EP3362483A1 - Anticorps anti-age et procédés d'utilisation correspondants - Google Patents

Anticorps anti-age et procédés d'utilisation correspondants

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Publication number
EP3362483A1
EP3362483A1 EP16731696.7A EP16731696A EP3362483A1 EP 3362483 A1 EP3362483 A1 EP 3362483A1 EP 16731696 A EP16731696 A EP 16731696A EP 3362483 A1 EP3362483 A1 EP 3362483A1
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EP
European Patent Office
Prior art keywords
seq
antibody
age
sequence identity
amino acid
Prior art date
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EP16731696.7A
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German (de)
English (en)
Inventor
Lewis S. Gruber
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Siwa Corp
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Siwa Corp
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Priority claimed from US14/974,561 external-priority patent/US10358502B2/en
Application filed by Siwa Corp filed Critical Siwa Corp
Publication of EP3362483A1 publication Critical patent/EP3362483A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/12—Ophthalmic agents for cataracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • Sarcopenia is the loss of muscle mass, quality and strength associated with aging. Humans begin to lose muscle mass and function at some point in the third decade of life. This loss of muscle mass typically accelerates around age 75.
  • Sarcopenia develops in both physically active and physically inactive people. As the average human lifespan continues to increase, sarcopenia is becoming a significant health concern. The loss of muscle mass from sarcopenia may lead to poor balance, reduced gait speed and frailty. Individuals suffering from sarcopenia are more susceptible to injury and disability, and may be unable to live independently as a result. The spread of sarcopenia will likely result in increases in health care and assisted living expenses.
  • sarcopenia has been considered to be an inevitable result of aging and the natural deterioration of the body over time.
  • the primary treatment for sarcopenia is exercise. Physical exercise, particularly resistance training or strength training, can reduce the impact of sarcopenia.
  • Testosterone, anabolic steroids, ghrelin, vitamin D, angiotensin converting enzyme inhibitors (ACE inhibitors), eicosapentaenoic acid (EPA), myostatin, selective androgen receptor modulators (SARMs), urocortin II (Ucn2) and hormone replacement therapy have been investigated or are being studied as potential treatments for sarcopenia.
  • ACE inhibitors angiotensin converting enzyme inhibitors
  • EPA eicosapentaenoic acid
  • SARMs selective androgen receptor modulators
  • Ucn2 urocortin II
  • FDA U.S. Food and Drug Administration
  • Senescent cells are cells that are partially-functional or non-functional and are in a state of irreversible 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 ⁇ -galactosidase.
  • AGEs Advanced glycation end-products
  • AGEs also referred to AGE-modified proteins, or glycation end-products
  • AGEs arise from a non-enzymatic reaction of sugars with protein side-chains in aging cells
  • 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, 23, 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.
  • the Amadori product undergoes further rearrangement to produce AGEs.
  • Hyperglycemia caused by diabetes mellitus (DM), and oxidative stress promote this post-translational modification of membrane proteins (Lindsey JB, et al., "Receptor For Advanced Glycation End-Products (RAGE) and soluble RAGE (sRAGE):
  • AGEs have been associated with several pathological conditions including diabetic complications, inflammation, retinopathy, nephropathy, 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 glycation end-product and senescence is well known in the art. See, for example, Gruber, L. (WO 2009/14341 1 , 26 Nov. 2009), 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)), Ahmed, E.K. et al. ("Protein Modification and Replicative Senescence of WI-38 Human Embryonic Fibroblasts" Aging Cells, vol. 9, 252, 260 (2010)), Vlassara, H.
  • 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 end- products is associated with senescence and lack of function.
  • AD Alzheimer's disease
  • Microglial cell senescence associated with normal aging is exacerbated by the presence of the amyloid plaques indicative of AD (Flanary, B. E. et al., "Evidence That Aging And Amyloid Promote Microglial Cell Senescence", Rejuvenation Research, Vol. 10(1 ), pp. 61-74 (March 2007)).
