/ml (45 μM) in triplicate. Progastrin concentration intervals were obtained as follows:

solution A: pre-dilution 1/10, 2 µl stock solution plus 18 µl buffer, solution B: pre-dilution 1/100, 10 µl A plus 90 µl buffer, solution C: pre-dilution 1/1000, 10 µl B plus 90 µl buffer solution D: 500 pM, 5.55 µl C plus 494.5 µl diluent,

- 19 042821 solution E: 250 pM, 250 µl D plus 250 µl diluent, solution F: 100 pM, 200 µl E plus 300 µl diluent, solution G: 50 pM, 250 µl F plus 250 µl diluent, solution H: 25 pM , 200 µl G plus 200 µl diluent, solution I: 10 pM, 100 µl H plus 150 µl diluent.

The recombinant PG concentration range is linear and therefore can be more or less wide according to the antibody used.

To obtain prototype samples, approximately 500 µl of each sample is set aside and stored until analysis (and confirmation, if necessary) of the results. 100 μl of each interval point and/or plasma are analyzed in pure form, diluted 1/2, 1/5 and 1/10, and incubated for 2 hours at 22°C on the plates.

Wash the plates 3 times with 300 µl 1x PBS / 0.1% Tween20 to detect assay results. A solution of a polyclonal rabbit antibody against progastrin, where these antibodies were obtained using the N-terminal part of progastrin as biotin-bound immunogen, at a concentration of 0.5 μg/ml, was diluted in 1x PBS/0.1% Tween-20/0, 1% BSA. 100 µl of this solution is added to each well. Incubation takes place for 1 hour at 22°C. Detection of streptavidin-HRP is performed by removing the detecting antibody and washing 3 times with 300 µl 1x PBS/0.1% Tween-20, then obtaining a solution of streptavidin-HRP at a concentration of 20 ng/ml diluted in 1x PBS/0.1% Tween-20 /0.1% BSA, where 100 μl of this solution is added to each well before incubation for 1 h at 22°C.

Detection consists of removing streptavidin-HRP and washing 3 times with 300 µl 1x PBS/0.1% Tween-20, then adding 100 µl chemiluminescent substrate solution per well. Substrate solution is prepared by mixing equal volumes of two SuperSignal ELISA Femto kit solutions, 20 ml plus 20 ml, 30 min prior to use and stored at room temperature in the dark. Luminescence is read after 5 min incubation at room temperature in the dark.

For each condition, the assay is run in triplicate and the interval results will be presented as a graph showing the change in luminescence as a function of progastrin concentration. For each plasma dilution, the progastrin concentration is determined using a direct linear regression equation for the corresponding interval (interval 1/10th for sample diluted to 1/10th).

Methods and results.

The median plasma concentration of progastrin is 0 pM in control patients (n is 103), while in patients with lung cancer, a significant plasma concentration of progastrin can be detected. Thus, patients with lung cancer have higher levels of progastrin in their plasma compared to healthy controls.

Example 2 Progastrin Concentration Detection Using Anti-Progastrin Monoclonal Antibodies.

The wells of Nunc MaxiSORP 96-well plates are coated with the first progastrin-specific antibody as follows. Anti-progastrin monoclonal antibodies specific for the carboxy-terminal region of progastrin are diluted to a concentration of 3 μg/ml in a solution of 50 mM sodium carbonate/bicarbonate buffer, pH 9.6, in MilliQ water.

A total of 100 μl of antibody solution is then added to each well of 96-well plates and incubated overnight at 4°C. After binding, the antibody solution is removed from the wells, which are then washed three times with 100 μl of wash buffer (1 x PBS/0.1% Tween-20). Then a total of 100 μl blocking buffer (1x PBS/0.1% Tween-20/0.1% BSA) is added to each well and incubated for 2 hours at 22°C. The blocking buffer is then removed and the wells are washed three times with wash buffer. Plasma or serum samples isolated from patients are then added to the wells in a volume of 100 µl in a series of dilutions - typically 1:1, 1:2, 1:5 and 1:10 dilutions - and then incubated for 2 hours at 22°C. . Plasma or serum samples are analyzed in duplicate.

