EP4673464A1 - Procédés et agents pour des interventions de maladies induites par des lésions protéiques - Google Patents

Procédés et agents pour des interventions de maladies induites par des lésions protéiques

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Publication number
EP4673464A1
EP4673464A1 EP24762836.5A EP24762836A EP4673464A1 EP 4673464 A1 EP4673464 A1 EP 4673464A1 EP 24762836 A EP24762836 A EP 24762836A EP 4673464 A1 EP4673464 A1 EP 4673464A1
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European Patent Office
Prior art keywords
isodgr
disease
antibody
seq
patient
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EP24762836.5A
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German (de)
English (en)
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Newman Siu-kwan SZE
Kalailingam PAZHANICHAMY
Neil Edward MCCARTHY
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Brock University
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Brock University
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Publication of EP4673464A1 publication Critical patent/EP4673464A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • A61K39/0005—Vertebrate antigens
    • 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
    • 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
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/16—(de-)amidation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7042—Aging, e.g. cellular aging

Definitions

  • the present disclosure relates to the development of isoDGR peptides and immunogens, anti-isoDGR antibodies and fragments thereof, and compositions comprising said immunogens or antibodies, for the diagnosis, prevention, and treatment of isoDGR-associated diseases such as cardiovascular disease.
  • BACKGROUND [0004] Aging is the primary risk factor for death from all age-related chronic diseases. As global society becomes more elderly, rates of age-related diseases such as chronic inflammation, dementia, pulmonary disorders and cardiovascular diseases are set to increase exponentially. Rapid population aging nowadays is therefore one of the biggest healthcare challenges facing the next century, e.g. Dementia-linked mortality rates in Canada are increasing rapidly ( ⁇ 59% over the past 10 years alone).
  • DPMs can greatly increase the diversity of biomolecules present in body tissues, 36 with a high chance of generating proteoforms capable of interacting with or binding to key biomolecules in novel ways. Indeed, it was recently reported that deamidation of the amino acid sequence NGR (Asn-Gly-Arg) in extracellular matrix (ECM) proteins results in ‘gain- of-function’ conformational switching to isoDGR (isoAsp-Gly-Arg) motifs 6,27,28 that can mimic integrin-binding RGD ligands.
  • NGR amino acid sequence
  • ECM extracellular matrix
  • the current drugs used to prevent blood clot formation, stroke, and heart attack include anti-platelet drugs such as Aspirin, Clopidogrel, Prasugrel, Abciximab, Eptifibatide, and Tirofiban, and anticoagulant drugs such as warfarin, dabigatran, rivaroxaban, apixaban, and edoxaban.
  • anti-platelet drugs such as Aspirin, Clopidogrel, Prasugrel, Abciximab, Eptifibatide, and Tirofiban
  • anticoagulant drugs such as warfarin, dabigatran, rivaroxaban, apixaban, and edoxaban.
  • a common problem with these drugs is that they significantly increase the risk of serious bleeding, which can be fatal.
  • the protein l-isoaspartate (d-aspartate)-O-methyltransferase (Pcmt1) enzyme is expressed in all mammalian tissues and mediates repair of age-linked protein damage by promoting conversion of abnormal aspartyl residues to l-aspartyl forms. 21,23 Previous studies have shown that global deletion of Pcmt1 in mice leads to the accumulation of isoaspartate in all body tissues and premature death at around 42 days, 21,24,38 which models the decline in protein repair function as animals age.
  • isoDGR modification of ECM components may represent the mechanistic link in common pathologies affecting both elderly humans and Pcmt1-KO mice.
  • isoDGR motifs are known to accumulate in blood plasma and body tissues from Pcmt1 +/- mice, which displayed infiltration of CD68+ monocyte- macrophages that expressed pro-inflammatory cytokines. 6
  • the aberrant aging-induced isoDGR-modified proteins can accumulate in key organs such as brain, blood vessel, lung, liver and blood that trigger various chronic age-related diseases.
  • isoDGR-modified proteins underpins this pathology, suggesting a potential role for this axis in the ‘inflammaging’ characteristics of advancing age. 39-41 Methods to detect, reduce, remove and prevent isoDGR formation are needed and may have the potential provide effective therapeutic and prophylactic strategies to increase human health-span.
  • anti-isoDGR immunotherapy can reduce low-grade inflammation and extend the lifespan of Pcmt1-KO mice. Weekly injection of 1mg/kg isoDGR-specific monoclonal antibody (mAb) significantly increased body weight, improved behaviour and coordination, and doubled the average lifespan of Pcmt1 -/- mice.
  • mAb treatment decreased levels of circulating pro-inflammatory cytokines and reduced tissue inflammation, strongly suggesting that isoDGR-modified proteins are at least partly responsible for the pathology observed in Pcmt1-deficient animals. Consistent with this concept, further in vitro and in vivo assays demonstrated that anti-isoDGR mAb can induce immune clearance of the target motif via antibody- dependent cell-mediated phagocytosis (ADCP). Since isoDGR-damaged proteins accumulate in body tissues with advancing age, motif-specific therapy is expected to provide an effective intervention for human age-linked disorders and in extending healthy lifespan.
  • ADCP antibody- dependent cell-mediated phagocytosis
  • anti-isoDGR therapy effectively induced immune clearance of isoDGR-modified proteins via antibody-dependent cell-mediated phagocytosis (ADCP), thereby reducing chronic inflammation, tissue damage and susceptibility to chronic age-related diseases, including chronic pulmonary disease, non-alcoholic fatty liver disease, atherosclerotic cardiovascular disease, cerebrovascular disease and vascular dementia.
  • ADCP antibody-dependent cell-mediated phagocytosis
  • isoDGR-modified proteins and/or isoDGR-specific autoantibody can be good biomarkers for chronic inflammation and age-related diseases.
  • anti-isoDGR immunotherapy is expected to treat and/or prevent isoDGR-induced chronic inflammation and age-related diseases.
  • isoDGR biomarkers are useful to differentiate patients with cardiovascular diseases (CVD), stroke, and vascular dementia (VaD). Furthermore, the immunotherapy potential of anti-isoDGR immunotherapy is demonstrated herein using rodent models of isoDGR accumulation with Pcmt1 knock out mouse, high-fat diet induced fatty liver disease and cardiovascular diseases, and vascular dementia. The results show that isoDGR-biomarkers can be used for diagnosis and prognosis of CVD, stroke and VaD.
