WO2023023654A1 - Methods and compositions for treating fibrotic diseases - Google Patents

Methods and compositions for treating fibrotic diseases Download PDF

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WO2023023654A1
WO2023023654A1 PCT/US2022/075226 US2022075226W WO2023023654A1 WO 2023023654 A1 WO2023023654 A1 WO 2023023654A1 US 2022075226 W US2022075226 W US 2022075226W WO 2023023654 A1 WO2023023654 A1 WO 2023023654A1
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age
seq
antibody
protein
cells
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Lewis S. Gruber
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Siwa Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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

Definitions

  • Fibrosis is the formation of excess fibrous connective tissue in an organ or in tissue.
  • Connective tissue such as extracellular matrix proteins, is produced by fibroblasts in response to pro-fibrotic factors including transforming growth factor beta (TGFp), connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF) and interleukin 4 (IL-4).
  • TGFp transforming growth factor beta
  • CGF connective tissue growth factor
  • PDGF platelet-derived growth factor
  • IL-4 interleukin 4
  • Fibroblasts may differentiate into myofibroblasts through an epithelial to mesenchymal transition largely mediated by TGF
  • 31 Transforming growth factor-p1 (TGF-
  • HA hyaluronan
  • EGFR epidermal growth factor receptor
  • CD44 co-localization in lipid rafts
  • Myofibroblasts are key mediators of profibrotic conditions and proliferate in response to TGFp signaling (Harris, W. T. et al., “Myofibroblast differentiation and enhanced TGF- signaling in cystic fibrosis lung disease”, PLoS One, Vol. 8, No. 8, 8 pages (2013)).
  • Fibrosis can result from specific trauma such as surgical complications, exposure to radiation, burns or physical injury.
  • Some therapeutic treatments such as the administration of chemotherapeutic drugs or exposure to ionizing radiation (radiotherapy) as used in treating cancer, are known to cause fibrosis as a side effect.
  • Certain cancers such as myelofibrosis (bone marrow cancer), can also cause fibrosis.
  • Fibrotic diseases can develop in many parts of the body in humans and animals and may affect organs including the lungs (interstitial lung disease, pulmonary fibrosis, acute lung injury), liver (liver fibrosis, cirrhosis), kidneys (kidney disease, nephrosclerosis), heart (cardiovascular disease) and eyes (macular degeneration, vitreal retinopathy) (Wynn, T. A., “Fibrotic disease and the TH1/TH2 paradigm”, Nature Reviews Immunology, Vol. 4, No. 8, p. 583-594 (2004)). Fibrotic diseases that are characterized by excessive connective tissue accumulation and progressive tissue contraction are known as fibroproliferative disorders (Bitterman, P.B. et al., “Fibroproliferative disorders”, Chest, Vol. 99, No. 3, pp. 81S-84S (1991)).
  • fibroproliferative disorders include scleroderma (systemic and local), hypertrophic scarring, keloid scarring, cirrhosis, nephrosis and restenosis.
  • Pulmonary fibrosis (scarring of the lungs) is a respiratory disease characterized by the development of fibrous tissue in the walls of the air sacs of the lungs. The formation of scar tissue impairs the ability of the lungs to oxygenate blood, which often presents as shortness of breath (dyspnea). Other symptoms of pulmonary fibrosis include a chronic dry or hacking cough, fatigue, weakness, chest pain or discomfort, loss of appetite, unexplained weight loss and clubbing of the tips of the fingers and toes. The scarring caused by pulmonary fibrosis is permanent and the loss of lung function cannot be restored.
  • Pulmonary fibrosis may result from a variety of different sources.
  • Known causes of pulmonary fibrosis include autoimmune diseases that produce inflammation or scarring in the lungs, such as rheumatoid arthritis, scleroderma and autoimmune muscle diseases; occupational exposure to small inorganic particles, such as asbestos, beryllium, coal dust, silica and heavy metal dusts; environmental exposure to small organic particles, such as animal proteins, bacteria and molds; radiation exposure, including environmental radiation and cancer radiotherapy; bacterial or viral infections; and exposure to medications, such as nitrofurantoin, sulfasalazine, amiodarone, propranolol, phenytoin, methotrexate, bleomycin and oxaliplatin (Pulmonary Fibrosis Foundation, “About PF”, available online at www.pulmonaryfibrosis.org/life-with-pf/about-pf, accessed on July 18, 2017).
  • Idiopathic pulmonary fibrosis may result from DNA abnormalities, and inheritable forms of idiopathic pulmonary fibrosis include familial pulmonary fibrosis (FPF) and familial interstitial pneumonia (FIP) (Id ).
  • FPF familial pulmonary fibrosis
  • FIP familial interstitial pneumonia
  • pulmonary fibrosis There is no standard treatment for pulmonary fibrosis. Lifestyle changes such as not smoking and maintaining a healthy weight can improve symptoms of pulmonary fibrosis. Pulmonary rehabilitation exercise programs can help to preserve lung function. Oxygen therapy may be used to improve oxygen delivery to the body.
  • Various pharmacotherapies have been investigated for treating pulmonary fibrosis including corticosteroids (immunosuppressant/anti-inflammatory), cyclophosphamide (anti-inflammatory), azathioprine (immunosuppressant), mycophenolate mofetil (immunosuppressant), N-acetylcysteine (antioxidant) and proton pump inhibitors (gastric acid reducer).
  • Nintedanib (OFEV®) and pirfenidone (ESBRIET®) are the only U.S. Food and Drug Administration-approved medications for treating idiopathic pulmonary fibrosis. However, these medications slow the progression of idiopathic pulmonary fibrosis, but do not treat the disease or prevent its onset. Severe cases of pulmonary fibrosis may be treated with lung transplantation. There are no pharmacotherapies for treating or preventing the onset of pulmonary fibrosis.
  • liver fibrosis is the formation of fibrous tissue in the liver.
  • Cirrhosis is the most well-known fibrotic liver disease and may be caused by alcoholism, hepatitis B, hepatitis C, non-alcoholic fatty liver disease, primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune hepatitis, hereditary hemochromatosis, Wilson’s disease, Indian childhood cirrhosis, alpha 1-antitrypsin deficiency (A1AD), cardiac cirrhosis, galactosemia, glycogen storage disease type IV, cystic fibrosis and exposure to hepatoxic drugs or toxins.
  • A1AD alpha 1-antitrypsin deficiency
  • cardiac cirrhosis galactosemia
  • glycogen storage disease type IV cystic fibrosis and exposure to hepatoxic drugs or toxins.
  • Cirrhosis may lead to complications such as ascites, esophageal variceal bleeding, hepatic encephalopathy, hepatorenal syndrome, spontaneous bacterial peritonitis, portal hypertensive gastropathy, infection and hepatocellular carcinoma.
  • cirrhosis may be reduced by vaccination against specific diseases that cause cirrhosis, such as hepatitis B or hepatitis C.
  • cirrhosis that develops as a side effect may be managed by treating the underlying disease, such as by administering interferon and corticosteroids to patients experiencing cirrhosis due to hepatitis.
  • Treatment options for cirrhosis are limited.
  • Further liver damage may be prevented with lifestyle changes including abstaining from alcohol, avoiding acetaminophen and maintaining a healthy diet. Liver transplantation may ultimately be necessary for patients who experience liver failure or cannot control the complications of cirrhosis.
  • Dermal fibrosis is the formation of fibrous tissue in the skin. Fibrotic skin disorders include keloid scarring, hypertrophic scarring, scleroderma, nephrogenic fibrosing dermopathy, mixed connective tissue disease, scleromyxedema, scleredema and eosinophilic fasciitis. Dermal fibrosis may be a minor aesthetic inconvenience, or may result in severe health complications.
  • scleroderma may affect major organs such as the lungs (loss of lung function, severe lung disease and lung tissue scarring), heart (scarring and weakness of the heart, swelling of the heart muscle and irregular heartbeat) and kidneys (high blood pressure and kidney failure) (“Scleroderma Living With It”, National Institute of Arthritis and Musculoskeletal and Skin Diseases, available online at www.niams.nih.gov/health-topics/scleroderma#tab-living-with (2016)).
  • keloid and hypertrophic scars may be treated with local steroid injections, pressure dressings, topical chemotherapy drugs, laser light therapy, pulsed-dye laser therapy, radiation therapy, surgical excision, silicone gel sheeting application and administration of hyaluronic acid (WO 2014/179262).
  • Other treatments for dermal fibrosis focus on the symptoms and complications of the specific fibrotic disorder.
  • scleroderma therapies include calcium channel blockers, angiotensin II receptor antagonists, angiotensin converting enzyme (ACE) inhibitors, non-steroidal anti-inflammatory drugs (NSAIDs), COX-2 inhibitors, acetaminophen, corticosteroids, narcotics, antacids, histamine H2-receptor antagonists, proton pump inhibitors, prokinetic agents, somatostatin agonists, antibiotics, prostaglandin derivatives, endothelin receptor antagonists, IP receptor agonists, phosphodiesterase type 5 (PDE5) inhibitors, anti-fibrotic agents, anti-inflammatory agents, tyrosine kinase inhibitors, immunosuppressants and alkylating agents (“Current treatments available for scleroderma patients”, Scleroderma Research Foundation, available online at www.srfcure.org/for-patients/current-treatments (2016)).
  • ACE angiotensin converting enzyme
  • NSAIDs non-steroidal
  • Kidney fibrosis may be further classified based on the location of the affected tissue, such as nephrosclerosis (the nephron), glomerulosclerosis (the glomerulus) or tubulointerstitial renal fibrosis (the interstitium).
  • Chronic kidney disease (CKD) a progressive disease, is frequently caused by kidney fibrosis.
  • CKD chronic kidney disease
  • Two primary factors that promote CKD are diabetes and high blood pressure (“About chronic kidney disease: symptoms and causes”, National Kidney Foundation, available online at www.kidney.org/atoz/content/about- chronic-kidney-disease (2020)).
  • urinary fibrosis The damage caused by urinary fibrosis is permanent. Further damage may be reduced by managing underlying conditions that contribute to urinary fibrosis, especially diabetes and high blood pressure. Lifestyle changes such as not smoking, limiting alcohol intake and maintaining a healthy weight can prevent further worsening of kidney fibrosis. Dietary changes such as adopting a low-salt and low- fat diet may also help. If untreated, urinary fibrosis may lead to loss of kidney function, end-stage renal disease or kidney failure. Individuals with kidney failure must undergo regular dialysis to cleanse the blood or receive a kidney transplant, if they are healthy enough for the procedure and are able to find a suitable donor. Pharmacotherapies for treating urinary fibrosis are limited and primarily focus on controlling symptoms, reducing complications and slowing the progression of the disease.
  • Desmoplasia refers to growth of dense connective tissue or stroma, and it may occur as result of injury or neoplasia.
  • the stromal reaction in cancer is similar to the stromal reaction induced by injury or wound repair, causing scar-like tissue to be built around the cancer.
  • the surrounding stroma plays a very important role in the progression of cancer. It has been suggested that tumor cells cause the proliferation of fibroblasts and subsequent secretion of collagen (El-Torky, M., et al., Collagens in scar carcinoma of the lung, The American Journal of Pathology, vol. 121, no. 2, pp. 322-326 (1985)).
  • Senescent cells are cells that are partially-functional or non-functional and are in a state of proliferative arrest. Senescence is a distinct state of a cell, and is associated with biomarkers, such as activation of the biomarker p16 lnk4a , and expression of p-galactosidase. Senescence begins with damage or stress (such as overstimulation by growth factors) of cells.
  • AGEs Advanced glycation end-products
  • AGEs also referred to as AGE-modified proteins or peptides, or glycation end-products
  • AGE-modified proteins or peptides or glycation end-products
  • Maho K. et al., Membrane Proteins of Human Erythrocytes Are Modified by Advanced Glycation End Products during Aging in the Circulation, Biochem Biophys Res Commun., Vol. 258, 123, 125 (1999)
  • This process begins with a reversible reaction between the reducing sugar and the amino group to form a Schiff base, which proceeds to form a covalently-bonded Amadori rearrangement product.
  • AGEs may also be formed from other processes.
  • the advanced glycation end product, N e -(carboxymethyl)lysine is a product of both lipid peroxidation and glycoxidation reactions.
  • AGEs have been associated with several pathological conditions including inflammation, atherosclerosis, stroke, endothelial cell dysfunction, and neurodegenerative disorders (Bierhaus A, “AGEs and their interaction with AGE-receptors in vascular disease and diabetes mellitus. I. The AGE concept,” Cardiovasc Res, Vol. 37(3), 586-600 (1998)).
  • AGE-modified proteins are also a marker of senescent cells. This association between AGEs and senescence is well known in the art. See, for example, Gruber, L. (WO 2009/143411, 26 Nov. 2009), Ando, K. etal. (Membrane Proteins of Human Erythrocytes Are Modified by Advanced Glycation End Products during Aging in the Circulation, Biochem Biophys Res Commun., Vol. 258, 123, 125 (1999)), Ahmed, E.K. etal. (“Protein Modification and Replicative Senescence of WI-38 Human Embryonic Fibroblasts” Aging Cells, vol. 9, 252, 260 (2010)), Vlassara, H. et al.
  • Ahmed, E.K. et al. indicates that glycation end-products are “one of the major causes of spontaneous damage to cellular and extracellular proteins” (Ahmed, E.K. et al., see above, page 353). Accordingly, the accumulation of glycation endproducts is associated with senescence and lack of function.
  • MG methyl glyoxal
  • Damage or stress to mitochondrial DNA also sets off a DNA damage response which induces the cell to produce cell cycle blocking proteins. These blocking proteins prevent the cell from dividing. Continued damage or stress causes mTOR production, which in turn activates protein synthesis and inactivates protein breakdown. Further stimulation of the cells leads to programmed cell death (apoptosis).
  • p16 is a protein involved in regulation of the cell cycle, by inhibiting the S phase (synthesis phase). It can be activated during ageing or in response to various stresses, such as DNA damage, oxidative stress or exposure to drugs. p16 is typically considered a tumor suppressor protein, causing a cell to become senescent in response to DNA damage and irreversibly preventing the cell from entering a hyperproliferative state. However, there has been some ambiguity in this regard, as some tumors show overexpression of p16, while others show downregulated expression. Evidence suggests that overexpression of p16 is some tumors results from a defective retinoblastoma protein (“Rb").
  • Rb defective retinoblastoma protein
  • p16 acts on Rb to inhibit the S phase, and Rb downregulates p16, creating negative feedback.
  • Defective Rb fails to both inhibit the S phase and downregulate p16, thus resulting in overexpression of p16 in hyperproliferating cells (Romagosa, C. et al., p16 lnk4a overexpression in cancer: a tumor suppressor gene associated with senescence and high-grade tumors, Oncogene, Vol. 30, 2087-2097 (2011)).
  • Senescent cells are associated with secretion of many factors involved in intercellular signaling, including pro-inflammatory factors; secretion of these factors has been termed the senescence-associated secretory phenotype, or SASP (Freund, A. “Inflammatory networks during cellular senescence: causes and consequences” Trends Mol Med. 2010 May;16(5):238-46). Autoimmune diseases, such as Crohn’s disease and rheumatoid arthritis, are associated with chronic inflammation (Ferraccioli, G. etal.
  • Chronic inflammation may be characterized by the presence of pro- inflammatory factors at levels higher than baseline near the site of pathology, but lower than those found in acute inflammation.
  • Senescent cells also upregulate genes with roles in inflammation including IL-1 p, IL-8, ICAM1, TNFAP3, ESM1 and CCL2 (Burton, D.G.A. et al., “Microarray analysis of senescent vascular smooth muscle cells: a link to atherosclerosis and vascular calcification”, Experimental Gerontology, Vol. 44, No. 10, pp. 659-665 (October 2009)).
  • ROS reactive oxygen species
  • the p16/Rb pathway leads to the induction of ROS, which in turn activates the protein kinase C delta creating a positive feedback loop that further enhance ROS, helping maintain the irreversible cell cycle arrest; it has even been suggested that exposing cancer cells to ROS might be effective to treat cancer by inducing cell phase arrest in hyperproliferating cells (Rayess, H. et al., Cellular senescence and tumor suppressor gene p16, Int J Cancer, Vol. 130, 1715-1725 (2012)).
  • mice that were treated to induce senescent cell elimination were found to have larger diameters of muscle fibers as compared to untreated mice. Treadmill exercise tests indicated that treatment also preserved muscle function. Continuous treatment of transgenic mice for removal of senescent cells had no negative side effects and selectively delayed age-related phenotypes that depend on cells. This data demonstrates that removal of senescent cells produces beneficial therapeutic effects and shows that these benefits may be achieved without adverse effects.
  • Vaccines have been widely used since their introduction by Edward Jenner in the 1770s to confer immunity against a wide range of diseases and afflictions.
  • Vaccine preparations contain a selected immunogenic agent capable of stimulating immunity to an antigen.
  • antigens are used as the immunogenic agent in vaccines, such as, for example, viruses, either killed or attenuated, and purified viral components.
  • Antigens used in the production of cancer vaccines include, for example, tumor-associated carbohydrate antigens (TACAs), dendritic cells, whole cells and viral vectors. Different techniques are employed to produce the desired amount and type of antigen being sought. For example, pathogenic viruses are grown either in eggs or cells. Recombinant DNA technology is often utilized to generate attenuated viruses for vaccines.
  • TACAs tumor-associated carbohydrate antigens
  • dendritic cells dendritic cells
  • whole cells whole cells
  • viral vectors Different techniques are employed to produce the desired amount and type of antigen being sought. For example, pathogenic viruses are grown either in eggs or cells. Recombinant DNA technology is often utilized to generate attenuated viruses for vaccines.
  • Vaccines may therefore be used to stimulate the production of antibodies in the body and provide immunity against antigens.
  • the immune system may destroy or remove cells that express the antigen.
  • the invention is a method of treating or preventing the onset of a fibrotic disease comprising administering to a subject a composition comprising an anti-AGE antibody.
  • the invention is a method of treating or preventing the onset of a fibrotic disease comprising administering to a subject a vaccine comprising an AGE antigen.
  • fibrotic disease means a disease or disorder characterized by the formation of fibrous tissue.
  • fibrotic diseases include interstitial lung disease, pulmonary fibrosis, liver fibrosis, cirrhosis, urinary fibrosis, kidney fibrosis, nephrosclerosis, nephrosis, cardiovascular disease, macular degeneration, vitreal retinopathy, scleroderma (systemic and local), hypertrophic scarring, keloid scarring, restenosis, myelofibrosis, nephrogenic fibrosing dermopathy, mixed connective tissue disease, scleromyxedema, scleredema, and eosinophilic fasciitis.
  • fibrotic disease does not include atherosclerosis.
  • Pulmonary fibrosis means a disease or disorder characterized by the formation of fibrous tissue in the lungs.
  • Pulmonary fibrosis includes idiopathic pulmonary fibrosis (IPF), idiopathic nonspecific interstitial pneumonia (NSIP), cryptogenic organizing pneumonia (COP), sarcoidosis, familial pulmonary fibrosis (FPF), familial interstitial pneumonia (FIP), asbestosis, silicosis, berylliosis, hypersensitivity pneumonitis, atypical pneumonia, pneumocystis pneumonia, tuberculosis, respiratory syncytial virus, acute interstitial pneumonitis/pneumonia (also known as Hamman-Rich syndrome), chronic obstructive pulmonary disease (COPD), emphysema and mesothelioma.
  • IPF idiopathic pulmonary fibrosis
  • NIP nonspecific interstitial pneumonia
  • COP cryptogenic organizing pneumonia
  • sarcoidosis familial
  • liver fibrosis means a disease or disorder characterized by the formation of fibrous tissue in the liver. Liver fibrosis includes cirrhosis.
  • dermal fibrosis means a disease or disorder characterized by the formation of fibrous tissue in the skin.
  • Dermal fibrosis includes keloid scarring, hypertrophic scarring, scleroderma, nephrogenic fibrosing dermopathy, mixed connective tissue disease, scleromyxedema, scleredema and eosinophilic fasciitis.
  • Urinary fibrosis means a disease or disorder characterized by the formation of fibrous tissue in the kidneys, ureter, bladder or the urethra.
  • Urinary fibrosis includes kidney fibrosis, nephrosclerosis, nephrosis, glomerulosclerosis, tubulointerstitial renal fibrosis, bladder fibrosis and urethral stricture. Urinary fibrosis may also be referred to as “renal fibrosis”.
  • peptide means a molecule composed of 2-50 amino acids.
  • protein means a molecule composed of more than 50 amino acids.
  • AGE-modified protein AGE-modified peptide
  • glycation end-product refers to modified proteins or peptides that are formed as the result of the reaction of sugars with protein side chains that further rearrange and form irreversible cross-links. This process begins with a reversible reaction between a reducing sugar and an amino group to form a Schiff base, which proceeds to form a covalently-bonded Amadori rearrangement product. Once formed, the Amadori product undergoes further rearrangement to produce AGEs.
  • AGE-modified proteins and antibodies to AGE-modified proteins are described in U.S.
  • AGEs may be identified by the presence of AGE modifications (also referred to as AGE epitopes or AGE moieties) such as 2-(2- furoyl)-4(5)-(2-furanyl)-1 H-imidazole (“FFI”); 5-hydroxymethyl-1-alkylpyrrole-2- carbaldehyde (“Pyrraline”); 1-alkyl-2-formyl-3,4-diglycosyl pyrrole (“AFGP”), a non- fluorescent model AGE; carboxymethyllysine; carboxyethyllysine; and pentosidine.
  • ALI another AGE, is described in Al-Abed.
  • AGE antigen means a substance that elicits an immune response against an AGE-modified protein or peptide of a cell.
  • the immune response against an AGE-modified protein or peptide of a cell does not include the production of antibodies to the non-AGE-modified protein or peptide.
  • an antibody that binds to an AGE-modified protein on a cell means an antibody, antibody fragment or other protein or peptide that binds to an AGE-modified protein or peptide which preferably includes a constant region of an antibody, where the protein or peptide which has been AGE-modified is a protein or peptide normally found bound on the surface of a cell, preferably a mammalian cell, more preferably a human, cat, dog, horse, camelid (for example, camel or alpaca), cattle, sheep, pig, or goat cell.
  • an antibody that binds to an AGE-modified protein on a cell does not include an antibody or other protein which binds with the same specificity and selectivity to both the AGE-modified protein or peptide, and the same non-AGE- modified protein or peptide (that is, the presence of the AGE modification does not increase binding).
  • AGE-modified albumin is not an AGE-modified protein on a cell, because albumin is not a protein normally found bound on the surface of cells.
  • An antibody that binds to an AGE-modified protein on a cell “anti-AGE antibody” or “AGE antibody” only includes those antibodies which lead to removal, destruction, or death of the cell.
  • antibodies which are conjugated, for example to a toxin, drug, or other chemical or particle Preferably, the antibodies are monoclonal antibodies, but polyclonal antibodies are also possible.
  • senescent cell means a cell which is in a state of proliferative arrest and expresses one or more biomarkers of senescence, such as activation of p16 lnk4a or expression of senescence-associated P-galactosidase. Also included are cells which express one or more biomarkers of senescence, do not proliferate in vivo, but may proliferate in vitro under certain conditions, such as some satellite cells found in the muscles of ALS patients.
  • the term “senolytic agent” means a small molecule with a molecular weight of less than 900 daltons that destroys senescent cells.
  • the term “senolytic agent” does not include antibodies, antibody conjugates, proteins, peptides or biologic therapies.
  • variant means a nucleotide, protein or amino acid sequence different from the specifically identified sequences, wherein one or more nucleotides, proteins or amino acid residues is deleted, substituted or added. Variants may be naturally-occurring allelic variants, or non-naturally-occurring variants. Variants of the identified sequences may retain some or all of the functional characteristics of the identified sequences.
  • percent (%) sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Preferably, % sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is publicly available from Genentech, Inc.
  • ALIGN-2 (South San Francisco, CA), or may be compiled from the source code, which has been filed with user documentation in the U.S. Copyright Office and is registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • the % sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B.
  • FIG. 1 is a graph of the response versus time in an antibody binding experiment.
  • FIG. 2 is a graph illustrating the effect of senescent cell clearance on peripheral capillary oxygen saturation (SpCh) in bleomycin exposed mice.
  • FIG. 3A is a graph illustrating the effect of senescent cell clearance with ganciclovir on lung elastance in 3MR mice exposed to bleomycin.
  • FIG. 3B is a graph illustrating the effect of senescent cell clearance with ganciclovir on dynamic lung compliance in 3MR mice exposed to bleomycin.
  • FIG. 3C is a graph illustrating the effect of senescent cell clearance with ganciclovir on static lung compliance in 3MR mice exposed to bleomycin.
  • FIG. 4 is a graph illustrating the effect of senescent cell clearance on peripheral capillary oxygen saturation (SpOa) in mice after 2 months and 4 months of cigarette smoke (CS) exposure.
  • AP AP20187;
  • GAN ganciclovir;
  • Navi Navitoclax (ABT-263);
  • Nutlin Nutlin 3A.
  • fibrotic diseases have established that inflammation is involved in fibrosis.
  • Pro-inflammatory factors such as TGF , PDGF, IL-1 p, IL-6, IL-10, IL-13 and IFN-y, and reactive oxygen species have been recognized as mediators of fibrosis (Bataller, R. et al., “Liver fibrosis”, The Journal of Clinical Investigation, Vol. 115, No. 2, p. 209-218 (2005)).