The analyzes also include two standard curves. A first standard curve was prepared using dilutions of recombinant progastrin to a final amount of 1 ng, 0.5 ng, 0.25 ng, 0.1 ng, 0.05 ng, 0.01 ng and 0 ng per well. A second standard curve, which serves as a negative control, is prepared from progastrin-negative human serum diluted in blocking buffer at the same dilutions as the test samples, ie. 1:1, 1:2, 1:5 and 1:10. Alternatively, when analyzing plasma samples, a second standard curve, which serves as a negative control, is prepared from progastrin-negative human plasma diluted in blocking buffer at the same dilutions as the test samples, i. 1:1, 1:2, 1:5 and 1:10.

After completion of incubation with plasma or serum samples, the contents of the wells are discarded and the wells are washed three times with wash buffer, 100 μl/well, after which the progastrin bound to the first antibody is detected using the second progastrin-specific antibody as follows.

- 20 042821

Biotin-bound anti-progastrin monoclonal antibodies specific for the amino-terminal region of progastrin are diluted in blocking buffer to a concentration of 0.1 to μg/ml, depending on the antibody. A total of 100 μl of antibody solution is then added to each well and incubated for 1 hour at 22°C.

After binding of the secondary antibody is complete, the plates are washed three times with wash buffer, 100 µl/well, after which 100 µl of streptavidin-HRP solution (25 ng/ml in blocking buffer) is added to each well and incubated for 1 hour at 22°C. After completion of incubation with streptavidin-HRP solution, the plates are washed three times with wash buffer, 100 μl/well. Then, 100 µl of chemiluminescent substrate prepared using the Pierce SuperSignal ELISA Femto Maximum Sensitivity Chemiluminescent Substrate Kit is added per well, incubated for 5 minutes at room temperature in the dark, and then read on a luminometer.

Based on the luminometer readings, a linear regression analysis is used to derive the equation of lines corresponding to the standard curve data. Using this equation, the concentration of progastrin in different patient samples is then calculated.

The median plasma concentration of progastrin is calculated in patients with lung cancer and compared with the median plasma concentration of progastrin in the plasma of control patients. Patients with lung cancer have elevated levels of progastrin in their plasma compared to healthy controls.

Example 3 Detection of plasma concentration of progastrin using a combination of polyclonal antibodies and monoclonal antibodies.

In the present example, plasma progastrin levels are quantified by ELISA using an antibody specific for human progastrin (hPG) coated on a 96-well plate. Standards and samples are added to the wells and any hPG present is bound to the immobilized capture antibody. The wells are washed and a horseradish peroxidase (HRP) detecting antibody against hPG is added to form an antibody-antigen sandwich. After the second wash, a TMB (tetramethylbenzidine) substrate solution is added, which gives a blue color in direct proportion to the amount of hPG present in the original sample. The stop solution turns blue to yellow and the wells are read at 450 nm using a microplate reader.

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

Monoclonal antibodies are generated using hybridomas producing antibodies against the N-terminus of progastrin (SEQ ID NO: 2) or against the C-terminus of progastrin corresponding to hPG amino acids 71-80 and having the sequence FGRRSAEDEN (SEQ ID NO: 40) according to standard protocols.

The binding characteristics of the polyclonal and monoclonal antibodies against progastrin used in this assay are as follows: no binding to G34-Gly, G34, G17Gly, G17, binding to full length progastrin.

For control analysis, ELISA is performed in the presence of a known concentration of progastrin, progastrin dilution is prepared as follows: uterine recombinant PG (full-length human progastrin produced in E. coli and affinity purified using glutathione-agarose/tag removal (Tev)/purification on the IMAC Counter/ dialysis, from the Pasteur Institute, Paris, France) are prepared at a concentration of 0.45 mg/ml (45 microM) in triplicate. Intervals of progastrin concentrations are obtained as follows:

solution A: pre-dilution 1/10, 2 µl stock solution plus 18 µl buffer, solution B: pre-dilution 1/100, 10 µl A plus 90 µl buffer, solution C: pre-dilution 1/1000, 10 µl B plus 90 µl buffer solution D: 500 pM, 5.55 µl C plus 494.5 µl diluent, solution E: 250 pM, 250 µl D plus 250 µl diluent, solution F: 100 pM, 200 µl E plus 300 µl diluent, solution G : 50 pM, 250 µl F plus 250 µl diluent, solution H: 25 pM, 200 µl G plus 200 µl diluent, solution I: 10 pM, 100 µl H plus 150 µl diluent.