  • anti-isoDGR therapies using either passive isoDGR-specific monoclonal antibody or active immunization using isoDGR-antigen can effectively reduce atherosclerotic cardiovascular disease, fatty liver disease, chronic lung inflammation/parenchymal lung disease and increase cognitive function in PCMT1-/- mice and mouse models of cardiovascular disease.
  • An aspect includes an isolated anti-isoDGR antibody comprising a light chain variable (VL) domain and a heavy chain variable (VH) domain, the VL domain comprising complementarity determining regions (CDRs) CDR-L1, CDR-L2, and CDR-L3, and the VH domain comprising CDRs CDR-H1, CDR-H2, and CDR-H3, wherein the amino acid sequences of said CDRs are as shown in any one of a) or b): a) C DR-L1 KSSQSVFYNSDQKNQLA SEQ ID NO: 1; CDR-L2 WASTRES SEQ ID NO: 2; CDR-L3 HQYFSSWT SEQ ID NO: 3; C DR-H1 NYAMS SEQ ID NO: 4; CDR-H2 SISNGDYTYYPDSVKG SEQ ID NO: 5; and CDR-H3 GYSNPWCFDV SEQ ID NO: 6; or b) C DR-L1 K
  • the VL domain and VH domain comprise i) a polypeptide having an amino acid sequence of a) SEQ ID NOs: 7 and/or 8; or b) SEQ ID NOs: 17 and/or 18; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to a) SEQ ID NOs: 7 and/or 8; or b) SEQ ID NOs: 17 and/or 18; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are those described herein.
  • the VL domain and VH domain comprise i) a polypeptide having an amino acid sequence of a) SEQ ID NOs: 26 and/or 27; or b) SEQ ID NOs: 30 and/or 31; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to a) SEQ ID NOs: 26 and/or 27 or b) SEQ ID NOs: 30 and/or 31; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are those described herein.
  • the antibody is a humanized or human antibody.
  • the antibody is an IgG, optionally IgG1, an scFv, or a Fab.
  • An aspect includes a nucleic acid molecule encoding a VL and/or a VH domain of an antibody described herein.
  • the nucleic acid molecule comprises a sequence of any one of SEQ ID NOs: 9, 10, 19, 20, 28, 29, 32, and 33, and functional variants of any thereof.
  • An aspect includes a cell comprising a nucleic acid molecule or expressing an antibody described herein.
  • An aspect includes an immunoconjugate comprising an antibody described herein and a detectable label.
  • An aspect includes a pharmaceutical composition comprising an antibody described herein and a pharmaceutically acceptable carrier or excipient.
  • An aspect includes an immunogen comprising an isoDGR peptide, optionally conjugated to a carrier protein or immunogenicity enhancing agent.
  • the isoDGR peptide comprises Ac-GC(isoD)GRCGK (SEQ ID NO: 25); GC(isoD)GRCGG-(CH2-CH2-NH2) (SEQ ID NO: 12); GC(isoD)GRCGK (SEQ ID NO: 38); Ac-C(isoD)GRCGGK (SEQ ID NO: 39); or Ac- GC(isoD)GRCGGK (SEQ ID NO: 40), and/or the carrier protein or immunogenicity enhancing agent comprises bovine serum albumin or keyhole limpet hemocyanin.
  • the isoDGR peptide comprises Ac-GC(isoD)GRCGK (SEQ ID NO: 25) and/or the carrier protein comprises keyhole limpet hemocyanin.
  • An aspect includes a pharmaceutical composition comprising an immunogen described herein and a pharmaceutically acceptable carrier and/or an adjuvant.
  • An aspect includes an anti-isoDGR antibody, optionally an antibody described herein, an immunogen described herein, or a composition comprising said antibody or immunogen, for use in treating or preventing an isoDGR-associated disease, optionally the isoDGR-associated disease is selected from cardiovascular disease, a cerebrovascular disease, a pulmonary disease, a liver disease, an inflammatory disease, a cancer, and a clotting disorder, in a patient in need thereof.
  • the patient is a human.
  • An aspect includes a use of an anti-isoDGR antibody, optionally an antibody described herein, an immunogen described herein, or a composition comprising said antibody or immunogen, for treating or preventing an isoDGR- associated disease, optionally the isoDGR-associated disease is selected from a cardiovascular disease, a cerebrovascular disease, a pulmonary disease, a liver disease, an inflammatory disease, a cancer, and a clotting disorder, in a patient in need thereof.
  • An aspect includes a use of an anti-isoDGR antibody, optionally an antibody described herein, an immunogen described herein, or a composition comprising said antibody or immunogen, in the manufacture of a medicament for treating or preventing an isoDGR-associated disease, optionally the isoDGR- associated disease is selected from cardiovascular disease, a cerebrovascular disease, a pulmonary disease, a liver disease, an inflammatory disease, a cancer, and a clotting disorder, in a patient in need thereof.
  • An aspect includes a method of identifying a patient who has or is at increased risk of developing an isoDGR-associated disease, the method comprising: providing a biological sample suspected of containing isoDGR-modified proteins from the patient; contacting the sample with an anti-isoDGR antibody, optionally an antibody or fragment thereof described herein, under conditions permissive for forming isoDGR:antibody complexes; detecting the presence of any isoDGR:antibody complexes; determining the level of one or more isoDGR-modified proteins based on the detected isoDGR:antibody complexes; and comparing the level of the one or more isoDGR-modified proteins to a control or reference value, wherein an increased level of one or more isoDGR-modified proteins relative to the control or reference value is indicative that the patient has, or is at increased risk of developing an isoDGR-associated disease.
  • the one or more isoDGR-modified proteins comprises apolipoprotein B100 (ApoB100), apolipoprotein (ApoD), complement component C6 (C6), complement factor B (CFB), complement factor H (CFH), coagulation factor IX (F9), coagulation factor XIII (F13), fibrinogen beta chain (FGB), fibrinogen gamma chain (FGG), plasminogen (PLG) and/or fibronectin (FN1).
  • the isoDGR-associated disease is stroke, and the patient is identified as having, or being at increased risk of developing, stroke, if the levels of isoDGR-modified FGB and/or FGG but not ApoB100 are increased relative to the control or reference value.