  • inflammatory cells and their secreted inflammatory factors have been recognized as the primary factors in activating dermal fibroblasts to become fibrotic (Shaw, T. J.
  • Senescent cells have been implicated in a number of fibrotic diseases.
  • Senescent biomarkers such as p16, p21 and senescence-associated - galactosidase (SA-0-gal) have been observed in fibroblasts and epithelial cells in human and mouse idiopathic lung fibrosis tissue (Schafer, M. J. et al., “Cellular senescence mediates fibrotic pulmonary disease”, Nature Communications, Vol. 8, No. 14532, 11 pages (2017)).
  • Pathogenic models of chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis both involve premature cellular senescence of progenitor cells, which results in stem cell exhaustion and disease progression (Chilosi, M.
  • Senescent cells in the lungs contribute to excess extracellular matrix deposition in an aged mouse model and in elderly human samples (Calhoun, C. et al., “Senescent cells contribute to the physiological remodeling of aged lungs”, Journals of Gerontology: Biological Sciences, Vol. 71 , No. 2, p. 153-160 (2016)).
  • the cell surface protein vimentin has been recognized as a marker of cellular senescence in mice that have been immunized with senescent mouse lung fibroblasts, and the IgM antibody clone 9H4 was found to bind to cell surface vimentin on senescent cells (Frescas, D. et al., “Senescent cells expose and secrete an oxidized form of membrane-bound vimentin as revealed by a natural polyreactive antibody", Proceedings of the National Academy of Sciences, p. E1668- E1677 (2017)).
  • CKD Cellular senescence and the SASP participate in the pathological process of CKD, and CKD accelerates the progression of cellular senescence and the secretion of inflammatory factors through the SASP (Wang, W-J. et al., “Cellular senescence, senescence-associated secretory phenotype, and chronic kidney disease", Oncotarget, Vol. 8, No. 38, pp. 64520-64533 (2017)).
  • Senescent cells increase in the glomeruli in response to renal injury and aging, and increased senescent markers have been detected in glomerulosclerosis (Valentijn, F.A. et al., “Cellular senescence in the aging and diseased kidney”, Journal of Cell Communication and Signaling, Vol.
  • CML and pentosidine have also been identified in glomerulosclerosis, FSGS, hypertensive nephrosclerosis and lupus nephritis (Tanji, N. et al., “Expression of advanced glycation end products and their cellular receptor RAGE in diabetic nephropathy and nondiabetic renal disease”, Journal of the American Society of Nephrology, Vol. 11 , pp. 1656-1666 (2000)).
  • Desmoplasia forms around tumors, acting as a barrier to drugs and even small molecules.
  • the tumor cells are maintained by the cancer-associated fibroblasts that form the desmoplasia, and the tumor cells contribute to the growth of the desmoplasia.
  • Senescent fibroblasts are present in the desmoplasia.
  • AGEs have been detected in higher staining of surrounding fibroblast foci in fibrotic lungs of IPF patients, compared to control patients (Machahua, C., et aL, Increased AGE-RAGE ratio in idiopathic pulmonary fibrosis, Respiratory Research, vol. 17, no. 144, pp. 1-
  • Eliminating or reducing the senescent fibroblasts would reduce the size of the desmoplasia, and it would enhance the permeability of the tumor to antibodies, natural killer cells (NK), immune effectors and other therapeutics, which would enhance the efficacy of the immune response or pharmaceuticals for treating cancer.
  • NK natural killer cells
  • senescent cells can be used to treat fibrotic diseases. Elimination of senescent fibroblasts in mice by administration of a combination of the senolytic agents dasatinib and quercetin improved lung function (Schafer, M. J. et al.).
  • the therapeutic benefits of removing senescent cells has been demonstrated in vivo in an art-accepted model in treating age-related diseases such as sarcopenia (US 9,161,810) and treating metastatic cancer (WO 2017/143073).
  • age-related diseases such as sarcopenia (US 9,161,810) and treating metastatic cancer (WO 2017/143073).
  • the identification of a link between cellular senescence and fibrotic diseases allows for similar treatment possibilities.
  • the present invention uses enhanced clearance of cells expressing AGE-modified proteins or peptides (AGE-modified cells) to treat, ameliorate or prevent the onset of fibrotic diseases by removing or killing senescent cells. This may be accomplished by administering anti-AGE antibodies to a subject.
  • Vaccination against AGE-modified proteins or peptides of a cell may also be used to control the presence of AGE-modified cells in a subject.
  • the continuous and virtually ubiquitous surveillance exercised by the immune system in the body in response to a vaccination allows maintaining low levels of AGE-modified cells in the body.
  • Vaccination against AGE-modified proteins or peptides of a cell removes or kills senescent cells.
  • the process of senescent cell removal or destruction allows vaccination against AGE-modified proteins or peptides of a cell to be used to treat or prevent the onset of fibrotic diseases.
  • Individuals may receive repeated vaccinations or boosters on a periodic basis to maintain their immunity.
  • Anti-AGE antibodies are known in the art and are commercially available. Examples include those described in U.S. 5,702,704 (Bucala) and U.S. 6,380,165 (Al-Abed et al.).
  • the antibody may bind to one or more AGE-modified proteins or peptides having an AGE modification such as FFI, pyrraline, AFGP, ALI, carboxymethyllysine (CML), carboxyethyllysine (CEL) and pentosidine, and mixtures of such antibodies.
  • the antibody is non-immunogenic to the animal in which it will be used, such as non-immunogenic to humans; companion animals including cats, dogs and horses; and commercially important animals, such camels (or alpaca), cattle (bovine), sheep, pig, and goats. More preferably, the antibody has the same species constant region as antibodies of the animal to reduce the immune response against the antibody, such as being humanized (for humans), felinized (for cats), caninized (for dogs), equuinized (for horses), camelized (for camels or alpaca), bovinized (for cattle), ovinized (for sheep), porcinized (for pigs), or caperized (for goats).
  • the antibody is identical to that of the animal in which it will be used (except for the variable region), such as a human antibody, a cat antibody, a dog antibody, a horse antibody, a camel antibody, a bovine antibody, a sheep antibody, a pig antibody, or a goat antibody. Details of the constant regions and other parts of antibodies for these animals are described below.
  • the antibody may be monoclonal or polyclonal.
  • the antibody is a monoclonal antibody.
  • Preferred anti-AGE antibodies include those which bind to proteins or peptides that exhibit a carboxymethyllysine or carboxyethyllysine AGE modification.
  • Carboxymethyllysine also known as N(epsilon)-(carboxymethyl)lysine, N(6)- carboxymethyllysine, or 2-Amino-6-(carboxymethylamino)hexanoic acid
  • carboxyethyllysine also known as N-epsilon-(carboxyethyl)lysine
  • CML- and CEL-modified proteins or peptides are recognized by the receptor RAGE which is expressed on a variety of cells. CML and CEL have been well-studied and CML- and CEL-related products are commercially available.
  • Cell Biolabs, Inc. sells CML-BSA antigens, CML polyclonal antibodies, CML immunoblot kits, and CML competitive ELISA kits (www.cellbiolabs.com/cml-assays) as well as CEL-BSA antigens and CEL competitive ELISA kits (www.cellbiolabs.com/cel-n- epsilon-carboxyethyl-lysine-assays-and-reagents).
  • a preferred antibody includes the variable region of the commercially available mouse anti-glycation end-product antibody raised against carboxymethyl lysine conjugated with keyhole limpet hemocyanin, the carboxymethyl lysine MAb (Clone 318003) available from R&D Systems, Inc. (Minneapolis, MN; catalog no. MAB3247), modified to have a human constant region (or the constant region of the animal into which it will be administered).
  • Commercially-available antibodies such as the carboxymethyl lysine antibody corresponding to catalog no. MAB3247 from R&D Systems, Inc., may be intended for diagnostic purposes and may contain material that is not suited for use in animals or humans.
  • commercially-available antibodies are purified and/or isolated prior to use in animals or humans to remove toxins or other potentially-harmful material.
  • the anti-AGE antibody preferably has a low rate of dissociation from the antibody-antigen complex, or ka (also referred to as kback or off-rate), preferably at most 9 x 10 -3 , 8 x 10 -3 , 7 x 10' 3 or 6 x 10 3 (sec 1 ).
  • the anti-AGE antibody preferably has a high affinity for the AGE-modified protein of a cell, which may be expressed as a low dissociation constant KD of at most 9 x 10’ 6 , 8 x 10' 6 , 7 x 10’ 6 , 6 x 10’ 6 , 5 x 10’ 6 , 4 x 10- 6 or 3 x 10‘ 6 (M).
  • the binding properties of the anti-AGE antibody are similar to, the same as, or superior to the carboxymethyl lysine MAb (Clone 318003) available from R&D Systems, Inc. (Minneapolis, MN; catalog no.
  • the anti-AGE antibody may destroy AGE-modified cells through antibodydependent cell-mediated cytotoxicity (ADCC).
  • ADCC is a mechanism of cell- mediated immune defense in which an effector cell of the immune system actively lyses a target cell whose membrane-surface antigens have been bound by specific antibodies.
  • ADCC may be mediated by natural killer (NK) cells, macrophages, neutrophils or eosinophils.
  • NK natural killer
  • the effector cells bind to the Fc portion of the bound antibody.
  • the anti-AGE antibody may also destroy AGE-modified cells through complement-dependent cytotoxicity (CDC). In CDC, the complement cascade of the immune system is triggered by an antibody binding to a target antigen.
  • CDC complement-dependent cytotoxicity
  • the anti-AGE antibody may be conjugated to an agent that causes the destruction of AGE-modified cells.
  • agents may be a toxin, a cytotoxic agent, magnetic nanoparticles, and magnetic spin-vortex discs.
  • a toxin such as pore-forming toxins (PFT) (Aroian R. et al., “Pore-Forming Toxins and Cellular Non-lmmune Defenses (CNIDs),” Current Opinion in Microbiology, 10:57-61 (2007)) conjugated to an anti-AGE antibody may be injected into a patient to selectively target and remove AGE-modified cells.
  • the anti-AGE antibody recognizes and binds to AGE-modified cells. Then, the toxin causes pore formation at the cell surface and subsequent cell removal through osmotic lysis.
  • Magnetic nanoparticles conjugated to the anti-AGE antibody may be injected into a patient to target and remove AGE-modified cells.
  • the magnetic nanoparticles can be heated by applying a magnetic field in order to selectively remove the AGE- modified cells.
  • Magnetic spin-vortex discs which are magnetized only when a magnetic field is applied to avoid self-aggregation that can block blood vessels, begin to spin when a magnetic field is applied, causing membrane disruption of target cells.
  • Magnetic spin-vortex discs, conjugated to anti-AGE antibodies specifically target AGE-modified cell types, without removing other cells.
  • Antibodies are Y-shaped proteins composed of two heavy chains and two light chains. The two arms of the Y shape form the fragment antigen-binding (Fab) region while the base or tail of the Y shape forms the fragment crystallizable (Fc) region of the antibody.
  • Antigen binding occurs at the terminal portion of the fragment antigen-binding region (the tips of the arms of the Y shape) at a location referred to as the paratope, which is a set of complementarity determining regions (also known as CDRs or the hypervariable region).
  • the complementarity determining regions vary among different antibodies and gives a given antibody its specificity for binding to a given antigen.
  • the fragment crystallizable region of the antibody determines the result of antigen binding and may interact with the immune system, such as by triggering the complement cascade or initiating antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • bi-specific antibodies When antibodies are prepared recombinantly, it is also possible to have a single antibody with variable regions (or complementary determining regions) that bind to two different antigens, with each tip of the Y shape being specific to one of the antigens; these are referred to as bi-specific antibodies.
  • a humanized anti-AGE antibody according to the present invention may have the human constant region sequence of amino acids shown in SEQ ID NO: 22.
  • the heavy chain complementarity determining regions of the humanized anti-AGE antibody may have one or more of the protein sequences shown in SEQ ID NO: 23 (CDR1H), SEQ ID NO: 24 (CDR2H) and SEQ ID NO: 25 (CDR3H).
  • the light chain complementarity determining regions of the humanized anti-AGE antibody may have one or more of the protein sequences shown in SEQ ID NO: 26 (CDR1L), SEQ ID NO: 27 (CDR2L) and SEQ ID NO: 28 (CDR3L).
  • the heavy chain of a humanized anti-AGE antibody may have or may include the protein sequence of SEQ ID NO: 1.
  • the variable domain of the heavy chain may have or may include the protein sequence of SEQ ID NO: 2.
  • the complementarity determining regions of the variable domain of the heavy chain (SEQ ID NO: 2) are shown in SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43.
  • the kappa light chain of a humanized anti-AGE antibody may have or may include the protein sequence of SEQ ID NO: 3.
  • the variable domain of the kappa light chain may have or may include the protein sequence of SEQ ID NO: 4.
  • the arginine (Arg or R) residue at position 128 of SEQ ID NO: 4 may be omitted.
  • variable domain of the light chain (SEQ ID NO: 4) are shown in SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46.
  • the variable regions may be codon-optimized, synthesized and cloned into expression vectors containing human immunoglobulin G1 constant regions.
  • the variable regions may be used in the preparation of non-human anti-AGE antibodies.
  • the antibody heavy chain may be encoded by the DNA sequence of SEQ ID NO: 12, a murine anti-AGE immunoglobulin G2b heavy chain.
  • the protein sequence of the murine anti-AGE immunoglobulin G2b heavy chain encoded by SEQ ID NO: 12 is shown in SEQ ID NO: 16.
  • the variable region of the murine antibody is shown in SEQ ID NO: 20, which corresponds to positions 25-142 of SEQ ID NO: 16.
  • the antibody heavy chain may alternatively be encoded by the DNA sequence of SEQ ID NO: 13, a chimeric anti-AGE human immunoglobulin G1 heavy chain.
  • the protein sequence of the chimeric anti-AGE human immunoglobulin G1 heavy chain encoded by SEQ ID NO: 13 is shown in SEQ ID NO: 17.
  • the chimeric anti-AGE human immunoglobulin includes the murine variable region of SEQ ID NO: 20 in positions 25-142.
  • the antibody light chain may be encoded by the DNA sequence of SEQ ID NO: 14, a murine anti-AGE kappa light chain.
  • the protein sequence of the murine anti-AGE kappa light chain encoded by SEQ ID NO: 14 is shown in SEQ ID NO: 18.
  • the variable region of the murine antibody is shown in SEQ ID NO: 21, which corresponds to positions 21-132 of SEQ ID NO: 18.
  • the antibody light chain may alternatively be encoded by the DNA sequence of SEQ ID NO: 15, a chimeric anti- AGE human kappa light chain.
  • the protein sequence of the chimeric anti-AGE human kappa light chain encoded by SEQ ID NO: 15 is shown in SEQ ID NO: 19.
  • the chimeric anti-AGE human immunoglobulin includes the murine variable region of SEQ ID NO: 21 in positions 21-132.
  • a humanized anti-AGE antibody according to the present invention may have or may include one or more humanized heavy chains or humanized light chains.
  • a humanized heavy chain may be encoded by the DNA sequence of SEQ ID NO: 30, 32 or 34.
  • the protein sequences of the humanized heavy chains encoded by SEQ ID NOs: 30, 32 and 34 are shown in SEQ ID NOs: 29, 31 and 33, respectively.
  • a humanized light chain may be encoded by the DNA sequence of SEQ ID NO: 36, 38 or 40.
  • the protein sequences of the humanized light chains encoded by SEQ ID NOs: 36, 38 and 40 are shown in SEQ ID NOs: 35, 37 and 39, respectively.
  • the humanized anti-AGE antibody maximizes the amount of human sequence while retaining the original antibody specificity.
  • a complete humanized antibody may be constructed that contains a heavy chain having a protein sequence chosen from SEQ ID NOs: 29, 31 and 33 and a light chain having a protein sequence chosen from SEQ ID NOs: 35, 37 and 39.
  • anti-AGE antibodies may be obtained by humanizing murine monoclonal anti-AGE antibodies.
  • Murine monoclonal anti-AGE antibodies have the heavy chain protein sequence shown in SEQ ID NO: 47 (the protein sequence of the variable domain is shown in SEQ ID NO: 52) and the light chain protein sequence shown in SEQ ID NO: 57 (the protein sequence of the variable domain is shown in SEQ ID NO: 62).
  • a preferred humanized heavy chain may have the protein sequence shown in SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 (the protein sequences of the variable domains of the humanized heavy chains are shown in SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, respectively).
  • a preferred humanized light chain may have the protein sequence shown in SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 or SEQ ID NO: 61 (the protein sequences of the variable domains of the humanized light chains are shown in SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, respectively).
  • a humanized anti-AGE monoclonal antibody is composed a heavy chain having a protein sequence selected from the group consisting of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51 and a light chain having a protein sequence selected from the group consisting of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61.
  • Humanized monoclonal anti-AGE antibodies composed of these protein sequences may have better binding and/or improved activation of the immune system, resulting in greater efficacy.
  • the protein sequence of an antibody from a non-human species may be modified to include the variable domain of the heavy chain having the sequence shown in SEQ ID NO: 2 or the kappa light chain having the sequence shown in SEQ ID NO: 4.
  • the non-human species may be a companion animal, such as the domestic cat or domestic dog, or livestock, such as cattle, the horse or the camel.
  • the non-human species is not the mouse.
  • the heavy chain of the horse (Equus caballus) antibody immunoglobulin gamma 4 may have or may include the protein sequence of SEQ ID NO: 5 (EMBL/GenBank accession number AY445518).
  • the heavy chain of the horse (Equus caballus) antibody immunoglobulin delta may have or may include the protein sequence of SEQ ID NO: 6 (EMBL/GenBank accession number AY631942).
  • the heavy chain of the dog (Canis familiaris) antibody immunoglobulin A may have or may include the protein sequence of SEQ ID NO: 7 (GenBank accession number L36871).
  • the heavy chain of the dog (Canis familiaris) antibody immunoglobulin E may have or may include the protein sequence of SEQ ID NO: 8 (GenBank accession number L36872).
  • the heavy chain of the cat (Felis catus) antibody immunoglobulin G2 may have or may include the protein sequence of SEQ ID NO: 9 (DDBJ/EMBL/GenBank accession number KF811175).
  • Camelus bactrianus Camelus bactrianus
  • llamas Lia glama, Lama paces and Lama vicugna
  • alpacas Vicugna paces
  • guanacos Liama guanicoe
  • camelids also have heavy chain immunoglobulin G antibodies that do not contain light chains and exist as heavy chain dimers. These antibodies are known as heavy chain antibodies, HCAbs, single-domain antibodies or sdAbs, and the variable domain of a camelid heavy chain antibody is known as the VHH.
  • the camelid heavy chain antibodies lack the heavy chain CH1 domain and have a hinge region that is not found in other species.
  • the variable region of the Arabian camel (Camelus dromedarius) single-domain antibody may have or may include the protein sequence of SEQ ID NO: 10 (GenBank accession number AJ245148).
  • the variable region of the heavy chain of the Arabian camel (Camelus dromedarius) tetrameric immunoglobulin may have or may include the protein sequence of SEQ ID NO: 11 (GenBank accession number AJ245184).
  • heavy chain antibodies are also found in cartilaginous fishes, such as sharks, skates and rays.
  • IgNAR immunoglobulin new antigen receptor
  • VNAR variable domain of an IgNAR
  • IgNAR immunoglobulin new antigen receptor
  • the IgNAR exists as two identical heavy chain dimers composed of one variable domain and five constant domains each. Like camelids, there is no light chain.
  • the protein sequences of additional non-human species may be readily found in online databases, such as the International ImMunoGeneTics Information System (www.imgt.org), the European Bioinformatics Institute (www.ebi.ac.uk), the DNA Databank of Japan (ddbj.nig.ac.jp/arsa) or the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).
  • online databases such as the International ImMunoGeneTics Information System (www.imgt.org), the European Bioinformatics Institute (www.ebi.ac.uk), the DNA Databank of Japan (ddbj.nig.ac.jp/arsa) or the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).
  • An anti-AGE antibody or a variant thereof may include a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51, including post-translational modifications thereof.
  • a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-AGE antibody including that sequence retains the ability to bind to AGE.
  • An anti-AGE antibody or a variant thereof may include a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, including post-translational modifications thereof.
  • variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-AGE antibody including that sequence retains the ability to bind to AGE.
  • substitutions, insertions, or deletions may occur in regions outside the variable region.
  • An anti-AGE antibody or a variant thereof may include a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 or SEQ ID NO: 61, including post-translational modifications thereof.
  • a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-AGE antibody including that sequence retains the ability to bind to AGE.
  • substitutions, insertions, or deletions may occur in regions outside the variable region.
  • An anti-AGE antibody or a variant thereof may include a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 or SEQ ID NO: 66, including post-translational modifications thereof.
  • variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-AGE antibody including that sequence retains the ability to bind to AGE.
  • substitutions, insertions, or deletions may occur in regions outside the variable region.
  • the antibody may have the complementarity determining regions of commercially available mouse anti-glycation end-product antibody raised against carboxymethyl lysine conjugated with keyhole limpet hemocyanin (CML-KLH), the carboxymethyl lysine MAb (Clone 318003) available from R&D Systems, Inc. (Minneapolis, MN; catalog no. MAB3247).
  • CML-KLH keyhole limpet hemocyanin
  • the antibody may have or may include constant regions which permit destruction of targeted cells by a subject’s immune system.
  • Bi-specific antibodies which are anti-AGE antibodies directed to two different epitopes, may also be used. Such antibodies will have a variable region (or complementary determining region) from those of one anti-AGE antibody, and a variable region (or complementary determining region) from a different antibody.
  • Antibody fragments may be used in place of whole antibodies.
  • immunoglobulin G may be broken down into smaller fragments by digestion with enzymes. Papain digestion cleaves the N-terminal side of inter-heavy chain disulfide bridges to produce Fab fragments.
  • Fab fragments include the light chain and one of the two N-terminal domains of the heavy chain (also known as the Fd fragment).
  • Pepsin digestion cleaves the C-terminal side of the inter-heavy chain disulfide bridges to produce F(ab’)2 fragments.
  • F(ab’)2 fragments include both light chains and the two N-terminal domains linked by disulfide bridges.
  • Pepsin digestion may also form the Fv (fragment variable) and Fc (fragment crystallizable) fragments.
  • the Fv fragment contains the two N-terminal variable domains.
  • the Fc fragment contains the domains which interact with immunoglobulin receptors on cells and with the initial elements of the complement cascade.
  • Pepsin may also cleave immunoglobulin G before the third constant domain of the heavy chain (CH3) to produce a large fragment F(abc) and a small fragment pFc’.
  • Antibody fragments may alternatively be produced recombinantly. Preferably, such antibody fragments are conjugated to an agent that causes the destruction of AGE-modified cells.
  • polyclonal antibodies can be raised in a mammalian host by one or more injections of an immunogen, and if desired, an adjuvant.
  • an immunogen and if desired, an adjuvant.
  • the immunogen (and adjuvant) is injected in a mammal by a subcutaneous or intraperitoneal injection.
  • the immunogen may be an AGE-modified protein of a cell, such as AGE-antithrombin III, AGE-calmodulin, AGE-insulin, AGE- ceruloplasmin, AGE-collagen, AGE-cathepsin B, AGE-albumin such as AGE-bovine serum albumin (AGE-BSA), AGE-human serum albumin and ovalbumin, AGE- crystallin, AGE-plasminogen activator, AGE-endothelial plasma membrane protein, AGE-aldehyde reductase, AGE-transferrin, AGE-fibrin, AGE-copper/zinc SOD, AGE- apo B, AGE-fibronectin, AGE-pancreatic ribose, AGE-apo A-l and II, AGE- hemoglobin, AGE-Na7K + -ATPase, AGE-plasminogen, AGE-myelin, AGE-lysozyme, AGE-
  • AGE- modified cells such as AGE-modified erythrocytes, whole, lysed, or partially digested, may also be used as AGE antigens.
  • adjuvants include Freund’s complete, monophosphoryl Lipid A synthetic-trehalose dicorynomycolate, aluminum hydroxide (alum), heat shock proteins HSP 70 or HSP96, squalene emulsion containing monophosphoryl lipid A, a2-macroglobulin and surface active substances, including oil emulsions, pleuronic polyols, polyanions and dinitrophenol.
  • an immunogen may be conjugated to a polypeptide that is immunogenic in the host, such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, cholera toxin, labile enterotoxin, silica particles or soybean trypsin inhibitor.
  • KLH keyhole limpet hemocyanin
  • serum albumin serum albumin
  • bovine thyroglobulin bovine thyroglobulin
  • cholera toxin cholera toxin
  • labile enterotoxin silica particles
  • silica particles silica particles
  • soybean trypsin inhibitor AGE- KLH.
  • pAbs may be made in chickens, producing IgY molecules.
  • Monoclonal antibodies may also be made by immunizing a host or lymphocytes from a host, harvesting the mAb-secreting (or potentially secreting) lymphocytes, fusing those lymphocytes to immortalized cells (for example, myeloma cells), and selecting those cells that secrete the desired mAb.
  • Other techniques may be used, such as the EBV-hybridoma technique.
  • Non-human antibodies may be made less immunogenic to humans by engineering the antibodies to contain a combination of non-human and human antibody components.
  • a chimeric antibody may be produced by combining the variable region of a non-human antibody with a human constant region.
