Antibodies and diagnostic methods for detecting bag3 protein in clinical samples

Abstract

The present invention relates to an anti-BAG3 monoclonal antibody or a fragment thereof and its use in a diagnostic method for detecting and quantifying BAG3 protein in human clinical samples. The invention also concerns a method or kit for diagnosing human fibrotic, inflammatory or oncological diseases through specific detection of BAG3 protein.

Description

[0001] Title: ANTIBODIES AND DIAGNOSTIC METHODS FOR DETECTING BAG3 PROTEIN IN CLINICAL SAMPLES

[0002] OBJECT OF THE INVENTION

[0003] The present invention relates to an anti-BAG3 monoclonal antibody or a fragment thereof and its use in a diagnostic method for detecting and quantifying the BAG3 protein in human clinical samples. The invention also concerns a method or kit for diagnosing human fibrotic, inflammatory or oncological diseases through specific detection of BAG3 protein.

[0004] STATE OF THE ART

[0005] The detection of BAG3 protein in patient serum or other clinical samples represents a challenging task due to the intrinsic characteristics of the protein itself. BAG3 is a member of the co-chaperone family characterized by the BAG domain, able to bind the ATPase domain of Hsc70 / Hsp70 proteins (heat shock protein 70). Through the WW domain and PXXP region (proline-rich motifs), BAG3 can also interact with other molecular partners (Doong et al., 2003). Recent literature suggests BAG3 may be considered a multifunctional protein, involved in modulating various biological processes such as apoptosis, proliferation, survival, cytoskeletal organization, cell differentiation and autophagy. Under physiological conditions, BAG3 expression is constitutive in cardiomyocytes and skeletal muscle. However, it has been demonstrated that bag3 gene expression can be induced by stress factors (e.g., oxidizing agents, high temperatures, heavy metals, serum deprivation, viral infections (HIV-1), exposure to ELF (electromagnetic field), pulsed ultrasound - in various normal cell types (leukocytes, epithelial and glial cells, retinal cells) (Rosati et al., 2007). The increased protein expression following stress stimuli is regulated by heat shock transcription factor HSF1 (Franceschelli et al., 2008). Growing evidence suggests an important role of BAG3 in cancer development; indeed, it has been shown that BAG3 is constitutively expressed in various cancer types (De Marco et al., 2018) and its expression directly correlates with aggressiveness in different tumors including pancreatic adenocarcinomas (Rosati et al., 2012). In fact, it has been reported that the anti-apoptotic BAG3 protein was expressed in 346 / 346 examined PDAC tumor tissue samples, while being absent in surrounding non- neoplastic tissues or healthy pancreas samples. Furthermore, in a cohort of 66 patients undergoing RO resection, BAG3 expression levels inversely correlated with patient survival. It has also been reported that BAG3 protein is released by pancreatic ductal adenocarcinoma (PDAC) cells (Rosati et al., 2015), and is detectable in serum samples from PDAC patients (Falco et al., 2013). BAG3 released by PDAC cells binds to its receptor IFITM-2 (Interferon-Induced TransMembrane protein-2) on macrophage plasma membranes and induces their activation and secretion of soluble factors able to promote PDAC cell proliferation (Rosati et al., 2015). Blocking this paracrine circuit with an anti-BAG3 antibody reduces tumor cell proliferation, tumor growth and metastasis spread. This evidence demonstrates BAG3's role in pancreatic cancer development and progression. On the other hand, the presence of BAG3 in PDAC patient serum and its expression in PDAC tumor samples suggest the use of BAG3 as a biomarker that could be useful and informative in a combined biomarker panel designed for early PDAC diagnosis, general population screening as well as monitoring of high-risk groups where PDAC incidence is higher. In a recent article, Firpo et al. (Firpo, M.A. et al. 2023) reported the identification of a subgroup of blood analytes related to Pancreatic Ductal Adenocarcinoma (PDAC) onset, such as CA-19, CEA and CA-125 antigens, in its early stages. The authors also measured BAG3 levels in a panel of 31 analytes, in serum from 837 subjects including healthy individuals, benign pancreatic diseases and early-stage PDAC samples. Using machine learning methods, they developed a classification model trained on 669 subjects. The model showed high accuracy in distinguishing PDAC from non-PDAC and healthy controls, with Area Under the Curve (AUC) of 0.920 and 0.944 respectively in the test set. The model was further validated on 146 additional patient samples and 40 more healthy controls, which confirmed previous results and showed similar AUC values. The study demonstrated that individually weak serum biomarkers can be integrated into a more powerful blood biomarker screening panel, whose combined results can be analyzed using a classification algorithm for early identification of subjects who may merit further investigations for more accurate PDAC diagnosis.

[0006] The complexity of BAG3 protein structure, its interactions and functions, along with its variable expression patterns, make the development of specific and sensitive assays for its detection and quantification in biological fluids difficult and challenging. This is because the protein structure and particularly the presence of different functional domains allow it to interact with various molecular partners, adding further difficulties to the analysis. Moreover, BAG3 presence in biological samples such as serum, plasma, urine, saliva or biopsy tissues can be influenced by various factors including disease stage, patient's general health status and individual genetic variations. These variables can affect BAG3 levels in analyzed samples, making it difficult to establish a reliable baseline value or detection threshold. Despite these difficulties, the solution for accurate BAG3 protein detection in patient serum or other clinical samples could have interesting and positive consequences for diagnosis and management of various pathologies. For example, as previously discussed, BAG3 is involved in development and progression of various cancer types, and its detection could potentially serve as a biomarker to monitor disease and help its characterization and staging. In the tumor microenvironment, moreover, specifically in cancer-associated fibroblasts, BAG3 regulates muscle actin (alpha-SMA) expression and local monocyte recruitment, and through a paracrine mechanism enhances intratumoral collagen deposition (Iorio V. et al., 2019 and De Marco M. et al., 2021). The formation of such inflammatory and fibrotic tumor microenvironment causes a more aggressive and drug-resistant neoplastic phenotype. However, the presence of inflammation and tissue fibrosis represents a complex biological phenomenon that is not exclusive to tumors, but manifests in a wide range of pathologies. These pathological processes are characterized by altered immune response and excessive extracellular matrix production and deposition, respectively. In particular, inflammation is a protective response of the immune system to tissue injury or infections. However, when inflammation becomes chronic, it can contribute to pathogenesis of many diseases including cardiovascular diseases, autoimmune and neurodegenerative diseases, besides tumors. In these conditions, immune cells such as macrophages and lymphocytes can release a series of pro-inflammatory mediators, including cytokines, chemokines and reactive oxygen species, which can damage tissues and alter their function. Tissue fibrosis, on the other hand, is a pathological process characterized by excessive production and deposition of extracellular matrix, particularly collagen, which can lead to tissue stiffness and dysfunction. This phenomenon is typical of many fibrotic diseases such as idiopathic pulmonary fibrosis, liver cirrhosis and systemic sclerosis. The identification of specific biomarkers of these processes, such as BAG3 protein, as well as the identification of specific and highly selective antibodies against BAG3 and diagnostic methods to identify and quantify this protein could therefore improve diagnosis and monitoring of these diseases, enabling intervention in both prevention and treatment.

[0007] Patent application WO2013 / 189778A1 describes the use of generic anti-BAG3 polyclonal or monoclonal antibodies as biological markers for diagnosis of cardiac diseases, cancers, diabetes and diseases related to inflammation of skin, nerves, bones, blood vessels and connective tissues. Patent application WO2011 / 067377A1 describes a method to determine presence and / or concentration of soluble BAG3 protein in a biological sample. This method is preferably performed through an ELISA assay based on polyclonal or monoclonal antibodies and is used to determine BAG3 protein presence in soluble form in cardiac diseases or pancreatic cancer. Some ELISA kits for BAG3 protein detection are also commercially available. However, these kits have shown poor diagnostic performance and are provided by manufacturers for research use only. In detail, currently available products #ABIN6954056, #ABIN6962016 (Antibodiesonline), #OKCD08090 (Aviva Systems Biology), show low sensitivity (detection limit 150 pg / ml); kit ab244047 (Abeam) shows low sensitivity and is optimized for cell and tissue extract analysis, not for serum, plasma or other body fluids where BAG3 protein concentrations are generally lower. The need is therefore evident to identify highly specific and selective anti-BAG3 monoclonal antibodies for BAG3 protein present even at low concentrations in various biological samples, which can be used to develop robust, precise and sensitive diagnostic methods and kits for BAG3 detection and quantification in biological samples such as serum, plasma, urine, saliva or biopsy tissues where B AG3 protein concentration may be very low. These methods must be sufficiently robust and modifiable to avoid or minimize bias due to protein complexes presence and variable protein expression levels, as well as potential influence and interference from biological and environmental factors. Such methods must also be able to minimize false positive or false negative detection that could compromise the final result obtained.