  • the presence of AGEs with astrocytes and microglial cells in AD is further evidence of the presence of senescent cells (Takeda, A., et al. "Advanced glycation end products co-localize with astrocyes and microglial cells in Alzheimer's disease brain", Acta Neuropathologica, Vol. 95, pp. 555-558 (1998)).
  • Chinta et al. proposed that environmental stressors associated with
  • Parkinson's disease may act in part by eliciting senescence within non-neuronal glial cells, contributing to the characteristic decline in neuronal integrity that occurs in this disorder (Chinta, S. J. et al. "Environmental stress, ageing and glial cell
  • senescence a novel mechanistic link to Parkinson's disease?", J Intern Med, Vol. 273, pp. 429-436 (2013)). Astrocyte senescence is also associated with PD (M. Mori, "The Parkinsonian Brain: Cellular Senescence and Neurodegeneration, SAGE (June 30, 2015) (sage.buckinstitute.org/the-parkinsonian-brain-cellular-senescence-and- neurodegeneration/). In a rodent model of familial amyotrophic lateral sclerosis (ALS) overexpressing mutant superoxide dismutase-1 (m-SOD1 ), the rate of astrocytes acquiring a senescent phenotype is accelerated (Das, M. M.
  • ALS familial amyotrophic lateral sclerosis
  • m-SOD1 mutant superoxide dismutase-1
  • Satellite cells also known as myosatellite cells, present in the muscle tissue of ALS patients exhibit an abnormal senescent-like morphology, although they may be capable of proliferating in vitro (Pradat, P.-F. et al., "Abnormalities of satellite cells function in amyotrophic lateral sclerosis” Amyotrophic Lateral Sclerosis, Vol. 12, pp. 264-271 (201 1 )). Satellite cells are small multipotent cells found in mature muscle, which are able to give rise to additional satellite cells, or differentiate into myoblasts as well as provide additional myonuclei.
  • Myoblasts are precursor cells which differentiate into myocytes (also referred to as muscle cells).
  • a characteristic of PD and Lewy body dementia is the formation of Lewy bodies that form inside nerve cells.
  • the primary structural component of the Lewy bodies is alpha-synuclein protein, in the form of fibrils.
  • the presence of tangles and plaques are a characteristic of AD, the presence of which is used to definitively diagnose the condition.
  • Plaques, composed of beta-amyloid protein also referred to as amyloid beta, ⁇ or Abeta
  • Prion diseases also known as transmissible spongiform encephalopathies (TSEs)
  • TSEs transmissible spongiform encephalopathies
  • TSEs include a variety of human and animal disorder such as Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease, bovine spongiform encephalopathy ("mad cow” disease), scrapie (in sheep and goats), chronic wasting disease (in deer and elk), kuru and fatal familial insomnia.
  • Prion protein is a misfolded protein molecule which may propagate by transmitting a misfolded protein state, resulting in the accumulation of the misfolded protein and causing tissue damage and cell death (Dobson, D.M., "The structural basis of protein folding and its links with human disease” Phil. Trans. R. Soc. Lond. B, Vol. 356, pp. 133-145 (2001)). In these diseases, it is believed the protein is a normal protein which misfolds or forms an abnormal aggregate. In the case of some patients with familial ALS, a mutated superoxide dismutase-1 (SOD1 ) forms inclusions and accumulates (Kato, S., et al.
  • SOD1 superoxide dismutase-1
  • Senescent cells are also known to fuel the growth of cancer cells. 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.
  • SASP senescence-associated secretory phenotype
  • the present invention is an anti-AGE antibody, comprising a protein or peptide comprising at least one 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
  • the antibody binds to a protein or peptide that exhibits a carboxymethyllysine modification.
  • the present invention is an anti-AGE antibody, comprising a protein or peptide comprising at least one 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
  • the antibody binds to a protein or
  • the present invention is an anti-AGE antibody comprising a heavy chain and a light chain.