  • a humanized antibody may be produced by replacing the complementarity determining regions (CDRs) of a human antibody with those of a non-human antibody.
  • CDRs complementarity determining regions
  • antibodies may be made less immunogenic to other species by being substantially “ized” to a given animal, such as cat, dog, horse, camel or alpaca, cattle, sheep, pig, or goat, at the amino acid level.
  • the mAbs may be purified from the culture medium or ascites fluid by conventional procedures, such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ammonium sulfate precipitation or affinity chromatography.
  • human monoclonal antibodies can be generated by immunization of transgenic mice containing a third copy IgG human trans-loci and silenced endogenous mouse Ig loci or using human-transgenic mice. Production of humanized monoclonal antibodies and fragments thereof can also be generated through phage display technologies.
  • a "pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • Preferred examples of such carriers or diluents include water, saline, Ringer’s solutions and dextrose solution. Supplementary active compounds can also be incorporated into the compositions.
  • Solutions and suspensions used for parenteral administration can include a sterile diluent, such as water for injection, saline solution, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • the antibodies may be administered systemically, such as by intravenous injection or infusion.
  • the antibodies may be administered locally with a pharmaceutically acceptable carrier suitable for the administration site, such as by percutaneous injection into an affected organ, topical administration at the site of dermal fibrosis or administration via nebulizer for pulmonary fibrosis.
  • compositions suitable for injection include sterile aqueous solutions or dispersions for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • Suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL® (BASF; Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid so as to be administered using a syringe.
  • Such compositions should be stable during manufacture and storage and must be preserved against contamination from microorganisms such as bacteria and fungi.
  • compositions that can delay absorption include agents such as aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating antibodies, and optionally other therapeutic components, in the required amount in an appropriate solvent with one or a combination of ingredients as required, followed by sterilization. Methods of preparation of sterile solids for the preparation of sterile injectable solutions include vacuum drying and freeze-drying to yield a solid.
  • the antibodies may be delivered as an aerosol spray from a nebulizer or a pressurized container that contains a suitable propellant, for example, a gas such as carbon dioxide.
  • a suitable propellant for example, a gas such as carbon dioxide.
  • Antibodies may also be delivered via inhalation as a dry powder, for example using the iSPERSETM inhaled drug delivery platform (PULMATRIX, Lexington, Mass.).
  • the use of anti-AGE antibodies which are chicken antibodies (IgY) may be non-immunogenic in a variety of animals, including humans, when administered by inhalation.
  • An appropriate dosage level of each type of antibody will generally be about 0.01 to 500 mg per kg patient body weight. Preferably, the dosage level will be about 0.1 to about 250 mg/kg; more preferably about 0.5 to about 100 mg/kg. A suitable dosage level may be about 0.01 to 250 mg/kg, about 0.05 to 100 mg/kg, or about 0.1 to 50 mg/kg. Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg.
  • each type of antibody may be administered on a regimen of 1 to 4 times per day, such as once or twice per day, antibodies typically have a long half-life in vivo. Accordingly, each type of antibody may be administered once a day, once a week, once every two or three weeks, once a month, or once every 60 to 90 days.
  • a subject that receives administration of an anti-AGE antibody may be tested to determine if the administration has been effective to treat fibrotic diseases.
  • a subject may be considered to have received an effective antibody treatment if he or she demonstrates an improvement in symptoms between subsequent measurements or over time.
  • the efficacy of treatment may be determined with a diagnostic test that is suitable for a given fibrotic disease. For example, liver fibrosis may be monitored with a liver biopsy and pulmonary fibrosis may be monitored with high-resolution computed tomography, pulmonary function tests, bronchoscopy or bronchoalveolar lavage. Alternatively, the concentration and/or number of senescent cells may be measured over time. Administration of antibody and subsequent testing may be repeated until the desired therapeutic result is achieved.
  • Unit dosage forms can be created to facilitate administration and dosage uniformity.
  • Unit dosage form refers to physically discrete units suited as single dosages for the subject to be treated, containing a therapeutically effective quantity of one or more types of antibodies in association with the required pharmaceutical carrier.
  • the unit dosage form is in a sealed container and is sterile.
  • Vaccines against AGE-modified proteins or peptides contain an AGE antigen, an adjuvant, optional preservatives and optional excipients.
  • AGE antigens include AGE-modified proteins or peptides such as AGE-antithrombin III, AGE-calmodulin, AGE-insulin, AGE-ceruloplasmin, AGE-collagen, AGE-cathepsin B, AGE-albumin such as AGE-bovine serum albumin (AGE-BSA), AGE-human serum albumin and ovalbumin, AGE-crystallin, AGE-plasminogen activator, AGE- endothelial plasma membrane protein, AGE-aldehyde reductase, AGE-transferrin, AGE-fibrin, AGE-copper/zinc SOD, AGE-apo B, AGE-fibronectin, AGE-pancreatic ribose, AGE-apo A-l and II, AGE-hemo
  • AGE-modified cells such as AGE-modified erythrocytes, whole, lysed, or partially digested, may also be used as AGE antigens.
  • Suitable AGE antigens also include proteins or peptides that exhibit AGE modifications (also referred to as AGE epitopes or AGE moieties) such as carboxymethyllysine (CML), carboxyethyllysine (CEL), pentosidine, pyrraline, FFI, AFGP and ALL
  • the AGE antigen may be an AGE-protein conjugate, such as AGE conjugated to keyhole limpet hemocyanin (AGE-KLH). Further details of some of these AGE-modified proteins or peptides and their preparation are described in Bucala.
  • Particularly preferred AGE antigens include proteins or peptides that exhibit a carboxymethyllysine or carboxyethyllysine AGE modification.
  • Carboxymethyllysine also known as N(epsilon)-(carboxymethyl)lysine, N(6)-carboxymethyllysine, or 2- Amino-6-(carboxymethylamino)hexanoic acid
  • carboxyethyllysine also known as N-epsilon-(carboxyethyl)lysine
  • proteins or peptides and lipids as a result of oxidative stress and chemical glycation and have been correlated with juvenile genetic disorders.
  • CML- and CEL-modified proteins or peptides are recognized by the receptor RAGE which is expressed on a variety of cells.
  • CML and CEL have been well-studied and CML- and CEL-related products are commercially available.
  • Cell Biolabs, Inc. sells CML-BSA antigens, CML polyclonal antibodies, CML immunoblot kits, and CML competitive ELISA kits (www.cellbiolabs.com/cml-assays) as well as CEL-BSA antigens and CEL competitive ELISA kits (www.cellbiolabs.com/cel-n-epsilon-carboxyethyl-lysine- assays-and-reagents).
  • AGE antigens may be conjugated to carrier proteins to enhance antibody production in a subject. Antigens that are not sufficiently immunogenic alone may require a suitable carrier protein to stimulate a response from the immune system.
  • suitable carrier proteins include keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, cholera toxin, labile enterotoxin, silica particles and soybean trypsin inhibitor.
  • KLH keyhole limpet hemocyanin
  • serum albumin serum albumin
  • bovine thyroglobulin cholera toxin
  • labile enterotoxin silica particles
  • soybean trypsin inhibitor e.g., the carrier protein is KLH (AGE-KLH).
  • KLH has been extensively studied and has been identified as an effective carrier protein in experimental cancer vaccines.
  • Preferred AGE antigen-carrier protein conjugates include CML-KLH and CEL-KLH.
  • Immunity is a long-term immune response, either cellular or humoral.
  • a cellular immune response is activated when an antigen is presented, preferably with a co-stimulator to a T-cell which causes it to differentiate and produce cytokines.
  • the cells involved in the generation of the cellular immune response are two classes of T-helper (Th) cells, Th1 and Th2.
  • Th1 cells stimulate B cells to produce predominantly antibodies of the lgG2A isotype, which activates the complement cascade and binds the Fc receptors of macrophages, while Th2 cells stimulate B cells to produce IgG 1 isotype antibodies in mice, lgG4 isotype antibodies in humans, and IgE isotype antibodies.
  • the human body also contains “professional” antigen-presenting cells such as dendritic cells, macrophages, and B cells.
  • a humoral immune response is triggered when a B cell selectively binds to an antigen and begins to proliferate, leading to the production of a clonal population of cells that produce antibodies that specifically recognize that antigen and which may differentiate into antibody-secreting cells, referred to as plasma-cells or memory-B cells.
  • Antibodies are molecules produced by B-cells that bind a specific antigen.
  • the antigen-antibody complex triggers several responses, either cell-mediated, for example by natural killers (NK) or macrophages, or serum-mediated, for example by activating the complement system, a complex of several serum proteins that act sequentially in a cascade that result in the lysis of the target cell.
  • Immunological adjuvants are the component(s) of a vaccine which augment the immune response to the immunogenic agent.
  • Adjuvants function by attracting macrophages to the immunogenic agent and then presenting the agent to the regional lymph nodes to initiate an effective antigenic response.
  • Adjuvants may also act as carriers themselves for the immunogenic agent.
  • Adjuvants may induce an inflammatory response, which may play an important role in initiating the immune response.
  • Adjuvants include mineral compounds such as aluminum salts, oil emulsions, bacterial products, liposomes, immunostimulating complexes and squalene.
  • Aluminum compounds are the most widely used adjuvants in human and veterinary vaccines. These aluminum compounds include aluminum salts such as aluminum phosphate (AIPO4) and aluminum hydroxide (AI(OH)3) compounds, typically in the form of gels, and are generically referred to in the field of vaccine immunological adjuvants as "alum.”
  • Aluminum hydroxide is a poorly crystalline aluminum oxyhydroxide having the structure of the mineral boehmite.
  • Aluminum phosphate is an amorphous aluminum hydroxyphosphate.
  • Negatively charged species can absorb onto aluminum hydroxide gels at neutral pH
  • positively charged species can absorb onto aluminum phosphate gels at neutral pH. It is believed that these aluminum compounds provide a depot of antigen at the site of administration, thereby providing a gradual and continuous release of antigen to stimulate antibody production. Aluminum compounds tend to more effectively stimulate a cellular response mediated by Th2, rather than Th1 cells.
  • Emulsion adjuvants include water-in-oil emulsions (for example, Freund's adjuvants, such as killed mycobacteria in oil emulsion) and oil-in-water emulsions (for example, MF-59).
  • Emulsion adjuvants include an immunogenic component, for example squalene (MF-59) or mannide oleate (Incomplete Freund's Adjuvants), which can induce an elevated humoral response, increased T cell proliferation, cytotoxic lymphocytes and cell-mediated immunity.
  • Liposomal or vesicular adjuvants include paucilamellar lipid vesicles
  • Paucilamellar vesicles can be prepared by mixing, under high pressure or shear conditions, a lipid phase comprising a nonphospholipid material (for example, an amphiphile surfactant; see U.S. Pat. Nos.
  • a sterol optionally a sterol, and any water-immiscible oily material to be encapsulated in the vesicles (for example, an oil such as squalene oil and an oil-soluble or oil-suspended antigen); and an aqueous phase such as water, saline, buffer or any other aqueous solution used to hydrate the lipids.
  • a sterol optionally a sterol, and any water-immiscible oily material to be encapsulated in the vesicles
  • an oil such as squalene oil and an oil-soluble or oil-suspended antigen
  • an aqueous phase such as water, saline, buffer or any other aqueous solution used to hydrate the lipids.
  • Liposomal or vesicular adjuvants are believed to promote contact of the antigen with immune cells, for example by fusion of the vesicle to the immune cell membrane, and preferentially stimulate
  • adjuvants include Mycobacterium bovis bacillus Calmette- Guerin (BCG), quill-saponin and unmethylated CpG dinucleotides (CpG motifs). Additional adjuvants are described in U.S. Patent Application Publication Pub. No. US 2010/0226932 (September 9, 2010) and Jiang, Z-H. et al. “Synthetic vaccines: the role of adjuvants in immune targeting”, Current Medicinal Chemistry, Vol. 10(15), pp. 1423-39 (2003). Preferable adjuvants include Freund’s complete adjuvant and Freund’s incomplete adjuvant.
  • the vaccine may optionally include one or more preservatives, such as antioxidants, antibacterial and antimicrobial agents, as well as combinations thereof.
  • preservatives such as antioxidants, antibacterial and antimicrobial agents, as well as combinations thereof.
  • examples include benzethonium chloride, ethylenediamine-tetraacetic acid sodium (EDTA), thimerosal, phenol, 2-phenoxyethanol, formaldehyde and formalin; antibacterial agents such as amphotericin B, chlortetracycline, gentamicin, neomycin, polymyxin B and streptomycin; antimicrobial surfactants such as polyoxyethylene-9, 10-nonyl phenol (Triton N-101 , octoxynol-9), sodium deoxycholate and polyoxyethylated octyl phenol (Triton X-I00).
  • the production and packaging of the vaccine may eliminate the need for a preservative. For example, a vaccine that has been sterilize
  • compositions include pharmaceutically acceptable excipients, such as stabilizers, thickening agents, toxin detoxifiers, diluents, pH adjusters, tonicity adjustors, surfactants, antifoaming agents, protein stabilizers, dyes and solvents.
  • pharmaceutically acceptable excipients such as stabilizers, thickening agents, toxin detoxifiers, diluents, pH adjusters, tonicity adjustors, surfactants, antifoaming agents, protein stabilizers, dyes and solvents.
  • excipients examples include hydrochloric acid, phosphate buffers, sodium acetate, sodium bicarbonate, sodium borate, sodium citrate, sodium hydroxide, potassium chloride, potassium chloride, sodium chloride, polydimethylsilozone, brilliant green, phenol red (phenolsulfon-phthalein), glycine, glycerin, sorbitol, histidine, monosodium glutamate, potassium glutamate, sucrose, urea, lactose, gelatin, sorbitol, polysorbate 20, polysorbate 80 and glutaraldehyde.
  • hydrochloric acid phosphate buffers, sodium acetate, sodium bicarbonate, sodium borate, sodium citrate, sodium hydroxide, potassium chloride, potassium chloride, sodium chloride, polydimethylsilozone, brilliant green, phenol red (phenolsulfon-phthalein), glycine, glycerin, sorbitol, histidine, monosodium glutamate, potassium glut
  • the vaccine may contain from 1 pg to 100 mg of at least one AGE antigen, including 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 400, 800 or 1000 pg, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80 or 90 mg.
  • the amount used for a single injection corresponds to a unit dosage.
  • the vaccine may be provided in unit dosage form or in multidosage form, such as 2-100 or 2-10 doses.
  • the unit dosages may be provided in a vial with a septum, or in a syringe with or without a needle.
  • the vaccine may be administered intravenously, subdermally or intraperitoneally.
  • the vaccine is sterile.
  • the vaccine may be administered one or more times, such as 1 to 10 times, including 2, 3, 4, 5, 6, 7, 8 or 9 times, and may be administered over a period of time ranging from 1 week to 1 year, 2-10 weeks or 2-10 months. Furthermore, booster vaccinations may be desirable, over the course of 1 year to 20 years, including 2, 5, 10 and 15 years.
  • a subject that receives a vaccine for AGE-modified proteins or peptides of a cell may be tested to determine if he or she has developed an immunity to the AGE- modified proteins or peptides. Suitable tests may include blood tests for detecting the presence of an antibody, such as immunoassays or antibody titers.
  • An immunity to AGE-modified proteins or peptides may also be determined by monitoring the concentration and/or number of senescent cells over time.
  • a subject may also be tested to determine if the vaccination has been effective to treat fibrotic diseases.
  • a subject may be considered to have received an effective vaccination if he or she demonstrates an improvement in symptoms between subsequent measurements or overtime, or by measuring the concentration and/or number of senescent cells. Vaccination and subsequent testing may be repeated until the desired therapeutic result is achieved.
  • the vaccination process may be designed to provide immunity against multiple AGE moieties.
  • a single AGE antigen may induce the production of AGE antibodies which are capable of binding to multiple AGE moieties.
  • the vaccine may contain multiple AGE antigens.
  • a subject may receive multiple vaccines, where each vaccine contains a different AGE antigen.
  • Any mammal that could develop fibrotic diseases may be treated by the methods herein described. Humans are a preferred mammal for treatment. Other mammals that may be treated include mice, rats, goats, sheep, pigs, cows, horses and companion animals, such as dogs or cats. Alternatively, any of the mammals or subjects identified above may be excluded from the patient population in need of treatment for fibrotic diseases.
  • a subject may be identified as in need of treatment based on a diagnosis with at least one fibrotic disease, or with a disease that is known to contribute to fibrosis.
  • Fibrotic diseases include interstitial lung disease, pulmonary fibrosis, liver fibrosis, cirrhosis, urinary fibrosis, kidney fibrosis, kidney disease, chronic kidney disease (CKD), nephrosclerosis, nephrosis, glomerulosclerosis, bladder fibrosis, urethral stricture, cardiovascular disease, macular degeneration, vitreal retinopathy, scleroderma (systemic and local), restenosis, myelofibrosis (bone marrow cancer), keloid scarring, hypertrophic scarring, nephrogenic fibrosing dermopathy, mixed connective tissue disease, scleromyxedema, scleredema and eosinophilic fasciitis.
  • CKD chronic kidney disease
  • nephrosclerosis ne
  • Fibrotic diseases include desmoplasia, as fibrotic tissue in the surrounding stroma plays a very important role in the progression of cancer. Diagnosis may involve any suitable diagnostic test or procedure for a given disease or disorder. A subject may also be identified as in need of treatment after experiencing physical trauma that is known to result in fibrosis, such as surgical complications, administration of chemotherapeutic drugs, exposure to radiation, burns or physical injury.
  • Subjects may also be identified as in need of treatment based on detection of advanced glycation end products in a sample obtained from the subject. Suitable samples include blood, skin, serum, saliva and urine. The diagnostic use of anti- AGE antibodies is discussed in more detail in International Publication No. WO 2018/204679.
  • the Present Application includes 66 nucleotide and amino acid sequences in the Sequence Listing filed herewith. Variants of the nucleotide and amino acid sequences are possible. Known variants include substitutions, deletions and additions to the sequences shown in SEQ ID NO: 4, 16 and 20. In SEQ ID NO: 4, the arginine (Arg or R) residue at position 128 may optionally be omitted. In SEQ ID NO: 16, the alanine residue at position 123 may optionally be replaced with a serine residue, and/or the tyrosine residue at position 124 may optionally be replaced with a phenylalanine residue. SEQ ID NO: 20 may optionally include the same substitutions as SEQ ID NO: 16 at positions 123 and 124. In addition, SEQ ID NO: 20 may optionally contain one additional lysine residue after the terminal valine residue.
  • Example 1 In vivo study of the administration of anti-glycation end-product antibody [115] To examine the effects of an anti-glycation end-product antibody, the antibody was administered to the aged CD1(ICR) mouse (Charles River Laboratories), twice daily by intravenous injection, once a week, for three weeks (Days 1, 8 and 15), followed by a 10 week treatment-free period.
  • the test antibody was a commercially available mouse anti-glycation end-product antibody raised against carboxymethyl lysine conjugated with keyhole limpet hemocyanin, the carboxymethyl lysine MAb (Clone 318003) available from R&D Systems, Inc. (Minneapolis, MN; catalog no. MAB3247). A control reference of physiological saline was used in the control animals.
  • mice referred to as “young” were 8 weeks old, while mice referred to as “old” were 88 weeks ( ⁇ 2 days) old. No adverse events were noted from the administration of the antibody.
  • the different groups of animals used in the study are shown in Table 1.
  • Pre Subset of animals euthanized prior to treatment start for collection of adipose tissue.
  • Example 2 Affinity and kinetics of test antibody
  • Example 1 The affinity and kinetics of the test antibody used in Example 1 were analyzed using Na,Na-bis(carboxymethyl)-L-lysine trifluoroacetate salt (Sigma-Aldrich, St. Louis, MO) as a model substrate for an AGE-modified protein of a cell. Label-free interaction analysis was carried out on a BIACORETM T200 (GE Healthcare, Pittsburgh, PA), using a Series S sensor chip CM5 (GE Healthcare, Pittsburgh, PA), with Fc1 set as blank, and Fc2 immobilized with the test antibody (molecular weigh of 150,000 Da).
  • the running buffer was an HBS-EP buffer (10 mM HEPES, 150 mM NaCI, 3 mM EDTA and 0.05% P-20, pH of 7.4), at a temperature of 25 °C.
  • Software was BIACORETM T200 evaluation software, version 2.0. A double reference (Fc2-1 and only buffer injection), was used in the analysis, and the data was fitted to a Langmuir 1:1 binding model.
  • Example 3 Construction and production of murine anti-AGE lgG2b antibody and chimeric anti-AGE lgG1 antibody
  • Murine and chimeric human anti-AGE antibodies were prepared.
  • the DNA sequence of murine anti-AGE antibody lgG2b heavy chain is shown in SEQ ID NO: 12.
  • the DNA sequence of chimeric human anti-AGE antibody lgG1 heavy chain is shown in SEQ ID NO: 13.
  • the DNA sequence of murine anti-AGE antibody kappa light chain is shown in SEQ ID NO: 14.
  • the DNA sequence of chimeric human anti- AGE antibody kappa light chain is shown in SEQ ID NO: 15.
  • the gene sequences were synthesized and cloned into high expression mammalian vectors. The sequences were codon optimized. Completed constructs were sequence confirmed before proceeding to transfection.
  • HEK293 cells were seeded in a shake flask one day before transfection, and were grown using serum-free chemically defined media.
  • the DNA expression constructs were transiently transfected into 0.03 liters of suspension HEK293 cells. After 20 hours, cells were sampled to obtain the viabilities and viable cell counts, and titers were measured (Octet QKe, ForteBio). Additional readings were taken throughout the transient transfection production runs. The cultures were harvested on day 5, and an additional sample for each was measured for cell density, viability and titer.
  • Example 3 The binding of the murine (parental) and chimeric anti-AGE antibodies described in Example 3 was investigated by a direct binding ELISA.
  • An anti- carboxymethyl lysine (CML) antibody (R&D Systems, MAB3247) was used as a control.
  • CML was conjugated to KLH (CML-KLH) and both CML and CML-KLH were coated overnight onto an ELISA plate.
  • HRP-goat anti-mouse Fc was used to detect the control and murine (parental) anti-AGE antibodies.
  • HRP-goat anti-human Fc was used to detect the chimeric anti-AGE antibody.
  • the antigens were diluted to 1 pg/mL in 1x phosphate buffer at pH 6.5.
  • a 96- well microtiter ELISA plate was coated with 100 pL/well of the diluted antigen and let sit at 4°C overnight. The plate was blocked with 1x PBS, 2.5% BSA and allowed to sit for 1-2 hours the next morning at room temperature.
  • the antibody samples were prepared in serial dilutions with 1x PBS, 1% BSA with the starting concentration of 50 pg/mL. Secondary antibodies were diluted 1 :5,000. 100 pL of the antibody dilutions was applied to each well. The plate was incubated at room temperature for 0.5-1 hour on a microplate shaker.
  • the plate was washed 3 times with 1x PBS. 100 pL/well diluted HRP-conjugated goat anti-human Fc secondary antibody was applied to the wells. The plate was incubated for 1 hour on a microplate shaker. The plate was then washed 3 times with 1x PBS. 100 pL HRP substrate TMB was added to each well to develop the plate. After 3-5 minutes elapsed, the reaction was terminated by adding 100 pL of 1N HCL A second direct binding ELISA was performed with only CML coating. The absorbance at OD450 was read using a microplate reader.
  • the OD450 absorbance raw data for the CML-only ELISA is shown in the plate map below. 24 of the 96 wells in the well plate were used. Blank wells in the plate map indicate unused wells.
  • control and chimeric anti-AGE antibodies showed binding to both CML and CML-KLH.
  • the murine (parental) anti-AGE antibody showed very weak to no binding to either CML or CML-KLH.
  • Data from repeated ELISA confirms binding of the control and chimeric anti-AGE to CML. All buffer control showed negative signal.
  • Humanized antibodies were designed by creating multiple hybrid sequences that fuse select parts of the parental (mouse) antibody sequence with the human framework sequences. Acceptor frameworks were identified based on the overall sequence identity across the framework, matching interface position, similarly classed CDR canonical positions, and presence of N-glycosylation sites that would have to be removed. Three humanized light chains and three humanized heavy chains were designed based on two different heavy and light chain human acceptor frameworks. The amino acid sequences of the heavy chains are shown in SEQ ID NO: 29, 31 and 33, which are encoded by the DNA sequences shown in SEQ ID NO: 30, 32 and 34, respectively.
  • the amino acid sequences of the light chains are shown in SEQ ID NO: 35, 37 and 39, which are encoded by the DNA sequences shown in SEQ ID NO: 36, 38 and 40, respectively.
  • the humanized sequences were methodically analyzed by eye and computer modeling to isolate the sequences that would most likely retain antigen binding. The goal was to maximize the amount of human sequence in the final humanized antibodies while retaining the original antibody specificity.
  • the light and heavy humanized chains could be combined to create nine variant fully humanized antibodies.
  • the three heavy chains and three light chains were analyzed to determine their humanness.
  • Antibody humanness scores were calculated according to the method described in Gao, S. H., et al., “Monoclonal antibody humanness score and its applications”, BMC Biotechnology, 13:55 (July 5, 2013).
  • the humanness score represents how human-like an antibody variable region sequence looks. For heavy chains a score of 79 or above is indicative of looking human-like; for light chains a score of 86 or above is indicative of looking human-like.