[0008] DETAILED DESCRIPTION OF THE INVENTION

[0009] In the present invention the inventors have identified new anti-BAG3 monoclonal antibodies, which have proven particularly effective and specific in detecting B AG3 protein presence in human clinical samples, even at low concentrations, when used in diagnostic methods. It is indeed known that antibodies used in such methods, which consist of capture and detection antibodies, must be highly specific and able to recognize the target protein with high sensitivity to be used in diagnostic assays / kits. In particular, a further characteristic of antibodies used in these diagnostic methods or kits is their ability to bind the protein in its native conformation, i.e., the conformation in which it is found in biological samples. This characteristic, for example, is not preserved in antibodies that recognize protein segments not accessible when the protein is in its native conformation. In fact, such antibodies can only bind the protein in its denatured form. It is therefore crucial that an antibody recognizes and binds to a protein in its native conformation in biological samples, since only in this form does the protein maintain its three- dimensional structure and biological functions. A protein's native conformation is determined by amino acid sequence and interactions between them, such as hydrogen bonds, hydrophobic interactions and disulfide bridges. When a protein is denatured, it loses this structure and, consequently, its functionality. Therefore, using antibodies that recognize the protein in its native form ensures that the diagnostic test is specific and accurate, since the antibody used in such tests will only bind to the correctly folded protein present in biological samples to be tested. This represents a significant advantage in building diagnostic methodologies, as it reduces the risk of false positives or negatives, improving result reliability and accuracy.

[0010] The invention disclosed here relates to monoclonal antibodies designed to recognize BAG3 protein in its native form with high sensitivity and specificity when present in serum, plasma, urine, saliva or biopsy tissue samples from subjects affected by fibrotic, inflammatory or oncological diseases. As evident from experimental data, the inventors selected the most suitable antigens for anti-BAG3 monoclonal antibody production assuming that monoclonal antibody selection based on immunogenic activity epitopes exposed in purified BAG3 protein was the most appropriate selection process to increase sensitivity and specificity of a diagnostic method and kit. As described in the experimental section, the study was based on identification of BAG3 protein epitopes recognized by a polyclonal antibody using peptide arrays and CLIPS technology, a technology that allowed identification and synthesis of a recombinant polypeptide CP2 (SEQ ID N:l) used for animal immunization protocol and production of the present invention's monoclonal antibodies. The monoclonal antibodies thus obtained have enabled construction of diagnostic methods or kits able to identify and / or quantify BAG3 protein in a biological sample with a particularly low determination limit of pg / ml. An embodiment of the present invention is therefore an anti-BAG3 monoclonal antibody or a fragment thereof, characterized by comprising a heavy chain variable region having at least 90% identity with amino acid sequence SEQ ID NO. 3, preferably at least 95% identity with amino acid sequence SEQ ID NO. 3, and a light chain variable region having at least 90% identity with amino acid sequence SEQ ID NO. 4, preferably at least 95% identity with amino acid sequence SEQ ID NO. 4.

[0011] Preferably said anti-BAG3 monoclonal antibody or fragment thereof is characterized by consisting of a heavy chain variable region having at least 90% identity with amino acid sequence SEQ ID NO. 3, preferably at least 95% identity with amino acid sequence SEQ ID NO. 3, and a light chain variable region having at least 90% identity with amino acid sequence SEQ ID NO. 4, preferably at least 95% identity with amino acid sequence SEQ ID NO. 4.

[0012] Preferably the heavy chain variable region has at least 98% identity with amino acid sequence SEQ ID NO. 3, and the light chain variable region has at least 98% identity with amino acid sequence SEQ ID NO. 4. Even more preferably in said anti-BAG3 monoclonal antibody the heavy chain variable region consists of amino acid sequence SEQ ID NO. 3, and the light chain variable region consists of amino acid sequence SEQ ID NO. 4.

[0013] In a further preferred embodiment said monoclonal antibody has a heavy chain constant region (HC) having nucleotide sequence SEQ ID NO. 19 and a light chain constant region (LC) having nucleotide sequence SEQ ID NO. 20. Preferably said monoclonal antibody is antibody 42F3D3.

[0014] Preferably said anti-BAG3 monoclonal antibody or fragment thereof is characterized in that the heavy chain variable region has the following H-CDR regions: H-CDR1 comprising amino acids of SEQ ID NO. 7, H-CDR2 comprising amino acids of SEQ ID NO. 8 and H-CDR3 comprising amino acids of SEQ ID NO. 9 and the light chain variable region has the following L-CDR regions: L- CDR1 comprising amino acids of SEQ ID NO. 10, L-CDR2 comprising amino acids of SEQ ID NO. 11 and L-CDR3 comprising amino acids of SEQ ID NO. 12. According to a further preferred embodiment the anti-B AG3 monoclonal antibody or fragment thereof according to the present invention is characterized in that said antibody is a natural or synthetic origin antibody, preferably an avian, mammalian or humanized antibody.

[0015] More preferably said antibody is an F(ab) fragment, F(ab') fragment, F(ab')2 fragment, Fv fragment, diabody, ScFv, affibody, avimer, nanobody, antibody domain and / or single chains.

[0016] The anti-BAG3 antibody or fragment thereof according to the present invention may be any natural, recombinant and / or synthetic origin antibody, an avian or mammalian origin or humanized antibody. Preferably, the constant domain, if present, is a humanized or human constant domain. The variable domain is preferably a mammalian variable domain, for example a humanized or human variable domain. Even more preferably said anti-BAG3 antibody is a recombinant antibody.

[0017] The anti-BAG3 antibodies or fragments thereof according to the invention are monoclonal antibodies. In particular, the antibodies of the present invention are preferably selected from the group consisting of recombinant antibodies, humanized or fully human antibodies, chimeric antibodies, or fragments thereof. The monoclonal antibodies of the present invention can be produced with any suitable method known in the art such as that of Kohler and Milstein (1975) or with recombinant DNA methods. Chimeric forms of antibodies can be generated according to methods known in the art, (Kettleborough C.A. et al., 1991), such as chimerization or CDR grafting. Alternative methods to produce humanized antibodies are well known in the art and are described for example in EP 0239400 and WO 90 / 07861. Humanized antibodies can also be derived with in vitro methods. Suitable examples include, but are not limited to, phage display, yeast display and similar.

[0018] According to the present invention "chimeric antibody" refers to antibodies comprising polypeptides from different species, such as mouse and human. Production of chimeric antibodies is described, for example, in WO 89 / 09622.

[0019] The term antibody includes "fragments" or "derivatives", which have at least one antigen binding site of the antibody.

[0020] The antibody of the invention is preferably of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD and IgE type. It will be appreciated that antibodies that are generated do not initially need to possess such isotype but, rather the antibody so generated can possess any isotype and that the antibody can be isotype switched.

[0021] A further embodiment of the present invention is a vector comprising the nucleic acid encoding the antibody of the invention. Said vector is selected from a phage, plasmid, viral or retroviral vector. Preferably, the vector of the invention is an expression vector in which the nucleic acid molecule is operatively linked to one or more control sequences allowing transcription and optionally expression in prokaryotic and / or eukaryotic host cells.

[0022] A further embodiment of the present invention is a host comprising the vector of the invention, selected from a prokaryotic or eukaryotic cell, preferably a mammalian or human cell, or a non -human transgenic animal.

[0023] A further embodiment of the present invention is the use of the anti-BAG3 monoclonal antibody or fragment thereof according to the present invention in a method for determining presence and / or concentration of soluble BAG3 protein in a biological sample.

[0024] Preferably said biological sample is selected from serum, plasma, urine, saliva or biopsy tissues.

[0025] Preferably said method is selected from ELISA (Enzyme linked immunosorbent Assays), CLIA (chemiluminescent assays), RIA (radioimmunoassays), FIA (immunofluorescence assays), more preferably said method is an ELISA method, even more preferably said ELISA is a sandwich ELISA.

[0026] Preferably said anti-BAG3 monoclonal antibody or fragment thereof is characterized by comprising a heavy chain variable region having at least 90% identity with amino acid sequence SEQ ID NO. 3, preferably at least 95% identity with amino acid sequence SEQ ID NO. 3, and a light chain variable region having at least 90% identity with amino acid sequence SEQ ID NO. 4, preferably at least 95% identity with amino acid sequence SEQ ID NO. 4.

[0027] More preferably the heavy chain variable region has at least 98% identity with amino acid sequence SEQ ID NO. 3, and the light chain variable region has at least 98% identity with amino acid sequence SEQ ID NO. 4. Even more preferably in said anti-BAG3 monoclonal antibody the heavy chain variable region consists of amino acid sequence SEQ ID NO. 3, and the light chain variable region consists of amino acid sequence SEQ ID NO. 4.

[0028] Preferably said monoclonal antibody is antibody 42F3D3.

[0029] Preferably said anti -BAG monoclonal antibody or fragment thereof is characterized in that the heavy chain variable region has the following H-CDR regions: H-CDR1 comprising amino acids of SEQ ID NO. 7, H-CDR2 comprising amino acids of SEQ ID NO. 8 and H-CDR3 comprising amino acids of SEQ ID NO. 9 and the light chain variable region has the following L-CDR regions: L-CDR1 comprising amino acids of SEQ ID NO. 10, L-CDR2 comprising amino acids of SEQ ID NO. 11 and L-CDR3 comprising amino acids of SEQ ID NO. 12.