  • 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 , and SEQ ID NO: 33, or 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, and SEQ ID NO: 39.
  • the antibody binds to a protein or peptide that exhibits a
  • the present invention is an anti-AGE antibody comprising a heavy chain and a light chain.
  • 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 , and SEQ ID NO: 33
  • 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, and SEQ ID NO: 39.
  • the antibody binds to a protein or peptide that exhibits a carboxymethyllysine modification.
  • the present invention is an anti-AGE antibody, comprising
  • the antibody binds to a protein or peptide that exhibits a carboxymethyllysine modification.
  • the antibody is substantially non- immunogenic to a species selected from the group consisting of mice, rats, goats, sheep, cows, horses, dogs and cats.
  • the present invention is an antibody conjugate, comprising an anti-AGE antibody fragment comprising a protein or peptide comprising at least one 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: 3, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO 35, SEQ ID NO: 37, and SEQ ID NO: 39, and an agent that causes the destruction of AGE-modified cells.
  • the agent that causes the destruction of AGE-modified cells is conjugated to the anti-AGE antibody fragment.
  • the antibody binds to a protein or peptide that exhibits a carboxymethyllysine modification.
  • peptide means a molecule composed of 2-50 amino acids.
  • protein means a molecule composed of more than 50 amino acids.
  • muscle mass means the syndrome characterized by the presence of (1) low muscle mass and (2) low muscle function (low muscle strength or reduced physical performance).
  • Muscle mass may be measured by body imaging techniques, such as computed tomography scanning (CT scan), magnetic resonance imaging (MRI) or dual energy X-ray absorptiometry (DXA or DEXA); bioimpedance analysis (BIA); body potassium measurement, such as total body potassium (TBK) or partial body potassium (PBK); or anthropometric measurements, such as mid-upper arm circumference, skin fold thickness or calf circumference.
  • CT scan computed tomography scanning
  • MRI magnetic resonance imaging
  • DEXA dual energy X-ray absorptiometry
  • BIOA bioimpedance analysis
  • body potassium measurement such as total body potassium (TBK) or partial body potassium (PBK)
  • anthropometric measurements such as mid-upper arm circumference, skin fold thickness or calf circumference.
  • muscle mass is measured by CT scan, MRI or DXA.
  • Muscle strength may
  • muscle strength is measured by handgrip strength.
  • Physical performance may be measured by the Short Physical Performance Battery, gait speed measurement, timed get-up-and-go (TGUG) or the stair climb power test.
  • TGUG timed get-up-and-go
  • physical performance is measured by gait speed measurement.
  • a subject may be identified as having sarcopenia or in need of treatment if (1) the subject is at least 25 years old and (2) his or her measured muscle mass and measured muscle function are two standard deviations or more below the mean value for healthy 25 year olds of the same gender and no alternative pathology has been identified to account for the reduced muscle mass and reduced muscle function.
  • a subject being treated for sarcopenia is at least 40 years old. More preferably, a subject being treated for sarcopenia is at least 50 years old. Most preferably, a subject being treated for sarcopenia is at least 60 years old.
  • a subject may be identified as having sarcopenia or in need of treatment if (1 ) his or her gait speed is less than 1.0 m/s across a 4 m course and (2) he or she has an objectively measured low muscle mass, such as, for example, an appendicular mass relative to the square of height less than or equal to 7.23 kg/m 2 for male subjects or less than or equal to 5.67 kg/m 2 for female subjects (Fielding, R.
  • neurodegenerative disorder means disorders which result in neurons loosing function and/or dying, in the central nervous system including the brain.
  • disorders included central nervous system neurodegenerative disorders such as AD, PD, Lewy body dementia, MS, prion diseases (also known as transmissible spongiform encephalopathies (TSEs), including Creutzfeldt-Jakob disease, variant Creutzfeldt- Jakob disease, bovine spongiform encephalopathy ("mad cow” disease), scrapie (in sheep and goats), chronic wasting disease (in deer and elk), kuru and fatal familial insomnia), and ALS.