  • the humanness of the three heavy chains, three light chains, a parental (mouse) heavy chain and a parental (mouse) light chain are shown below in Table 6:
  • variable region sequences were designed by first synthesizing the variable region sequences. The sequences were optimized for expression in mammalian cells. These variable region sequences were then cloned into expression vectors that already contain human Fc domains; for the heavy chain, the lgG1 was used.
  • the binding of the humanized antibodies may be evaluated, for example, by dose-dependent binding ELISA or cell-based binding assay.
  • Example 6 An AGE-RNAse containing vaccine in a human subject.
  • AGE-RNAse is prepared by incubating RNAse in a phosphate buffer solution containing 0.1-3 M glucose, glucose-6-phosphate, fructose or ribose for 10-100 days. The AGE-RNAse solution is dialyzed and the protein content is measured. Aluminum hydroxide or aluminum phosphate, as an adjuvant, is added to 100 pg of the AGE-RNAse. Formaldehyde or formalin is added as a preservative to the preparation. Ascorbic acid is added as an antioxidant.
  • the vaccine also includes phosphate buffer to adjust the pH and glycine as a protein stabilizer. The composition is injected intravenously into a subject with idiopathic pulmonary fibrosis.
  • Example 7 Injection regimen for an AGE-RNAse containing vaccine in a human subject.
  • Example 8 An AGE-hemoglobin containing vaccine in a human subject
  • AGE-hemoglobin is prepared by incubating human hemoglobin in a phosphate buffer solution containing 0.1-3 M glucose, glucose-6-phosphate, fructose or ribose for 10-100 days. The AGE-hemoglobin solution is dialyzed and the protein content is measured. All vaccine components are the same as in Example 6, except AGE-hemoglobin is substituted for AGE-RNAse. Administration is carried out as in Example 6, or as in Example 7.
  • Example 9 An AGE-human serum albumin containing vaccine in a human subject.
  • AGE-human serum albumin is prepared by incubating human serum albumin in a phosphate buffer solution containing 0.1-3 M glucose, glucose-6-phosphate, fructose or ribose for 10-100 days. The AGE-human serum albumin solution is dialyzed and the protein content is measured. All vaccine components are the same as in Example 6, except AGE-human serum albumin is substituted for AGE-RNAse. Administration is carried out as in Example 6, or as in Example 7.
  • Example 10 Carboxymethyllysine-modified protein vaccine for a human subject
  • a vaccine is prepared by combining a carboxymethyllysine-modified protein as an AGE antigen, aluminum hydroxide as an adjuvant, formaldehyde as a preservative, ascorbic acid as an antioxidant, a phosphate buffer to adjust the pH of the vaccine and glycine as a protein stabilizer.
  • the vaccine is injected subcutaneously into a subject with myelofibrosis.
  • Example 11 Carboxyethyllysine-modified peptide vaccine for a human subject
  • a vaccine is prepared by combining a carboxyethyllysine-modified peptide conjugated to KLH as an AGE antigen, aluminum hydroxide as an adjuvant, formaldehyde as a preservative, ascorbic acid as an antioxidant, a phosphate buffer to adjust the pH of the vaccine and glycine as a protein stabilizer.
  • the vaccine is injected subcutaneously into a subject with chronic kidney disease.
  • Example 12 In vivo study of the administration of a carboxymethyl lysine monoclonal antibody
  • 4T1 murine breast tumor cells (ATCC CRL-2539) were cultured in RPM1 1640 medium containing 10% fetal bovine serum, 2 mM glutamine, 25 pg/mL gentamicin, 100 units/mL penicillin G Na and 100 pg/mL streptomycin sulfate. Tumor cells were maintained in tissue culture flasks in a humidified incubator at 37 °C in an atmosphere of 5% CO2 and 95% air.
  • the cultured breast cancer cells were then implanted in the mice.
  • 4T1 cells were harvested during log phase growth and re-suspended in phosphate buffered saline (PBS) at a concentration of 1 x 10 6 cells/mL on the day of implant.
  • Tumors were initiated by subcutaneously implanting 1 x 10 5 4T1 cells (0.1 mL suspension) into the right flank of each test animal. Tumors were monitored as their volumes approached a target range of 80-120 mm 3 .
  • Tumor weight was approximated using the assumption that 1 mm 3 of tumor volume has a weight of 1 mg.
  • the four treatment groups are shown in Table 8 below:
  • An anti-carboxymethyl lysine monoclonal antibody was used as a therapeutic agent.
  • 250 mg of carboxymethyl lysine monoclonal antibody was obtained from R&D Systems (Minneapolis, MN).
  • Dosing solutions of the carboxymethyl lysine monoclonal antibody were prepared at 1 and 0.5 mg/mL in a vehicle (PBS) to provide the active dosages of 10 and 5 pg/g, respectively, in a dosing volume of 10 mL/kg.
  • Dosing solutions were stored at 4 °C protected from light.
  • %TGI Percent tumor growth inhibition
  • %lnhibition (1-Mean Count of Focitreated/Mean Count of Focicontroi) x 100.
  • Treatment efficacy was also evaluated by the incidence and magnitude of regression responses observed during the study.
  • Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal.
  • PR partial regression
  • CR complete regression
  • the tumor volume was 50% or less of its Day 1 volume for three consecutive measurements during the course of the study, and equal to or greater than 13.5 mm 3 for one or more of these three measurements.
  • the tumor volume was less than 13.5 mm 3 for three consecutive measurements during the course of the study.
  • Example 13 Treatment of fibrotic lung diseases by removal of senescent cells
  • mice develop lung fibrosis within 7-14 days after bleomycin treatment (see, e.g., Limjunyawong et al., 2014, Physiological Reports 2:e00249; Daniels et al, 2004, J. Clin. Invest. 114: 1308- 1316).
  • Bleomycin was administered to anesthetized 6-8 week old 3MR mice by intratracheal aspiration (2.5U/kg of bleomycin in 50 pT PBS) using a microsprayer syringe (Penn- Century, Inc.) as described in Daniels et al. (2004, J. Clin. Invest 114: 1308-1316). Control mice were administered saline. The day following bleomycin treatment, ganciclovir (GCV) (25mg/kg in PBS) was administered. 3MR mice were treated via intraperitoneal injection with ganciclovir for 5 consecutive days, followed by 5 days of rest, followed by a second treatment cycle of 5 consecutive days. Untreated mice received an equal volume of vehicle.
  • GCV ganciclovir
  • BAL Bronchoalveolar lavage
  • INK- ATT AC pl6 lnk4a apoptosis through targeted activation of caspase
  • transgenic mice have an FK506- binding protein (FKBP)-caspase 8 (Casp8) fusion polypeptide under the control of the pl6 lnk4a promoter (see, e.g., Baker et al, Nature, supra; Int'l Patent Application Publication No. WO/2012/177927).
  • FKBP FK506- binding protein
  • Casp8 caspase 8
  • a synthetic drug that induces dimerization of a membrane bound myristoylated FKBP-Casp8 fusion protein senescent cells specifically expressing the FKBP-Casp8 fusion protein via the pl6 lnk4a promoter undergo programmed cell death (apoptosis) (see, e.g., Baker, Nature, supra, Figure 1 therein).
  • a second study also assesses the effect of clearance of senescence cells using a senolytic agent in C57BL6/J mice that have bleomycin induced lung injury.
  • Bleomycin is administered to 6 week old C57BL6/J mice as described above.
  • a senolytic agent is administered during the first and third week post-bleomycin treatment. Control mice are treated with vehicle.
  • clearance of senescent cells and lung function/histopathology is assessed.
  • mice were exposed to cigarette smoke.
  • the effect of a senolytic agent on the mice exposed to smoke is assessed by senescent cell clearance, lung function, and histopathology.
  • Each lighted cigarette (3R4F research cigarettes containing 10.9 mg of total particulate matter (TPM), 9.4 mg of tar, and 0.726 mg of nicotine, and 11.9 mg carbon monoxide per cigarette [University of Kentucky, Lexington, KY]) was puffed for 2 seconds and once every minute for a total of 8 puffs, with the flow rate of 1.05 L/min, to provide a standard puff of 35 cm 3 .
  • the smoke machine was adjusted to produce a mixture of side stream smoke (89%) and mainstream smoke (11%) by smoldering 2 cigarettes at one time.
  • the smoke chamber atmosphere was monitored for total suspended particulates (80-120 mg/m 3 ) and carbon monoxide (350 ppm).
  • An additional 70 animals that did not receive exposure to cigarette smoke were used as controls for the experiment.
  • mice are killed by i.p. injection of pentobarbital for in-depth analysis of lung histopathology as previously described (Rangasamy et al, 2004, J. Clin. Invest. 114: 1248-1259). Briefly, lungs are inflated with 0.5% low-melting agarose at a constant pressure of 25 cm. Part of the lung tissue is collected for RNA extraction and qRT-PCR analysis of senescent markers. Other parts of lungs are fixed in 10% buffered formalin and embedded in paraffin. Sections (5 urn) are stained with hematoxylin and eosin. Mean alveolar diameter, alveolar length, and mean linear intercepts are determined by computer- assisted morphometry with Image Pro Plus software (Media Cybernetics).
  • 3MR or INK-ATTAC mice The potential therapeutic effect of clearance of senescent cells after COPD is fully developed may be assessed in 3MR or INK-ATTAC mice.
  • Six week-old 3MR or INK-ATTAC mice are chronically exposed to cigarette smoke for 6 months as described above.
  • 3MR or INK- ATTAC mice are treated with ganciclovir (5 consecutive days of treatment followed by 16 days off drug) or AP20187 (3x/week), respectively, until 9 months following the start of smoke exposure, when assessment of senescent cell clearance, lung function, and histopathology is performed.
  • Example 14 Treatment of idiopathic pulmonary fibrosis
  • a patient presents with idiopathic pulmonary fibrosis. She is administered an anti-AGE antibody by inhalation using a nebulizer.
  • the anti-AGE antibody will specifically bind to cells expressing cell-surface AGEs, such as senescent myofibroblasts, and allow her immune system to destroy those cells. Killing and removing senescent cells will treat her idiopathic pulmonary fibrosis and prevent the disease from worsening.
  • Example 15 T reatment of scleroderma
  • a patient presents with scleroderma on his right arm. He is administered a topical cream containing an anti-AGE antibody.
  • the anti-AGE antibody will specifically bind to cells expressing cell-surface AGEs, such as senescent myofibroblasts, and allow his immune system to destroy those cells. Killing and removing senescent cells will treat his scleroderma and prevent further progression of the disease.
  • TGF-p1 Transforming growth factor-
  • HA hyaluronan
  • EGFR epidermal growth factor receptor

Abstract

A method of treating or preventing the onset of a fibrotic disease comprises administering to a subject a composition comprising an anti-AGE antibody. The anti- AGE antibody binds an AGE antigen comprising at least one protein or peptide that exhibits AGE modifications selected from the group consisting of FFI, pyrraline, AFGP, ALI, carboxymethyllysine, carboxyethyllysine and pentosidine. A method of treating or preventing the onset of a fibrotic disease comprises administering to a subject a vaccine comprising an AGE antigen.

Description

METHODS AND COMPOSITIONS FOR TREATING FIBROTIC DISEASES
BACKGROUND
[01] Fibrosis is the formation of excess fibrous connective tissue in an organ or in tissue. Connective tissue, such as extracellular matrix proteins, is produced by fibroblasts in response to pro-fibrotic factors including transforming growth factor beta (TGFp), connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF) and interleukin 4 (IL-4). Fibroblasts may differentiate into myofibroblasts through an epithelial to mesenchymal transition largely mediated by TGF|3 (Midgley, A. C. et al., “Transforming growth factor-p1 (TGF-|31)-stimulated fibroblast to myofibroblast differentiation is mediated by hyaluronan (HA)-facilitated epidermal growth factor receptor (EGFR) and CD44 co-localization in lipid rafts”, Journal of Biological Chemistry, Vol. 288, No. 21 , p. 14824-14838 (2013)). Myofibroblasts are key mediators of profibrotic conditions and proliferate in response to TGFp signaling (Harris, W. T. et al., “Myofibroblast differentiation and enhanced TGF- signaling in cystic fibrosis lung disease”, PLoS One, Vol. 8, No. 8, 8 pages (2013)).
[02] Fibrosis can result from specific trauma such as surgical complications, exposure to radiation, burns or physical injury. Some therapeutic treatments, such as the administration of chemotherapeutic drugs or exposure to ionizing radiation (radiotherapy) as used in treating cancer, are known to cause fibrosis as a side effect. Certain cancers, such as myelofibrosis (bone marrow cancer), can also cause fibrosis.
[03] Fibrotic diseases can develop in many parts of the body in humans and animals and may affect organs including the lungs (interstitial lung disease, pulmonary fibrosis, acute lung injury), liver (liver fibrosis, cirrhosis), kidneys (kidney disease, nephrosclerosis), heart (cardiovascular disease) and eyes (macular degeneration, vitreal retinopathy) (Wynn, T. A., “Fibrotic disease and the TH1/TH2 paradigm”, Nature Reviews Immunology, Vol. 4, No. 8, p. 583-594 (2004)). Fibrotic diseases that are characterized by excessive connective tissue accumulation and progressive tissue contraction are known as fibroproliferative disorders (Bitterman, P.B. et al., “Fibroproliferative disorders”, Chest, Vol. 99, No. 3, pp. 81S-84S (1991)).
Examples of fibroproliferative disorders include scleroderma (systemic and local), hypertrophic scarring, keloid scarring, cirrhosis, nephrosis and restenosis.
[04] Pulmonary fibrosis (scarring of the lungs) is a respiratory disease characterized by the development of fibrous tissue in the walls of the air sacs of the lungs. The formation of scar tissue impairs the ability of the lungs to oxygenate blood, which often presents as shortness of breath (dyspnea). Other symptoms of pulmonary fibrosis include a chronic dry or hacking cough, fatigue, weakness, chest pain or discomfort, loss of appetite, unexplained weight loss and clubbing of the tips of the fingers and toes. The scarring caused by pulmonary fibrosis is permanent and the loss of lung function cannot be restored.
[05] Pulmonary fibrosis may result from a variety of different sources. Known causes of pulmonary fibrosis include autoimmune diseases that produce inflammation or scarring in the lungs, such as rheumatoid arthritis, scleroderma and autoimmune muscle diseases; occupational exposure to small inorganic particles, such as asbestos, beryllium, coal dust, silica and heavy metal dusts; environmental exposure to small organic particles, such as animal proteins, bacteria and molds; radiation exposure, including environmental radiation and cancer radiotherapy; bacterial or viral infections; and exposure to medications, such as nitrofurantoin, sulfasalazine, amiodarone, propranolol, phenytoin, methotrexate, bleomycin and oxaliplatin (Pulmonary Fibrosis Foundation, “About PF”, available online at www.pulmonaryfibrosis.org/life-with-pf/about-pf, accessed on July 18, 2017). When the specific cause of pulmonary fibrosis cannot be identified, the condition is referred to as idiopathic pulmonary fibrosis (IPF). Idiopathic pulmonary fibrosis may result from DNA abnormalities, and inheritable forms of idiopathic pulmonary fibrosis include familial pulmonary fibrosis (FPF) and familial interstitial pneumonia (FIP) (Id ).
[06] There is no standard treatment for pulmonary fibrosis. Lifestyle changes such as not smoking and maintaining a healthy weight can improve symptoms of pulmonary fibrosis. Pulmonary rehabilitation exercise programs can help to preserve lung function. Oxygen therapy may be used to improve oxygen delivery to the body. Various pharmacotherapies have been investigated for treating pulmonary fibrosis including corticosteroids (immunosuppressant/anti-inflammatory), cyclophosphamide (anti-inflammatory), azathioprine (immunosuppressant), mycophenolate mofetil (immunosuppressant), N-acetylcysteine (antioxidant) and proton pump inhibitors (gastric acid reducer). Nintedanib (OFEV®) and pirfenidone (ESBRIET®) are the only U.S. Food and Drug Administration-approved medications for treating idiopathic pulmonary fibrosis. However, these medications slow the progression of idiopathic pulmonary fibrosis, but do not treat the disease or prevent its onset. Severe cases of pulmonary fibrosis may be treated with lung transplantation. There are no pharmacotherapies for treating or preventing the onset of pulmonary fibrosis.
[07] Liver fibrosis is the formation of fibrous tissue in the liver. Cirrhosis is the most well-known fibrotic liver disease and may be caused by alcoholism, hepatitis B, hepatitis C, non-alcoholic fatty liver disease, primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune hepatitis, hereditary hemochromatosis, Wilson’s disease, Indian childhood cirrhosis, alpha 1-antitrypsin deficiency (A1AD), cardiac cirrhosis, galactosemia, glycogen storage disease type IV, cystic fibrosis and exposure to hepatoxic drugs or toxins. Cirrhosis may lead to complications such as ascites, esophageal variceal bleeding, hepatic encephalopathy, hepatorenal syndrome, spontaneous bacterial peritonitis, portal hypertensive gastropathy, infection and hepatocellular carcinoma.
[08] The possibility of developing cirrhosis may be reduced by vaccination against specific diseases that cause cirrhosis, such as hepatitis B or hepatitis C. Similarly, cirrhosis that develops as a side effect may be managed by treating the underlying disease, such as by administering interferon and corticosteroids to patients experiencing cirrhosis due to hepatitis. Treatment options for cirrhosis are limited. Further liver damage may be prevented with lifestyle changes including abstaining from alcohol, avoiding acetaminophen and maintaining a healthy diet. Liver transplantation may ultimately be necessary for patients who experience liver failure or cannot control the complications of cirrhosis. There are no pharmacotherapies for treating or preventing the onset of cirrhosis.
[09] Dermal fibrosis is the formation of fibrous tissue in the skin. Fibrotic skin disorders include keloid scarring, hypertrophic scarring, scleroderma, nephrogenic fibrosing dermopathy, mixed connective tissue disease, scleromyxedema, scleredema and eosinophilic fasciitis. Dermal fibrosis may be a minor aesthetic inconvenience, or may result in severe health complications. For example, scleroderma may affect major organs such as the lungs (loss of lung function, severe lung disease and lung tissue scarring), heart (scarring and weakness of the heart, swelling of the heart muscle and irregular heartbeat) and kidneys (high blood pressure and kidney failure) (“Scleroderma Living With It”, National Institute of Arthritis and Musculoskeletal and Skin Diseases, available online at www.niams.nih.gov/health-topics/scleroderma#tab-living-with (2016)).
[10] Many treatments for dermal fibrosis address only the local fibrous tissue. For example, keloid and hypertrophic scars may be treated with local steroid injections, pressure dressings, topical chemotherapy drugs, laser light therapy, pulsed-dye laser therapy, radiation therapy, surgical excision, silicone gel sheeting application and administration of hyaluronic acid (WO 2014/179262). Other treatments for dermal fibrosis focus on the symptoms and complications of the specific fibrotic disorder. For example, scleroderma therapies include calcium channel blockers, angiotensin II receptor antagonists, angiotensin converting enzyme (ACE) inhibitors, non-steroidal anti-inflammatory drugs (NSAIDs), COX-2 inhibitors, acetaminophen, corticosteroids, narcotics, antacids, histamine H2-receptor antagonists, proton pump inhibitors, prokinetic agents, somatostatin agonists, antibiotics, prostaglandin derivatives, endothelin receptor antagonists, IP receptor agonists, phosphodiesterase type 5 (PDE5) inhibitors, anti-fibrotic agents, anti-inflammatory agents, tyrosine kinase inhibitors, immunosuppressants and alkylating agents (“Current treatments available for scleroderma patients”, Scleroderma Research Foundation, available online at www.srfcure.org/for-patients/current-treatments (2018)). These various treatments fail to address the underlying causes of dermal fibrosis. [11] The formation of fibrous tissue in the urinary system may occur in the kidneys, ureter, bladder or the urethra. Kidney fibrosis may be further classified based on the location of the affected tissue, such as nephrosclerosis (the nephron), glomerulosclerosis (the glomerulus) or tubulointerstitial renal fibrosis (the interstitium). Chronic kidney disease (CKD), a progressive disease, is frequently caused by kidney fibrosis. Two primary factors that promote CKD are diabetes and high blood pressure (“About chronic kidney disease: symptoms and causes”, National Kidney Foundation, available online at www.kidney.org/atoz/content/about- chronic-kidney-disease (2020)).
[12] The damage caused by urinary fibrosis is permanent. Further damage may be reduced by managing underlying conditions that contribute to urinary fibrosis, especially diabetes and high blood pressure. Lifestyle changes such as not smoking, limiting alcohol intake and maintaining a healthy weight can prevent further worsening of kidney fibrosis. Dietary changes such as adopting a low-salt and low- fat diet may also help. If untreated, urinary fibrosis may lead to loss of kidney function, end-stage renal disease or kidney failure. Individuals with kidney failure must undergo regular dialysis to cleanse the blood or receive a kidney transplant, if they are healthy enough for the procedure and are able to find a suitable donor. Pharmacotherapies for treating urinary fibrosis are limited and primarily focus on controlling symptoms, reducing complications and slowing the progression of the disease.
[13] Desmoplasia refers to growth of dense connective tissue or stroma, and it may occur as result of injury or neoplasia. The stromal reaction in cancer is similar to the stromal reaction induced by injury or wound repair, causing scar-like tissue to be built around the cancer. Thus, the surrounding stroma plays a very important role in the progression of cancer. It has been suggested that tumor cells cause the proliferation of fibroblasts and subsequent secretion of collagen (El-Torky, M., et al., Collagens in scar carcinoma of the lung, The American Journal of Pathology, vol. 121, no. 2, pp. 322-326 (1985)). The newly secreted collagen is similar to that of collagen in scar formation, and it inhibits infiltration of cells to the site of injury. [14] Senescent cells are cells that are partially-functional or non-functional and are in a state of proliferative arrest. Senescence is a distinct state of a cell, and is associated with biomarkers, such as activation of the biomarker p16lnk4a, and expression of p-galactosidase. Senescence begins with damage or stress (such as overstimulation by growth factors) of cells.
[15] Advanced glycation end-products (AGEs; also referred to as AGE-modified proteins or peptides, or glycation end-products) arise from a non-enzymatic reaction of sugars with protein side-chains (Ando, K. et al., Membrane Proteins of Human Erythrocytes Are Modified by Advanced Glycation End Products during Aging in the Circulation, Biochem Biophys Res Commun., Vol. 258, 123, 125 (1999)). This process begins with a reversible reaction between the reducing sugar and the amino group to form a Schiff base, which proceeds to form a covalently-bonded Amadori rearrangement product. Once formed, the Amadori product undergoes further rearrangement to produce AGEs. Hyperglycemia and oxidative stress promote this post-translational modification of membrane proteins (Lindsey JB, etal., “Receptor For Advanced Glycation End-Products (RAGE) and soluble RAGE (sRAGE): Cardiovascular Implications,” Diabetes Vascular Disease Research, Vol. 6(1), 7-14, (2009)). AGEs may also be formed from other processes. For example, the advanced glycation end product, Ne-(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions. AGEs have been associated with several pathological conditions including inflammation, atherosclerosis, stroke, endothelial cell dysfunction, and neurodegenerative disorders (Bierhaus A, “AGEs and their interaction with AGE-receptors in vascular disease and diabetes mellitus. I. The AGE concept,” Cardiovasc Res, Vol. 37(3), 586-600 (1998)).
[16] AGE-modified proteins are also a marker of senescent cells. This association between AGEs and senescence is well known in the art. See, for example, Gruber, L. (WO 2009/143411, 26 Nov. 2009), Ando, K. etal. (Membrane Proteins of Human Erythrocytes Are Modified by Advanced Glycation End Products during Aging in the Circulation, Biochem Biophys Res Commun., Vol. 258, 123, 125 (1999)), Ahmed, E.K. etal. (“Protein Modification and Replicative Senescence of WI-38 Human Embryonic Fibroblasts” Aging Cells, vol. 9, 252, 260 (2010)), Vlassara, H. et al. (Advanced Glycosylation Endproducts on Erythrocyte Cell Surface Induce Receptor- Mediated Phagocytosis by Macrophages, J. Exp. Med., Vol. 166, 539, 545 (1987)) and Vlassara et al. (“High-affinity-receptor-mediated Uptake and Degradation of Glucose-modified Proteins: A Potential Mechanism for the Removal of Senescent Macromolecules” Proc. Natl. Acad. Sci. USAI, Vol. 82, 5588, 5591 (1985)).
Furthermore, Ahmed, E.K. et al. indicates that glycation end-products are “one of the major causes of spontaneous damage to cellular and extracellular proteins” (Ahmed, E.K. et al., see above, page 353). Accordingly, the accumulation of glycation endproducts is associated with senescence and lack of function.
[17] The damage or stress that causes cellular senescence also negatively impacts mitochondrial DNA in the cells to cause them to produce free radicals which react with sugars in the cell to form methyl glyoxal (MG). MG in turn reacts with proteins or lipids to generate advanced glycation end products. In the case of the protein component lysine, MG reacts to form carboxymethyllysine, which is an AGE.
[18] Damage or stress to mitochondrial DNA also sets off a DNA damage response which induces the cell to produce cell cycle blocking proteins. These blocking proteins prevent the cell from dividing. Continued damage or stress causes mTOR production, which in turn activates protein synthesis and inactivates protein breakdown. Further stimulation of the cells leads to programmed cell death (apoptosis).