[0030] A further embodiment of the present invention is a method for determining presence and / or concentration of BAG3 protein in a biological sample, characterized by using a first anti-BAG3 monoclonal antibody or fragment thereof comprising a heavy chain variable region having at least 90% identity with amino acid sequence SEQ ID NO. 3, preferably at least 95% identity with amino acid sequence SEQ ID NO. 3, and a light chain variable region having at least 90% identity with amino acid sequence SEQ ID NO. 4, preferably at least 95% identity with amino acid sequence SEQ ID NO. 4 and a second anti-BAG3 monoclonal antibody comprising a heavy chain variable region having amino acid sequence SEQ ID NO. 5 and a light chain variable region having amino acid sequence SEQ ID NO. 6.

[0031] Preferably said BAG3 protein is soluble BAG3 protein.

[0032] Preferably in said first antibody the heavy chain variable region has at least 98% identity with amino acid sequence SEQ ID NO. 3, and the light chain variable region has at least 98% identity with amino acid sequence SEQ ID NO. 4.

[0033] Preferably said first anti-B AG3 antibody or fragment thereof is characterized in that the heavy chain variable region has the following H-CDR regions: H-CDR1 comprising amino acids of SEQ ID NO. 7, H-CDR2 comprising amino acids of SEQ ID NO. 8 and H-CDR3 comprising amino acids of SEQ ID NO. 9 and the light chain variable region has the following L-CDR regions: L-CDR1 comprising amino acids of SEQ ID NO. 10, L-CDR2 comprising amino acids of SEQ ID NO. 11 and L-CDR3 comprising amino acids of SEQ ID NO. 12.

[0034] In a further preferred embodiment said first monoclonal antibody has a heavy chain constant region (HC) having nucleotide sequence SEQ ID NO. 19 and a light chain constant region (LC) having nucleotide sequence SEQ ID NO. 20.

[0035] Preferably said monoclonal antibody is antibody 42F3D3.

[0036] In a preferred embodiment said method is characterized in that the heavy chain variable region of the second anti-BAG3 monoclonal antibody has the following H- CDR regions: H-CDR1 comprising amino acids of SEQ ID NO. 13, H-CDR2 comprising amino acids of SEQ ID NO. 14, H-CDR3 comprising amino acids of SEQ ID NO. 15 and the light chain variable region of the anti-BAG3 antibody has the following L-CDR regions: L-CDR1 comprising amino acids of SEQ ID NO. 16, L-CDR2 comprising amino acids of SEQ ID NO. 17 and L-CDR3 comprising amino acids of SEQ ID NO. 18.

[0037] In a further preferred embodiment said second monoclonal antibody has a heavy chain constant region (HC) having nucleotide sequence SEQ ID NO. 21 and a light chain constant region (LC) having nucleotide sequence SEQ ID NO. 22.

[0038] According to a further preferred embodiment in the method of the present invention the first anti-BAG3 monoclonal antibody is characterized in that the heavy chain variable region consists of the following H-CDR regions: H-CDR1 comprising amino acids of SEQ ID NO. 7, H-CDR2 comprising amino acids of SEQ ID NO. 8 and H-CDR3 comprising amino acids of SEQ ID NO. 9 and the light chain variable region consists of the following L-CDR regions: L-CDR1 comprising amino acids of SEQ ID NO. 10, L-CDR2 comprising amino acids of SEQ ID NO. 11 and L- CDR3 comprising amino acids of SEQ ID NO. 12, and the second anti-BAG3 monoclonal antibody is characterized in that the heavy chain variable region consists of the following H-CDR regions: H-CDR1 comprising amino acids of SEQ ID NO. 13, H-CDR2 comprising amino acids of SEQ ID NO. 14, H-CDR3 comprising amino acids of SEQ ID NO. 15 and the light chain variable region consists of the following L-CDR regions: L-CDR1 comprising amino acids of SEQ ID NO. 16, L-CDR2 comprising amino acids of SEQ ID NO. 17 and L-CDR3 comprising amino acids of SEQ ID NO. 18.

[0039] Preferably said anti-BAG3 monoclonal antibody is antibody 27F1B9E3.

[0040] According to a further preferred embodiment said method is characterized in that the first anti-BAG3 monoclonal antibody is antibody 42F3D3 and the second anti- BAG3 monoclonal antibody is antibody 27F1B9E3.

[0041] In a further preferred embodiment the method according to the present invention is characterized by the following steps: a) determining presence and / or concentration of BAG3 protein in a previously obtained biological sample consisting of serum, plasma, urine, saliva or biopsy tissues; b) comparing obtained values with reference values or values obtained from a reference biological sample; and c) associating presence and / or concentration of BAG3 protein with a pathological condition selected from fibrotic diseases, inflammatory diseases or tumors. Preferably said BAG3 protein is soluble BAG3 protein.

[0042] Preferably said fibrotic diseases are selected from scleroderma, systemic sclerosis, pulmonary fibrosis, liver fibrosis, cystic fibrosis, cardiac fibrosis or nephrogenic fibrosis, preferably systemic sclerosis.

[0043] Preferably said inflammatory diseases are selected from rheumatoid arthritis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis, psoriasis, psoriatic arthritis, autoimmune hepatitis, vasculitis, idiopathic pulmonary fibrosis or infectious diseases causing inflammation such as COVID-19, viral hepatitis, HIV / AIDS, tuberculosis or sepsis, preferably COVID-19.

[0044] Preferably said tumors are selected from carcinomas, sarcomas, leukemias, lymphomas, melanomas or central nervous system tumors, preferably breast, ovarian or pancreatic carcinoma.

[0045] According to a further preferred embodiment the method according to the present invention is used to determine presence and / or concentration of BAG3 protein in tumors, selected from carcinomas, sarcomas, leukemias, lymphomas, melanomas or central nervous system tumors, preferably breast, ovarian or pancreatic carcinoma.

[0046] According to a further preferred embodiment the method of the present invention is a direct, indirect, competitive or sandwich ELISA method.

[0047] More preferably the ELISA method of the present invention is a sandwich or direct ELISA method.

[0048] Preferably in said method step a) is performed by an ELISA method characterized by the following steps: i) immobilization of the first anti-BAG3 monoclonal antibody according to the present invention in wells of an ELISA plate by incubation for 2-20 hours; ii) addition of biological sample and blocking solution, iii) incubation for 2-20 hours; iv) addition of the second anti-BAG3 monoclonal antibody according to the present invention conjugated with HRP; v) incubation for 10-60 minutes; vi) incubation with a solution containing chromogenic substrate for 5-10 minutes at room temperature; vii) stopping chromogenic reaction by adding a denaturing solution, preferably sulfuric acid; viii) measuring optical density of the solution and calculating quantity or concentration of BAG3 protein in the biological sample.

[0049] According to a further preferred embodiment in said method step a) is performed by an ELISA method characterized by the following steps: i) immobilization of the first anti-BAG3 monoclonal antibody characterized in that the heavy chain variable region consists of the following H-CDR regions: H-CDR1 comprising amino acids of SEQ ID NO. 7, H-CDR2 comprising amino acids of SEQ ID NO. 8 and H-CDR3 comprising amino acids of SEQ ID NO. 9 and the light chain variable region consists of the following L-CDR regions: L-CDR1 comprising amino acids of SEQ ID NO. 10, L-CDR2 comprising amino acids of SEQ ID NO. 11 and L-CDR3 comprising amino acids of SEQ ID NO. 12, in wells of an ELISA plate by incubation for 2-20 hours; ii) addition of biological sample and blocking solution, iii) incubation for 2-20 hours; iv) addition of the second anti-BAG3 monoclonal antibody conjugated with HRP, characterized in that the heavy chain variable region consists of the following H- CDR regions: H-CDR1 comprising amino acids of SEQ ID NO. 13, H-CDR2 comprising amino acids of SEQ ID NO. 14, H-CDR3 comprising amino acids of SEQ ID NO. 15 and the light chain variable region consists of the following L-CDR regions: L-CDR1 comprising amino acids of SEQ ID NO. 16, L-CDR2 comprising amino acids of SEQ ID NO. 17 and L-CDR3 comprising amino acids of SEQ ID NO. 18; v) incubation for 10-60 minutes; vi) incubation with a solution containing chromogenic substrate for 5-10 minutes at room temperature; vii) stopping chromogenic reaction by adding a denaturing solution, preferably sulfuric acid; viii) measuring optical density of the solution and calculating quantity or concentration of BAG3 protein in the biological sample.

[0050] Preferably said first anti-BAG3 monoclonal antibody is antibody 42F3D3.

[0051] Preferably said second anti-BAG3 monoclonal antibody is antibody 27F1B9E3. Preferably incubations at steps i) and iii) are performed at 4°C.

[0052] More preferably the blocking solution at step ii) contains fish gelatin, bovine IgG and tween 20 in PBS.

[0053] Preferably incubation at step v) is at room temperature, preferably at 20-24°C.

[0054] Preferably the antibody used at point i) is an anti-BAG3 monoclonal antibody or fragment thereof comprising a heavy chain variable region having at least 90% identity with amino acid sequence SEQ ID NO. 3, preferably at least 95% identity with amino acid sequence SEQ ID NO. 3, and a light chain variable region having at least 90% identity with amino acid sequence SEQ ID NO. 4, preferably at least 95% identity with amino acid sequence SEQ ID NO. 4.

[0055] Preferably in said first antibody the heavy chain variable region has at least 98% identity with amino acid sequence SEQ ID NO. 3, and the light chain variable region has at least 98% identity with amino acid sequence SEQ ID NO. 4.

