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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/303—Liver or Pancreas
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
  • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

• the present invention relates to the use of Anti-BAG3 antibodies as a medicament, in particular for use in the treatment of pancreatic tumours or other pathologies of an immune, inflammatory, neoplastic and/or degenerative nature.
• BAG3 protein is a 74 kDa cytoplasmic protein which belongs to the family of co- chaperonins that interact with the ATPase domain of the protein HSP70 (Heat Shock Protein) through the structural domain known as the BAG domain (amino acids 1 10- 124). Furthermore, BAG3 protein contains a WW domain (Trp-Trp), a proline-rich region (PXXP), and two conserved motifs IPV (IIe-Pro- Val), which can mediate binding to other proteins. Thanks to the nature of BAG3 protein as an adapter, attributable to the presence of many functional domains, such protein can therefore interact with different proteins.
• bag3 gene expression is constitutive for a few kinds of normal cells, including myocytes, while mutations thereof are associated with diseases of the skeletal and cardiac muscles. Furthermore, BAG3 protein is expressed in many types of primary tumours or tumour cell lines (lymphoid or myeloid leukemias, neuroblastoma, pancreatic cancer, thyroid cancer, breast cancer and prostate cancer, melanoma, osteosarcoma, glioblastoma and tumours of the kidney, colon, and ovary).
• primary tumours or tumour cell lines lymphoid or myeloid leukemias, neuroblastoma, pancreatic cancer, thyroid cancer, breast cancer and prostate cancer, melanoma, osteosarcoma, glioblastoma and tumours of the kidney, colon, and ovary.
• bag3 gene expression can be induced by stressors, such as oxidants, high temperatures, lack of serum, heavy metals, HIV-1 infections, etc.
• stressors such as oxidants, high temperatures, lack of serum, heavy metals, HIV-1 infections, etc.
• BAG3 protein influences cell survival in different types of cells, interacting with different molecular partners.
• BAG3 protein cooperates in a positive way with HSP70 protein to maintain BAX protein in the cytosol and prevent the translocation thereof into the mitochondria (Festa M. et al, Am J Pathol 178 (201 1) 2504-25). Finally, in some tumours, BAG3 has been shown to regulate proteins that modulate cell adhesion.
• cytoplasmic BAG3 protein has also been described in many different cellular systems and has been associated, not only with various tumours, but also in pathologies in general related to cell survival.
• patent application n. WO201 1/067377 describes soluble BAG3 protein, secreted externally to the cell, as a biochemical marker in serum, which is highly specific for the diagnosis of certain pathological conditions, such as cardiac pathologies and pancreatic tumour.
• concentration of (extra-cellular) soluble BAG3 is generally greater than 10 ng/ml.
• BAG3 protein is expressed in 346/346 patients with pancreatic ductal adenocarcinoma (PDAC) and is released by the cells of the pancreatic tumour, but such protein is not expressed in either the surrounding nonneoplastic tissues or in a normal pancreas; likewise, it has been reported that the levels of BAG3 expression are related to patient survival.
• PDAC pancreatic ductal adenocarcinoma
• the results of the study demonstrate that the use of specific siRNA molecules for BAG3 mRNA can silence bag3 gene expression and induce cell death, confirming that BAG3 protein is an important survival factor for pancreatic tumour cells and that the down-regulation thereof, when combined with gentamicin, may contribute to the eradication of the tumor cells (Rosati et al., Am J Pathol. 2012 Nov: 181 (5): 1524-9).
• any of the anti-BAG3 antibodies described in patent application n. WO03/055908 able to inhibit, specifically, the activity of soluble BAG3 protein (i.e. extra-cellular) on macrophages, that are considered the target cells proves particularly effective in the treatment of those pathologies characterised by the activation of macrophages, such as neoplastic diseases and diseases of an inflammatory, immune, or degenerative nature.
• these specific antibodies for BAG3 can bind and block, in a highly selective and targeted manner, pathological effects related to BAG3 protein when secreted by cells.
• anti-BAG3 antibodies in this process has the surprising advantage of being more specific for the selected pathological states characterised by the over-expression and release of BAG3 protein, and also less damaging in terms of side effects.
• soluble BAG3 protein is understood as extra-cellular BAG3 protein, i.e. the protein secreted externally to the cell.
• One aim of the present invention is therefore the use of anti-BAG3 antibodies as a medicament.
• the antibodies useable in accordance with the present invention may be either monoclonal or polyclonal antibodies, and are preferably monoclonal antibodies.
• said monoclonal antibodies may be chosen from the following: murinr antibodies, humanised antibodies, chimeric antibodies, recombinant antibodies, conjugated antibodies, scFv fragments (diabody, triabody and tetrabody), Fab fragments, and fragments F (ab') 2.
• polyclonal antibody refers to a mixture of antibodies which are genetically different since produced by different plasma cells and which recognise a different epitope of the same antigen.