[19] p16 is a protein involved in regulation of the cell cycle, by inhibiting the S phase (synthesis phase). It can be activated during ageing or in response to various stresses, such as DNA damage, oxidative stress or exposure to drugs. p16 is typically considered a tumor suppressor protein, causing a cell to become senescent in response to DNA damage and irreversibly preventing the cell from entering a hyperproliferative state. However, there has been some ambiguity in this regard, as some tumors show overexpression of p16, while others show downregulated expression. Evidence suggests that overexpression of p16 is some tumors results from a defective retinoblastoma protein (“Rb"). p16 acts on Rb to inhibit the S phase, and Rb downregulates p16, creating negative feedback. Defective Rb fails to both inhibit the S phase and downregulate p16, thus resulting in overexpression of p16 in hyperproliferating cells (Romagosa, C. et al., p16lnk4a overexpression in cancer: a tumor suppressor gene associated with senescence and high-grade tumors, Oncogene, Vol. 30, 2087-2097 (2011)).
[20] Senescent cells are associated with secretion of many factors involved in intercellular signaling, including pro-inflammatory factors; secretion of these factors has been termed the senescence-associated secretory phenotype, or SASP (Freund, A. “Inflammatory networks during cellular senescence: causes and consequences” Trends Mol Med. 2010 May;16(5):238-46). Autoimmune diseases, such as Crohn’s disease and rheumatoid arthritis, are associated with chronic inflammation (Ferraccioli, G. etal. “Interleukin-1 p and lnterleukin-6 in Arthritis Animal Models: Roles in the Early Phase of Transition from Acute to Chronic Inflammation and Relevance for Human Rheumatoid Arthritis” Mol Med. 2010 Nov-Dec; 16(11-12): 552-557). Chronic inflammation may be characterized by the presence of pro- inflammatory factors at levels higher than baseline near the site of pathology, but lower than those found in acute inflammation. Examples of these factors include TNF, IL-1a, IL-1 p, IL-5, IL-6, IL-8, IL-12, IL-23, CD2, CD3, CD20, CD22, CD52, CD80, CD86, C5 complement protein, BAFF, APRIL, IgE, a4 1 integrin and a4|37 integrin. Senescent cells also upregulate genes with roles in inflammation including IL-1 p, IL-8, ICAM1, TNFAP3, ESM1 and CCL2 (Burton, D.G.A. et al., “Microarray analysis of senescent vascular smooth muscle cells: a link to atherosclerosis and vascular calcification”, Experimental Gerontology, Vol. 44, No. 10, pp. 659-665 (October 2009)).
[21] Senescent cells secrete reactive oxygen species (“ROS”) as part of the SASP. ROS are believed to play an important role in maintaining senescence of cells. The secretion of ROS creates a bystander effect, where senescent cells induce senescence in neighboring cells: ROS create the very cellular damage known to activate p16 expression, leading to senescence (Nelson, G., A senescent cell bystander effect: senescence-induced senescence, Aging Cell, Vo. 11, 345-349 (2012)). The p16/Rb pathway leads to the induction of ROS, which in turn activates the protein kinase C delta creating a positive feedback loop that further enhance ROS, helping maintain the irreversible cell cycle arrest; it has even been suggested that exposing cancer cells to ROS might be effective to treat cancer by inducing cell phase arrest in hyperproliferating cells (Rayess, H. et al., Cellular senescence and tumor suppressor gene p16, Int J Cancer, Vol. 130, 1715-1725 (2012)).
[22] Recent research demonstrates the therapeutic benefits of removing senescent cells. In vivo animal studies at the Mayo Clinic in Rochester, Minnesota, found that elimination of senescent cells in transgenic mice carrying a biomarker for elimination delayed age-related disorders associated with cellular senescence. Eliminating senescent cells in fat and muscle tissues substantially delayed the onset of sarcopenia and cataracts and reduced senescence indicators in skeletal muscle and the eye (Baker, D. J. et al., “Clearance of p16lnk4a-positive senescent cells delays ageing-associated disorders", Nature, Vol. 479, pp. 232-236, (2011)). Mice that were treated to induce senescent cell elimination were found to have larger diameters of muscle fibers as compared to untreated mice. Treadmill exercise tests indicated that treatment also preserved muscle function. Continuous treatment of transgenic mice for removal of senescent cells had no negative side effects and selectively delayed age-related phenotypes that depend on cells. This data demonstrates that removal of senescent cells produces beneficial therapeutic effects and shows that these benefits may be achieved without adverse effects.
[23] Additional In vivo animal studies in mice found that removing senescent cells using senolytic agents treats aging-related disorders and atherosclerosis. Shortterm treatment with senolytic drugs in chronologically aged or progeroid mice alleviated several aging-related phenotypes (Zhu, Y. etal., “The Achilles’ heel of senescent cells: from transcriptome to senolytic drugs”, Aging Cell, vol. 14, pp. 644- 658 (2015)). Long-term treatment with senolytic drugs improved vasomotor function in mice with established atherosclerosis and reduced intimal plaque calcification (Roos, C.M. et al., “Chronic senolytic treatment alleviates established vasomotor dysfunction in aged or atherosclerotic mice”, Aging Cell (2016)). This data further demonstrates the benefits of removing senescent cells. [24] Vaccines have been widely used since their introduction by Edward Jenner in the 1770s to confer immunity against a wide range of diseases and afflictions. Vaccine preparations contain a selected immunogenic agent capable of stimulating immunity to an antigen. Typically, antigens are used as the immunogenic agent in vaccines, such as, for example, viruses, either killed or attenuated, and purified viral components. Antigens used in the production of cancer vaccines include, for example, tumor-associated carbohydrate antigens (TACAs), dendritic cells, whole cells and viral vectors. Different techniques are employed to produce the desired amount and type of antigen being sought. For example, pathogenic viruses are grown either in eggs or cells. Recombinant DNA technology is often utilized to generate attenuated viruses for vaccines.
[25] Vaccines may therefore be used to stimulate the production of antibodies in the body and provide immunity against antigens. When an antigen is introduced to a subject that has been vaccinated and developed immunity to that antigen, the immune system may destroy or remove cells that express the antigen.
SUMMARY
[26] In a first aspect, the invention is a method of treating or preventing the onset of a fibrotic disease comprising administering to a subject a composition comprising an anti-AGE antibody.
[27] In a second aspect, the invention is a method of treating or preventing the onset of a fibrotic disease comprising administering to a subject a vaccine comprising an AGE antigen.
[28] DEFINITIONS
[29] The term “fibrotic disease” means a disease or disorder characterized by the formation of fibrous tissue. Examples of fibrotic diseases include interstitial lung disease, pulmonary fibrosis, liver fibrosis, cirrhosis, urinary fibrosis, kidney fibrosis, nephrosclerosis, nephrosis, cardiovascular disease, macular degeneration, vitreal retinopathy, scleroderma (systemic and local), hypertrophic scarring, keloid scarring, restenosis, myelofibrosis, nephrogenic fibrosing dermopathy, mixed connective tissue disease, scleromyxedema, scleredema, and eosinophilic fasciitis. As used herein, “fibrotic disease” does not include atherosclerosis.
[30] The term “pulmonary fibrosis” means a disease or disorder characterized by the formation of fibrous tissue in the lungs. Pulmonary fibrosis includes idiopathic pulmonary fibrosis (IPF), idiopathic nonspecific interstitial pneumonia (NSIP), cryptogenic organizing pneumonia (COP), sarcoidosis, familial pulmonary fibrosis (FPF), familial interstitial pneumonia (FIP), asbestosis, silicosis, berylliosis, hypersensitivity pneumonitis, atypical pneumonia, pneumocystis pneumonia, tuberculosis, respiratory syncytial virus, acute interstitial pneumonitis/pneumonia (also known as Hamman-Rich syndrome), chronic obstructive pulmonary disease (COPD), emphysema and mesothelioma.
[31] The term “liver fibrosis" means a disease or disorder characterized by the formation of fibrous tissue in the liver. Liver fibrosis includes cirrhosis.
[32] The term “dermal fibrosis” means a disease or disorder characterized by the formation of fibrous tissue in the skin. Dermal fibrosis includes keloid scarring, hypertrophic scarring, scleroderma, nephrogenic fibrosing dermopathy, mixed connective tissue disease, scleromyxedema, scleredema and eosinophilic fasciitis.
[33] The term “urinary fibrosis” means a disease or disorder characterized by the formation of fibrous tissue in the kidneys, ureter, bladder or the urethra. Urinary fibrosis includes kidney fibrosis, nephrosclerosis, nephrosis, glomerulosclerosis, tubulointerstitial renal fibrosis, bladder fibrosis and urethral stricture. Urinary fibrosis may also be referred to as “renal fibrosis”.
[34] The term “peptide” means a molecule composed of 2-50 amino acids.
[35] The term “protein” means a molecule composed of more than 50 amino acids.
[36] The terms “advanced glycation end-product”, “AGE”, “AGE-modified protein”, “AGE-modified peptide” and “glycation end-product” refer to modified proteins or peptides that are formed as the result of the reaction of sugars with protein side chains that further rearrange and form irreversible cross-links. This process begins with a reversible reaction between a reducing sugar and an amino group to form a Schiff base, which proceeds to form a covalently-bonded Amadori rearrangement product. Once formed, the Amadori product undergoes further rearrangement to produce AGEs. AGE-modified proteins and antibodies to AGE-modified proteins are described in U.S. 5,702,704 to Bucala (“Bucala”) and U.S. 6,380,165 to Al-Abed et al. (“Al-Abed”). Glycated proteins or peptides that have not undergone the necessary rearrangement to form AGEs, such as N-deoxyfructosyllysine found on glycated albumin, are not AGEs. AGEs may be identified by the presence of AGE modifications (also referred to as AGE epitopes or AGE moieties) such as 2-(2- furoyl)-4(5)-(2-furanyl)-1 H-imidazole ("FFI"); 5-hydroxymethyl-1-alkylpyrrole-2- carbaldehyde ("Pyrraline"); 1-alkyl-2-formyl-3,4-diglycosyl pyrrole ("AFGP"), a non- fluorescent model AGE; carboxymethyllysine; carboxyethyllysine; and pentosidine. ALI, another AGE, is described in Al-Abed.
[37] The term “AGE antigen” means a substance that elicits an immune response against an AGE-modified protein or peptide of a cell. The immune response against an AGE-modified protein or peptide of a cell does not include the production of antibodies to the non-AGE-modified protein or peptide.
[38] “An antibody that binds to an AGE-modified protein on a cell”, “anti-AGE antibody” or “AGE antibody” means an antibody, antibody fragment or other protein or peptide that binds to an AGE-modified protein or peptide which preferably includes a constant region of an antibody, where the protein or peptide which has been AGE-modified is a protein or peptide normally found bound on the surface of a cell, preferably a mammalian cell, more preferably a human, cat, dog, horse, camelid (for example, camel or alpaca), cattle, sheep, pig, or goat cell. “An antibody that binds to an AGE-modified protein on a cell”, “anti-AGE antibody” or “AGE antibody” does not include an antibody or other protein which binds with the same specificity and selectivity to both the AGE-modified protein or peptide, and the same non-AGE- modified protein or peptide (that is, the presence of the AGE modification does not increase binding). AGE-modified albumin is not an AGE-modified protein on a cell, because albumin is not a protein normally found bound on the surface of cells. “An antibody that binds to an AGE-modified protein on a cell”, “anti-AGE antibody” or “AGE antibody” only includes those antibodies which lead to removal, destruction, or death of the cell. Also included are antibodies which are conjugated, for example to a toxin, drug, or other chemical or particle. Preferably, the antibodies are monoclonal antibodies, but polyclonal antibodies are also possible.
[39] The term “senescent cell” means a cell which is in a state of proliferative arrest and expresses one or more biomarkers of senescence, such as activation of p16lnk4a or expression of senescence-associated P-galactosidase. Also included are cells which express one or more biomarkers of senescence, do not proliferate in vivo, but may proliferate in vitro under certain conditions, such as some satellite cells found in the muscles of ALS patients.
[40] The term “senolytic agent” means a small molecule with a molecular weight of less than 900 daltons that destroys senescent cells. The term “senolytic agent" does not include antibodies, antibody conjugates, proteins, peptides or biologic therapies.
[41] The term “variant” means a nucleotide, protein or amino acid sequence different from the specifically identified sequences, wherein one or more nucleotides, proteins or amino acid residues is deleted, substituted or added. Variants may be naturally-occurring allelic variants, or non-naturally-occurring variants. Variants of the identified sequences may retain some or all of the functional characteristics of the identified sequences.
[42] The term "percent (%) sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Preferably, % sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is publicly available from Genentech, Inc. (South San Francisco, CA), or may be compiled from the source code, which has been filed with user documentation in the U.S. Copyright Office and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
[43] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. Where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program.
BRIEF DESCRIPTION OF THE DRAWINGS
[44] FIG. 1 is a graph of the response versus time in an antibody binding experiment.
[45] FIG. 2 is a graph illustrating the effect of senescent cell clearance on peripheral capillary oxygen saturation (SpCh) in bleomycin exposed mice.
[46] FIG. 3A is a graph illustrating the effect of senescent cell clearance with ganciclovir on lung elastance in 3MR mice exposed to bleomycin. [47] FIG. 3B is a graph illustrating the effect of senescent cell clearance with ganciclovir on dynamic lung compliance in 3MR mice exposed to bleomycin.
[48] FIG. 3C is a graph illustrating the effect of senescent cell clearance with ganciclovir on static lung compliance in 3MR mice exposed to bleomycin.
[49] FIG. 4 is a graph illustrating the effect of senescent cell clearance on peripheral capillary oxygen saturation (SpOa) in mice after 2 months and 4 months of cigarette smoke (CS) exposure. AP=AP20187; GAN=ganciclovir; Navi=Navitoclax (ABT-263); and Nutlin=Nutlin 3A.
DETAILED DESCRIPTION
[50] Studies of fibrotic diseases have established that inflammation is involved in fibrosis. Pro-inflammatory factors, such as TGF , PDGF, IL-1 p, IL-6, IL-10, IL-13 and IFN-y, and reactive oxygen species have been recognized as mediators of fibrosis (Bataller, R. et al., “Liver fibrosis”, The Journal of Clinical Investigation, Vol. 115, No. 2, p. 209-218 (2005)). Similarly, inflammatory cells and their secreted inflammatory factors have been recognized as the primary factors in activating dermal fibroblasts to become fibrotic (Shaw, T. J. et al., “Wound-associated skin fibrosis: mechanisms and treatments based on modulating the inflammatory response”, Endocrine, Metabolic & Immune Disorders, Vol. 10, No. 4, pp. 320-330 (2010)). These studies have led to the development of therapies that treat fibrotic diseases by targeting pro-inflammatory factors. Administration of resveratrol, a known anti-inflammatory agent, and an inhibitor of the inflammatory cytokine monocyte chemoattractant protein-1 (MCP-1) was shown to inhibit fibroplasia in wound healing and reduce or prevent scar formation (WO 2016/057831). Similarly, interference with TGF signaling was found to influence liver fibrosis in mouse models (Weiler-Normann, C., et al., “Mouse models of liver fibrosis”, Zeitschrift fur Gastroenterologie, Vol. 45, p. 43-50 (2007)). However, treatments that target the inflammatory cascade have been unsuccessful (Wynn). Recent research into the pathogenesis of fibrotic diseases suggests that senescent cells, which secrete pro- inflammatory factors as part of the SASP, may be a more appropriate therapeutic target than pro-inflammatory factors.
[51] Senescent cells have been implicated in a number of fibrotic diseases.
Senescent biomarkers such as p16, p21 and senescence-associated - galactosidase (SA-0-gal) have been observed in fibroblasts and epithelial cells in human and mouse idiopathic lung fibrosis tissue (Schafer, M. J. et al., “Cellular senescence mediates fibrotic pulmonary disease”, Nature Communications, Vol. 8, No. 14532, 11 pages (2017)). Pathogenic models of chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis both involve premature cellular senescence of progenitor cells, which results in stem cell exhaustion and disease progression (Chilosi, M. et al., “Premature lung aging and cellular senescence in the pathogenesis of idiopathic pulmonary fibrosis and COPD/emphysema”, Translational Research, Vol. 162, p. 156-173 (2013)). Senescent cells in the lungs contribute to excess extracellular matrix deposition in an aged mouse model and in elderly human samples (Calhoun, C. et al., “Senescent cells contribute to the physiological remodeling of aged lungs”, Journals of Gerontology: Biological Sciences, Vol. 71 , No. 2, p. 153-160 (2016)). The cell surface protein vimentin has been recognized as a marker of cellular senescence in mice that have been immunized with senescent mouse lung fibroblasts, and the IgM antibody clone 9H4 was found to bind to cell surface vimentin on senescent cells (Frescas, D. et al., “Senescent cells expose and secrete an oxidized form of membrane-bound vimentin as revealed by a natural polyreactive antibody", Proceedings of the National Academy of Sciences, p. E1668- E1677 (2017)). Cellular senescence and the SASP participate in the pathological process of CKD, and CKD accelerates the progression of cellular senescence and the secretion of inflammatory factors through the SASP (Wang, W-J. et al., “Cellular senescence, senescence-associated secretory phenotype, and chronic kidney disease", Oncotarget, Vol. 8, No. 38, pp. 64520-64533 (2017)). Senescent cells increase in the glomeruli in response to renal injury and aging, and increased senescent markers have been detected in glomerulosclerosis (Valentijn, F.A. et al., “Cellular senescence in the aging and diseased kidney”, Journal of Cell Communication and Signaling, Vol. 12, pp. 69-82 (2018)). [52] Advanced glycation end-products, known markers of senescent cells, have also been implicated in fibrotic diseases. RAGE, a receptor for advanced glycation end-products, is expressed by hepatic stellate cells and myofibroblasts, both of which are involved in the pathogenesis of liver fibrosis and cirrhosis (Yagmur, E. et al., “Elevation of A/£-(carboxymethyl)lysine-modified advanced glycation end products in chronic liver disease is an indicator of liver cirrhosis”, Clinical Biochemistry, Vol. 39, p. 39-45 (2006)). In addition, serum levels of carboxymethyllysine, the most well-studied advanced glycation end-product, were significantly affected by the stage of liver cirrhosis and were closely associated with liver function capacity (Yagmur et al.). AGE exposure followed by binding to RAGE results in tubulointerstitial fibrosis in the diabetic kidney (Oldfield, M.D. et al., “Advanced glycation end products cause epithelial-myofibroblast transdifferentiation via the receptor for advanced glycation end products (RAGE)”, The Journal of Clinical Investigation, Vol. 108, No. 12, pp. 1853-1863 (2001)). CML and pentosidine have also been identified in glomerulosclerosis, FSGS, hypertensive nephrosclerosis and lupus nephritis (Tanji, N. et al., “Expression of advanced glycation end products and their cellular receptor RAGE in diabetic nephropathy and nondiabetic renal disease”, Journal of the American Society of Nephrology, Vol. 11 , pp. 1656-1666 (2000)).
[53] Desmoplasia forms around tumors, acting as a barrier to drugs and even small molecules. The tumor cells are maintained by the cancer-associated fibroblasts that form the desmoplasia, and the tumor cells contribute to the growth of the desmoplasia. Senescent fibroblasts are present in the desmoplasia. AGEs have been detected in higher staining of surrounding fibroblast foci in fibrotic lungs of IPF patients, compared to control patients (Machahua, C., et aL, Increased AGE-RAGE ratio in idiopathic pulmonary fibrosis, Respiratory Research, vol. 17, no. 144, pp. 1-
11 (2016)). Eliminating or reducing the senescent fibroblasts would reduce the size of the desmoplasia, and it would enhance the permeability of the tumor to antibodies, natural killer cells (NK), immune effectors and other therapeutics, which would enhance the efficacy of the immune response or pharmaceuticals for treating cancer. [541 Recent studies have found that elimination of senescent cells can be used to treat fibrotic diseases. Elimination of senescent fibroblasts in mice by administration of a combination of the senolytic agents dasatinib and quercetin improved lung function (Schafer, M. J. et al.). Dasatinib alone attenuated bleomycin-induced lung fibrosis in vivo in mice, and suppressed TGFp-induced endothelial mesenchymal transition in vitro (Kato, M. et al., “Dasatinib suppresses TFG0-induced epithelial mesenchymal transition and inhibits pulmonary fibrosis”, European Respiratory Journal, Vol. 44, No. Suppl. 58, p. 742 (2014); Yilmaz, O. et al., “Dasatinib attenuated bleomycin-induced pulmonary fibrosis in mice”, Growth Factors, Vol. 33, No. 5, p. 366-375 (2015)). Ablation of p19ARF-expressing cells in transgenic mice using a toxin receptor-mediated cell knockout system improved aging-associated lung hypofunction (Hashimoto, M. etal., “Elimination of p19ARF-expressing cells enhances pulmonary function in mice”, Journal of Clinical Investigation Insight, Vol.
1 , No. 12, 15 pages (2016)). In addition, the elimination of p19ARF reversed the expression of other aging-associated genes. Administration of oral rapamycin to mice prevented cellular senescence in the vasculature and limited collagen deposition in the lungs (Calhoun, C. et al.). Removing senescent cells by administration of senolytic agents treated pulmonary fibrosis resulting from ionizing radiation (Pan, J. etal., “Inhibition of Bcl-2/xl with ABT-263 selectively kills senescent type II pneumocytes and reverses pulmonary fibrosis induced by ionizing radiation in mice”, International Journal of Radiation Oncology Biology Physics, Vol. 99, No. 2, pp. 353-361 (2017)). Clearance of senescent cells by induction of apoptosis in transgenic animal models attenuated glomerulosclerosis (Valentijn, F.A. etal.).
These studies validate the selection of senescent cells as a target for treating fibrotic diseases.
[55] The therapeutic benefits of removing senescent cells has been demonstrated in vivo in an art-accepted model in treating age-related diseases such as sarcopenia (US 9,161,810) and treating metastatic cancer (WO 2017/143073). The identification of a link between cellular senescence and fibrotic diseases allows for similar treatment possibilities. The present invention uses enhanced clearance of cells expressing AGE-modified proteins or peptides (AGE-modified cells) to treat, ameliorate or prevent the onset of fibrotic diseases by removing or killing senescent cells. This may be accomplished by administering anti-AGE antibodies to a subject.
[56] Vaccination against AGE-modified proteins or peptides of a cell may also be used to control the presence of AGE-modified cells in a subject. The continuous and virtually ubiquitous surveillance exercised by the immune system in the body in response to a vaccination allows maintaining low levels of AGE-modified cells in the body. Vaccination against AGE-modified proteins or peptides of a cell removes or kills senescent cells. The process of senescent cell removal or destruction allows vaccination against AGE-modified proteins or peptides of a cell to be used to treat or prevent the onset of fibrotic diseases. Individuals may receive repeated vaccinations or boosters on a periodic basis to maintain their immunity.
[57] Anti-AGE antibodies are known in the art and are commercially available. Examples include those described in U.S. 5,702,704 (Bucala) and U.S. 6,380,165 (Al-Abed et al.). The antibody may bind to one or more AGE-modified proteins or peptides having an AGE modification such as FFI, pyrraline, AFGP, ALI, carboxymethyllysine (CML), carboxyethyllysine (CEL) and pentosidine, and mixtures of such antibodies. Preferably, the antibody is non-immunogenic to the animal in which it will be used, such as non-immunogenic to humans; companion animals including cats, dogs and horses; and commercially important animals, such camels (or alpaca), cattle (bovine), sheep, pig, and goats. More preferably, the antibody has the same species constant region as antibodies of the animal to reduce the immune response against the antibody, such as being humanized (for humans), felinized (for cats), caninized (for dogs), equuinized (for horses), camelized (for camels or alpaca), bovinized (for cattle), ovinized (for sheep), porcinized (for pigs), or caperized (for goats). Most preferably, the antibody is identical to that of the animal in which it will be used (except for the variable region), such as a human antibody, a cat antibody, a dog antibody, a horse antibody, a camel antibody, a bovine antibody, a sheep antibody, a pig antibody, or a goat antibody. Details of the constant regions and other parts of antibodies for these animals are described below. The antibody may be monoclonal or polyclonal. Preferably, the antibody is a monoclonal antibody. [58] Preferred anti-AGE antibodies include those which bind to proteins or peptides that exhibit a carboxymethyllysine or carboxyethyllysine AGE modification. Carboxymethyllysine (also known as N(epsilon)-(carboxymethyl)lysine, N(6)- carboxymethyllysine, or 2-Amino-6-(carboxymethylamino)hexanoic acid) and carboxyethyllysine (also known as N-epsilon-(carboxyethyl)lysine) are found on proteins or peptides and lipids as a result of oxidative stress and chemical glycation. CML- and CEL-modified proteins or peptides are recognized by the receptor RAGE which is expressed on a variety of cells. CML and CEL have been well-studied and CML- and CEL-related products are commercially available. For example, Cell Biolabs, Inc. sells CML-BSA antigens, CML polyclonal antibodies, CML immunoblot kits, and CML competitive ELISA kits (www.cellbiolabs.com/cml-assays) as well as CEL-BSA antigens and CEL competitive ELISA kits (www.cellbiolabs.com/cel-n- epsilon-carboxyethyl-lysine-assays-and-reagents). A preferred antibody includes the variable region of the commercially available mouse anti-glycation end-product antibody raised against carboxymethyl lysine conjugated with keyhole limpet hemocyanin, the carboxymethyl lysine MAb (Clone 318003) available from R&D Systems, Inc. (Minneapolis, MN; catalog no. MAB3247), modified to have a human constant region (or the constant region of the animal into which it will be administered). Commercially-available antibodies, such as the carboxymethyl lysine antibody corresponding to catalog no. MAB3247 from R&D Systems, Inc., may be intended for diagnostic purposes and may contain material that is not suited for use in animals or humans. Preferably, commercially-available antibodies are purified and/or isolated prior to use in animals or humans to remove toxins or other potentially-harmful material.