[0056] Preferably said first anti-B AG3 antibody or fragment thereof is characterized in that the heavy chain variable region has the following H-CDR regions: H-CDR1 comprising amino acids of SEQ ID NO. 7, H-CDR2 comprising amino acids of SEQ ID NO. 8 and H-CDR3 comprising amino acids of SEQ ID NO. 9 and the light chain variable region has the following L-CDR regions: L-CDR1 comprising amino acids of SEQ ID NO. 10, L-CDR2 comprising amino acids of SEQ ID NO. 11 and L-CDR3 comprising amino acids of SEQ ID NO. 12. In a preferred embodiment said ELISA method is characterized in that the heavy chain variable region of the anti-BAG3 monoclonal antibody used at point iv) has the following H-CDR regions: H-CDR1 comprising amino acids of SEQ ID NO. 13, H-CDR2 comprising amino acids of SEQ ID NO. 14, H-CDR3 comprising amino acids of SEQ ID NO. 15 and the light chain variable region of the anti-B AG3 antibody has the following L-CDR regions: L-CDR1 comprising amino acids of SEQ ID NO. 16, L-CDR2 comprising amino acids of SEQ ID NO. 17 and L-CDR3 comprising amino acids of SEQ ID NO. 18.

[0057] According to a further preferred embodiment in the method of the present invention the first anti-BAG3 antibody is used at a concentration between 1-8 pg / ml, preferably 4 pg / ml and the second anti-BAG3 antibody is used at a concentration between 0.01-0.10 pg / ml, preferably 0.05 pg / ml.

[0058] According to a preferred embodiment the ELISA method of the present invention has a determination limit greater than or equal to 15.0 pg / ml.

[0059] A further embodiment of the present invention is a kit comprising the first anti- BAG3 antibody and the second anti-BAG3 antibody according to the present invention.

[0060] Preferably said kit is for use in diagnosis of a pathological condition selected from fibrotic diseases, inflammatory diseases or tumors.

[0061] Preferably the first antibody used in said kit is an anti-BAG3 monoclonal antibody or fragment thereof comprising a heavy chain variable region having at least 90% identity with amino acid sequence SEQ ID NO. 3, preferably at least 95% identity with amino acid sequence SEQ ID NO. 3, and a light chain variable region having at least 90% identity with amino acid sequence SEQ ID NO. 4, preferably at least 95% identity with amino acid sequence SEQ ID NO. 4. Preferably in said first antibody the heavy chain variable region has at least 98% identity with amino acid sequence SEQ ID NO. 3, and the light chain variable region has at least 98% identity with amino acid sequence SEQ ID NO. 4.

[0062] Preferably said first anti-B AG3 antibody or fragment thereof is characterized in that the heavy chain variable region has the following H-CDR regions: H-CDR1 comprising amino acids of SEQ ID NO. 7, H-CDR2 comprising amino acids of SEQ ID NO. 8 and H-CDR3 comprising amino acids of SEQ ID NO. 9 and the light chain variable region has the following L-CDR regions: L-CDR1 comprising amino acids of SEQ ID NO. 10, L-CDR2 comprising amino acids of SEQ ID NO. 11 and L-CDR3 comprising amino acids of SEQ ID NO. 12.

[0063] In a preferred embodiment said second anti-BAG3 monoclonal antibody has the following H-CDR regions: H-CDR1 comprising amino acids of SEQ ID NO. 13, H-CDR2 comprising amino acids of SEQ ID NO. 14, H-CDR3 comprising amino acids of SEQ ID NO. 15 and the light chain variable region has the following L- CDR regions: L-CDR1 comprising amino acids of SEQ ID NO. 16, L-CDR2 comprising amino acids of SEQ ID NO. 17 and L-CDR3 comprising amino acids of SEQ ID NO. 18.

[0064] Preferably said first anti-BAG3 monoclonal antibody is antibody 42F3D3. Preferably said second anti-BAG3 monoclonal antibody is antibody 27F1B9E3. According to a further preferred embodiment the first and second antibody according to the present invention can be used interchangeably in the method or kit of the present invention.

[0065] That is, the first antibody can be used in place of the second and vice versa.

[0066] DEFINITIONS

[0067] Unless otherwise specified, all terms known in the art, notations and other scientific terminology used in this document are intended to have the meanings commonly understood by those skilled in the art to which this communication refers. In some cases, terms with commonly understood meanings are defined herein for clarity and / or quick reference; therefore, the inclusion of such definitions in this document should not be interpreted to represent a substantial difference from what is generally known in the art.

[0068] The terms "comprising", "having", "including" and "containing" are to be understood as open-ended terms (i.e., "including, but not limited to"), and are also considered to support terms such as "consisting of', "consisting essentially of, "consisting substantially of', "composed of or "constituted by".

[0069] The terms "consisting essentially of', "constituted essentially by" are to be understood as semi-closed terms, meaning that no other ingredients affecting the new characteristics of the invention are included (optional excipients could therefore be included).

[0070] The terms "consisting of', "composed of', "constituted by" are to be understood as closed terms.

[0071] In the present invention, "serum" means the blood component that is neither a blood cell nor a clotting factor; it is blood plasma with fibrinogens removed. Serum includes all proteins not used in blood clotting (coagulation) and all electrolytes, antibodies, antigens, hormones and any exogenous substances.

[0072] In the present invention, "plasma" means the liquid straw-colored / pale yellow component of blood that normally holds blood cells in whole blood in suspension. It contains clotting factors, such as fibrinogens.

[0073] The term "biopsy tissues" means biological tissue samples taken from biopsies.

[0074] The term "antibody" as used here includes "fragments" or "derivatives", which have at least one antigen binding site of the antibody and / or show the same biological activity. An antibody preferably comprises at least one immunoglobulin heavy chain and at least one immunoglobulin light chain. An immunoglobulin chain comprises a variable domain and optionally a constant domain. A variable domain may comprise complementarity determining regions (CDRs), e.g., a CDR1, CDR2 and / or CDR3 region, and flanking regions.

[0075] The term "humanized antibody" refers to an antibody of human origin whose hypervariable region has been replaced by the homologous region of non-human monoclonal antibodies.

[0076] The term "chimeric antibody" refers to an antibody containing portions derived from different antibodies.

[0077] The term "recombinant antibody" refers to an antibody obtained using recombinant DNA methods.

[0078] The term "scFv fragment" (single-chain variable fragment) refers to immunoglobulin fragments capable only of binding to the target antigen. scFv fragments can also be synthesized as dimers (diabodies), trimers (triabodies) and tetramers (tetrabodies) using peptide linkers.

[0079] The terms "Fab fragment" (antigen-binding fragment) and "F(ab')2 fragment" refer to immunoglobulin fragments consisting of a light chain linked to the adjacent heavy chain Fc fragment, and such fragments are monovalent antibodies. When Fab portions are paired, the fragment is called F(ab')2.

[0080] With the definition "associating presence and / or concentration of BAG3 protein with a pathological condition" it is meant associating presence and / or concentration of BAG3 protein with diagnosis of a pathological condition based on measured BAG3 protein levels / quantities in a patient's biological sample.

[0081] The term "ELISA method" means an enzyme-linked immunosorbent assay, which is a method used to quantitatively detect a specific antigen (protein) in a sample. The characteristics of different ELISA tests known in the art are described below. In a direct ELISA assay, the antigen is immobilized on the bottom of the microplate well and then binds to an antibody that is specific for the antigen and is conjugated to an enzyme or another molecule that allows its detection.

[0082] In an indirect ELISA assay, the antigen is immobilized on the bottom of the microplate well, then a specific antibody for the antigen is added. Subsequently, a secondary antibody specific for the antigen and conjugated to an enzyme or another detection molecule binds to the first antibody.

[0083] In a competitive ELISA assay, a reference antigen is immobilized on the bottom of the microplate well. Then the sample and antibody are added to the well so that any antigen present in the sample competes with the reference antigen for antibody binding. Unbound material is removed by washing. The greater the amount of antigen present in the sample, the less antibody will bind to the reference antigen on the well bottom and thus the lower the signal.

[0084] In a sandwich ELISA assay, two antibodies specific for two different epitopes on the target antigen are used. The capture antibody is immobilized on the bottom of the microplate well and binds to one epitope of the antigen. The detection antibody, which is conjugated to an enzyme that allows its presence to be determined, binds to a different epitope of the antigen. (If the detection antibody is not conjugated, a secondary detection antibody conjugated to an enzyme is required).

[0085] By "CLIA method" (Chemiluminescent Immunoassays) is meant a chemiluminescent immunological test, i.e., a type of immunoassay that uses chemiluminescence.

[0086] By "FIA method" (Fluorescent Immunoassays) is meant a fluorescence immunoassay based on the same principle as ELISA but using fluorescence-labeled antibodies. By "RIA method" (Radio Immunoassays) is meant a radioimmunoassay method, used to assay any immunogenic compound available in pure form and radioactively labeled.

[0087] The examples that follow are provided to better detail the present invention, without in any way limiting it.