  • the isoDGR-associated disease is coronary artery disease, and the patient is identified as having, or being at risk of developing, coronary artery disease if the levels of isoDGR-modified ApoB100, and optionally FGB and/or FGG, are increased relative to the control or reference value.
  • the isoDGR-associated disease is vascular dementia
  • the patient is identified as having, or being at risk of developing, vascular dementia if the levels of isoDGR-modified C6, CFB, and/or CFH (and optionally FGB and/or FGG) are increased relative to the control or reference value.
  • An aspect includes a method of identifying whether a patient has, or is at increased risk of developing, an isoDGR-associated disease, the method comprising: providing a biological sample suspected of containing anti-isoDGR autoantibodies from the patient; contacting the sample with an isoDGR peptide under conditions permissive for forming isoDGR:autoantibody complexes; detecting the presence of any isoDGR:autoantibody complexes; determining the level of autoantibodies based on the detected isoDGR:antibody complexes; and comparing the level of the autoantibodies to a control or reference value, wherein the patient is identified as having or being at increased risk of developing an isoDGR-associated disease depending on the level of autoantibodies relative to the control or reference value.
  • An aspect includes a method of treating or preventing an isoDGR- associated disease, optionally selected from a cardiovascular disease, cerebrovascular disease, a pulmonary disease, a liver disease, an inflammatory disease, a cancer, and a clotting disorder, the method comprising administering an effective amount of an anti-isoDGR antibody, optionally an antibody described herein, an immunogen described herein, or a composition comprising said antibody or immunogen, to a patient in need thereof, optionally the patient is a human.
  • an anti-isoDGR antibody optionally an antibody described herein, an immunogen described herein, or a composition comprising said antibody or immunogen
  • the cardiovascular disease is endothelial dysfunction, atherosclerosis, coronary heart disease (CHD), coronary artery disease (CAD), heart failure, myocardial ischemia, myocardial infarction, hypertrophic cardiomyopathy, or left ventricular hypertrophy;
  • the cerebrovascular disease is stroke, transient ischemic attack (TIA), carotid artery disease, neurovascular inflammation, blood brain barrier dysfunctions, vascular cognitive impairment, or dementia including vascular dementia and Alzheimer’s disease;
  • the pulmonary disease is chronic lung inflammation, parenchymal lung disease, emphysema, chronic obstructive pulmonary disease (COPD), asthma, or lung fibrosis;
  • the liver disease is chronic liver inflammation, fatty liver disease, non- alcoholic fatty liver disease, or non-alcoholic steatohepatitis (NASH);
  • the cancer is lung cancer, liver cancer, colon cancer, breast cancer, skin cancer, prostate cancer, ovarian cancer, kidney cancer, pancreatic cancer;
  • the method further comprises detecting isoDGR- modified proteins and/or anti-isoDGR autoantibodies in a biological sample according to a method described herein, wherein the biological sample is obtained from the subject, wherein the detecting is done before, during, or following administering the antibody, immunoconjugate, or composition.
  • (D) Dot plot shows average body weight (male or female) for each genotype at 6 weeks and 165 days of age (n 5).
  • Fig.2 shows anti-isoDGR mAb treatment reduces motif level in brain and liver from Pcmt1 -/- mice. Protein lysates from brain (A) and liver (B) of Pcmt1 +/+ , Pcmt1 +/- , Pcmt1 -/- , and mAb-treated Pcmt1 -/- mice were subjected to western blot using isoDGR-specific antibody. Protein loadings were visualized by Ponceau S.
  • Graphs show quantification of isoDGR-modified protein levels in mouse brain (C) and liver (D), ** p ⁇ 0.01, * p ⁇ 0.05.
  • Fig. 3 shows isoDGR co-localization with CD68+ monocyte- macrophages in liver from Pcmt1-KO mice.
  • FIG. 4 shows increased isoDGR levels/co-localization with CD68+ macrophages in Pcmt1 -/- mouse spleen.
  • B IsoDGR or
  • C CD68 fluorescence in 50 randomized regions from 3 images of 3 independent spleen sections for each genotype were quantified using image J (graphs shows averaged values for the same region from 3 images). Results shown are mean values ⁇ SE (*** p ⁇ 0.001, ** p ⁇ 0.01). [0045] Fig.
  • FIG. 5 shows increased isoDGR levels/co-localization with CD68+ macrophages in Pcmt1 -/- mouse thymus.
  • A Representative immunostaining images showing isoDGR distribution and co-localisation with CD68+ macrophages in cryosectioned thymus tissue (Pcmt1 +/+ , Pcmt1 +/- , Pcmt1 -/- , and mAb-treated Pcmt1 -/- mice at 5 weeks).
  • B IsoDGR or
  • C CD68 fluorescence in 50 randomized regions from 3 images of 3 independent thymus sections for each genotype were quantified using image J (graphs shows averaged values for the same region from 3 images).
  • Fig. 6 shows elevated levels of local and systemic inflammation in Pcmt1 -/- mice.
  • FIG. 7 shows isoDGR-modified plasma proteins induce systemic inflammation in wild-type C57BL/6 mice.
  • FIG. 8 shows isoDGR-peptides promote inflammatory cytokine release in wild-type C57BL/6 mice.
  • Fig. 9 shows age-induced accumulation of isoDGR co-localizes with CD68+ monocyte-macrophage infiltration of liver from PCMT1 +/+ and Pcmt1 +/- mice.
  • A Representative immunostaining of isoDGR protein distribution and co-localisation with CD68+ macrophages in cryosectioned liver tissue from Pcmt1 +/+ and Pcmt1 +/- mice at 4, 15 and 24 months.
  • IsoDGR B or CD68 (C) fluorescence intensities in a set of 50 random regions taken from 3 images representing 3 independent liver sections per genotype (quantified by image J software with average values plotted in the dot / bar graph).
  • D Plot showing the accumulation of isoDGR-motif with age in both WT and PCMT+/- animals. Results indicate that isoDGR accumulation is accelerated in older animal of both WT and PCMT+/- mice.
  • E CD68+ cells infiltration to tissues are proportional to the isoDGR levels. Results shown are mean values ⁇ SE (**** p ⁇ 0.0001, *** p ⁇ 0.001,** p ⁇ 0.01, * p ⁇ 0.05).
  • Fig.10 shows partial deletion of Pcmt1 leads to systemic elevation of proinflammatory cytokines in mice.