[59] The anti-AGE antibody preferably has a low rate of dissociation from the antibody-antigen complex, or ka (also referred to as kback or off-rate), preferably at most 9 x 10-3, 8 x 10-3, 7 x 10'3 or 6 x 103 (sec1). The anti-AGE antibody preferably has a high affinity for the AGE-modified protein of a cell, which may be expressed as a low dissociation constant KD of at most 9 x 10’6, 8 x 10'6, 7 x 10’6, 6 x 10’6, 5 x 10’6, 4 x 10-6 or 3 x 10‘6 (M). Preferably, the binding properties of the anti-AGE antibody are similar to, the same as, or superior to the carboxymethyl lysine MAb (Clone 318003) available from R&D Systems, Inc. (Minneapolis, MN; catalog no.
MAB3247), illustrated in FIG. 1.
[60] The anti-AGE antibody may destroy AGE-modified cells through antibodydependent cell-mediated cytotoxicity (ADCC). ADCC is a mechanism of cell- mediated immune defense in which an effector cell of the immune system actively lyses a target cell whose membrane-surface antigens have been bound by specific antibodies. ADCC may be mediated by natural killer (NK) cells, macrophages, neutrophils or eosinophils. The effector cells bind to the Fc portion of the bound antibody. The anti-AGE antibody may also destroy AGE-modified cells through complement-dependent cytotoxicity (CDC). In CDC, the complement cascade of the immune system is triggered by an antibody binding to a target antigen.
[61] The anti-AGE antibody may be conjugated to an agent that causes the destruction of AGE-modified cells. Such agents may be a toxin, a cytotoxic agent, magnetic nanoparticles, and magnetic spin-vortex discs.
[62] A toxin, such as pore-forming toxins (PFT) (Aroian R. et al., “Pore-Forming Toxins and Cellular Non-lmmune Defenses (CNIDs),” Current Opinion in Microbiology, 10:57-61 (2007)), conjugated to an anti-AGE antibody may be injected into a patient to selectively target and remove AGE-modified cells. The anti-AGE antibody recognizes and binds to AGE-modified cells. Then, the toxin causes pore formation at the cell surface and subsequent cell removal through osmotic lysis.
[63] Magnetic nanoparticles conjugated to the anti-AGE antibody may be injected into a patient to target and remove AGE-modified cells. The magnetic nanoparticles can be heated by applying a magnetic field in order to selectively remove the AGE- modified cells.
[64] As an alternative, magnetic spin-vortex discs, which are magnetized only when a magnetic field is applied to avoid self-aggregation that can block blood vessels, begin to spin when a magnetic field is applied, causing membrane disruption of target cells. Magnetic spin-vortex discs, conjugated to anti-AGE antibodies specifically target AGE-modified cell types, without removing other cells. [65] Antibodies are Y-shaped proteins composed of two heavy chains and two light chains. The two arms of the Y shape form the fragment antigen-binding (Fab) region while the base or tail of the Y shape forms the fragment crystallizable (Fc) region of the antibody. Antigen binding occurs at the terminal portion of the fragment antigen-binding region (the tips of the arms of the Y shape) at a location referred to as the paratope, which is a set of complementarity determining regions (also known as CDRs or the hypervariable region). The complementarity determining regions vary among different antibodies and gives a given antibody its specificity for binding to a given antigen. The fragment crystallizable region of the antibody determines the result of antigen binding and may interact with the immune system, such as by triggering the complement cascade or initiating antibody-dependent cell-mediated cytotoxicity (ADCC). When antibodies are prepared recombinantly, it is also possible to have a single antibody with variable regions (or complementary determining regions) that bind to two different antigens, with each tip of the Y shape being specific to one of the antigens; these are referred to as bi-specific antibodies.
[66] A humanized anti-AGE antibody according to the present invention may have the human constant region sequence of amino acids shown in SEQ ID NO: 22. The heavy chain complementarity determining regions of the humanized anti-AGE antibody may have one or more of the protein sequences shown in SEQ ID NO: 23 (CDR1H), SEQ ID NO: 24 (CDR2H) and SEQ ID NO: 25 (CDR3H). The light chain complementarity determining regions of the humanized anti-AGE antibody may have one or more of the protein sequences shown in SEQ ID NO: 26 (CDR1L), SEQ ID NO: 27 (CDR2L) and SEQ ID NO: 28 (CDR3L).
[67] The heavy chain of a humanized anti-AGE antibody may have or may include the protein sequence of SEQ ID NO: 1. The variable domain of the heavy chain may have or may include the protein sequence of SEQ ID NO: 2. The complementarity determining regions of the variable domain of the heavy chain (SEQ ID NO: 2) are shown in SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43. The kappa light chain of a humanized anti-AGE antibody may have or may include the protein sequence of SEQ ID NO: 3. The variable domain of the kappa light chain may have or may include the protein sequence of SEQ ID NO: 4. Optionally, the arginine (Arg or R) residue at position 128 of SEQ ID NO: 4 may be omitted. The complementarity determining regions of the variable domain of the light chain (SEQ ID NO: 4) are shown in SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46. The variable regions may be codon-optimized, synthesized and cloned into expression vectors containing human immunoglobulin G1 constant regions. In addition, the variable regions may be used in the preparation of non-human anti-AGE antibodies.
[68] The antibody heavy chain may be encoded by the DNA sequence of SEQ ID NO: 12, a murine anti-AGE immunoglobulin G2b heavy chain. The protein sequence of the murine anti-AGE immunoglobulin G2b heavy chain encoded by SEQ ID NO: 12 is shown in SEQ ID NO: 16. The variable region of the murine antibody is shown in SEQ ID NO: 20, which corresponds to positions 25-142 of SEQ ID NO: 16. The antibody heavy chain may alternatively be encoded by the DNA sequence of SEQ ID NO: 13, a chimeric anti-AGE human immunoglobulin G1 heavy chain. The protein sequence of the chimeric anti-AGE human immunoglobulin G1 heavy chain encoded by SEQ ID NO: 13 is shown in SEQ ID NO: 17. The chimeric anti-AGE human immunoglobulin includes the murine variable region of SEQ ID NO: 20 in positions 25-142. The antibody light chain may be encoded by the DNA sequence of SEQ ID NO: 14, a murine anti-AGE kappa light chain. The protein sequence of the murine anti-AGE kappa light chain encoded by SEQ ID NO: 14 is shown in SEQ ID NO: 18. The variable region of the murine antibody is shown in SEQ ID NO: 21, which corresponds to positions 21-132 of SEQ ID NO: 18. The antibody light chain may alternatively be encoded by the DNA sequence of SEQ ID NO: 15, a chimeric anti- AGE human kappa light chain. The protein sequence of the chimeric anti-AGE human kappa light chain encoded by SEQ ID NO: 15 is shown in SEQ ID NO: 19. The chimeric anti-AGE human immunoglobulin includes the murine variable region of SEQ ID NO: 21 in positions 21-132.
[69] A humanized anti-AGE antibody according to the present invention may have or may include one or more humanized heavy chains or humanized light chains. A humanized heavy chain may be encoded by the DNA sequence of SEQ ID NO: 30, 32 or 34. The protein sequences of the humanized heavy chains encoded by SEQ ID NOs: 30, 32 and 34 are shown in SEQ ID NOs: 29, 31 and 33, respectively. A humanized light chain may be encoded by the DNA sequence of SEQ ID NO: 36, 38 or 40. The protein sequences of the humanized light chains encoded by SEQ ID NOs: 36, 38 and 40 are shown in SEQ ID NOs: 35, 37 and 39, respectively. Preferably, the humanized anti-AGE antibody maximizes the amount of human sequence while retaining the original antibody specificity. A complete humanized antibody may be constructed that contains a heavy chain having a protein sequence chosen from SEQ ID NOs: 29, 31 and 33 and a light chain having a protein sequence chosen from SEQ ID NOs: 35, 37 and 39.
[70] Particularly preferred anti-AGE antibodies may be obtained by humanizing murine monoclonal anti-AGE antibodies. Murine monoclonal anti-AGE antibodies have the heavy chain protein sequence shown in SEQ ID NO: 47 (the protein sequence of the variable domain is shown in SEQ ID NO: 52) and the light chain protein sequence shown in SEQ ID NO: 57 (the protein sequence of the variable domain is shown in SEQ ID NO: 62). A preferred humanized heavy chain may have the protein sequence shown in SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51 (the protein sequences of the variable domains of the humanized heavy chains are shown in SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, respectively). A preferred humanized light chain may have the protein sequence shown in SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 or SEQ ID NO: 61 (the protein sequences of the variable domains of the humanized light chains are shown in SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, respectively). Preferably, a humanized anti-AGE monoclonal antibody is composed a heavy chain having a protein sequence selected from the group consisting of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51 and a light chain having a protein sequence selected from the group consisting of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61. Humanized monoclonal anti-AGE antibodies composed of these protein sequences may have better binding and/or improved activation of the immune system, resulting in greater efficacy.
[71] The protein sequence of an antibody from a non-human species may be modified to include the variable domain of the heavy chain having the sequence shown in SEQ ID NO: 2 or the kappa light chain having the sequence shown in SEQ ID NO: 4. The non-human species may be a companion animal, such as the domestic cat or domestic dog, or livestock, such as cattle, the horse or the camel. Preferably, the non-human species is not the mouse. The heavy chain of the horse (Equus caballus) antibody immunoglobulin gamma 4 may have or may include the protein sequence of SEQ ID NO: 5 (EMBL/GenBank accession number AY445518). The heavy chain of the horse (Equus caballus) antibody immunoglobulin delta may have or may include the protein sequence of SEQ ID NO: 6 (EMBL/GenBank accession number AY631942). The heavy chain of the dog (Canis familiaris) antibody immunoglobulin A may have or may include the protein sequence of SEQ ID NO: 7 (GenBank accession number L36871). The heavy chain of the dog (Canis familiaris) antibody immunoglobulin E may have or may include the protein sequence of SEQ ID NO: 8 (GenBank accession number L36872). The heavy chain of the cat (Felis catus) antibody immunoglobulin G2 may have or may include the protein sequence of SEQ ID NO: 9 (DDBJ/EMBL/GenBank accession number KF811175).
[72] Animals of the camelid family, such as camels (Camelus dromedarius and
Camelus bactrianus), llamas (Lama glama, Lama paces and Lama vicugna), alpacas (Vicugna paces) and guanacos (Lama guanicoe), have a unique antibody that is not found in other mammals. In addition to conventional immunoglobulin G antibodies composed of heavy and light chain tetramers, camelids also have heavy chain immunoglobulin G antibodies that do not contain light chains and exist as heavy chain dimers. These antibodies are known as heavy chain antibodies, HCAbs, single-domain antibodies or sdAbs, and the variable domain of a camelid heavy chain antibody is known as the VHH. The camelid heavy chain antibodies lack the heavy chain CH1 domain and have a hinge region that is not found in other species. The variable region of the Arabian camel (Camelus dromedarius) single-domain antibody may have or may include the protein sequence of SEQ ID NO: 10 (GenBank accession number AJ245148). The variable region of the heavy chain of the Arabian camel (Camelus dromedarius) tetrameric immunoglobulin may have or may include the protein sequence of SEQ ID NO: 11 (GenBank accession number AJ245184). [73] In addition to camelids, heavy chain antibodies are also found in cartilaginous fishes, such as sharks, skates and rays. This type of antibody is known as an immunoglobulin new antigen receptor or IgNAR, and the variable domain of an IgNAR is known as the VNAR. The IgNAR exists as two identical heavy chain dimers composed of one variable domain and five constant domains each. Like camelids, there is no light chain.
[74] The protein sequences of additional non-human species may be readily found in online databases, such as the International ImMunoGeneTics Information System (www.imgt.org), the European Bioinformatics Institute (www.ebi.ac.uk), the DNA Databank of Japan (ddbj.nig.ac.jp/arsa) or the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).
[75] An anti-AGE antibody or a variant thereof may include a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51, including post-translational modifications thereof. A heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-AGE antibody including that sequence retains the ability to bind to AGE.
[76] An anti-AGE antibody or a variant thereof may include a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, including post-translational modifications thereof. A variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-AGE antibody including that sequence retains the ability to bind to AGE. The substitutions, insertions, or deletions may occur in regions outside the variable region.
[77] An anti-AGE antibody or a variant thereof may include a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 or SEQ ID NO: 61, including post-translational modifications thereof. A light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-AGE antibody including that sequence retains the ability to bind to AGE. The substitutions, insertions, or deletions may occur in regions outside the variable region.
[78] An anti-AGE antibody or a variant thereof may include a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 or SEQ ID NO: 66, including post-translational modifications thereof. A variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-AGE antibody including that sequence retains the ability to bind to AGE. The substitutions, insertions, or deletions may occur in regions outside the variable region.
[79] Alternatively, the antibody may have the complementarity determining regions of commercially available mouse anti-glycation end-product antibody raised against carboxymethyl lysine conjugated with keyhole limpet hemocyanin (CML-KLH), the carboxymethyl lysine MAb (Clone 318003) available from R&D Systems, Inc. (Minneapolis, MN; catalog no. MAB3247). [80] The antibody may have or may include constant regions which permit destruction of targeted cells by a subject’s immune system.
[81] Mixtures of antibodies that bind to more than one type AGE of AGE-modified proteins may also be used.
[82] Bi-specific antibodies, which are anti-AGE antibodies directed to two different epitopes, may also be used. Such antibodies will have a variable region (or complementary determining region) from those of one anti-AGE antibody, and a variable region (or complementary determining region) from a different antibody.
[83] Antibody fragments may be used in place of whole antibodies. For example, immunoglobulin G may be broken down into smaller fragments by digestion with enzymes. Papain digestion cleaves the N-terminal side of inter-heavy chain disulfide bridges to produce Fab fragments. Fab fragments include the light chain and one of the two N-terminal domains of the heavy chain (also known as the Fd fragment). Pepsin digestion cleaves the C-terminal side of the inter-heavy chain disulfide bridges to produce F(ab’)2 fragments. F(ab’)2 fragments include both light chains and the two N-terminal domains linked by disulfide bridges. Pepsin digestion may also form the Fv (fragment variable) and Fc (fragment crystallizable) fragments. The Fv fragment contains the two N-terminal variable domains. The Fc fragment contains the domains which interact with immunoglobulin receptors on cells and with the initial elements of the complement cascade. Pepsin may also cleave immunoglobulin G before the third constant domain of the heavy chain (CH3) to produce a large fragment F(abc) and a small fragment pFc’. Antibody fragments may alternatively be produced recombinantly. Preferably, such antibody fragments are conjugated to an agent that causes the destruction of AGE-modified cells.
[84] If additional antibodies are desired, they can be produced using well-known methods. For example, polyclonal antibodies (pAbs) can be raised in a mammalian host by one or more injections of an immunogen, and if desired, an adjuvant. Typically, the immunogen (and adjuvant) is injected in a mammal by a subcutaneous or intraperitoneal injection. The immunogen may be an AGE-modified protein of a cell, such as AGE-antithrombin III, AGE-calmodulin, AGE-insulin, AGE- ceruloplasmin, AGE-collagen, AGE-cathepsin B, AGE-albumin such as AGE-bovine serum albumin (AGE-BSA), AGE-human serum albumin and ovalbumin, AGE- crystallin, AGE-plasminogen activator, AGE-endothelial plasma membrane protein, AGE-aldehyde reductase, AGE-transferrin, AGE-fibrin, AGE-copper/zinc SOD, AGE- apo B, AGE-fibronectin, AGE-pancreatic ribose, AGE-apo A-l and II, AGE- hemoglobin, AGE-Na7K+-ATPase, AGE-plasminogen, AGE-myelin, AGE-lysozyme, AGE-immunoglobulin, AGE-red cell Glu transport protein, AGE-p-N-acetyl hexokinase, AGE-apo E, AGE-red cell membrane protein, AGE-aldose reductase, AGE-ferritin, AGE-red cell spectrin, AGE-alcohol dehydrogenase, AGE-haptoglobin, AGE-tubulin, AGE-thyroid hormone, AGE-fibrinogen, AGE-fh-microglobulin, AGE- sorbitol dehydrogenase, AGE-ai -antitrypsin, AGE-carbonate dehydratase, AGE- RNAse, AGE-hexokinase, AGE-apo C-l, AGE-hemoglobin such as AGE-human hemoglobin, AGE-low density lipoprotein (AGE-LDL) and AGE-collagen IV. AGE- modified cells, such as AGE-modified erythrocytes, whole, lysed, or partially digested, may also be used as AGE antigens. Examples of adjuvants include Freund’s complete, monophosphoryl Lipid A synthetic-trehalose dicorynomycolate, aluminum hydroxide (alum), heat shock proteins HSP 70 or HSP96, squalene emulsion containing monophosphoryl lipid A, a2-macroglobulin and surface active substances, including oil emulsions, pleuronic polyols, polyanions and dinitrophenol. To improve the immune response, an immunogen may be conjugated to a polypeptide that is immunogenic in the host, such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, cholera toxin, labile enterotoxin, silica particles or soybean trypsin inhibitor. A preferred immunogen conjugate is AGE- KLH. Alternatively, pAbs may be made in chickens, producing IgY molecules.
[85] Monoclonal antibodies (mAbs) may also be made by immunizing a host or lymphocytes from a host, harvesting the mAb-secreting (or potentially secreting) lymphocytes, fusing those lymphocytes to immortalized cells (for example, myeloma cells), and selecting those cells that secrete the desired mAb. Other techniques may be used, such as the EBV-hybridoma technique. Non-human antibodies may be made less immunogenic to humans by engineering the antibodies to contain a combination of non-human and human antibody components. A chimeric antibody may be produced by combining the variable region of a non-human antibody with a human constant region. A humanized antibody may be produced by replacing the complementarity determining regions (CDRs) of a human antibody with those of a non-human antibody. Similarly, antibodies may be made less immunogenic to other species by being substantially “ized” to a given animal, such as cat, dog, horse, camel or alpaca, cattle, sheep, pig, or goat, at the amino acid level. If desired, the mAbs may be purified from the culture medium or ascites fluid by conventional procedures, such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ammonium sulfate precipitation or affinity chromatography. Additionally, human monoclonal antibodies can be generated by immunization of transgenic mice containing a third copy IgG human trans-loci and silenced endogenous mouse Ig loci or using human-transgenic mice. Production of humanized monoclonal antibodies and fragments thereof can also be generated through phage display technologies.
[86] A "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Preferred examples of such carriers or diluents include water, saline, Ringer’s solutions and dextrose solution. Supplementary active compounds can also be incorporated into the compositions. Solutions and suspensions used for parenteral administration can include a sterile diluent, such as water for injection, saline solution, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
[87] The antibodies may be administered systemically, such as by intravenous injection or infusion. Alternatively, the antibodies may be administered locally with a pharmaceutically acceptable carrier suitable for the administration site, such as by percutaneous injection into an affected organ, topical administration at the site of dermal fibrosis or administration via nebulizer for pulmonary fibrosis.
Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions for the extemporaneous preparation of sterile injectable solutions or dispersion. Various excipients may be included in pharmaceutical compositions of antibodies suitable for injection. Suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL® (BASF; Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid so as to be administered using a syringe. Such compositions should be stable during manufacture and storage and must be preserved against contamination from microorganisms such as bacteria and fungi. Various antibacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal, can contain microorganism contamination. Isotonic agents such as sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride can be included in the composition. Compositions that can delay absorption include agents such as aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating antibodies, and optionally other therapeutic components, in the required amount in an appropriate solvent with one or a combination of ingredients as required, followed by sterilization. Methods of preparation of sterile solids for the preparation of sterile injectable solutions include vacuum drying and freeze-drying to yield a solid.
[88] For administration by inhalation, the antibodies may be delivered as an aerosol spray from a nebulizer or a pressurized container that contains a suitable propellant, for example, a gas such as carbon dioxide. Antibodies may also be delivered via inhalation as a dry powder, for example using the iSPERSE™ inhaled drug delivery platform (PULMATRIX, Lexington, Mass.). The use of anti-AGE antibodies which are chicken antibodies (IgY) may be non-immunogenic in a variety of animals, including humans, when administered by inhalation.
[89] An appropriate dosage level of each type of antibody will generally be about 0.01 to 500 mg per kg patient body weight. Preferably, the dosage level will be about 0.1 to about 250 mg/kg; more preferably about 0.5 to about 100 mg/kg. A suitable dosage level may be about 0.01 to 250 mg/kg, about 0.05 to 100 mg/kg, or about 0.1 to 50 mg/kg. Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg. Although each type of antibody may be administered on a regimen of 1 to 4 times per day, such as once or twice per day, antibodies typically have a long half-life in vivo. Accordingly, each type of antibody may be administered once a day, once a week, once every two or three weeks, once a month, or once every 60 to 90 days.
[90] A subject that receives administration of an anti-AGE antibody may be tested to determine if the administration has been effective to treat fibrotic diseases. A subject may be considered to have received an effective antibody treatment if he or she demonstrates an improvement in symptoms between subsequent measurements or over time. The efficacy of treatment may be determined with a diagnostic test that is suitable for a given fibrotic disease. For example, liver fibrosis may be monitored with a liver biopsy and pulmonary fibrosis may be monitored with high-resolution computed tomography, pulmonary function tests, bronchoscopy or bronchoalveolar lavage. Alternatively, the concentration and/or number of senescent cells may be measured over time. Administration of antibody and subsequent testing may be repeated until the desired therapeutic result is achieved.
[91] Unit dosage forms can be created to facilitate administration and dosage uniformity. Unit dosage form refers to physically discrete units suited as single dosages for the subject to be treated, containing a therapeutically effective quantity of one or more types of antibodies in association with the required pharmaceutical carrier. Preferably, the unit dosage form is in a sealed container and is sterile.
[92] Vaccines against AGE-modified proteins or peptides contain an AGE antigen, an adjuvant, optional preservatives and optional excipients. Examples of AGE antigens include AGE-modified proteins or peptides such as AGE-antithrombin III, AGE-calmodulin, AGE-insulin, AGE-ceruloplasmin, AGE-collagen, AGE-cathepsin B, AGE-albumin such as AGE-bovine serum albumin (AGE-BSA), AGE-human serum albumin and ovalbumin, AGE-crystallin, AGE-plasminogen activator, AGE- endothelial plasma membrane protein, AGE-aldehyde reductase, AGE-transferrin, AGE-fibrin, AGE-copper/zinc SOD, AGE-apo B, AGE-fibronectin, AGE-pancreatic ribose, AGE-apo A-l and II, AGE-hemoglobin, AGE-Na+/K+-ATPase, AGE- plasminogen, AGE-myelin, AGE-lysozyme, AGE-immunoglobulin, AGE-red cell Glu transport protein, AGE-0-N-acetyl hexokinase, AGE-apo E, AGE-red cell membrane protein, AGE-aldose reductase, AGE-ferritin, AGE-red cell spectrin, AGE-alcohol dehydrogenase, AGE-haptoglobin, AGE-tubulin, AGE-thyroid hormone, AGE- fibrinogen, AGE- 2-microglobulin, AGE-sorbitol dehydrogenase, AGE-cu -antitrypsin, AGE-carbonate dehydratase, AGE-RNAse, AGE-hexokinase, AGE-apo C-l, AGE- hemoglobin such as AGE-human hemoglobin, AGE-low density lipoprotein (AGE- LDL) and AGE-collagen IV. AGE-modified cells, such as AGE-modified erythrocytes, whole, lysed, or partially digested, may also be used as AGE antigens. Suitable AGE antigens also include proteins or peptides that exhibit AGE modifications (also referred to as AGE epitopes or AGE moieties) such as carboxymethyllysine (CML), carboxyethyllysine (CEL), pentosidine, pyrraline, FFI, AFGP and ALL The AGE antigen may be an AGE-protein conjugate, such as AGE conjugated to keyhole limpet hemocyanin (AGE-KLH). Further details of some of these AGE-modified proteins or peptides and their preparation are described in Bucala.
[93] Particularly preferred AGE antigens include proteins or peptides that exhibit a carboxymethyllysine or carboxyethyllysine AGE modification. Carboxymethyllysine (also known as N(epsilon)-(carboxymethyl)lysine, N(6)-carboxymethyllysine, or 2- Amino-6-(carboxymethylamino)hexanoic acid) and carboxyethyllysine (also known as N-epsilon-(carboxyethyl)lysine) are found on proteins or peptides and lipids as a result of oxidative stress and chemical glycation, and have been correlated with juvenile genetic disorders. CML- and CEL-modified proteins or peptides are recognized by the receptor RAGE which is expressed on a variety of cells. CML and CEL have been well-studied and CML- and CEL-related products are commercially available. For example, Cell Biolabs, Inc. sells CML-BSA antigens, CML polyclonal antibodies, CML immunoblot kits, and CML competitive ELISA kits (www.cellbiolabs.com/cml-assays) as well as CEL-BSA antigens and CEL competitive ELISA kits (www.cellbiolabs.com/cel-n-epsilon-carboxyethyl-lysine- assays-and-reagents).