[0088] EXAMPLES

[0089] The study of the present invention began with identification of BAG3 protein epitopes recognized by a polyclonal antibody mixture able to efficiently bind BAG3 protein in its native conformation using peptide arrays and CLIPS technology. This technology fixes peptides in defined three-dimensional structures, allowing functional imitation of complex binding sites. The screening process involved converting the target protein into a library of overlapping peptide constructs. Constructs presenting the complete epitope in the correct conformation bind the antibody with high affinity, while those presenting an incomplete epitope bind with lower affinity. Affinity information was analyzed to define epitope sequence and conformation. The obtained data showed that the anti-BAG3 polyclonal antibody binds several peptides, and eight peptides were recognized and classified as three dominant motifs and five minor motifs. These data were used to design and synthesize a polypeptide used for animal immunization protocol and production of monoclonal antibodies able to bind portions of BAG3 protein exposed and accessible to antibodies when BAG3 protein is in its native conformation within biological samples.

[0090] The recombinant polypeptide, called CP2 (SEQ ID NO. 1) and containing the main epitopes of B AG3 protein, was used to immunize four mice. As will be explained later, this recombinant polypeptide does not overlap with peptides contained in BAG3 protein described in WO2013 / 189778. Anti-CP2 antibody titers were monitored in mouse sera tested with ELISA method. The next step involved fusion of myeloma cells with splenocytes from sacrificed mice, followed by screening, selection and expansion of positive clones producing antibodies against the antigen protein. With this procedure, clone 42F3D3 was obtained.

[0091] In said 42F3D3 antibody the heavy chain variable region has the VH region of SEQ ID NO. 3 and the light chain variable region has the VL region of SEQ ID NO. 4 and the following H-CDR regions: H-CDR1 comprising amino acids of SEQ ID NO. 7, H-CDR2 comprising amino acids of SEQ ID NO. 8 and H-CDR3 comprising amino acids of SEQ ID NO. 9 and the light chain variable region comprises the following L-CDR regions: L-CDR1 comprising amino acids of SEQ ID NO. 10, L-CDR2 comprising amino acids of SEQ ID NO. 11 and L-CDR3 comprising amino acids of SEQ ID NO. 12.

[0092] A second monoclonal antibody was obtained by immunizing mice with the entire sequence of recombinant BAG3 protein (SEQ ID NO.2) and was selected among the obtained clones for its ability to recognize BAG3 protein in cell lysates through immunoprecipitation. With this procedure, clone 27F1B9E3 producing the monoclonal antibody was chosen.

[0093] In said antibody the heavy chain variable region has the VH region of SEQ ID NO. 5 and the light chain variable region has the VL region of SEQ ID NO. 6 and the following H-CDR regions: H-CDR1 comprising amino acids of SEQ ID NO. 13, H-CDR2 comprising amino acids of SEQ ID NO. 14, H-CDR3 comprising amino acids of SEQ ID NO. 15 and the light chain variable region of the anti-BAG3 antibody has the following L-CDR regions: L-CDR1 comprising amino acids of SEQ ID NO. 16, L-CDR2 comprising amino acids of SEQ ID NO. 17 and L-CDR3 comprising amino acids of SEQ ID NO. 18.

[0094] Example 1. Screening of anti-BAG3 monoclonal and polyclonal antibodies through IP / WB of recombinant BAG3 protein. To identify a pair of monoclonal antibodies capable of recognizing soluble BAG3 protein in sandwich ELISA tests for diagnostic use, 4 different murine anti-BAG3 monoclonal antibodies were tested: monoclonal antibody AC-2 described in WO201 1 / 067377A1 (and in WO2013 / 189778) (AC-2 mAb) obtained from animal immunization with PEP-2 peptide; monoclonal antibodies AC-rb2 mAb and AC- rb3 mAb described in WO2014 / 147503A2 and monoclonal antibody 27F1B9E3, obtained from immunization of animals with full-length human BAG3 protein, and a rabbit-generated anti-BAG3 polyclonal antibody, for their ability to immunoprecipitate recombinant rBAG3 protein in the indicated amounts.

[0095] As observed in Figure 1, the most efficient antibodies in immunoprecipitating BAG3 protein are clone 27F1B9E3 and the polyclonal antibody (reference antibody), as demonstrated by presence of B AG3 protein immunoprecipitated by said antibodies in quantities of 600 and 300 pg and detected in WB. The other antibodies immunoprecipitate much lower quantities under identical experimental conditions.

[0096] The polyclonal antibody was used as reference antibody because it is particularly efficient in binding BAG3 protein in its native conformation. However, polyclonal antibody mixtures are not used in immunometric diagnostic devices mainly due to their intrinsic variability. Polyclonal antibodies are produced by different B cell clones and recognize various epitopes on an antigen. This diversity makes it difficult to obtain polyclonal antibody batches that are identical over time, compromising test reproducibility and reliability. In immunometric devices, highly specific and consistent reagents are essential to ensure accurate and comparable results. Therefore, monoclonal antibodies are preferred, as they are derived from a single B cell clone and offer superior specificity and uniformity.

[0097] Monoclonal antibody 27F1B9E3, unlike other tested antibodies, shows greater efficiency in immunoprecipitating BAG3 protein in its native conformation, both in its recombinant form and in its naturally occurring form in tumor cell lysates, similarly to the polyclonal antibody. The results demonstrate that monoclonal antibody 27F1B9E3 is particularly suitable for use in diagnostic assays where it is necessary to capture even small soluble quantities of BAG3 protein. This was highlighted by comparing its efficiency in immunoprecipitation techniques to that of other monoclonal antibodies already described in literature.

[0098] Example 2. Screening of anti-BAG3 monoclonal and polyclonal antibodies through IP / WB of BAG3 protein in a cell lysate.

[0099] Four different murine anti-BAG3 monoclonal antibodies: monoclonal antibody AC-2 described in WO2011 / 067377A1 (and in WO2013 / 189778) (AC-2 mAb or anti-BAG-3 mAbl) obtained from animal immunization with PEP-2 peptide; monoclonal antibodies AC-rb2 mAb (anti-BAG3 mAb3) and AC-rb3 mAb (anti- BAG3 mAb2) described in WO2014 / 147503A2 and antibody 27F1B9E3, obtained from immunization of animals with full-length human BAG3 protein, and a rabbitgenerated anti-BAG3 polyclonal antibody, were tested for their ability to immunoprecipitate B AG3 protein from a MiaPaCA-2 cell lysate.

[0100] As can be observed from Figure 2, the most efficient antibodies in immunoprecipitating BAG3 protein are clone 27F1B9E3 and the polyclonal antibody (reference antibody). The other antibodies are much less efficient as demonstrated by presence of BAG3 protein in the non-bound fraction of total proteins precipitated in acetone. Also in this case, the results demonstrate that antibody 27F1B9E3 is particularly suitable for use in diagnostic assays where it is necessary to capture even small soluble quantities of BAG3 protein. This was highlighted by comparing its efficiency in immunoprecipitation techniques to that of other monoclonal antibodies already described in literature.

[0101] Example 3. Identification of BAG3 protein epitopes recognized by the anti- BAG3 polyclonal antibody. To obtain a sandwich ELISA with two monoclonal antibodies, a coating antibody and a signal detection antibody, a study was conducted on epitopes recognized by the anti-BAG3 polyclonal antibody. For the study, peptide arrays were designed and used. The study was conducted at Pepscan Presto BV (Zuidersluisweg 2, 8243RC Lelystad, Netherlands). For the study, CLIPS technology was used (Timmerman, P; et al. (2009). "Functional reconstruction of structurally complex epitopes using CLIPS™ technology". The Open Vaccine Journal. 2 (1): 56-67) which consists in structurally fixing peptides in defined three-dimensional structures. CLIPS technology is routinely used to create peptide libraries in single, double or triple loop structures, as well as beta-sheet or alpha-helix structures. CLIPS library screening begins with conversion of the target protein into a library of up to 10,000 overlapping peptide constructs, using a combinatorial matrix design. On a solid support, a matrix of linear peptides (set 1) is synthesized, which are subsequently modeled into spatially defined CLIPS constructs (set 5). Constructs presenting the complete epitope in the correct conformation bind the antibody with high affinity, which is detected and quantified. Constructs presenting an incomplete epitope bind the antibody with lower affinity, while constructs not containing the epitope do not bind at all. Affinity information is analyzed to define in detail epitope sequence and conformation. In Figure 3, intensity profiles recorded for the anti-BAG3 polyclonal are reported as a function of B AG3 protein sequence. Signals were distinguished for two peptide sets: 1) set 1 peptides (linear peptides, intensity signals reported as line graph); 2) set 5 peptides (CLIPS peptides, intensity signals reported as dots).

[0102] In conclusion, the obtained data showed that the polyclonal antibody binds several peptides, a typical characteristic of a polyclonal antibody. In particular, 8 peptides were recognized and based on recorded signal intensity, 3 were classified as dominant epitopes and 5 as minor. The (dominant) peptides were located in the amino-terminal portion of BAG3 protein. Based on these results, peptide CP2 (SEQ. ID NO. 1) was synthesized, comprising a BAG3 protein region (aa. 89-213) containing the dominant epitopes found through the experiment shown in this example, and used as immunogen to obtain a new murine monoclonal antibody. The selected murine monoclonal antibody is 42F3D3.

[0103] These dominant epitopes are present in a BAG3 protein region (aa. 89-213) different from that of PEP1-PEP4 peptides with which monoclonal antibodies described in WO2011 / 067377A1, WO2013 / 189778 or in W02003055908A2 were obtained.