The recombinant PG concentration range is linear and therefore can be more or less wide according to the antibody used.

Methods and results.

Progastrin levels are determined in plasma samples from subjects later known to have developed lung cancer. Progastrin is captured by the anti-C-terminal monoclonal antibody mAb 14 produced by the 2H9F4B7 hybridoma described in WO 2011/083088 (hybridoma

- 21 042821

2H9F4B7 deposited under the Budapest Agreement at CNCM, Pasteur Institute, 25-28 rue du

Docteur Roux, 75724 Paris CEDEX 15, France, December 27, 2016, reference I-5158). Detection is carried out with labeled polyclonal antibodies specific for the N-terminus.

The control is composed of plasma samples from the general population.

The data demonstrate that patients with lung cancer have detectable levels of progastrin in their plasma, while healthy controls do not.

Example 4 Determination of Plasma Concentration of Progastrin Using the DECODE Lab Kit.

This assay provides a measurement of plasma hPG-EDTA (ethylenediaminetetraacetic acid) by ELISA.

This kit uses an hPG-specific capture antibody that is pre-coated on a 96-well plate. hPG present in the standards and samples added to the wells binds to the immobilized capture antibody. These wells are washed and a horseradish peroxidase (HRP) conjugate of anti-hPG detecting antibody is added, resulting in the formation of an antibody-antigen-antibody complex. After the second wash, 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution is added to the well, producing a blue color in direct proportion to the amount of hPG present in the original sample. The color of the stop solution changes from blue to yellow and the wells are read at 450 nm using a microplate reader.

Methods and results.

To measure progastrin concentration, 40 plasma samples from patients with lung cancer and 119 plasma samples from healthy donors were used using the DECODE Lab ELISA kit (capture antibody: mAb14, detection antibody: polyclonal against hPG) following the manufacturer's recommendation.

Briefly.

1. Prepare all reagents, controls, and samples as described in the previous section, except for the 1x conjugate.

2. Remove the excess strip from the frame of the microtiter plate, return it to the plate package and store at 2-8°C.

3. Samples and controls should be analyzed in duplicate. Prepare preload controls and samples by adding 65 µl/rep to wells of 96-well deep well polypropylene microplates.

4. Add 50 µl of sample dilution buffer to all wells to be used from strips of the pre-coated 96-well plate included in the kit.

5. Transfer 50 µl of controls and samples with a multichannel pipette (8 channels) from the preloaded 96-well deep-well polypropylene microplates to the strips of the pre-coated 96-well plate included in the kit. Download time should not exceed 10 minutes.

6. Cover the plate with plastic paraffin and incubate for 3 hours plus/minus 5 minutes at 37°C (plus/minus 2°C).

7. Prepare 1x conjugate as described in section 10.2.

8. At the end of the incubation step, discard all liquid from the wells by inverting the plate. Proceed to the thorough washing step by adding 300 µl per well of 1x wash solution. Discard the 1x wash solution by inverting the plate and thoroughly blot the frame of the microtiter plate on absorbent paper by turning it upside down. Repeat the washing step 6 times. At the end of the wash steps, ensure that all liquid has been removed from the wells: all liquid has been successfully removed when no sign of liquid remains on the paper towel. The flushing procedure is critical. Insufficient washing can lead to poor accuracy and falsely elevated absorbances.

9. Add 100 µl of 1x conjugate to each well.

10. Cover the plate with plastic paraffin and incubate for 30 min plus/minus 3 min at 21°C (plus/minus 5°C).

11. At the end of the incubation step, discard all liquid from the wells by inverting the plate. Proceed to the thorough washing step by adding 300 µl per well of 1x wash solution. Discard the 1x wash solution by inverting the plate and thoroughly blot the frame of the microtiter plate on absorbent paper by turning it upside down. Repeat the washing step 6 times. At the end of the wash steps, ensure that all liquid has been removed from the wells: all liquid has been successfully removed when no sign of liquid remains on the paper towel. The flushing procedure is critical. Insufficient washing will result in poor accuracy and falsely elevated absorbances.