  • Fig. 11 shows isoDGR-specific mAb promotes ADCP of isoDGR- modified fibronectin and fibrinogen.
  • Fig.12 shows isoDGR-specific mAb enhances phagocytosis of isoDGR- modified fibronectin.
  • A Representative immunostaining images showing that phagocytosis of isoDGR-FN by RAW macrophages increases with motif-specific mAb concentration (1-5 ⁇ g/ml).
  • B Bar graph shows average number of phagocytic RAW macrophages. Fluorescent signal in 50 random regions taken from 3 images in 3 independent experiments were quantified by image J software (graph shows averaged values).
  • FIG. 13 shows vascular pathology due to isoDGR accumulation in PCMT1-KO mice
  • A Blood from PCMT1 knockout (KO) and heterozygous (HET) animals is less oxygenated / darker than in WT mice.
  • B and
  • C When KO mice were injected biweekly with 3 mg/kg isoDGR-mAb from birth up to 2 months (KO+mAb), the blood became oxygenated similar to WT. Note: in (B) Blood from WT and KO+mAb mice spreads out in Eppendorf tubes since it is thinner than in KO and HET animals.
  • FIG.14 shows (A) Immunofluorescent images of brain tissue from WT and PCMT1-KO mice with accumulated isoDGR-modified proteins. (B) Immunofluorescent imaging of brain tissue from PCMT KO mice. Glucose transporter 1 (GLUT1) was found to concentrate in endothelial cells at blood-tissue barriers, thus allowing clear detection of brain capillaries when used in conjunction with DAPI.
  • Erythroid-specific marker TERT119 indicates potential bleeding or thrombosis. Discrete patches of TERT119 fluorescence signal were observed in different brain regions from PMCT1 KO mice, indicating the presence of erythroid clusters characteristic of microbleeds and thrombus. Using confocal microscopy, red blood cells leaking from the endothelium of the capillary were detected.
  • Fig. 15 shows atherosclerotic arterial lesions in (A) WT mice fed with western diet (WD) for 3 months. (B) WT mice fed WD and injected with isoDGR-mAb for 3 months. (C) apoE -/- mice fed WD for 12-weeks.
  • FIG.16 shows WT mice fed HFD and treated with PBS or 3mg/kg mAb for 3 months.
  • A Representative images of livers from WT control mice and 14G7 mAb-treated mice fed HFD. Inset shows an image of a representative liver from a WT mouse fed a normal diet.
  • B Hepatic lipid droplets stained with ORO.
  • C LDL levels, (D) Liver weight, and (E) Body weight of WT+chow diet, WD+PBS, and WD+isoDGR mAb.
  • FIG.17 shows detailed atherosclerotic arterial lesions in Apoe -/- mice fed WD for 12-weeks (left); and Apoe -/- mice fed WD and injected with isoDGR-mAb for 12 weeks (right). Results show that isoDGR-mAb can reduce/prevent atherosclerosis.
  • Fig.18 shows increased IsoDGR protein accumulation and colocalized with CD68 positive immune cells in the lung of PCMT-/- mice.
  • FIG. 19 shows isoDGR mAb treatment improves the emphysema phenotype in lung of PCMT-/- mice.
  • Fig.20 shows eleven isoDGR-modified plasma biomarkers were pulled down from patients with coronary artery disease (CAD), stroke or vascular dementia (VaD). These biomarkers are known to be closely associated with key aspects of vascular pathology, including lipid metabolism (ApoB100, ApoD), inflammation (C6, CFB, CFH), coagulopathy (F9, F13, FGB, FGG and PLG) and vessel disease (FN1). Relative abundance of the individual biomarkers generated characteristic profiles for CAD, stroke, and VaD.
  • Fig. 21 shows levels of isoDGR autoantibodies measured in patient plasma using ELISA method. Healthy donors displayed significantly higher levels of isoDGR-autoantibody in blood plasma.
  • Fig. 22 shows active immunization using isoDGR immunogens stimulates production of isoDGR-specific antibodies and extends the lifespan of PCMT -/- mice.
  • A Concentrations of plasma anti-isoDGR antibody in 40-day-old mice from the three groups as measured by indirect ELISA.
  • B Average body weight of vaccinated PCMT -/- mice compared to that of untreated PCMT -/- and WT mice.
  • Fig.23 shows isoDGR-mAb inhibits pancreatic cancer cell growth and tumour development in mice.
  • A Pancreatic cancer development is inhibited in anti- isoDGR antibody-treated mice (top) compared to PBS-treated controls (bottom).
  • FIG.24 shows structural models of fibrinogen.
  • A Two isoDGR motifs in fibrinogen beta or fibrinogen gamma chains were detected by LC-MS/MS in plasma of patients with CVD. These motifs are located in the D-domain of fibrinogen.
  • B The isoDGRs in both beta and gamma chains are localized in the D-domain of fibrinogen trimers where the molecule binds to ⁇ IIb ⁇ 3 integrins on platelets, which is known to trigger outside-in signalling and induce platelet activation, spreading and aggregation.
  • Fig. 25 shows platelet adhesion to various substrates.
  • A Platelet adhesion to a plastic plate (without any coating);
  • B Platelet spreading on native fibrinogen coated over a plastic plate;
  • C Increased platelet clumping on isoDGR- modified fibrinogen.
  • Fig. 26 shows isoDGR-modified plasma proteins in PCMT1 KO mice enhance platelet activation/aggregation in the presence of 2uM ADP relative to WT mice, as determined using a multiplate platelet function analyzer.
  • Fig. 27 shows isoDGR-specific mAb blocks isoDGR enhanced coagulation.
  • A Graph showing bleeding times.
  • transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively.
  • the term “consisting” and its derivatives as used herein are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
  • the term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of these features, elements, components, groups, integers, and/or steps.
  • the phrase "at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.
  • Immunogens Antibodies, Nucleic acids, and Cells
  • the inventors show herein the development of isoDGR peptides and immunogens, and monoclonal anti-isoDGR antibodies that specifically bind isoDGR epitopes (e.g. isoDGR peptides or proteins comprising an isoDGR-modification) which are useful for diagnostic and/or therapeutic purposes.
  • an isoDGR immunogen comprising an isoDGR peptide, optionally conjugated to a carrier protein or immunogenicity enhancing agent.
  • An "immunogen" as used herein means a substance which provokes an immune response and causes production of an antibody.