[0104] This difference in epitope position can be observed from the following SEQ ID. NO. 2, relating to BAG3 protein amino acid sequence, the sequence of PEP 1-4 peptides is indicated in bold while the sequence of dominant epitopes in region 89- 213 is indicated in bold / italic:

[0105] MSAATHSPMMQVASGNGDRDPLPPGWEIKIDPQTGWPFFVDHNSRTTT WNDPRVPSEGPKETPSSANGPSREGSRLPPAREGHPVYPQZPPGT / P / PEL HEGAENRQVHPFHVYPQPGMQRFRTEAAAAAPQRSQSPLRGMPETTQP DKQCGQVAAAAAAQPPASHGPERSQSPAASDCSSSSSSASLPSSGRSSLGS H^ZPPGTZS / PEt EQNVTRPAAQPSFHQAQKTHYPAQQGEYQTHQPVYH KIQGDD WEPRPLRAASPFRS S VQGAS SREGSP ARS STPLHSP SPIRVHT VVD RPQQPMTHRETAPVSQPENKPESKPGPVGPELPPGHIPIQVIRKEV DSKPVSQKPPPPSEKVEVKVPPAPVPCPPPSPGPSAVPSSPKSVATEERAA PSTAPAEATPPKPGEAEAPPKHPGVLKVEAILEKVQGLEQAVDNFEGKKT DKKYLMIEEYLTKELLALDSVDPEGRADVRQARRDGVRKVQTILEKLEQ KAIDVPGQVQVYELQPSNLEADQPLQAIMEMGAVAADKGKKNAGNAE DPHTETQQPEATAAATSNPSSMTDTPGNPAAP PEP1 : DRDPLPPGWEIKIDPQ

[0106] PEP2: SSPKSVATEERAAPS

[0107] PEP3: DKGKKNAGNAEDPHT

[0108] PEP4: NPSSMTDTPGNPAAP.

[0109] From the above, it is evident that the immunogen selected for generating a new anti- BAG3 antibody, designed to show greater affinity towards soluble BAG3 protein compared to other antibodies already described, is different from peptides used as immunogens and reported in patent applications WO2011 / 067377A1 and WO2013 / 189778A1 and from full-length recombinant BAG3 protein.

[0110] Example 4. Comparison of binding capacity of new antibody produced by clone 42F3D3 to recombinant BAG3 protein through indirect ELISA assay.

[0111] The monoclonal antibody obtained by immunizing animals with CP2 peptide is clone 42F3D3. Binding activity to recombinant BAG3 protein of antibody 42F3D3 was verified through an indirect ELISA assay and compared to other monoclonal antibodies described in known art. A 96-well microplate was incubated overnight at 4°C with recombinant BAG3 proteins. After coating, wells were washed with wash buffer (PBS 1X / O.1% Tween) and subsequently blocked for 1 hour at room temperature (RT) with Blocking buffer (PBS 1X / O.5% fish gelatin). Next, different concentrations of various anti-BAG3 antibodies were incubated in wells for 1 hour at RT (range 200 - 3.2 ng / ml).

[0112] After incubation, the plate was washed 5 times with wash buffer and then incubated for 30 minutes at RT with anti-mouse IgG antibody conjugated with peroxidase (HRP) (Bio-Rad). Finally, TMB solution was added for chromogenic reaction development, which was subsequently stopped with 0.5 M EESCk Absorbance (O.D.) was measured at 450 nm. As shown in the graph in Fig.4, antibody 42F3D3 shows more efficient binding compared to antibodies AC-2 mAb (see Examples 1 and 2) and AC-4 mAb described in WO2011 / 067377A1 and WO2013 / 189778A1 and capable of binding B AG3 peptides described as PEP-2 and PEP-4, respectively, and antibody AC-rb3 described in WO2014 / 147503A2 and obtained from immunization of animals with full-length human BAG3 protein.

[0113] Example 5. Comparison of immunoprecipitation capacity of new antibody produced by clone 42F3D3 through IP / WB assay.

[0114] The antibody selected to develop a sandwich ELISA assay aimed at detecting very low BAG3 protein concentrations in clinical samples must possess excellent ability to immunoprecipitate BAG3 protein both in its soluble form and within complex protein mixtures, such as cell lysates or supernatants containing secreted proteins. To this end, performance of monoclonal antibody produced by clone 42F3D3 was compared to that of other antibodies described in known art, evaluating its effectiveness in binding both purified recombinant BAG3 protein in solution and native BAG3 protein present in pancreatic tumor cell lysates and respective supernatants, where it is secreted. For this purpose, proteins were immunoprecipitated with 3 different anti-BAG3 monoclonal antibodies conjugated to magnetic beads (20 pl): the three antibodies used in this example are 42F3D3 compared to antibodies AC-4 and AC-rb3 (see Example 4). In detail, 100 pg of protein extract, 1 ml of supernatant or 1 ng of rBAG3 were incubated overnight at 4°C with 20 pl of antibodies conjugated to magnetic beads.

[0115] After incubation, immunocomplexes were washed four times with IP buffer IX (Invitrogen). Immunoprecipitated proteins, along with 10 pg of total proteins, were separated by SDS-PAGE and subsequently transferred to nitrocellulose membrane. The membrane was then incubated with an anti-B AG3 polyclonal antibody to detect BAG3 protein presence by immunoblot. Results reported in figure 5 clearly demonstrate that antibody 42F3D3 (indicated in figure as IP 42) is able to capture both purified soluble BAG3 protein and that present in total cell lysates (LT) and supernatants (Sur). In contrast, other antibodies used for comparison, such as those described in examples 1 and 2, fail to efficiently capture BAG3 protein under these conditions.

[0116] Example 6. Comparison of ELISA test performance with antibodies 42F3D3 and 27F1B9E3 with other anti-BAG3 monoclonal antibody pairs.

[0117] Figure 6 shows results obtained using different pairs of anti-BAG3 monoclonal antibodies. In particular, scalar concentrations of rBAG3 protein were tested. Antibodies used in the 4 ELISA test formats (performed according to ELISA method described below in Materials and Methods) are anti-BAG3 antibody AC -2, anti-BAG3 antibody AC-rb3, antibody 42F3D3 and antibody 27F1B9E3, using the following antibody combinations:

[0118] 1. Antibody 42F3D3 as primary coating antibody and AC-2 as secondary antibody;

[0119] 2. Antibody 42F3D3 as primary coating antibody and antibody 27F1B9E3 as secondary antibody;

[0120] 3. Antibody AC-rb3 as primary coating antibody and anti-BAG3 antibody AC -2 as secondary antibody;

[0121] 4. Anti-BAG3 antibody AC-rb3 as primary coating antibody and antibody 27F1B9E3 as secondary antibody.

[0122] As described above, antibody AC -2 is described in WO2011 / 067377 (and in WO2013 / 189778) and is capable of binding PEP-2 peptide and AC-rb3 described in WO2014147503A2 is obtained from immunization of animals with full-length human B AG3 protein.

[0123] Data obtained in Figure 6 clearly show that using antibody 42F3D3 in combination with anti-BAG3 antibody AC-2 shows poor signals in the test, while it shows signals increasing in relation to increasing BAG3 protein concentration when used in pair with antibody 27F1B9E3. Anti-BAG3 antibodies AC-2 and anti-BAG3 AC- rb3, when used in pair, do not show good sensitivity in an ELISA test, just as anti- BAG3 antibody AC -2 used in pair with 27F1B9E3.

[0124] Data reported in this example show that using the combination of antibodies 42F3D3 with 27F 1B9E3 in an ELISA test detects soluble B AG3 protein much more sensitively and specifically compared to other tested antibody pairs.

[0125] This effect is due to the fact that antibody 42F3D3 was obtained by immunizing animals with dominant epitopes that allow recognition and immunoprecipitation of native BAG3 protein present in biological samples with greater affinity and specificity compared to other tested antibodies known in the art.

[0126] Example 7. Specificity and performance of antibodies 42F3D3 and 27F1B9E3 in sandwich ELISA.

[0127] Figure 7 shows results obtained using monoclonal antibody 42F3D3 as first antibody (capture) and monoclonal antibody 27F1B9E3 as signal detection antibody (second antibody) in sandwich ELISA. Scalar concentrations of rBAG3 protein and control pancreatic adenocarcinoma PANC-1 cell lysates or transfected with a specific siRNA to negatively modulate BAG3 protein levels were tested. Results show that the described antibody pair detects rBAG3 protein up to a limit equal to 15.6 pg / ml and that in cells with reduced BAG3 levels, ELISA signal decreases compared to signal from cells not transfected with BAG3 -specific siRNA.

[0128] The obtained result therefore shows that the ELISA test performed using antibody 42F3D3 and antibody 27F1B9E3 detects rBAG3 protein up to a determination limit equal to 15.6 pg / ml and that in cell lysates containing a heterogeneous protein mixture it specifically detects reduction in BAG3 protein levels when cells are transfected with a specific siRNA that reduces intracellular BAG3 protein levels. It was observed that this determination limit is better than that observed with currently available anti-BAG3 antibodies on the market, such as #ABIN6954056, #ABIN6962016 (Antibodiesonline), #OKCD08090 (Aviva Systems Biology) which show low sensitivity (detection limit 150 pg / ml). While kit ab244047 (Abeam) shows low sensitivity and is optimized for cell and tissue extract analysis, not for serum, plasma or other body fluids where BAG3 protein concentrations are generally lower.