12. Add 100 µl of substrate solution to each well. When the substrate solution is added, the contents of the positive control 1 and positive control 2 wells should turn blue.

-

Claims (4)

13. Incubate for 15 min plus/minus 2 min at 21°C (plus/minus 5°C) in the dark. 14. Without removing the contents of the wells, add 100 µl of stop solution to each well to stop the reaction. When the stop solution is added, the contents of the positive control 1 and positive control 2 wells should turn yellow. 15. Read and write down the OD (optical density) at 450 nm. As shown in FIG. 1, the median plasma progastrin concentration was 0 pM in control patients (n = 119), while in patients with lung cancer (n = 40), a significant plasma progastrin concentration could be detected. Thus, patients with lung cancer have higher levels of progastrin in their plasma compared to healthy controls. Bibliographic references Yanaoka et al., Cancer Epidemiol Biomarkers Prev, 2008, 17(4). Pepe et al., J Natl Cancer Inst, 2008, Oct., 100(20). Leja et al., Best Practice & Research Clinical Gastroenterology, 2014, Dec, 28(6). CLAIM 1. A method for diagnosing lung cancer in vitro in a subject, comprising the steps of: a) contacting a biological sample from said subject with at least one progastrin-binding antibody or antigen-binding fragment thereof, wherein the biological sample is selected from the following: blood, serum and plasma; b) detecting the binding of said progastrin-binding antibody, or antigen-binding fragment thereof, to progastrin in said sample, wherein said binding indicates the presence of lung cancer in said subject and said progastrin-binding antibody is a monoclonal antibody selected from the group, consisting of the following: monoclonal antibody containing a heavy chain containing CDR-H1, CDR-H2 and CDR-H3 amino acid sequences SEQ ID NO: 4, 5 and 6, respectively, and a light chain containing CDR-L1, CDR-L2 and CDR-L3 amino acid sequences SEQ ID NOs: 7, 8 and 9, respectively, a monoclonal antibody containing a heavy chain containing CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively, and a light chain containing CDR- L1, CDR-L2 and CDR-L3 of the amino acid sequences of SEQ ID NOs: 13, 14 and 15, respectively, a monoclonal antibody containing a heavy chain containing CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequences of SEQ ID NOs: 16, 17 and 18, respectively, and a light chain containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequences of SEQ ID NOs: 19, 20, and 21, respectively; a heavy chain-containing monoclonal antibody containing CDR-H1, CDR-H2, and CDR -H3 amino acid sequences of SEQ ID NOs: 22, 23, and 24, respectively, and a light chain containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequences of SEQ ID NOs: 25, 26, and 27, respectively, a monoclonal antibody containing a heavy chain containing CDR-H1, CDR-H2 and CDR-H3 amino acid sequences SEQ ID NO: 28, 29 and 30, respectively, and a light chain containing CDR-L1, CDR-L2 and CDR-L3 amino acid sequences SEQ ID NO: 31, 32 and 33, respectively, a monoclonal antibody containing a heavy chain containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequences of SEQ ID NOs: 34, 35, and 36, respectively, and a light chain containing CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of SEQ ID NOs: 37, 38 and 39, respectively, and a monoclonal antibody produced by a hybridoma deposited with CNCM, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris CEDEX 15, France, December 27, 2016. , according to reference I5158. 2. The method of claim 1, wherein in step b) the progastrin concentration is further determined, and wherein a progastrin concentration of at least 10 pM in said biological sample indicates the presence of lung cancer in said subject. 3. The method according to claim 2, including additional stages, in which: c) determine the control concentration of progastrin in the control sample, d) comparing the concentrations of progastrin in the specified biological sample with the specified control concentration of progastrin, e) determine from the comparison stage d) the presence of lung cancer. 4. The method according to any one of claims 1 to 3, wherein said antibody or antigen-binding fragment thereof is selected from monoclonal antibodies against the N-terminus of progastrin and monoclonal antibodies against the C-terminus of progastrin. -