  • the immunogen may comprise for example an isoDGR peptide in the context of an isolated protein or fragment thereof, an isoDGR peptide conjugated to a carrier protein or immunogenicity enhancing agent, or an isoDGR peptide as part of a multiple antigenic peptide (MAP).
  • a protein, or fragment thereof which naturally comprises an NGR sequence e.g. fibronectin or fibrinogen
  • an isoDGR peptide in the context of an isolated protein or fragment thereof.
  • isoDGR peptide is used to refer to a peptide comprising an isoAsp-Gly-Arg motif, and optionally one or more additional N- and/or C-terminal amino acid residues.
  • the isoDGR peptide comprises Ac- GC(isoD)GRCGK (SEQ ID NO: 25); GC(isoD)GRCGG-(CH2-CH2-NH2) (SEQ ID NO: 12); GC(isoD)GRCGK (SEQ ID NO: 38); Ac-C(isoD)GRCGGK (SEQ ID NO: 39); or Ac-GC(isoD)GRCGGK (SEQ ID NO: 40).
  • Common carrier proteins include for example albumins such as bovine serum albumin (BSA) or modified BSA (e.g. cationized BSA), ovalbumin, and hemocyanin (e.g. keyhole limpet hemocyanin (KLH), or Blue CarrierTM), diphtheria toxin variants (e.g. CRM197), diphtheria toxoid, tetanus toxoid, meningococcal outer membrane protein complex (OMPC), and H. influenzae protein D (HiD).
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • virus-like particles or nanoparticles may be used as immunogenicity enhancing agents.
  • the carrier protein or immunogenicity enhancing agent can be coupled to the compound either directly, such as through an amide bond, disulfide bond, or indirectly through a linker, depending on the functional groups available.
  • the immunogen can be produced by conjugating the peptide and optionally a linker comprising a functionalizable moiety such as cysteine to the carrier protein using, for example and without limitation, 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), maleimide (or sulfhydryl), or glutaraldehyde. Other cross-linkers may also be used.
  • the immunogen comprises multiple peptides, each peptide comprising isoDGR, wherein the multiple peptides are synthesized as a multiple antigenic peptide (MAP).
  • MAP is a branched poly-lysine dendrimer. Multiple epitope peptides are attached for example to one or both of the amino terminus and side-chain of the lysines.
  • anti-isoDGR antibodies which specifically bind an isoDGR epitope comprising the complementarity determining regions (CDRs) of a): C DR-L1 KSSQSVFYNSDQKNQLA SEQ ID NO: 1; CDR-L2 WASTRES SEQ ID NO: 2; CDR-L3 HQYFSSWT SEQ ID NO: 3; CDR-H1 NYAMS SEQ ID NO: 4; C DR-H2 SISNGDYTYYPDSVKG SEQ ID NO: 5; and CDR-H3 GYSNPWCFDV SEQ ID NO: 6; or b): C DR-L1 KSSQSLLNSRNRKNYLA SEQ ID NO: 11; CDR-L2 WASTRES SEQ ID NO: 2; CDR-L3 KQSYNLWT SEQ ID NO: 13; CDR-H1 TSGMGIS SEQ ID NO: 14; C DR-H2 HIYWDDDNRYNP
  • the antibodies may comprise light chain variable (VL) and heavy chain variable (VH) domains set out in i) SEQ ID NOs: 7 and 8 (corresponding to clone 6E11), or ii) SEQ ID NOs: 17 and 18 (corresponding to clone 14G7); or variants (typically functional variants) thereof having the CDR sequences specified in a) or b), above.
  • the antibodies may further comprise a signal peptide, for example a signal peptide of any one of SEQ ID NOs: 34-37.
  • the antibody may comprise VL and VH domains set out in i) SEQ ID NOs: 26 and 27 (corresponding to clone 6E11), or ii) SEQ ID NOs: 30 and 31 (corresponding to clone 14G7), or variants (typically functional variants) thereof having the CDR sequences specified in a) or b), above.
  • the variants have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to either instance of i) or ii), or have a conservatively substituted amino acid sequence of either instance of i) or ii), wherein the CDR sequences are those indicated in a) or b) above.
  • antibodies, antibody fragments, peptides, and small molecules which compete for binding to an isoDGR epitope with the antibodies described herein, for example in a competitive binding assay.
  • the basic antibody structural unit is known to comprise a tetramer composed of two identical pairs of polypeptide chains, each pair having one light (“L”) (about 25 kDa) and one heavy (“H”) chain (about 50-70 kDa).
  • L light
  • H heavy
  • the amino-terminal portion of the light chain forms a light chain variable domain (VL)
  • VH heavy chain variable domain
  • VH and VL domains form the antibody variable region (Fv) which is primarily responsible for antigen recognition/binding.
  • each of the VH and VL domains are three hypervariable regions or complementarity determining regions (CDRs, commonly denoted CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR- L3).
  • CDRs complementarity determining regions
  • the carboxy-terminal portions of the heavy and light chains together form a constant region (Fc domain) primarily responsible for effector function.
  • Fc domain constant region primarily responsible for effector function.
  • the term "antibody” as used herein is intended to include monoclonal antibodies, chimeric and humanized or fully human antibodies, and binding fragments thereof, including for example a single chain Fab fragment, Fab’2 fragment, or single chain Fv fragment (scFv).
  • the antibody may be from recombinant sources and/or produced in transgenic animals.
  • Humanized or other chimeric antibodies may include sequences from one or more than one isotype, class, or species.
  • the isotype or class is determined by the heavy chain constant region or Fc domain.
  • Antibodies may be any class of immunoglobulins including: IgG, IgM, IgD, IgA, or IgE; and any isotype thereof, including IgG1, IgG2 (e.g. IgG2a, IgG2b), IgG3 and IgG4.
  • the antibody is an IgG, more typically an IgG1.
  • these antibodies may be produced as binding fragments as described herein.
  • the binding fragment is a Fab or scFv.
  • the antibodies may include sequences from any suitable species including human.
  • the antibodies may exist in monomeric or polymeric form.
  • the antibodies suitably comprise an Fc domain which is isotype- and species-matched to the immune cell.
  • the antibody suitably comprises an Fc domain from e.g. a gamma heavy chain (corresponding to an IgG class) from the same species as the leukocytes.