[0129] The method of the present invention has therefore proven particularly effective in determining B AG3 protein in biological samples such as serum, plasma, blood and other body fluids where BAG3 protein concentration is very low.

[0130] Example 8. Inter-assay performance of antibodies 27F1B9E3 and 42F3D3 in sandwich ELISA.

[0131] Figure 8 shows results obtained using monoclonal antibody 42F3D3 and monoclonal antibody 27F1B9E3 used in sandwich ELISA. Recorded signals were analyzed to verify inter-assay repeatability of generated sandwich ELISA.

[0132] Results show a low CV% (range 2.38-6.38) for each tested concentration. Moreover, different dilutions of rBAG3 protein detected in the reference curve are significantly different compared to blank sample reading. The identified assay therefore has a determination limit equal to 15.6 pg / ml.

[0133] Example 9. ELISA method performance in detecting BAG3 protein in serum.

[0134] In Figure 9, recombinant B AG3 protein was tested in serum spiking experiments at high (1000 pg / ml), intermediate (250 pg / ml) and low (62.5 pg / ml) concentrations (panel A).

[0135] Spiking experiments of the analyte in serum (or other matrices) are fundamental for technical validation of an ELISA kit (Enzyme-Linked Immunosorbent Assay) for several reasons: 1) Accuracy and Precision: by adding a known amount of analyte to serum, it can be verified whether the ELISA kit is able to correctly and precisely detect that amount. This helps determine test accuracy and precision.

[0136] 2) Matrix Effects: serum, like other biological matrices, may contain components that interfere with analyte detection. Adding, in spiking experiments, the analyte to serum helps identify and quantify these interference effects, ensuring the kit works correctly even in presence of such components.

[0137] 3) Linearity and Dynamic Range: by testing different analyte concentrations in serum, ELISA kit response linearity and its dynamic range can be determined, i.e., the concentration range in which the kit provides reliable results.

[0138] 4) Robustness: these experiments also help evaluate kit robustness, i.e., its ability to provide consistent results under different operating conditions.

[0139] These aspects are crucial to ensure the ELISA kit is reliable and can be used in analyte detection in clinical samples.

[0140] To evaluate the above parameters, recovery percentage of recombinant BAG3 protein signal within human serum, a complex matrix, is calculated using monoclonal antibody 42F3D3 and monoclonal antibody 27F1B9E3 used in sandwich ELISA. Recovery provides a direct index of kit effectiveness in detecting the protein without significant losses. In panel B (Figure 9), recombinant BAG3 signal recovery added to serum is reported, compared to that obtained in a buffer without human serum. At all tested concentration levels, signal recovery remained satisfactory, exceeding 90%.

[0141] Example 10. Analytical specificity performance of ELISA method in detecting BAG3 protein in serum.

[0142] In Figure 10, the ELISA method was tested on recombinant or purified proteins or molecules constitutively present in different biological samples. Results show that the described method does not present false positivity in presence of molecules and / or proteins present in biological samples such as hemoglobin, bilirubin and others as described in figure. Moreover, in presence of such molecules, detection of rBAG3 protein at intermediate concentration of 250 pg / ml is not subject to interference. These results demonstrate that said ELISA method is highly specific.

[0143] Example 11. Presence of BAG3 protein in serum samples from patients with oncological diseases.

[0144] Sera from patients with ovarian carcinoma (N=10), pancreatic carcinoma (N=10) and liver carcinoma (N=l 0) were tested for BAG3 protein levels through the ELISA KIT of the present invention. In Figure 11, BAG3 values found in these patients' sera are represented compared to healthy donors (N=4). Median BAG3 protein values found in carcinoma patients' sera are higher than in healthy donors. These are: 88 pg / ml in liver carcinoma patients, 65 pg / ml in pancreatic carcinoma patients and 33 pg / ml in ovarian carcinoma patients; in donors the reported median is 15 pg / ml, value at the kit's minimum determination limit.

[0145] Example 12. Presence of BAG3 protein in plasma samples from COVID-19 patients.

[0146] Blood was collected in sodium citrate tubes and kept at room temperature until processing. Blood samples were centrifuged at 3000 rpm at 4°C for 10 minutes. Subsequently, plasma supernatant was collected and stored in aliquots at -80°C until later use. Plasma B AG3 concentration was determined using an ELISA kit of the present invention. A total of 30 plasma samples were analyzed and results shown in Figure 12. COVID-19 patients, before any treatment, had significantly higher plasma BAG3 concentration (***p<0.0001) (mean 142.5 ± st. dev. 166.7 pg / mL) compared to healthy controls (mean 7.15 ± st. dev. 13.41 pg / mL).

[0147] Example 13. Analysis of serum BAG3 protein levels in patients at risk of heart failure. Serum levels of circulating BAG3 protein were also studied in different stages of heart failure risk population (according to UDHF staging - Universal Definition for Heart Failure) using the ELISA test according to the present invention, characterized by using antibody 42F3D3 as primary antibody and antibody 27F1B9E3 as secondary antibody, demonstrating that said test is able to determine with high sensitivity and specificity BAG3 protein presence in biological samples from patients at risk of heart failure and thus allows associating BAG3 protein quantity with different heart failure stages identified in patients.

[0148] Hereinafter the clinical characteristics associated with various stages. At risk (stage A): patients at risk of heart failure (HF), but without current or previous symptoms or signs of HF and without structural cardiac alterations nor elevated cardiac disease biomarkers.

[0149] Pre-HF (stage B): patients without current or previous symptoms or signs of HF, but with evidence of at least one of the following conditions: structural heart disease; abnormal cardiac function; elevated natriuretic peptide levels.

[0150] HF (stage C): patients with current or previous HF symptoms and / or signs caused by a structural and / or functional heart abnormality.

[0151] Advanced HF (stage D): patients with severe HF symptoms and / or signs at rest, recurrent hospitalizations despite optimal medical therapy (GDMT), refractory or intolerant to GDMT, requiring advanced therapies such as evaluation for transplant, mechanical circulatory support or palliative care.

[0152] Plasma samples from 1121 individuals undergoing coronary and / or peripheral angiography, distributed in various UDHF stages, were analyzed using the BAG3 test. Patients were followed for 3.7 years (CASABLANCA study, NCT00842868). Higher BAG3 concentrations predicted heart failure / cardiovascular death in subjects with stage A / B heart failure (HR = 1.13; p = 0.05), predicting an increase in total event burden in these individuals. In patients with stage C / D heart failure, higher BAG3 concentrations predicted death from cardiovascular events (HR = 1.24; p < 0.001).

[0153] Circulating BAG3 protein therefore represents a prognostic biomarker in cardiovascular diseases. Its stage-specific prognostic relevance highlights a potential role in heart failure progression and use of a diagnostic test like the ELISA test according to the present invention with high sensitivity and specificity allows identifying BAG3's clinical utility for risk stratification and cardiovascular disease management.

[0154] MATERIALS AND METHODS

[0155] BAG3 ELISA Test

[0156] 96-well microplates (MaxiSorp™, ThermoScientific, Waltham, MA, USA) were treated with 200 pl of solutions containing coating anti-BAG3 monoclonal antibody 42F3D3 (4pg / ml in PBS IX) and left at 4°C overnight. The next day, wells were washed with PBS IX, and non-specific site blocking was performed for 2 hours at room temperature using PBS IX containing 1% Fish gelatin (Sigma). When blocking buffer was removed, 70 microliters of BAG3 standard protein or 70 microliters of serum samples were distributed in wells with 70 microliters of PBS IX. Then 60 microliters of adsorbent diluent (1.65% Fish gelatin + 1.65 mg / ml bovine IgG diluted in 0.165% tween 20-3.3X PBS) were added to each well.

[0157] Plates were incubated overnight at 4°C, then washed six times for 1 minute with wash buffer, and subsequently a solution containing detecting antibody 27F1B9E3 (0.5% Fish gelatin+ 500pg / ml bovine IgG + 0.05pg / ml recombinant anti-BAG3 antibody 27F1B9E3 conjugated with HRP diluted in 0.05% tween 20-1X PBS) (200ul / pz) was added. Plates were again incubated at room temperature for 30 minutes and then washed six times for 1 minute. Subsequently, IX TMB solution (eBioscience, San Diego, CA, USA) was added to wells (200ul / pz) and chromogenic reaction was stopped by adding 0.5 M sulfuric acid. Optical density (OD) values were detected by spectrophotometer at wavelength of 450nm.

[0158] Production of Recombinant Antibodies

[0159] Sequences encoding antibodies were cloned into expression vector Evi-5 (Evitria AG, Switzerland) and expressed in CHO-K1 cells, using methods known in the art. For chimeric antibody production, murine constant regions were replaced with human constant regions, using methods known in the art. (Liu AY, Robinson RR, Murray ED Jr, Ledbetter JA, Hellstrom I, Hellstrom KE (1987) Production of a mouse— human chimeric monoclonal antibody to CD20 with potent Fc-dependent biologic activity. J Immunol 139:3521—3526.