• the term "monoclonal antibody” refers to a set of antibodies which are all identical since produced by cell lines from only one type of immune cell (i.e. a cell clone).
• humanized antibody refers to an antibody of human origin, whose hypervariable region has been replaced by the homologous region of non-human monoclonal antibodies.
• chimeric antibody refers to an antibody containing portions derived from different antibodies.
• recombinant antibody refers to an antibody obtained using recombinant DNA methods.
• conjugated antibody refers to antibodies conjugated with drugs, toxins, radioactive substances or other agents.
• scFv fragment single chain variable fragment refers to immunoglobulin fragments only capable of binding with the antigen concerned. ScFv fragments can also be synthesised into dimers (diabodies), trimers (triabodies) and tetramers (tetrabodies) using peptide linkers.
• Fab fragment (antigen-binding fragment) and "Fab2 fragment” refer to immunoglobulin fragments consisting of a light chain linked to the Fc fragment of the adjacent heavy chain, and such fragments are monovalent antibodies. When the Fab portions are in pairs, the fragment is called Fab2.
• hybrida refers to a cell producing monoclonal antibodies.
• the monoclonal antibodies used in the examples were obtained by immunising mice against four distinct BAG3 protein peptides using any method known to a person skilled in the art. Such peptides were chosen because they are BAG3 protein-specific and are not shared with any other protein, including BAG proteins.
• sequences of the four peptides are included in the BAG3 amino acid sequence
• SEQ ID NO 2 SSPKSVATEERAAPS; (includes BAG3 protein amino acids 385-399);
• SEQ ID NO 3 DKGKKNAGNAEDPHT; (includes BAG3 protein amino acids 533-
• NPSSMTDTPGNPAAP NPSSMTDTPGNPAAP; (includes BAG3 protein amino acids 561 -575).
• said antibodies may be obtained by means of the Multiple Antigene Peptide approach (MAP) (Keah HH et a!., J Pept Res (1988); 51 : 2. Tarn JP; Proc Natl acad Sci USA (1988), 85: 5409. Ota S, et al., Cancer Res (2002), 62: 1471), using the following map constructs:
• MAP Multiple Antigene Peptide approach
• MAP-BAG3-1 nh2- DRDPLPPGWEIKIDPQ- MAP (which contains sequence SEQ ID NO: 1);
• MAP-BAG3-2 nh2- SSPKSVATEERAAPS - MAP (which contains sequence SEQ ID NO: 2);
• MAP-BAG3-3 nh2- DKGKKNAGNAEDPHT - MAP (which contains sequence SEQ ID NO: 3);
• MAP-BAG3-4 nh2- NPSSMTDTPGNPAAP - MAP (which contains sequence SEQ ID NO: 4);
• said polyclonal anti- BAG3 antibodies are obtained by immunising the animals against one of the four peptides of the sequences SEQ ID NO. 1 - 4 stated above.
• the monoclonal anti-BAG3 antibodies of the present invention are obtained by means of a standard procedure (Tassone P., et al., Tissue Antigens 51 : 671 (1998)) using the four MAP-BAG3 peptides described above and are produced by at least one of the nine mother clones chosen from the following: AC-1 , AC-2, AC-3, AC-4, AC-5, AC-6, AC-7, AC-8, or AC-9 (described in WO03/055908), which contain specific hybridomas for each of the four MAP-BAG3 constructs used.
• the antibodies used are monoclonal anti-BAG3 antibodies obtained from at least one of the aforesaid mother clones, and preferably at least one chosen from the following: AC-1 , AC-2, AC-3, AC-4, or AC-5. More preferably, said monoclonal antibodies are obtained from at least one mother clone chosen from the following: AC-1 , AC-2, and AC-3.
• the monoclonal anti-BAG3 antibodies envisaged in the present invention are obtained from at least one of the following clones: AC-rb1 , AC-rb2, AC-rb3 and AC.rb4, and/or at least one of the following subclones: AC-rb1 a, AC-rb1b, AC-rb2a, AC-rb2b, AC-rb3a, AC-rb3b, AC-rb4a, and AC-rb4b.
• the monoclonal antibodies produced by all these clones and subclones recognise the BAG3 recombinant protein in an ELISA test. (Example 2).
• said monoclonal anti-BAG3 antibodies are those that recognise epitopes in the BAG3 protein amino acid sequence, which include at least one of the following fragments: 18-33, 385-399, 533-547 or 562-575.
• a further aim of the present invention is the use of the aforesaid anti-BAG3 antibodies in the treatment of a particular pathological state which involves the activation of macrophages.
• pathological state can be chosen from: neoplastic diseases, inflammatory diseases, immune diseases, and/or degenerative diseases.
• such neoplastic diseases may be either pancreatic tumour or bladder tumor, more preferably pancreatic tumour.