  • TSEs transmissible spongiform encephalopathies
  • Neurodegenerative proteins are proteins which accumulate in a patient having a neurodegenerative disorders and which are associated with the neurodegenerative disorder. Examples include, beta-amyloid protein plaques (associated with AD), tau protein tangles (associated with AD), mutated superoxide dismutase-1 (associated with ALS), prion protein aggregates (associated with TSEs) and alpha-synuclein protein fibrils (associated with PD and Lewy Body dementia).
  • a “neurodegenerative protein” is the form of the protein which accumulates during the neurodegenerative disorder, typically a mutant or mis-folded form.
  • AGE-modified protein or peptide 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.
  • Glycated proteins or peptides that have not undergone the necessary rearrangement to form AGEs 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; and pentosidine.
  • ALI another AGE, is described in Al-Abed.
  • 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, 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 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 irreversible proliferative arrest and expresses one or more biomarkers of senescence, such as activation of p16 lnk4a or expression of senescence-associated ⁇ -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.
  • age-related phenotypes include, for example, sarcopenia, cataracts, loss of adipose tissue and lordokyphosis.
  • 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.
  • the identification of a link between cellular senescence and sarcopenia allows for new treatment possibilities. For example, if anti-AGE antibodies are administered to a subject, the antibodies will specifically and selectively target senescent cells, and kill or induce apoptosis in such cells expressing an AGE-modified protein or peptide.
  • the present invention makes use of the discovery that enhanced clearance of cells expressing AGE-modified proteins or peptides (AGE-modified cells) is beneficial in treating or ameliorating sarcopenia. This may be accomplished by administering anti- AGE antibodies to a subject.
  • Administering anti-AGE antibodies to a subject may also be used for increasing health span. Health span may be increased by reducing age-related phenotypes. Administering anti-AGE antibodies may be used, for example, to prevent or delay the onset of cataracts, lordokyphosis or loss of adipose tissue.
  • anti-AGE antibodies may be used therapeutically to treat neurodegenerative disorders or cancer.
  • anti-AGE antibody or "AGE antibody”
  • Examples include those described in U.S.
  • Examples include an antibody that binds to one or more AGE-modified proteins having an AGE modification such as FFI, pyrraline, AFGP, ALI, carboxymethyllysine, carboxyethyllysine and pentosidine, and mixtures of such antibodies.
  • the antibody binds carboxymethyllysine- modified proteins.
  • 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, and goats.
  • 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), 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 or a goat antibody. Details of the constant regions and other parts of antibodies for these animals are described below.
  • the antibody is a monoclonal antibody.
  • a particularly preferred anti-AGE antibody is an antibody which binds to a protein or peptide that exhibits a carboxymethyllysine modification.
  • Carboxymethyllysine also known as CML, N(epsilon)-(carboxymethyl)lysine, N(6)-carboxymethyllysine, or 2- Amino-6-(carboxymethylamino)hexanoic acid
  • CML-modified proteins or peptides are recognized by the receptor RAGE which is expressed on a variety of cells. CML has been well-studied and CML-related products are commercially available.
  • CML-BSA antigens 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).
  • a particularly 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.
  • the anti-AGE antibody has low rate of dissociation from the antibody-antigen complex, or k d (also referred to as k back 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 has a high affinity for the AGE- modified protein of a cell, which may be expressed as a low dissociation constant K D of at most 9 x 10 '6 , 8 x 10 '6 , 7 x 10 "6 , 6 x 10 " *, 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. MAB3247), illustrated in FIG. 1.
  • carboxymethyl lysine MAb Clone 318003 available from R&D Systems, Inc. (Minneapolis, MN; catalog no. MAB3247), illustrated in FIG. 1.