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[0166] - Rosati A, Bersani S, Tavano F, Dalia Pozza E, De Marco M, Palmieri M, De Laurenzi V, Franco R, Scognamiglio G, Palaia R et al. Expression of the antiapoptotic protein BAG3 is a feature of pancreatic adenocarcinoma and its overexpression is associated with poorer survival. Am J Pathol. 2012b, 181,1524— 1529.

[0167] - Rosati A, Basile A, D'Auria R, d'Avenia M, De Marco M, Falco A, Festa M, Guerriero L, Iorio V, Parente R et al. BAG3 promotes pancreatic ductal adenocarcinoma growth by activating stromal macrophages. Nat Commun. 2015, 6, 8695.

[0168] - Rosati A, Ammirante M, Gentilella A, Basile A, Festa M, Pascale M, et al. 2007. Apoptosis inhibition in cancer cells: a novel molecular pathway that involves BAG3 protein. Int J Biochem Cell Biol. 2007, 39: 1337—1342.

[0169] - Iorio V, De Marco M, Basile A, Eletto D, Capunzo M, Remondelli P, et al, CAF- derived IL6 and GM-CSF cooperate to induce M2-like TAMs. Clin. Cancer Res. 2019; 15: 892-893.

[0170] - De Marco M, Del Papa N, Reppucci F, Iorio V, Basile A, Falco A, et al. BAG3 induces a-SMA expression in human fibroblasts and its over-expression correlates with poorer survival in fibrotic cancer patients. Journal of Cellular Biochemistry 2021; 123: 91-101. - Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495-7. doi: 10.1038 / 256495a0. PMID: 1172191.

Claims

CLAIMS1. An anti-BAG3 monoclonal antibody or a fragment thereof characterized by comprising a heavy chain variable region having at least 90% identity with amino acid sequence SEQ ID NO. 3, preferably at least 95% identity with amino acid sequence SEQ ID NO. 3, and a light chain variable region having at least 90% identity with amino acid sequence SEQ ID NO. 4, preferably at least 95% identity with amino acid sequence SEQ ID NO. 4.

2. An anti-BAG3 monoclonal antibody or a fragment thereof according to claim 1, characterized by comprising a heavy chain variable region having at least 98% identity with amino acid sequence SEQ ID NO. 3 and a light chain variable region having at least 98% identity with amino acid sequence SEQ ID NO. 4.

3. An anti-BAG3 monoclonal antibody or a fragment thereof according to any of claims 1 or 2, characterized in that the heavy chain variable region has the following H-CDR regions: H-CDR1 comprising amino acids of SEQ ID NO. 7, H-CDR2 comprising amino acids of SEQ ID NO. 8, H-CDR3 comprising amino acids of SEQ ID NO. 9 and the light chain variable region has the following L-CDR regions: L-CDR1 comprising amino acids of SEQ ID NO. 10, L-CDR2 comprising amino acids of SEQ ID NO. 11 and L-CDR3 comprising amino acids of SEQ ID NO. 12.

4. An anti-BAG3 monoclonal antibody or a fragment thereof according to any of claims 1-3 characterized in that said antibody is a natural or synthetic origin antibody, preferably an avian, mammalian or humanized antibody, more preferably said antibody is an F(ab) fragment, F(ab') fragment, F(ab')2 fragment, Fv fragment, diabody, ScFv, affibody, avimer, nanobody, antibody domain and / or single chains.

5. Use of the anti-BAG3 monoclonal antibody or a fragment thereof according to any of claims 1-4 in a method for determining presence and / or concentration ofsoluble BAG-3 protein in a biological sample, preferably said biological sample is selected from serum, plasma, urine, saliva or biopsy tissues.

6. Method for determining presence and / or concentration of BAG3 protein in a biological sample, characterized by using a first anti-BAG3 monoclonal antibody or a fragment thereof according to any of claims 1-4 and a second anti-BAG3 monoclonal antibody comprising a heavy chain variable region having amino acid sequence SEQ ID NO. 5 and a light chain variable region having amino acid sequence SEQ ID NO. 6.

7. Method according to claim 6, characterized in that the heavy chain variable region of the second anti-BAG3 monoclonal antibody has the following H-CDR regions: H-CDR1 comprising amino acids of SEQ ID NO. 13, H-CDR2 comprising amino acids of SEQ ID NO. 14, H-CDR3 comprising amino acids of SEQ ID NO. 15 and the light chain variable region of the second anti-BAG3 antibody comprises the following L-CDR regions: L-CDR1 comprising amino acids of SEQ ID NO. 16, L-CDR2 comprising amino acids of SEQ ID NO. 17 and L-CDR3 comprising amino acids of SEQ ID NO. 18.

8. Method according to any of claims 6 and 7, wherein the first anti-BAG3 monoclonal antibody is characterized in that the heavy chain variable region consists of the following H-CDR regions: H-CDR1 comprising amino acids of SEQ ID NO. 7, H-CDR2 comprising amino acids of SEQ ID NO. 8 and H-CDR3 comprising amino acids of SEQ ID NO. 9 and the light chain variable region consists of the following L-CDR regions: L-CDR1 comprising amino acids of SEQ ID NO. 10, L-CDR2 comprising amino acids of SEQ ID NO. 11 and L-CDR3 comprising amino acids of SEQ ID NO. 12, and the second anti-BAG3 monoclonal antibody is characterized in that the heavy chain variable region consists of the following H-CDR regions: H-CDR1 comprising amino acids of SEQ ID NO. 13, H-CDR2 comprising amino acids of SEQ ID NO. 14, H-CDR3 comprising aminoacids of SEQ ID NO. 15 and the light chain variable region consists of the following L-CDR regions: L-CDR1 comprising amino acids of SEQ ID NO. 16, L-CDR2 comprising amino acids of SEQ ID NO. 17 and L-CDR3 comprising amino acids of SEQ ID NO. 18.

9. Method according to any of claims 6-8, characterized in that the first anti-BAG3 monoclonal antibody is antibody 42F3D3 and the second anti-BAG3 monoclonal antibody is antibody 27F1B9E3.

10. Method according to any of claims 6-9 characterized by the following steps: a) determining presence and / or concentration of BAG3 protein in a previously obtained biological sample consisting of serum, plasma, urine, saliva or biopsy tissues; b) comparing obtained values with reference values or values obtained from a reference biological sample; and c) associating presence and / or concentration of BAG3 protein with a pathological condition selected from fibrotic diseases, inflammatory diseases or tumors.

11. Method according to any of claims 6-10, characterized by being an ELISA, CLIA (chemiluminescent assays), RIA (radioimmunoassays) or FIA (immunofluorescence assays) method, preferably said method is an ELISA method, more preferably said ELISA method is a sandwich ELISA.

12. Method according to claim 11, wherein step a) is performed by an ELISA method characterized by the following steps: i) immobilization of the first anti-BAG3 monoclonal antibody according to claim 3 in wells of an ELISA plate by incubation for 2-20 hours; ii) addition of biological sample and blocking solution; iii) incubation for 2-20 hours;iv) addition of the second anti-BAG3 monoclonal antibody according to claim 7 conjugated with HRP; v) incubation for 10-60 minutes; vi) incubation with a solution containing chromogenic substrate for 5-10 minutes at room temperature; vii) stopping chromogenic reaction by adding a denaturing solution, preferably sulfuric acid; viii) measuring optical density of the solution and calculating quantity or concentration of BAG3 protein in the biological sample.

13. Method according to claim 12, characterized in that the first anti-BAG3 antibody according to claim 3 is used at point iv) and the second anti-BAG3 antibody according to claims 7 is used at point i).

14. Method according to claim 10, characterized in that said fibrotic diseases are selected from scleroderma, systemic sclerosis, pulmonary fibrosis, liver fibrosis, cystic fibrosis, cardiac fibrosis or nephrogenic fibrosis, preferably systemic sclerosis; said inflammatory diseases are selected from rheumatoid arthritis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis, psoriasis, psoriatic arthritis, autoimmune hepatitis, vasculitis, idiopathic pulmonary fibrosis or infectious diseases causing inflammation such as COVID-19, viral hepatitis, HIV / AIDS, tuberculosis or sepsis, preferably COVID-19 and said tumors are selected from carcinomas, sarcomas, leukemias, lymphomas, melanomas or central nervous system tumors, more preferably breast, ovarian or pancreatic carcinoma.

15. Method according to claim 10, characterized in that in said method presence and / or concentration of soluble BAG3 protein is associated with presence of tumors, selected from carcinomas, sarcomas, leukemias, lymphomas, melanomasor central nervous system tumors, preferably breast, ovarian or pancreatic carcinoma.

16. Method according to any of claims 6-15 characterized in that the first anti- BAG3 antibody is used at a concentration between 1-8 pg / ml, preferably 4 pg / ml and the second anti-BAG3 antibody is used at a concentration between 0.01-0.10 pg / ml, preferably 0.05 pg / ml.

17. Method according to any of claims 6-16, characterized in that the first and second anti-BAG3 antibody can be used interchangeably.

18. Kit comprising a first anti-BAG3 antibody according to any of claims 1-3 and a second anti-BAG3 antibody according to any of claims 6 or 7.

19. Kit according to claim 18 for use in diagnosis of a pathological condition selected from fibrotic diseases, inflammatory diseases and tumors.

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