  • the antibody may be a neutralizing antibody.
  • Neutralizing antibodies may be for example an immunoglobulin, or may be an antibody fragment such as a Fab or scFv, that binds to an isoDGR epitope and is suitably species-matched to the patient.
  • the term "antibody fragment” or “binding fragment” as used herein is intended to include without limitations Fab, Fab', F(ab')2, scFab, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof, and Domain Antibodies.
  • Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and other fragments can also be synthesized by recombinant techniques. [0097] The term “complementarity determining region” or “CDR” as used herein refers to particular hypervariable regions of antibodies that are commonly presumed to contribute to epitope binding.
  • the CDRs listed in the present disclosure are identified using IMGT.
  • a person skilled in the art having regard to the sequences comprised herein would be able to identify CDR sequences based on any of Kabat, IMGT, and Chothia etc.
  • Such antibodies are similarly encompassed.
  • the phrase "isolated antibody” refers to antibody produced in vivo or in vitro that has been removed from the source that produced the antibody, for example, an animal, hybridoma, phage display, B-cells and plasma cells, or other cell lines (such as recombinant insect, yeast or bacteria cells that produce antibody). In some embodiments the antibody is an isolated antibody.
  • the isolated antibody is optionally "purified", which means at least: 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% purity.
  • epitope as commonly used means an antibody binding site, typically a polypeptide segment having a particular structural conformation, in an antigen that is specifically recognized by the antibody.
  • an antibody generated or selected against a recombinant protein comprising the identified target region e.g. isoDGR
  • the epitope is typically represented herein by a linear amino acid sequence or the region of the peptide or protein recognized by the antibody.
  • greater affinity refers to a relative degree of antibody binding where an antibody X binds to target Y more strongly (Kon) and/or with a smaller dissociation constant (Koff) than does comparator antibody Z, and in this context antibody X has a greater affinity for target Y than Z.
  • lesser affinity refers to a degree of antibody binding where an antibody X binds to target Y less strongly and/or with a larger dissociation constant than does antibody Z, and in this context antibody X has a lesser affinity for target Y than Z.
  • the affinity of binding between an antibody and its target antigen can be expressed quantitatively as KA equal to 1/ KD where KD is equal to kon/koff.
  • a greater affinity corresponds to a lower KD.
  • the kon and koff values can be measured using surface plasmon resonance technology, and/or as described herein. Binding affinity can also be assessed using other techniques such as flow cytometry.
  • the term “functional variant” as used herein includes modifications of the polypeptide sequences disclosed herein that perform substantially the same function as the polypeptide molecules disclosed herein in substantially the same way.
  • the functional variant may comprise sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequences disclosed herein provided that the variant retains at least or about the same binding affinity for isoDGR.
  • the functional variant may also comprise conservatively substituted amino acid sequences of the sequences disclosed herein.
  • sequence identity refers to the percentage of sequence identity between two amino acid sequences or two nucleic acid sequences.
  • the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
  • One non- limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877.
  • Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402.
  • PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules.
  • the default parameters of the respective programs e.g. of XBLAST and NBLAST
  • Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • IMGT immunoglobulin G protein sequence
  • ImMunoGeneTics database numbering refer to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or antigen binding portion thereof.
  • IMGT and other alignment systems can also be used to identify or annotate CDRs in an antibody sequence.
  • a "conservative amino acid substitution” or “conservatively substituted amino acid sequence” as used herein, is one in which one amino acid residue is replaced with another amino acid residue without abolishing the protein's desired properties.
  • Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another.
  • conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as alanine, isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
  • “conservative substitution” or “conservatively substituted amino acid sequence” also includes the use of a chemically derivatized residue or non- natural amino acid in place of a non-derivatized residue provided that such polypeptide displays the requisite activity.
  • Competition between antibodies, antibody fragments, peptides, small molecules, etc. can be determined for example using an assay in which an antibody, antibody fragment, peptide, small molecule, etc. under test is assessed for its ability to inhibit specific binding of a reference antibody to the common antigen. A test antibody, antibody fragment, peptide, small molecule, etc.
  • the antibodies described herein may be provided as immunoconjugates. Accordingly, also provided herein are immunoconjugates comprising an antibody described herein and a suitable reagent such as a therapeutic, cytotoxic agent, or detectable label.
  • Suitable reagents can be identified by the skilled person depending on the application. Detectable labels including radionuclides, fluorescent dyes, enzymes, or biotin may be used depending on the application, and are contemplated herein.
  • Immunoconjugates may be generated using any suitable technique. Common conjugation techniques include N-hydroxysuccinimide ester (NHS ester) or maleimide crosslinking, but other techniques are known in the art.
  • a further aspect is an isolated nucleic acid encoding an antibody or fragment thereof described herein.
  • Nucleic acids encoding a heavy chain or a light chain or parts thereof are also provided, for example encoding a heavy chain variable domain comprising CDR-H1, CDR-H2 and/or CDR-H3 regions described herein or encoding a light chain variable domain comprising CDR-L1, CDR-L2 and/or CDR-L3 regions described herein, variable heavy and light domains described herein, and codon optimized and codon degenerate versions thereof.
  • an aspect is an isolated nucleic acid encoding an antibody described herein, for example the nucleic acids shown in any of SEQ ID NOs: 9, 10, 19, 20, 28, 29, 32, and 33, or functional variants thereof.
  • the present disclosure also provides variants of the nucleic acid sequences that encode for the antibody and/or binding fragment thereof disclosed herein.
  • the variants include nucleotide sequences that hybridize to the nucleic acid sequences encoding the antibody and/or binding fragment thereof disclosed herein under at least moderately stringent hybridization conditions or codon degenerate or optimized sequences
  • the variant nucleic acid sequences have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to nucleic acid sequences encoding any of the amino acid sequences described herein for example as shown in any of SEQ ID NOs: 9, 10, 19, 20, 28, 29, 32, and 33, or functional variants thereof.
  • expression cassette refers to a DNA molecule encoding an RNA or protein operably linked to a promoter and a transcriptional termination signal (e.g. polyadenylation signal), such that certain portions of the expression cassette are capable of being transcribed into RNA such as a messenger RNA that is subsequently translated into protein by cellular machinery.
  • transcriptional termination signal e.g. polyadenylation signal
  • operably linked refers to a relationship between two components that allows them to function in an intended manner.