  • the anti-AGE antibody may destroy AGE-modified cells through antibody- dependent 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 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 a/., "Pore-Forming Toxins and Cellular Non-Immune 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 typically comprise two heavy chains and two light chains of polypeptides joined to form a ⁇ " shaped molecule.
  • the constant region determines the mechanism used to target the antigen.
  • the amino acid sequence in the tips of the "Y" (the variable region) varies among different antibodies. This variation gives the antibody its specificity for binding antigen.
  • the variable region which includes the ends of the light and heavy chains, is further subdivided into hypervariable (HV - also sometimes referred to as complementarity determining regions, or CDRs) and framework (FR) regions.
  • 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 " being specific to each antigen; 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 (CDR1 H), 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 (CDR1 L), SEQ ID NO: 27 (CDR2L) and SEQ ID NO: 28 (CDR3L).
  • the heavy chain of human (Homo sapiens) antibody immunoglobulin G1 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 human (Homo sapiens) antibody immunoglobulin G1 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.
  • 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.
  • the variable regions may be used in the humanization of non-human 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.
  • 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).
  • camelids Animals of the camelid family, such as camels (Camelus dromedarius and Camelus bactrianus), llamas (Lama glama, Lama pacos and Lama vicugna), alpacas (Vicugna pacos) and guanacos (Lama guanicoe), have a unique antibody that is not found in other mammals.
  • camelids 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.
  • 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
  • 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.
  • This type of antibody is known as an immunoglobulin new antigen receptor or IgNAR
  • 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.
  • 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).
  • 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 or SEQ ID NO:
  • 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 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 or SEQ ID NO:
  • 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.
  • CML-KLH keyhole limpet hemocyanin
  • CDN carboxymethyl lysine MAb
  • 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
  • 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 (C H 3) to produce a large fragment F(abc) and a small fragment pFc'.
  • Antibody fragments may alternatively be produced recombinantly. If additional antibodies are desired, they can be produced using well-known methods.
  • 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, 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-ace
  • 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
  • pAbs may be made in chickens, producing IgY molecules.
  • 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.
  • chimeric antibodies that are substantially human (humanized) or substantially “ized” to another animal (such as cat, dog, horse, camel or alpaca, cattle, sheep, 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, hydroxylapatite
  • 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.
  • 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.
  • 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 manitol, 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 agents, and optionally other therapeutic agents, and optionally other therapeutic agents, and optionally other therapeutic agents, and optionally other therapeutic agents.
  • 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.
  • 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 it has been effective to treat the sarcopenia, by measuring changes in muscle mass over time. For example, a baseline muscle mass in a subject may be measured followed by administration of the anti-AGE antibody. The effectiveness of the treatment may be determined by periodically measuring muscle mass in the subject and comparing the subsequent measurements to the baseline measurement. A subject may be considered to have effective treatment of sarcopenia if he or she does not demonstrate loss of muscle mass between subsequent measurements or over time. Alternatively, the concentration and/or number of senescent cells in fat or muscle tissue may also be monitored. 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.
  • Any mammal that could develop sarcopenia or other diseases or disorders associated with cellular senescence 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, cows, horses and companion animals, such as dogs or cats.
  • a subject in need of treatment may be identified by the diagnosis of a disease or disorder that is known to cause elevated levels of AGEs such as, for example, diabetes (both Type 1 and Type 2), or the presence of a pathological condition associated with AGEs such as, for example, atherosclerosis, inflammation, retinopathy, nephropathy, stroke, endothelial cell dysfunction, neurodegenerative disorders or cancer.
  • subjects may be identified for treatment based on their age.
  • a human over 75 years of age may be treated for sarcopenia, while a human under 30 years of age might not be identified as in need of treatment for sarcopenia.
  • any of the mammals or subjects identified above may be excluded from the patient population in need of treatment for sarcopenia.
  • a subject may be identified as having sarcopenia or in need of treatment if (1) the subject is at least 25 years old and (2) his or her measured muscle mass and measured muscle function are two standard deviations or more below the mean value for healthy 25 year olds of the same gender and no alternative pathology has been identified to account for the reduced muscle mass and reduced muscle function.