[94] AGE antigens may be conjugated to carrier proteins to enhance antibody production in a subject. Antigens that are not sufficiently immunogenic alone may require a suitable carrier protein to stimulate a response from the immune system. Examples of suitable carrier proteins include keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, cholera toxin, labile enterotoxin, silica particles and soybean trypsin inhibitor. Preferably, the carrier protein is KLH (AGE-KLH). KLH has been extensively studied and has been identified as an effective carrier protein in experimental cancer vaccines. Preferred AGE antigen-carrier protein conjugates include CML-KLH and CEL-KLH.
[95] The administration of an AGE antigen allows the immune system to develop immunity to the antigen. Immunity is a long-term immune response, either cellular or humoral. A cellular immune response is activated when an antigen is presented, preferably with a co-stimulator to a T-cell which causes it to differentiate and produce cytokines. The cells involved in the generation of the cellular immune response are two classes of T-helper (Th) cells, Th1 and Th2. Th1 cells stimulate B cells to produce predominantly antibodies of the lgG2A isotype, which activates the complement cascade and binds the Fc receptors of macrophages, while Th2 cells stimulate B cells to produce IgG 1 isotype antibodies in mice, lgG4 isotype antibodies in humans, and IgE isotype antibodies. The human body also contains “professional” antigen-presenting cells such as dendritic cells, macrophages, and B cells.
[96] A humoral immune response is triggered when a B cell selectively binds to an antigen and begins to proliferate, leading to the production of a clonal population of cells that produce antibodies that specifically recognize that antigen and which may differentiate into antibody-secreting cells, referred to as plasma-cells or memory-B cells. Antibodies are molecules produced by B-cells that bind a specific antigen. The antigen-antibody complex triggers several responses, either cell-mediated, for example by natural killers (NK) or macrophages, or serum-mediated, for example by activating the complement system, a complex of several serum proteins that act sequentially in a cascade that result in the lysis of the target cell.
[97] Immunological adjuvants (also referred to simply as “adjuvants”) are the component(s) of a vaccine which augment the immune response to the immunogenic agent. Adjuvants function by attracting macrophages to the immunogenic agent and then presenting the agent to the regional lymph nodes to initiate an effective antigenic response. Adjuvants may also act as carriers themselves for the immunogenic agent. Adjuvants may induce an inflammatory response, which may play an important role in initiating the immune response.
[98] Adjuvants include mineral compounds such as aluminum salts, oil emulsions, bacterial products, liposomes, immunostimulating complexes and squalene. Aluminum compounds are the most widely used adjuvants in human and veterinary vaccines. These aluminum compounds include aluminum salts such as aluminum phosphate (AIPO4) and aluminum hydroxide (AI(OH)3) compounds, typically in the form of gels, and are generically referred to in the field of vaccine immunological adjuvants as "alum." Aluminum hydroxide is a poorly crystalline aluminum oxyhydroxide having the structure of the mineral boehmite. Aluminum phosphate is an amorphous aluminum hydroxyphosphate. Negatively charged species (for example, negatively charged antigens) can absorb onto aluminum hydroxide gels at neutral pH, whereas positively charged species (for example, positively charged antigens) can absorb onto aluminum phosphate gels at neutral pH. It is believed that these aluminum compounds provide a depot of antigen at the site of administration, thereby providing a gradual and continuous release of antigen to stimulate antibody production. Aluminum compounds tend to more effectively stimulate a cellular response mediated by Th2, rather than Th1 cells.
[99] Emulsion adjuvants include water-in-oil emulsions (for example, Freund's adjuvants, such as killed mycobacteria in oil emulsion) and oil-in-water emulsions (for example, MF-59). Emulsion adjuvants include an immunogenic component, for example squalene (MF-59) or mannide oleate (Incomplete Freund's Adjuvants), which can induce an elevated humoral response, increased T cell proliferation, cytotoxic lymphocytes and cell-mediated immunity.
[100] Liposomal or vesicular adjuvants (including paucilamellar lipid vesicles) have lipophilic bilayer domains and an aqueous milieu which can be used to encapsulate and transport a variety of materials, for example an antigen. Paucilamellar vesicles (for example, those described in U.S. Pat. No. 6,387,373) can be prepared by mixing, under high pressure or shear conditions, a lipid phase comprising a nonphospholipid material (for example, an amphiphile surfactant; see U.S. Pat. Nos. 4,217,344; 4,917,951 ; and 4,911 ,928), optionally a sterol, and any water-immiscible oily material to be encapsulated in the vesicles (for example, an oil such as squalene oil and an oil-soluble or oil-suspended antigen); and an aqueous phase such as water, saline, buffer or any other aqueous solution used to hydrate the lipids. Liposomal or vesicular adjuvants are believed to promote contact of the antigen with immune cells, for example by fusion of the vesicle to the immune cell membrane, and preferentially stimulate the Th1 sub-population of T-helper cells.
[101] Other types of adjuvants include Mycobacterium bovis bacillus Calmette- Guerin (BCG), quill-saponin and unmethylated CpG dinucleotides (CpG motifs). Additional adjuvants are described in U.S. Patent Application Publication Pub. No. US 2010/0226932 (September 9, 2010) and Jiang, Z-H. et al. “Synthetic vaccines: the role of adjuvants in immune targeting”, Current Medicinal Chemistry, Vol. 10(15), pp. 1423-39 (2003). Preferable adjuvants include Freund’s complete adjuvant and Freund’s incomplete adjuvant.
[102] The vaccine may optionally include one or more preservatives, such as antioxidants, antibacterial and antimicrobial agents, as well as combinations thereof. Examples include benzethonium chloride, ethylenediamine-tetraacetic acid sodium (EDTA), thimerosal, phenol, 2-phenoxyethanol, formaldehyde and formalin; antibacterial agents such as amphotericin B, chlortetracycline, gentamicin, neomycin, polymyxin B and streptomycin; antimicrobial surfactants such as polyoxyethylene-9, 10-nonyl phenol (Triton N-101 , octoxynol-9), sodium deoxycholate and polyoxyethylated octyl phenol (Triton X-I00). The production and packaging of the vaccine may eliminate the need for a preservative. For example, a vaccine that has been sterilized and stored in a sealed container may not require a preservative.
[103] Other components of vaccines include pharmaceutically acceptable excipients, such as stabilizers, thickening agents, toxin detoxifiers, diluents, pH adjusters, tonicity adjustors, surfactants, antifoaming agents, protein stabilizers, dyes and solvents. Examples of such excipients include hydrochloric acid, phosphate buffers, sodium acetate, sodium bicarbonate, sodium borate, sodium citrate, sodium hydroxide, potassium chloride, potassium chloride, sodium chloride, polydimethylsilozone, brilliant green, phenol red (phenolsulfon-phthalein), glycine, glycerin, sorbitol, histidine, monosodium glutamate, potassium glutamate, sucrose, urea, lactose, gelatin, sorbitol, polysorbate 20, polysorbate 80 and glutaraldehyde. A variety of these components of vaccines, as well as adjuvants, are described in www.cdc.gov/vaccines/pubs/pinkbook/downloads/appendices/B/excipient-table-2.pdf and Vogel, F. R. et al., “A compendium of vaccine adjuvants and excipients”, Pharmaceutical Biotechnology, Vol. 6, pp. 141-228 (1995).
[104] The vaccine may contain from 1 pg to 100 mg of at least one AGE antigen, including 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 400, 800 or 1000 pg, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80 or 90 mg. The amount used for a single injection corresponds to a unit dosage.
[105] The vaccine may be provided in unit dosage form or in multidosage form, such as 2-100 or 2-10 doses. The unit dosages may be provided in a vial with a septum, or in a syringe with or without a needle. The vaccine may be administered intravenously, subdermally or intraperitoneally. Preferably, the vaccine is sterile.
[106] The vaccine may be administered one or more times, such as 1 to 10 times, including 2, 3, 4, 5, 6, 7, 8 or 9 times, and may be administered over a period of time ranging from 1 week to 1 year, 2-10 weeks or 2-10 months. Furthermore, booster vaccinations may be desirable, over the course of 1 year to 20 years, including 2, 5, 10 and 15 years. [107] A subject that receives a vaccine for AGE-modified proteins or peptides of a cell may be tested to determine if he or she has developed an immunity to the AGE- modified proteins or peptides. Suitable tests may include blood tests for detecting the presence of an antibody, such as immunoassays or antibody titers. An immunity to AGE-modified proteins or peptides may also be determined by monitoring the concentration and/or number of senescent cells over time. In addition to testing for the development of an immunity to AGE-modified proteins or peptides, a subject may also be tested to determine if the vaccination has been effective to treat fibrotic diseases. A subject may be considered to have received an effective vaccination if he or she demonstrates an improvement in symptoms between subsequent measurements or overtime, or by measuring the concentration and/or number of senescent cells. Vaccination and subsequent testing may be repeated until the desired therapeutic result is achieved.
[108] The vaccination process may be designed to provide immunity against multiple AGE moieties. A single AGE antigen may induce the production of AGE antibodies which are capable of binding to multiple AGE moieties. Alternatively, the vaccine may contain multiple AGE antigens. In addition, a subject may receive multiple vaccines, where each vaccine contains a different AGE antigen.
[109] Any mammal that could develop fibrotic diseases may be treated by the methods herein described. Humans are a preferred mammal for treatment. Other mammals that may be treated include mice, rats, goats, sheep, pigs, cows, horses and companion animals, such as dogs or cats. Alternatively, any of the mammals or subjects identified above may be excluded from the patient population in need of treatment for fibrotic diseases.
[110] A subject may be identified as in need of treatment based on a diagnosis with at least one fibrotic disease, or with a disease that is known to contribute to fibrosis. Fibrotic diseases include interstitial lung disease, pulmonary fibrosis, liver fibrosis, cirrhosis, urinary fibrosis, kidney fibrosis, kidney disease, chronic kidney disease (CKD), nephrosclerosis, nephrosis, glomerulosclerosis, bladder fibrosis, urethral stricture, cardiovascular disease, macular degeneration, vitreal retinopathy, scleroderma (systemic and local), restenosis, myelofibrosis (bone marrow cancer), keloid scarring, hypertrophic scarring, nephrogenic fibrosing dermopathy, mixed connective tissue disease, scleromyxedema, scleredema and eosinophilic fasciitis. Fibrotic diseases include desmoplasia, as fibrotic tissue in the surrounding stroma plays a very important role in the progression of cancer. Diagnosis may involve any suitable diagnostic test or procedure for a given disease or disorder. A subject may also be identified as in need of treatment after experiencing physical trauma that is known to result in fibrosis, such as surgical complications, administration of chemotherapeutic drugs, exposure to radiation, burns or physical injury.
[111] Subjects may also be identified as in need of treatment based on detection of advanced glycation end products in a sample obtained from the subject. Suitable samples include blood, skin, serum, saliva and urine. The diagnostic use of anti- AGE antibodies is discussed in more detail in International Publication No. WO 2018/204679.
[112] The Present Application includes 66 nucleotide and amino acid sequences in the Sequence Listing filed herewith. Variants of the nucleotide and amino acid sequences are possible. Known variants include substitutions, deletions and additions to the sequences shown in SEQ ID NO: 4, 16 and 20. In SEQ ID NO: 4, the arginine (Arg or R) residue at position 128 may optionally be omitted. In SEQ ID NO: 16, the alanine residue at position 123 may optionally be replaced with a serine residue, and/or the tyrosine residue at position 124 may optionally be replaced with a phenylalanine residue. SEQ ID NO: 20 may optionally include the same substitutions as SEQ ID NO: 16 at positions 123 and 124. In addition, SEQ ID NO: 20 may optionally contain one additional lysine residue after the terminal valine residue.
[113] EXAMPLES
[114] Example 1 : In vivo study of the administration of anti-glycation end-product antibody [115] To examine the effects of an anti-glycation end-product antibody, the antibody was administered to the aged CD1(ICR) mouse (Charles River Laboratories), twice daily by intravenous injection, once a week, for three weeks (Days 1, 8 and 15), followed by a 10 week treatment-free period. The test antibody was a commercially available mouse anti-glycation end-product antibody raised against carboxymethyl lysine conjugated with keyhole limpet hemocyanin, the carboxymethyl lysine MAb (Clone 318003) available from R&D Systems, Inc. (Minneapolis, MN; catalog no. MAB3247). A control reference of physiological saline was used in the control animals.
[116] Mice referred to as “young” were 8 weeks old, while mice referred to as “old” were 88 weeks (±2 days) old. No adverse events were noted from the administration of the antibody. The different groups of animals used in the study are shown in Table 1.
[117] Table 1 : The different groups of animals used in the study
Figure imgf000041_0001
- = Not Applicable, Pre = Subset of animals euthanized prior to treatment start for collection of adipose tissue.
[118] P16lNK4a mRNA, a marker for senescent cells, was quantified in adipose tissue of the groups by Real Time-qPCR. The results are shown in Table 2. In the table AACt = ACt mean control Group (2) - ACt mean experimental Group (1 or 3 or 5); Fold Expression= 2 ~AACt.
[119] Table 2: pi6INK4a mRNA quantified in adipose tissue
Figure imgf000041_0002
Figure imgf000042_0001
[120] The table above indicates that untreated old mice (Control Group 2) express 2.55-fold more p16lnk4a mRNA than the untreated young mice (Control Group 1), as expected. This was observed when comparing Group 2 untreated old mice euthanized at end of recovery Day 85 to Group 1 untreated young mice euthanized at end of treatment Day 22. When results from Group 2 untreated old mice were compared to results from Group 3 treated old mice euthanized Day 85, it was observed that p16lnk4a mRNA was 1.23-fold higher in Group 2 than in Group 3. Therefore, the level of p16lnk4a mRNA expression was lower when the old mice were treated with 2.5 pg/gram/BID/week of antibody.
[121] When results from Group 2 (Control) untreated old mice were compared to results from Group 5 (5 pg/gram) treated old mice euthanized Day 22, it was observed that p16lnk4a mRNA was 3.03-fold higher in Group 2 (controls) than in Group 5 (5 pg/gram). This comparison indicated that the Group 5 animals had lower levels of p16lnk4a mRNA expression when they were treated with 5.0 pg/gram/BID/week, providing pl6lnk4a mRNA expression levels comparable to that of the young untreated mice (i.e. Group 1). Unlike Group 3 (2.5 pg/gram) mice that were euthanized at end of recovery Day 85, Group 5 mice were euthanized at end of treatment Day 22.
[122] These results indicate the antibody administration resulted in the killing of senescent cells.
[123] The mass of the gastrocnemius muscle was also measured, to determine the effect of antibody administration on sarcopenia. The results are provided in Table 3. The results indicate that administration of the antibody increased muscle mass as compared to controls, but only at the higher dosage of 5.0 pg/gm/BID/ week. [124] Table 3: Effect of antibody administration on mass of the gastrocnemius muscle
Summary Absolute weight of Weight relative to body mass of
Group Information Gastrocnemius Muscle (g) Gastrocnemius Muscle (%)
1 Mean 0.3291 1.1037
SD 0.0412 0.1473
N 20 20
2 Mean 0.3304 0.7671
SD 0.0371 0.1246
N 20 20
3 Mean 0.3410 0.7706
SD 0.0439 0.0971
N 19 19
5 Mean 0.4074 0.9480
SD 0.0508 0.2049
N 9 9
[125] These results demonstrate that administration of antibodies that bind to AGEs of a cell resulted in a reduction of cells expressing p16,nk4a, a biomarker of senescence. The data show that reducing senescent cells leads directly to an increase in muscle mass in aged mice. These results indicate that the loss of muscle mass, a classic sign of sarcopenia, can be treated by administration of antibodies that bind to AGEs of a cell. The results suggest that administration of the antibodies would be effective in treating or preventing the onset of fibrotic diseases by removing senescent cells.
[126] Example 2: Affinity and kinetics of test antibody
[127] The affinity and kinetics of the test antibody used in Example 1 were analyzed using Na,Na-bis(carboxymethyl)-L-lysine trifluoroacetate salt (Sigma-Aldrich, St. Louis, MO) as a model substrate for an AGE-modified protein of a cell. Label-free interaction analysis was carried out on a BIACORE™ T200 (GE Healthcare, Pittsburgh, PA), using a Series S sensor chip CM5 (GE Healthcare, Pittsburgh, PA), with Fc1 set as blank, and Fc2 immobilized with the test antibody (molecular weigh of 150,000 Da). The running buffer was an HBS-EP buffer (10 mM HEPES, 150 mM NaCI, 3 mM EDTA and 0.05% P-20, pH of 7.4), at a temperature of 25 °C. Software was BIACORE™ T200 evaluation software, version 2.0. A double reference (Fc2-1 and only buffer injection), was used in the analysis, and the data was fitted to a Langmuir 1:1 binding model.
[128] Table 4: Experimental set-up of affinity and kinetics analysis
Figure imgf000044_0001
[129] A graph of the response versus time is illustrated in FIG. 1. The following values were determined from the analysis: ka (1/Ms) = 1.857 x 103; kd (1/s) = 6.781 x 10'3; KD (M) = 3.651 x 10'6; Rmax (RU) = 19.52; and Chi2 = 0.114. Because the Chi2 value of the fitting is less than 10% of Rmax, the fit is reliable.
[130] Example 3: Construction and production of murine anti-AGE lgG2b antibody and chimeric anti-AGE lgG1 antibody
[131] Murine and chimeric human anti-AGE antibodies were prepared. The DNA sequence of murine anti-AGE antibody lgG2b heavy chain is shown in SEQ ID NO: 12. The DNA sequence of chimeric human anti-AGE antibody lgG1 heavy chain is shown in SEQ ID NO: 13. The DNA sequence of murine anti-AGE antibody kappa light chain is shown in SEQ ID NO: 14. The DNA sequence of chimeric human anti- AGE antibody kappa light chain is shown in SEQ ID NO: 15. The gene sequences were synthesized and cloned into high expression mammalian vectors. The sequences were codon optimized. Completed constructs were sequence confirmed before proceeding to transfection.
[132] HEK293 cells were seeded in a shake flask one day before transfection, and were grown using serum-free chemically defined media. The DNA expression constructs were transiently transfected into 0.03 liters of suspension HEK293 cells. After 20 hours, cells were sampled to obtain the viabilities and viable cell counts, and titers were measured (Octet QKe, ForteBio). Additional readings were taken throughout the transient transfection production runs. The cultures were harvested on day 5, and an additional sample for each was measured for cell density, viability and titer.
[133] The conditioned media for murine and chimeric anti-AGE antibodies were harvested and clarified from the transient transfection production runs by centrifugation and filtration. The supernatants were run over a Protein A column and eluted with a low pH buffer. Filtration using a 0.2 pm membrane filter was performed before aliquoting. After purification and filtration, the protein concentrations were calculated from the OD280 and the extinction coefficient A summary of yields and aliquots is shown in Table 5:
[134] Table 5: Yields and aliquots
Figure imgf000045_0001
[135] Antibody purity was evaluated by capillary electrophoresis sodium-dodecyl sulfate (CE-SDS) analysis using LabChip® GXII, (PerkinElmer).
[136] Example 4: Binding of murine (parental) and chimeric anti-AGE antibodies
[137] The binding of the murine (parental) and chimeric anti-AGE antibodies described in Example 3 was investigated by a direct binding ELISA. An anti- carboxymethyl lysine (CML) antibody (R&D Systems, MAB3247) was used as a control. CML was conjugated to KLH (CML-KLH) and both CML and CML-KLH were coated overnight onto an ELISA plate. HRP-goat anti-mouse Fc was used to detect the control and murine (parental) anti-AGE antibodies. HRP-goat anti-human Fc was used to detect the chimeric anti-AGE antibody.
[138] The antigens were diluted to 1 pg/mL in 1x phosphate buffer at pH 6.5. A 96- well microtiter ELISA plate was coated with 100 pL/well of the diluted antigen and let sit at 4°C overnight. The plate was blocked with 1x PBS, 2.5% BSA and allowed to sit for 1-2 hours the next morning at room temperature. The antibody samples were prepared in serial dilutions with 1x PBS, 1% BSA with the starting concentration of 50 pg/mL. Secondary antibodies were diluted 1 :5,000. 100 pL of the antibody dilutions was applied to each well. The plate was incubated at room temperature for 0.5-1 hour on a microplate shaker. The plate was washed 3 times with 1x PBS. 100 pL/well diluted HRP-conjugated goat anti-human Fc secondary antibody was applied to the wells. The plate was incubated for 1 hour on a microplate shaker. The plate was then washed 3 times with 1x PBS. 100 pL HRP substrate TMB was added to each well to develop the plate. After 3-5 minutes elapsed, the reaction was terminated by adding 100 pL of 1N HCL A second direct binding ELISA was performed with only CML coating. The absorbance at OD450 was read using a microplate reader.
[139] The OD450 absorbance raw data for the CML and CML-KLH ELISA is shown in the plate map below. 48 of the 96 wells in the well plate were used. Blank wells in the plate map indicate unused wells.
[140] Plate map of CML and CML-KLH ELISA: Cone.
(pg/mL) 1 2 3 4 5 6 7
Figure imgf000047_0001
[141] The OD450 absorbance raw data for the CML-only ELISA is shown in the plate map below. 24 of the 96 wells in the well plate were used. Blank wells in the plate map indicate unused wells.
[142] Plate map of CML-only ELISA:
Cone.
(pg/mL) 1 2 3 4 5 6 7
Figure imgf000047_0002
Figure imgf000048_0001
[143] The control and chimeric anti-AGE antibodies showed binding to both CML and CML-KLH. The murine (parental) anti-AGE antibody showed very weak to no binding to either CML or CML-KLH. Data from repeated ELISA confirms binding of the control and chimeric anti-AGE to CML. All buffer control showed negative signal.
[144] Example 5: Humanized antibodies
[145] Humanized antibodies were designed by creating multiple hybrid sequences that fuse select parts of the parental (mouse) antibody sequence with the human framework sequences. Acceptor frameworks were identified based on the overall sequence identity across the framework, matching interface position, similarly classed CDR canonical positions, and presence of N-glycosylation sites that would have to be removed. Three humanized light chains and three humanized heavy chains were designed based on two different heavy and light chain human acceptor frameworks. The amino acid sequences of the heavy chains are shown in SEQ ID NO: 29, 31 and 33, which are encoded by the DNA sequences shown in SEQ ID NO: 30, 32 and 34, respectively. The amino acid sequences of the light chains are shown in SEQ ID NO: 35, 37 and 39, which are encoded by the DNA sequences shown in SEQ ID NO: 36, 38 and 40, respectively. The humanized sequences were methodically analyzed by eye and computer modeling to isolate the sequences that would most likely retain antigen binding. The goal was to maximize the amount of human sequence in the final humanized antibodies while retaining the original antibody specificity. The light and heavy humanized chains could be combined to create nine variant fully humanized antibodies.
[146] The three heavy chains and three light chains were analyzed to determine their humanness. Antibody humanness scores were calculated according to the method described in Gao, S. H., et al., “Monoclonal antibody humanness score and its applications”, BMC Biotechnology, 13:55 (July 5, 2013). The humanness score represents how human-like an antibody variable region sequence looks. For heavy chains a score of 79 or above is indicative of looking human-like; for light chains a score of 86 or above is indicative of looking human-like. The humanness of the three heavy chains, three light chains, a parental (mouse) heavy chain and a parental (mouse) light chain are shown below in Table 6:
[147] Table 6: Antibody humanness
Figure imgf000049_0001
Figure imgf000050_0001
[148] Full-length antibody genes were constructed by first synthesizing the variable region sequences. The sequences were optimized for expression in mammalian cells. These variable region sequences were then cloned into expression vectors that already contain human Fc domains; for the heavy chain, the lgG1 was used.
[149] Small scale production of humanized antibodies was carried out by transfecting plasmids for the heavy and light chains into suspension HEK293 cells using chemically defined media in the absence of serum. Whole antibodies in the conditioned media were purified using MabSelect SuRe Protein A medium (GE Healthcare).
[150] Nine humanized antibodies were produced from each combination of the three heavy chains having the amino acid sequences shown in SEQ ID NO: 29, 31 and 33 and three light chains having the amino acid sequences shown in SEQ ID NO: 35, 37 and 39. A comparative chimeric parental antibody was also prepared. The antibodies and their respective titers are shown below in Table 7:
[151] Table 7: Antibody titers
Figure imgf000050_0002
Figure imgf000051_0001
[152] The binding of the humanized antibodies may be evaluated, for example, by dose-dependent binding ELISA or cell-based binding assay.
[153] Example 6 (Prophetic): An AGE-RNAse containing vaccine in a human subject.
[154] AGE-RNAse is prepared by incubating RNAse in a phosphate buffer solution containing 0.1-3 M glucose, glucose-6-phosphate, fructose or ribose for 10-100 days. The AGE-RNAse solution is dialyzed and the protein content is measured. Aluminum hydroxide or aluminum phosphate, as an adjuvant, is added to 100 pg of the AGE-RNAse. Formaldehyde or formalin is added as a preservative to the preparation. Ascorbic acid is added as an antioxidant. The vaccine also includes phosphate buffer to adjust the pH and glycine as a protein stabilizer. The composition is injected intravenously into a subject with idiopathic pulmonary fibrosis.
[155] Example 7 (Prophetic): Injection regimen for an AGE-RNAse containing vaccine in a human subject.