  • promoter actuates expression of the RNA encoded therein.
  • promoter or “promoter sequence” generally refers to a regulatory DNA sequence capable of being bound by an RNA polymerase to initiate transcription of a downstream (i.e. 3’) sequence to generate an RNA.
  • Suitable promoters may be derived from any organism and may be bound or recognized by any RNA polymerase. Suitable promoters will be known to the skilled person.
  • the promoter is an inducible promoter and/or comprises a binding sequence for a transactivator or a repressor that will activate or inhibit transcription respectively, for example isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG)- inducible promoters are commonly used for expression in E. coli.
  • IPTG isopropyl ⁇ -D-1-thiogalactopyranoside
  • suitable promoters will depend on the expression system and/or host cell being used.
  • Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes.
  • regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, and/or a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector.
  • the nucleic acid molecules may be incorporated in a known manner into an appropriate expression vector which ensures expression of the protein. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses).
  • the vector should be compatible with the host cell used.
  • the expression vectors are "suitable for transformation of a host cell", which means that the expression vectors contain a nucleic acid molecule encoding the peptides corresponding to antibodies described herein.
  • the vector can be any vector, including vectors suitable for producing an antibody and/or binding fragment thereof described herein. In an embodiment, the vector is an isolated vector.
  • the recombinant expression vectors may also contain a marker gene which facilitates the selection of host cells transformed, infected or transfected with a vector for expressing an antibody or epitope peptide described herein.
  • the recombinant expression vectors may also contain expression cassettes which encode a fusion moiety (i.e.
  • fusion protein which provides increased expression or stability of the recombinant peptide; increased solubility of the recombinant peptide; and/or aid in the purification of the target recombinant peptide by acting as a ligand in affinity purification, including for example tags and labels described herein.
  • a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the recombinant protein.
  • GST glutathione S-transferase
  • a cell optionally an isolated and/or recombinant cell, expressing an antibody described herein or comprising a nucleic acid or vector herein disclosed.
  • the recombinant cell can be generated using any cell suitable for producing a polypeptide, for example suitable for producing an antibody and/or binding fragment thereof.
  • a nucleic acid e.g. a vector
  • the cell may be transfected, transformed or infected, depending upon the vector employed.
  • Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells.
  • the proteins described herein may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells, or mammalian cells. III.
  • compositions and Kits [00124]
  • the isoDGR peptide, immunogen, anti-isoDGR antibodies or fragments thereof described herein are useful in the diagnosis, prevention, and/or treatment of any of the diseases, disorders, or conditions described herein, in particular isoDGR- associated diseases, and may be suitably formulated in a conventional manner into compositions using one or more carriers, excipients, or diluents.
  • the present description also includes a composition comprising one or more isoDGR immunogens described herein or one or more anti-isoDGR antibodies, optionally the antibodies described herein, and a carrier, excipient, or diluent.
  • the immunogens or antibodies described herein are suitably formulated into pharmaceutical compositions or dosage forms for administration to patients in a biologically compatible form suitable for administration in vivo. Accordingly, the present description further includes a pharmaceutical composition comprising an immunogen or antibody described herein, and a pharmaceutically acceptable carrier, excipient, or diluent. Also provided herein are dosage forms comprising an immunogen or antibody described herein.
  • pharmaceutical composition and “composition” are used interchangeably herein, unless the context clearly dictates otherwise.
  • the pharmaceutical compositions or dosage forms are used in the treatment of any of the diseases, disorders or conditions described herein, for example isoDGR-associated diseases.
  • the term “dosage form” as used herein refers to the physical form of a dose for example comprising an immunogen or antibody described herein, and includes without limitation injectable dosage forms, including, for example, sterile solutions and sterile powders for reconstitution, and the like, that are suitably formulated for injection, resuspendable powders, liquids and solutions.
  • the injectable dosage form can be a subcutaneous, intradermal, or intramuscular depot injection that allows the compound to be released in a controlled and consistent way over a period of time, for example over one month. Methods for making depot injections are described, for example, in U.S.
  • compositions or dosage forms described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions that can be administered to patients, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.
  • Pharmaceutical compositions include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially compatible with the tissues or the blood of an intended recipient.
  • compositions include water, surfactants (such as Tween), alcohols, polyols, glycerin and vegetable oils, for example.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions.
  • the composition may be supplied, for example but not by way of limitation, as a lyophilized powder which is reconstituted with sterile water or saline prior to administration to the patient.
  • Pharmaceutical compositions may comprise a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition.
  • Suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), and liposomes.
  • DOTMA N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride
  • DOPE diolesylphosphotidyl-ethanolamine
  • liposomes examples include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), and liposomes.
  • DOTMA N-(1(2,3-dio
  • the composition may be in the form of a pharmaceutically acceptable salt which includes, without limitation, those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylarnino ethanol, etc..
  • the composition comprises an immunogen described herein.
  • the composition comprises an immunogen described herein and an adjuvant.
  • Adjuvants are immunomodulators which are typically non- covalently linked to antigens and are formulated to enhance the host immune response.
  • FCA complete adjuvant
  • FIA incomplete adjuvant
  • squalene e.g. MF59, AS03
  • saponins e.g. QS21 and Matrix-M
  • lipopolysaccharides or derivatives thereof e.g. monophosphoryl lipid A
  • muramyl dipeptide MDP
  • aluminum salts collectively referred to as alums
  • the adjuvant may be administered with an immuogen as a single composition.
  • an adjuvant may be administered before, concurrent and/or after administration of the immunogen.
  • the composition comprises an antibody described herein.
  • the composition comprises an antibody described herein and a diluent.
  • the composition is a sterile composition.
  • the immunogens, antibodies, compositions, etc. described herein may be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art.
  • the immunogens, antibodies, compositions etc. described herein may be administered by parenteral administration and the pharmaceutical compositions formulated accordingly. In some embodiments, administration is by means of a pump for periodic or continuous delivery.
  • Parenteral administration includes systemic delivery routes other than the gastrointestinal (GI) tract, and includes, for example intravenous, intra-arterial, intraperitoneal, subcutaneous, and intramuscular modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.
  • GI gastrointestinal
  • the immunogen, antibodies, compositions etc. described herein are administered parenterally. For example, solutions of one or more immunogens, antibodies, compositions etc.
  • dispersions are prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.