  • a subject being treated for sarcopenia is at least 40 years old. More preferably, a subject being treated for sarcopenia is at least 50 years old. Most preferably, a subject being treated for sarcopenia is at least 60 years old.
  • a subject may be identified as having sarcopenia or in need of treatment if (1 ) his or her gait speed is less than 1.0 m/s across a 4 m course and (2) he or she has an objectively measured low muscle mass, such as, for example, an appendicular mass relative to the square of height less than or equal to 7.23 kg/m 2 for male subjects or less than or equal to 5.67 kg/m 2 for female subjects.
  • an objectively measured low muscle mass such as, for example, an appendicular mass relative to the square of height less than or equal to 7.23 kg/m 2 for male subjects or less than or equal to 5.67 kg/m 2 for female subjects.
  • a composition containing the anti-AGE antibody directly into the central nervous system.
  • administration include intrathecal administration; administration into the ventricular system of the brain (intraventricular administration), for example, through a catheter or a permanent shunt, or other administration device which may be placed during a ventriculostomy (see, for example, Takami, A. et al. "Treatment of primary central nervous system lymphoma with induction of complement-dependent cytotoxicity by intraventricular administration of autologous- serum-supplemented rituximab", Cancer Sci. Vol. 97, pp.
  • central nervous system administration may optionally also include administration of a serum supplement (such as autologous serum), to enhance the cell killing properties of the AGE antibody; administration of serum supplement may be prior to, simultaneous with, or subsequent to, the administration of the AGE antibody.
  • a serum supplement such as autologous serum
  • any of the composition containing AGE antibodies described herein may further contain a serum supplement (such as an autologous serum supplement).
  • a serum supplement such as an autologous serum supplement
  • purified immune system cells may also be used, either autologous immune system cells, or immune system cells from a donor; examples of such cells include natural killer cells.
  • artificial natural killer cells such as those of NANTKWEST®, engineered to bind directly to antibodies, or engineered to bind directly to an AGE antigen (such as carboxymethyllysine) (see www.nantkwest.com).
  • a mammal that could develop metastatic cancer 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, cows, horses and companion animals, such as dogs or cats.
  • a subject in need of treatment may be identified by the diagnosis of a cancer.
  • Cancers which are particularly subject to metastasis include lung cancer, melanoma, colon cancer, renal cell carcinoma, prostate cancer, cancer of the cervix, bladder cancer, rectal cancer, esophageal cancer, liver cancer, mouth and throat cancer, multiple myeloma, ovarian cancer, and stomach cancer. Treatment may be of patients experiencing metastatic cancer.
  • Treatment may also be administered to patients who have cancer, but prior to any identified metastasis, in order to prevent metastasis.
  • a subject that receives administration of an anti-AGE antibody may be tested to determine if it has been effective to treat the cancer by examining the patient for the spread of the cancer to different parts of the body, particularly in lymph nodes. Administration of antibody and subsequent testing may be repeated until the desired therapeutic result is achieved.
  • the anti-AGE antibodies may be used in cellular purification processes, such as immunopanning and immunoadsorption. Purification processes are useful in isolating desirable or unwanted cells from tissue cultures, cell cultures or blood. Cellular purification may be used in transplantations, such as a bone marrow transplant, or transfusions, such as a blood transfusion. Cellular purification is especially useful in autologous stem cell transplantation during chemotherapy to remove malignant cells and concentrate beneficial stem cells, such as hematopoietic cells expressing the CD34 protein (CD34 + cells). Immunopanning or immunoadsorption using an anti-AGE antibody may isolate partially-functional or non-functional cells, such as senescent cells, from a tissue culture, cell culture or blood sample.
  • cellular purification processes are useful in isolating desirable or unwanted cells from tissue cultures, cell cultures or blood.