• said inflammatory diseases can be chosen from diseases related to inflammation of the skin, nerves, bones, blood vessels, and connective tissues, and more preferably, psoriasis, arthritis, neuritis, connectivitis.
• said immune diseases can be chosen from autoimmune diseases such as rheumatic diseases, connective tissue diseases, neuromuscular diseases, endocrine diseases, gastrointestinal diseases, haematologic diseases, skin diseases, and vasculitis, and more preferably, rheumatoid arthritis, multiple sclerosis, connectivitis, lupus erythematosus, endometriosis, and ulcerative colitis.
• said degenerative diseases can be chosen from neurodegenerative diseases and muscular degenerative diseases, and more preferably Alzheimer's disease, Parkinson's disease, and muscular dystrophy.
• the anti-BAG3 antibodies are used in the treatment of neoplastic diseases, inflammatory diseases, immune diseases, and degenerative diseases.
• a further aim of the present invention is a pharmaceutical composition
• a pharmaceutical composition comprising the aforesaid anti-BAG3 antibody in association with at least one pharmaceutically acceptable excipient.
• a further object of the present invention is the use of said composition as a medicament.
• a preferred embodiment of the present invention is the use of the composition in the treatment of neoplastic diseases and diseases of an inflammatory, immune and/or degenerative nature.
• composition of the present invention can be formulated in a form suitable for oral administration or in a form suitable for parenteral or topical administration.
• said oral form can be chosen from the following: tablets, capsules, solutions, suspensions, granules, and oily capsules.
• said topical form can be chosen from the following: cream, ointment, ointment, solution, suspension, eye drops, pessary, nebuliser solution, spray, powder, or gel.
• said parenteral form can be either an aqueous buffer solution or an oily suspension.
• Said parenteral administration include administration by intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intranodal, or intrasplenic means.
• Figure 1 Picture showing the reduction of tumour growth in mice treated with AC-rb2.
• Example 1 Treatment of BALB/c mice with anti-BAG3 antibody (AC- rb2). Nude mice (nu/nu) female BALB/c 6 weeks (Charles Rivers Wilmington, MA, USA) were caged (3 per cage) with food and water ad libitum and kept in 12 h light/dark cycles in standard, pathogen-free conditions. The research protocol was approved by the Ethics Committee in accordance with Italian Ministry of Health official guidelines. After one week of acclimatisation, the mice were subjected to inoculation of cancer cells. 10 mice were used in total, each one individually identified. The entire experiment was conducted in laminar flow hoods and all surgical procedures were performed in strict compliance with aseptic techniques.
• AC- rb2 anti-BAG3 antibody
• mice were anesthetised with 100 mg/ml of ketamine HC1 and 20 mg/ml of xylazine injected intraperitoneally; they were then subjected to laparotomy and the tail of the pancreas was gently exteriorised.
• MlA-PaCa 2 RFP cells (2.5 ⁇ 10 6 ) were resuspended in 40 microlitres of PBS 1 X in a 1 ml syringe; using a 25G needle, the cells were injected into the tail of the pancreas and the injection point was swabbed with sterile cotton. Once homeostasis was ascertained, the tail of the pancreas was repositioned in the abdomen and the wound closed.
• mice were randomised into two groups: the first received an intraperitoneal injection of 100 mg/kg of mouse IgG and the second 100 mg/kg of a murine monoclonal anti-BAG3 antibody (AC- rb2). This treatment was performed twice a week in total and the mice were then anesthetised again to check the tumour area using Macro Fluo and LAS V3.7 software, by Leica Mycrosystems Ltd. In each imaging, the tumour mass was determined by quantification of the fluorescent area. Results
• Example 2 ELISA test on the antibodies obtained from the subclones AC-rbla, AC-rblb, AC-rb2a, AC-rb2b, AC-rb3a, AC-rb3b, AC-rb4a and AC-rb4b.
• the NUNC Maxisorp 96-well microtitre plates were functionalised with BAG3 recombinant protein 1 ⁇ g/ml in PBS 1X pH 7 (50 ⁇ l/well) and incubated for 18 hours at 4°C. The plates were then washed twice with a buffer wash (PBS 1X + 0.05% Tween- 20), and then kept in place for one hour at room temperature with 0.5% fish gelatin in PBS 1X (150 ⁇ l/well).
• the plates were buffer-washed twice and the supernatants of the eight subclones were diluted 1/10, 1/100, and 1/1000, in a 0.5% fish gelatin in PBS 1X and then incubated (50 ⁇ /well) in triplicate and incubated at room temperature for 2 hours. The plates were then buffer-washed six times. To develop the signal, murine anti-lgG antibodies (H + L) (Sigma Aldrich) were used, diluted in the same buffer 1/20,000, then added to the plate (50 ⁇ l/well) and incubated at 4°C for 30 minutes.
• H + L murine anti-lgG antibodies

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