[156] The same vaccine as described in Example 6 is injected intraperitoneally into a subject with liver cirrhosis. The titer of antibodies to AGE-RNAse is determined by ELISA after two weeks. Additional injections are performed after three weeks and six weeks, respectively. Further titer determination is performed two weeks after each injection. [157] Example 8 (Prophetic): An AGE-hemoglobin containing vaccine in a human subject
[158] AGE-hemoglobin is prepared by incubating human hemoglobin in a phosphate buffer solution containing 0.1-3 M glucose, glucose-6-phosphate, fructose or ribose for 10-100 days. The AGE-hemoglobin solution is dialyzed and the protein content is measured. All vaccine components are the same as in Example 6, except AGE-hemoglobin is substituted for AGE-RNAse. Administration is carried out as in Example 6, or as in Example 7.
[159] Example 9 (Prophetic): An AGE-human serum albumin containing vaccine in a human subject.
[160] AGE-human serum albumin is prepared by incubating human serum albumin in a phosphate buffer solution containing 0.1-3 M glucose, glucose-6-phosphate, fructose or ribose for 10-100 days. The AGE-human serum albumin solution is dialyzed and the protein content is measured. All vaccine components are the same as in Example 6, except AGE-human serum albumin is substituted for AGE-RNAse. Administration is carried out as in Example 6, or as in Example 7.
[161] Example 10 (Prophetic): Carboxymethyllysine-modified protein vaccine for a human subject
[162] A vaccine is prepared by combining a carboxymethyllysine-modified protein as an AGE antigen, aluminum hydroxide as an adjuvant, formaldehyde as a preservative, ascorbic acid as an antioxidant, a phosphate buffer to adjust the pH of the vaccine and glycine as a protein stabilizer. The vaccine is injected subcutaneously into a subject with myelofibrosis.
[163] Example 11 (Prophetic): Carboxyethyllysine-modified peptide vaccine for a human subject
[164] A vaccine is prepared by combining a carboxyethyllysine-modified peptide conjugated to KLH as an AGE antigen, aluminum hydroxide as an adjuvant, formaldehyde as a preservative, ascorbic acid as an antioxidant, a phosphate buffer to adjust the pH of the vaccine and glycine as a protein stabilizer. The vaccine is injected subcutaneously into a subject with chronic kidney disease.
[165] Example 12: In vivo study of the administration of a carboxymethyl lysine monoclonal antibody
[166] The effect of a carboxymethyl lysine antibody on tumor growth, metastatic potential and cachexia was investigated. In vivo studies were carried out in mice using a murine breast cancer tumor model. Female BALB/c mice (BALB/cAnNCrl, Charles River) were eleven weeks old on Day 1 of the study.
[167] 4T1 murine breast tumor cells (ATCC CRL-2539) were cultured in RPM1 1640 medium containing 10% fetal bovine serum, 2 mM glutamine, 25 pg/mL gentamicin, 100 units/mL penicillin G Na and 100 pg/mL streptomycin sulfate. Tumor cells were maintained in tissue culture flasks in a humidified incubator at 37 °C in an atmosphere of 5% CO2 and 95% air.
[168] The cultured breast cancer cells were then implanted in the mice. 4T1 cells were harvested during log phase growth and re-suspended in phosphate buffered saline (PBS) at a concentration of 1 x 106 cells/mL on the day of implant. Tumors were initiated by subcutaneously implanting 1 x 1054T1 cells (0.1 mL suspension) into the right flank of each test animal. Tumors were monitored as their volumes approached a target range of 80-120 mm3. Tumor volume was determined using the formula: tumor volume = (tumor width)2(tumor length)/2. Tumor weight was approximated using the assumption that 1 mm3 of tumor volume has a weight of 1 mg. Thirteen days after implantation, designated as Day 1 of the study, mice were sorted into four groups (n=15/group) with individual tumor volumes ranging from 108 to 126 mm3 and a group mean tumor volume of 112 mm3. The four treatment groups are shown in Table 8 below:
[169] Table 8: Treatment groups
Figure imgf000053_0001
Figure imgf000054_0001
[170] An anti-carboxymethyl lysine monoclonal antibody was used as a therapeutic agent. 250 mg of carboxymethyl lysine monoclonal antibody was obtained from R&D Systems (Minneapolis, MN). Dosing solutions of the carboxymethyl lysine monoclonal antibody were prepared at 1 and 0.5 mg/mL in a vehicle (PBS) to provide the active dosages of 10 and 5 pg/g, respectively, in a dosing volume of 10 mL/kg. Dosing solutions were stored at 4 °C protected from light.
[171] All treatments were administered intravenously (i.v.) twice daily for 21 days, except on Day 1 of the study where the mice were administered one dose. On Day 19 of the study, i.v. dosing was changed to intraperitoneal (i.p.) dosing forthose animals that could not be dosed i.v. due to tail vein degradation. The dosing volume was 0.200 mL per 20 grams of body weight (10 mL/kg), and was scaled to the body weight of each individual animal.
[172] The study continued for 23 days. Tumors were measured using calipers twice per week. Animals were weighed daily on Days 1-5, then twice per week until the completion of the study. Mice were also observed for any side effects. Acceptable toxicity was defined as a group mean body weight loss of less than 20% during the study and not more than 10% treatment-related deaths. Treatment efficacy was determined using data from the final day of the study (Day 23). [173] The ability of the anti-carboxymethyl lysine antibody to inhibit tumor growth was determined by comparing the median tumor volume (MTV) for Groups 1-3. Tumor volume was measured as described above. Percent tumor growth inhibition (%TGI) was defined as the difference between the MTV of the control group (Group 1) and the MTV of the drug-treated group, expressed as a percentage of the MTV of the control group. %TGI may be calculated according to the formula: %TGI = (1- MTVtreated/MTVcontrol) X 100.
[174] The ability of the anti-carboxymethyl lysine antibody to inhibit cancer metastasis was determined by comparing lung cancer foci for Groups 1-3. Percent inhibition (%lnhibition) was defined as the difference between the mean count of metastatic foci of the control group and the mean count of metastatic foci of a drug- treated group, expressed as a percentage of the mean count of metastatic foci of the control group. %l nhibition may be calculated according to the following formula: %lnhibition = (1-Mean Count of Focitreated/Mean Count of Focicontroi) x 100.
[175] The ability of the anti-carboxymethyl lysine antibody to inhibit cachexia was determined by comparing the weights of the lungs and gastrocnemius muscles for Groups 1-3. Tissue weights were also normalized to 100 g body weight.
[176] Treatment efficacy was also evaluated by the incidence and magnitude of regression responses observed during the study. Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal. In a PR response, the tumor volume was 50% or less of its Day 1 volume for three consecutive measurements during the course of the study, and equal to or greater than 13.5 mm3 for one or more of these three measurements. In a CR response, the tumor volume was less than 13.5 mm3 for three consecutive measurements during the course of the study.
[177] Statistical analysis was carried out using Prism (GraphPad) for Windows 6.07. Statistical analyses of the differences between Day 23 mean tumor volumes (MTVs) of two groups were accomplished using the Mann-Whitney (7 test. Comparisons of metastatic foci were assessed by ANOVA-Dunnett. Normalized tissue weights were compared by ANOVA. Two-tailed statistical analyses were conducted at significance level P = 0.05. Results were classified as statistically significant or not statistically significant.
[178] The results of the study are shown below in Table 9:
[179] Table 9: Results
Figure imgf000056_0001
[180] All treatment regimens were acceptably tolerated with no treatment-related deaths. The only animal deaths were non-treatment-related deaths due to metastasis. The %TGI trended towards significance (P > 0.05, Mann-Whitney) for the 5 pg/g (Group 2) or 10 pg/g treatment group (Group 3). The %lnhibition trended towards significance (P > 0.05, ANOVA-Dunnett) for the 5 pg/g treatment group. The %lnhibition was statistically significant (P < 0.01, ANOVA-Dunnett) for the 10 pg/g treatment group. The ability of the carboxymethyl lysine antibody to treat cachexia trended towards significance (P > 0.05, ANOVA) based on a comparison of the organ weights of the lung and gastrocnemius between treatment groups and the control group. The results indicate that administration of an anti-carboxymethyl lysine monoclonal antibody is able to reduce cancer metastases. This data provides additional evidence that in vivo administration of anti-AGE antibodies can provide therapeutic benefits safely and effectively. [181] Example 13: Treatment of fibrotic lung diseases by removal of senescent cells
[182] The following example was obtained from International Publication no. WO 2015/116740 and describes research carried out by parties other than the inventor and applicant of the Present Application regarding therapeutic removal of senescent cells in vivo by administration of senolytic agents in a mouse model, as well as potential future studies that could be carried out.
[183] One animal model study assessed the effect of clearance of senescence cells in the transgenic mouse strain 3MR that has bleomycin induced lung injury. In the bleomycin injury model for idiopathic pulmonary fibrosis, mice develop lung fibrosis within 7-14 days after bleomycin treatment (see, e.g., Limjunyawong et al., 2014, Physiological Reports 2:e00249; Daniels et al, 2004, J. Clin. Invest. 114: 1308- 1316). Bleomycin was administered to anesthetized 6-8 week old 3MR mice by intratracheal aspiration (2.5U/kg of bleomycin in 50 pT PBS) using a microsprayer syringe (Penn- Century, Inc.) as described in Daniels et al. (2004, J. Clin. Invest 114: 1308-1316). Control mice were administered saline. The day following bleomycin treatment, ganciclovir (GCV) (25mg/kg in PBS) was administered. 3MR mice were treated via intraperitoneal injection with ganciclovir for 5 consecutive days, followed by 5 days of rest, followed by a second treatment cycle of 5 consecutive days. Untreated mice received an equal volume of vehicle. At 7, 14, and 21 days post-bleomycin treatment, lung function was assessed by monitoring oxygen saturation using the MouseSTAT PhysioSuite pulse oximeter (Kent Scientific). Animals were anesthetized with isoflurane (1.5%) and a toe clip was applied. Mice were monitored for 30 seconds and the average peripheral capillary oxygen saturation (SpOz) measurement over this duration was calculated. As shown in Figure 2, bleomycin administration significantly reduced SpOa levels in vehicle treated mice, and removal of senescent cells resulted in higher Sp02 levels, which approached normal levels at 21 days post bleomycin administration. At 21 days post- bleomycin treatment, airway hyper-reactivity (AHR) of mice was examined. AHR of mice was measured by methacholine challenge while other parameters of lung function (airway mechanics, lung volume and lung compliance) were determined using a SCIREQ flexiVent ventilator. While under ketamine/xylazine anesthesia and subjected to cannulation of the trachea via a tracheostomy (19Fr blunt Luer cannula), airway resistance (elastance) and compliance of mice were assessed at baseline and in response to increasing concentrations of methacholine (0 to 50 mg/ml in PBS) delivered via nebulization (AeroNeb) as described in Aravamudan et al. (Am. J. Physiol. Lung Cell. Mol. Physiol. (2012) 303:L669-L681). Animals were maintained at 37°C, and while under muscle paralysis (pancuronium); airway function was measured by using the FlexiVent™ ventilator and lung mechanics system (SCIREQ, Montreal, Quebec, Canada), which was housed on Stabile 8. As shown in Figure 3A, in vehicle treated mice, bleomycin administration increased lung elastance, whereas ganciclovir treatment reduced lung elastance. As shown in Figures 3B-C, bleomycin administration reduced static compliance and (dynamic) compliance in vehicle treated mice. Clearance of senescent cells with ganciclovir in bleomycin exposed mice improved compliance values significantly (Figures 3B-C). Although not statistically significant because the animal group size was too small, data suggested that clearance of senescent cells with a senolytic agent (Nutlin-3A) also reduced lung elastance and increased lung compliance in a bleomycin exposed mouse. Mice were euthanized by i.p injection of pentobarbital.
[184] Bronchoalveolar lavage (BAL) fluids and lungs is obtained and analyzed.
Hydroxyproline content of lungs is measured as described in Christensen et al. (1999, Am . J. Pathol. 155: 1773-1779), and quantitative histopathology is performed. RNA is extracted from lung tissue to measure senescence cell markers by qRT-PCR in treated and control mice. The effect of clearance of senescence cells in the bleomycin induced lung injury model of IPF may also be studied in INK- ATT AC transgenic mice in the study design described above. INK- ATT AC (pl6lnk4a apoptosis through targeted activation of caspase) transgenic mice have an FK506- binding protein (FKBP)-caspase 8 (Casp8) fusion polypeptide under the control of the pl6lnk4a promoter (see, e.g., Baker et al, Nature, supra; Int'l Patent Application Publication No. WO/2012/177927). In the presence of AP20187, a synthetic drug that induces dimerization of a membrane bound myristoylated FKBP-Casp8 fusion protein, senescent cells specifically expressing the FKBP-Casp8 fusion protein via the pl6lnk4a promoter undergo programmed cell death (apoptosis) (see, e.g., Baker, Nature, supra, Figure 1 therein).
[185] A second study also assesses the effect of clearance of senescence cells using a senolytic agent in C57BL6/J mice that have bleomycin induced lung injury. Bleomycin is administered to 6 week old C57BL6/J mice as described above. A senolytic agent is administered during the first and third week post-bleomycin treatment. Control mice are treated with vehicle. At 21 days post-bleomycin treatment, clearance of senescent cells and lung function/histopathology is assessed.
[186] In a second animal model for pulmonary diseases (e.g., COPD), mice were exposed to cigarette smoke. The effect of a senolytic agent on the mice exposed to smoke is assessed by senescent cell clearance, lung function, and histopathology.
[187] Six week-old 3 MR (n=35) or INK-ATTAC (n=35) mice were chronically exposed to cigarette smoke generated from a Teague TE-10 system, an automatically-controlled cigarette smoking machine that produces a combination of side-stream and mainstream cigarette smoke in a chamber, which is transported to a collecting and mixing chamber where varying amounts of air is mixed with the smoke mixture. The COPD protocol was adapted from the COPD core facility at Johns Hopkins University (at Internet site web
.jhu.edu/Biswal/exposure_core/copd. html#Cigarette_Smoke) (Rangasamy et al, 2004, J. Clin. Invest. 114: 1248-1259; Yao et al, 2012, J. Clin. Invest. 122:2032- 2045). Mice received a total of 6 hours of cigarette smoke exposure per day, 5 days a week for 6 months. Each lighted cigarette (3R4F research cigarettes containing 10.9 mg of total particulate matter (TPM), 9.4 mg of tar, and 0.726 mg of nicotine, and 11.9 mg carbon monoxide per cigarette [University of Kentucky, Lexington, KY]) was puffed for 2 seconds and once every minute for a total of 8 puffs, with the flow rate of 1.05 L/min, to provide a standard puff of 35 cm3. The smoke machine was adjusted to produce a mixture of side stream smoke (89%) and mainstream smoke (11%) by smoldering 2 cigarettes at one time. The smoke chamber atmosphere was monitored for total suspended particulates (80-120 mg/m3) and carbon monoxide (350 ppm). Beginning at day 7, (10) INK-ATTAC and (10) 3 MR mice were treated with AP20187 (3x per week) or ganciclovir (5 consecutive days of treatment followed by 16 days off drug, repeated until the end of the experiment), respectively. An equal number of mice received the corresponding vehicle. The remaining 30 mice (15 INK- ATTAC and 15 3 MR) were evenly split with 5 of each genetically modified strain placed into three different treatment groups. One group (n=10) received Nutlin-3A (25 mg/kg dissolved in 10% DMSO/3%) Tween-20 in PBS, treated 14 days consecutively followed by 14 days off drug, repeated until the end of the experiment). One group (n=10) received ABT-263 (Navitoclax) (100 mg/kg dissolved in 15% DMSO/5% Tween-20, treated 7 days consecutively followed by 14 days off drug, repeated until the end of the experiment), and the last group (n=10) received only the vehicle used for ABT-263 (15% DMSO/5% Tween-20), following the same treatment regimen as ABT-263. An additional 70 animals that did not receive exposure to cigarette smoke were used as controls for the experiment.
[188] After two months of cigarette smoke exposure, lung function was assessed by monitoring oxygen saturation using the MouseSTAT PhysioSuite pulse oximeter (Kent Scientific). Animals were anesthetized with isoflurane (1.5%) and the toe clip was applied. Mice were monitored for 30 seconds and the average peripheral capillary oxygen saturation (SpOz) measurement over this duration was calculated. As shown in Figure 4, clearance of senescent cells via AP20187, ganciclovir, ABT- 263 (Navi), or Nutlin-3A resulted in statistically significant increases in Sp02 levels in mice after 2 months of cigarette smoke exposure compared to untreated controls.
[189] At the end of the experimental period, airway hyper-reactivity (AHR) of mice to methacholine challenge using a SCIREQ flexiVent ventilator and lung mechanics system is examined as described above. After AHR measurement, mice are killed by i.p. injection of pentobarbital for in-depth analysis of lung histopathology as previously described (Rangasamy et al, 2004, J. Clin. Invest. 114: 1248-1259). Briefly, lungs are inflated with 0.5% low-melting agarose at a constant pressure of 25 cm. Part of the lung tissue is collected for RNA extraction and qRT-PCR analysis of senescent markers. Other parts of lungs are fixed in 10% buffered formalin and embedded in paraffin. Sections (5 urn) are stained with hematoxylin and eosin. Mean alveolar diameter, alveolar length, and mean linear intercepts are determined by computer- assisted morphometry with Image Pro Plus software (Media Cybernetics).
[190] The potential therapeutic effect of clearance of senescent cells after COPD is fully developed may be assessed in 3MR or INK-ATTAC mice. Six week-old 3MR or INK-ATTAC mice are chronically exposed to cigarette smoke for 6 months as described above. At 6 months following the start of smoke exposure, 3MR or INK- ATTAC mice are treated with ganciclovir (5 consecutive days of treatment followed by 16 days off drug) or AP20187 (3x/week), respectively, until 9 months following the start of smoke exposure, when assessment of senescent cell clearance, lung function, and histopathology is performed.
[191] These studies demonstrate that fibrotic lung diseases may be treated by the removal of senescent cells. In addition, these studies confirm that senescent cells are appropriate targets for treating fibrotic diseases.
[192] Example 14 (Prophetic): Treatment of idiopathic pulmonary fibrosis
[193] A patient presents with idiopathic pulmonary fibrosis. She is administered an anti-AGE antibody by inhalation using a nebulizer. The anti-AGE antibody will specifically bind to cells expressing cell-surface AGEs, such as senescent myofibroblasts, and allow her immune system to destroy those cells. Killing and removing senescent cells will treat her idiopathic pulmonary fibrosis and prevent the disease from worsening.
[194] Example 15 (Prophetic): T reatment of scleroderma
[195] A patient presents with scleroderma on his right arm. He is administered a topical cream containing an anti-AGE antibody. The anti-AGE antibody will specifically bind to cells expressing cell-surface AGEs, such as senescent myofibroblasts, and allow his immune system to destroy those cells. Killing and removing senescent cells will treat his scleroderma and prevent further progression of the disease. REFERENCES
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Claims

WHAT IS CLAIMED IS:
1. A method of treating or preventing the onset of a fibrotic disease, comprising administering to a subject a composition comprising an anti-AGE antibody.
2. A method of treating or preventing the onset of a fibrotic disease, comprising administering to a subject a vaccine comprising an AGE antigen.
3. The method of any of the preceding claims, wherein the composition further comprises a second anti-AGE antibody.
4. The method of any of the preceding claims, wherein the composition further comprises a pharmaceutically acceptable carrier.
5. The method of any of the preceding claims, wherein the subject is selected from the group consisting of humans, goats, sheep, pigs, cows, horses, camels, alpacas, dogs and cats.
6. The method of any of the preceding claims, wherein the subject is a human.
7. The method of any of the preceding claims, wherein the anti-AGE antibody is non-immunogenic to a species selected from the group consisting of humans, cats, dogs, horses, camels, alpaca, cattle, sheep, pigs, and goats.
8. The method of any of the preceding claims, wherein the anti-AGE antibody binds an AGE antigen comprising at least one protein or peptide that exhibits AGE modifications selected from the group consisting of FFI, pyrraline, AFGP, ALI, carboxymethyllysine, carboxyethyl lysine and pentosidine.
- 65 -
9. The method of any of the preceding claims, wherein the anti-AGE antibody binds a carboxymethyllysine-modified protein or peptide.
10. The method of any of the preceding claims, wherein the composition is in unit dosage form.
11. The method of any of the preceding claims, wherein the composition is sterile.
12. The method of any of the preceding claims, wherein the vaccine further comprises an adjuvant, optionally, a preservative, and optionally, an excipient.
13. The method of any of the preceding claims, wherein the AGE antigen is an AGE-modified protein or AGE-modified peptide selected from the group consisting of AGE-RNAse, AGE-human hemoglobin, AGE-albumin, AGE-BSA, AGE- human serum albumin, AGE-ovalbumin, AGE-low density lipoprotein, AGE-collagen IV, AGE-antithrombin III, AGE-calmodulin, AGE-insulin, AGE-ceruloplasmin, AGE- collagen, AGE-cathepsin B, AGE-crystallin, AGE-plasminogen activator, AGE- endothelial plasma membrane protein, AGE-aldehyde reductase, AGE-transferrin, AGE-fibrin, AGE-copper/zinc SOD, AGE-apo B, AGE-fibronectin, AGE-pancreatic ribose, AGE-apo A-l and II, AGE-hemoglobin, AGE-Na+/K+-ATPase, AGE- plasminogen, AGE-myelin, AGE-lysozyme, AGE-immunoglobulin, AGE-red cell Glu transport protein, AGE-P-N-acetyl hexokinase, AGE-apo E, AGE-red cell membrane protein, AGE-aldose reductase, AGE-ferritin, AGE-red cell spectrin, AGE-alcohol dehydrogenase, AGE-haptoglobin, AGE-tubulin, AGE-thyroid hormone, AGE- fibrinogen, AGE-02-microglobulin, AGE-sorbitol dehydrogenase, AGE-ai-antitrypsin, AGE-carbonate dehydratase, AGE-hexokinase, AGE-apo C-l, AGE-KLH and mixtures thereof.
- 66 -
14. The method of any of the preceding claims, wherein the AGE antigen comprises at least one protein or peptide that exhibits AGE modifications selected from the group consisting of carboxymethyllysine, carboxyethyllysine, pentosidine, pyrraline, FFI, AFGP, and ALL
15. The method of any of the preceding claims, wherein the AGE antigen comprises a carboxymethyllysine-modified protein or peptide.
16. The method of any of the preceding claims, wherein the vaccine is sterile.
17. The method of any of the preceding claims, wherein the vaccine is in unit dosage form.
18. The method of any of the preceding claims, further comprising testing the patient to determine if the fibrotic disease has been ameliorated, and repeating the administering, if necessary.
19. The method of any of the previous claims, wherein the antibody comprises a first complementary determining region comprising the amino acid sequence of SEQ ID NO: 23, a second complementary determining region comprising the amino acid sequence of SEQ ID NO: 24, a third complementary determining region comprising the amino acid sequence of SEQ ID NO: 25, a fourth complementary determining region comprising the amino acid sequence of SEQ ID NO: 26, a fifth complementary determining region comprising the amino acid sequence of SEQ ID NO: 27, and a sixth complementary determining region comprising the amino acid sequence of SEQ ID NO: 28.
- 67 -
20. The method of any of the preceding claims, wherein the antibody comprises a heavy chain, and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, and the light chain comprises an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61.
21. The method of any of the previous claims, wherein the antibody comprises a constant region from a species selected from the group consisting of humans, goats, sheep, pigs, cows, horses, camels, alpacas, dogs and cats.
22. The method of any of the preceding claims, wherein the antibody is a humanized antibody.
23. The method of any of the preceding claims, wherein the antibody is monoclonal.
24. The method of any of the preceding claims, wherein the antibody is substantially non-immunogenic to humans.
25. The method of any of the preceding claims, wherein the antibody has a rate of dissociation (k ) of at most 9 x 10'3 sec-1.
- 68 -
26. The method of any of the preceding claims, wherein the antibody is conjugated to an agent that causes the destruction of AGE-modified cells, wherein the agent is selected from the group consisting of a toxin, a cytotoxic agent, magnetic nanoparticles, and magnetic spin-vortex discs.
27. The method of any of the preceding claims, wherein the antibody includes constant regions which permit destruction of targeted cells by a subject’s immune system.
28. The method of any of the preceding claims, wherein the AGE antigen comprises carboxymethyllysine conjugated with keyhole limpet hemocyanin (CML- KLH).
29. The method of any of the preceding claims, wherein the fibrotic disease comprises at least one disease or disorder selected from the group consisting of interstitial lung disease, pulmonary fibrosis, liver fibrosis, cirrhosis, kidney fibrosis, kidney disease, nephrosclerosis, nephrosis, glomerulosclerosis, bladder fibrosis, urethral stricture, cardiovascular disease, macular degeneration, vitreal retinopathy, scleroderma, hypertrophic scarring, keloid scarring, restenosis, myelofibrosis, nephrogenic fibrosing dermopathy, mixed connective tissue disease, scleromyxedema, scleredema, and eosinophilic fasciitis.
30. The method of any of the preceding claims, wherein the fibrotic disease is a fibroproliferative disorder.
31. The method of any of the preceding claims, wherein the anti-AGE antibody is administered by inhalation.
32. A method of reducing desmoplasia, comprising: administering to a subject a composition comprising an anti-AGE antibody.
- 69 -
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