  • Cellular purification may be used in transplantations, such as a bone marrow transplant, or transfusions, such as a blood transfusion. Cellular purification is especially useful in autolog
  • an immunopanning process may involve immobilizing the anti-AGE antibody on a surface, such as a cell culture plate.
  • a tissue culture or cell culture may then be applied to the surface. Any senescent cells present in the tissue culture or cell culture will bind to the anti-AGE antibody, leaving a purified tissue culture or cell culture that is free from senescent cells.
  • an immunoadsorption process may involve binding the anti-AGE antibody to senescent cells in a cell culture.
  • the cells may then be passed through a column packed with beads that are coated with a protein that binds to the anti-AGE labeled senescent cells.
  • the cells which pass through the column without binding will be cells that do not express AGE, such as fully-functional cells.
  • An immunoadsorption process may be carried out with a CEPRATE SC Stem Cell Concentration System (CellPro, Inc., Bothell, WA) or similar apparatus.
  • CellPro, Inc. Bothell, WA
  • the one-letter amino acid sequence that corresponds to SEQ ID NO: 1 is shown below:
  • Positions 16-133 of the above amino acid sequence correspond to SEQ ID NO: 2. Positions 46-50 of the above amino acid sequence correspond to SEQ ID NO: 41 . Positions 65-81 of the above amino acid sequence correspond to SEQ ID NO: 42. Positions 1 14-122 of the above amino acid sequence correspond to SEQ ID NO: 43.
  • Positions 16-128 of the above amino acid sequence correspond to SEQ ID NO: 4.
  • the arginine (Arg or R) residue at position 128 of SEQ ID NO: 4 may be omitted.
  • Positions 39-54 of the above amino acid sequence correspond to SEQ ID NO: 44.
  • Positions 70-76 of the above amino acid sequence correspond to SEQ ID NO: 45.
  • Positions 109-1 17 of the above amino acid sequence correspond to SEQ ID NO: 46.
  • VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK
  • the one-letter amino acid sequence that corresponds to SEQ ID NO: 31 is MDPKGSLSWRILLFLSLAFELSYGQVQLVQSGAEVKKPGASVKVSCKASGYLFTTYW MHWVRQAPGQGLEWMGEISPTNGRAYYNAKFQGRVTMTVDKSTNTAYMELSSLRSE DTAVYYCARAYGNYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNH
  • Example 1 In vivo study of the administration of anti-glycation end-product
  • 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
  • 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 immodilized with the test antibody (molecular weigh of 150,000 Da).
  • the running buffer was a 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
  • 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.
  • CE-SDS sulfate
  • CML-KLH KLH 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 1 N HCI. 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 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.
  • 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
  • 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:
  • 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.
  • AGE glycation end product
  • VHHs camelid heavy chain antibodies

Abstract

L'invention concerne un anticorps anti-AGE, comprenant une protéine ou un peptide qui comprend au moins une séquence d'acides aminés présentant une identité de séquence d'au moins 90 %, de préférence une identité de séquence d'au moins 95 %, plus préférablement une identité de séquence d'au moins 98 %, par rapport à des séquences d'acides aminés spécifiques. L'anticorps anti-AGE se lie à une protéine ou à un peptide qui présente une modification carboxyméthyllysine. L'anticorps anti-AGE peut être utilisé pour tuer des cellules sénescentes, pour tuer des cellules partiellement fonctionnelles ou non fonctionnelles, pour traiter la sarcopénie, pour favoriser la régénération d'organes ou de tissus, pour favoriser des processus de régénération ou surmonter les effets de vieillissement, pour traiter l'athérosclérose, pour prévenir ou retarder l'apparition de la cataracte, pour prévenir ou retarder l'apparition d'une perte de tissu adipeux, pour augmenter la durée de vie en santé, pour prévenir ou retarder l'apparition de la lordo-cyphose, pour traiter l'inflammation ou les maladies auto-immunes, pour traiter des troubles neurodégénératifs ou pour traiter le cancer.
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