  • dispersions are prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • suitable formulations For parenteral administration, sterile solutions of the immunogens, antibodies, compositions, etc. described herein are usually prepared, and the pH of the solutions are suitably adjusted and buffered. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.
  • ointments or droppable liquids are delivered, for example, by ocular delivery systems known to the art such as applicators or eye droppers.
  • such compositions include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or polyvinyl alcohol, preservatives such as sorbic acid, EDTA or benzyl chromium chloride, and the usual quantities of diluents or carriers.
  • diluents or carriers will be selected to be appropriate to allow the formation of an aerosol.
  • Formulations for injection are, for example, presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions take such forms as sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulating agents such as suspending, stabilizing and/or dispersing agents. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. Alternatively, immunogens, antibodies, compositions, etc.
  • immunogens, antibodies, compositions, etc. described herein are suitably in a sterile powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the immunogens, antibodies, compositions, etc. described herein, including pharmaceutically acceptable salts and/or solvates thereof, are suitably used on their own but will generally be administered in the form of a pharmaceutical composition in which the one or more immunogens, antibodies, compositions, etc. described herein (the active ingredient) is in association with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition will comprise from about 0.05 wt% to about 99 wt% or about 0.10 wt% to about 70 wt%, of the active ingredient, and from about 1 wt% to about 99.95 wt% or about 30 wt% to about 99.90 wt% of a pharmaceutically acceptable carrier, all percentages by weight being based on the total composition.
  • a further aspect relates to a kit comprising i) an isoDGR peptide or immunogen described herein, ii) an antibody and/or binding fragment thereof described herein, iii) a nucleic acid encoding said antibody or a part thereof described herein, iv) composition comprising an immunogen, antibody, composition etc.
  • the kit is for diagnosing or monitoring an isoDGR- associated disease or disorder.
  • the kit further comprises one or more of a collection vial, standard buffer, detection antibodies (for example for capturing or detecting one or more of ApoB100, ApoD, C6, CFB, CFH, F9, F13, FGB, FGG, PLG, and FN1), and/or one or more detection reagents for detecting patient autoantibodies.
  • the kit further comprises one or more pre-coated microwell strips, one or more buffers, including, for example, a standard wash buffer (e.g. PBS + detergent + blocking agent) and specimen dilutant (e.g. PBS + blocking agent, optionally including a visual dye), positive and negative controls, detection reagents (such as substrate and substate buffer (e.g. p ⁇ nitrophenylphosphate and DEA buffer), and/or plastic plate sealers.
  • a standard wash buffer e.g. PBS + detergent + blocking agent
  • specimen dilutant e.g. PBS + blocking agent, optionally including a visual dye
  • detection reagents such as substrate and substate buffer (e.g. p ⁇ nitrophenylphosphate and DEA buffer)
  • plastic plate sealers e.g. a standard wash buffer
  • detection reagents such as substrate and substate buffer (e.g. p ⁇ nitrophenylphosphate and DEA buffer)
  • plastic plate sealers e.g.
  • a number of age-related and/or inflammatory diseases are associated with increased levels of isoDGR-modified proteins.
  • a number of isoDGR-modified proteins can be detected in patient plasma using the anti-isoDGR antibodies described herein, and increased levels of specific combinations of one or more isoDGR-modified proteins are associated with coronary artery disease, stroke, and/or vascular dementia.
  • anti-isoDGR antibodies such as the antibodies and fragments thereof described herein, can be used in diagnostic assays to identify patients with increased levels of one or more isoDGR-modified proteins and/or who have, or are at increased risk of developing, an isoDGR-associated disease.
  • an isoDGR-associated disease optionally coronary artery disease, stroke, and/or vascular dementia. Also shown in the Examples, healthy patients have higher levels of isoDGR-autoantibody in blood plasma compared to cardiovascular disease patients. Accordingly, the isoDGR antigens described herein can be used to identify patients with increased levels of isoDGR-autoantibodies and/or who have a decreased risk (or do not have an increased risk) of developing an isoDGR-associated disease such as cardiovascular disease. Provided herein are methods for identifying patients with increased levels of isoDGR-autoantibodies.
  • isoDGR-associated disease encompasses diseases associated with increased levels of isoDGR protein modifications. These include, for example and without limitation, age-related and/or inflammatory diseases such as cardiovascular diseases (e.g. endothelial dysfunction, atherosclerosis, coronary heart disease (CHD), coronary artery disease (CAD), heart failure, myocardial ischemia, myocardial infarction, hypertrophic cardiomyopathy, left ventricular hypertrophy), cerebrovascular diseases (e.g.
  • cardiovascular diseases e.g. endothelial dysfunction, atherosclerosis, coronary heart disease (CHD), coronary artery disease (CAD), heart failure, myocardial ischemia, myocardial infarction, hypertrophic cardiomyopathy, left ventricular hypertrophy
  • cerebrovascular diseases e.g.
  • TIA transient ischemic attack
  • carotid artery disease neurovascular inflammation
  • blood brain barrier dysfunctions vascular cognitive impairment
  • dementia dementia including vascular dementia and Alzheimer’s disease
  • pulmonary disease e.g. chronic lung inflammation, parenchymal lung disease, emphysema, chronic obstructive pulmonary disease (COPD), asthma, lung fibrosis
  • liver disease e.g. chronic liver inflammation, fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH)
  • clotting disorders e.g. thrombosis
  • primary and/or metastatic cancers e.g.
  • the isoDGR-associated disease is a cardiovascular disease, cerebrovascular disease, pulmonary disease, liver disease, clotting disorder, cancer, or inflammatory disease.
  • the isoDGR-associated disease is chronic inflammation, chronic pulmonary disease, non-alcoholic fatty liver disease, atherosclerotic cardiovascular disease, cerebrovascular disease and vascular dementia.
  • the isoDGR-associated disease is a cardiovascular disease, stroke, and/or vascular dementia.
  • the isoDGR-associated disease is coronary artery disease, stroke, and/or vascular dementia.
  • the isoDGR-associated disease is atherosclerotic cardiovascular disease, fatty liver disease, chronic lung inflammation/parenchymal lung disease.
  • the isoDGR-associated disease is atherosclerotic cardiovascular disease, liver disease, pulmonary disease, clotting disorders, and/or cognitive impairment.
  • Various aspects of the methods described comprise methods of detecting whether a sample comprises isoDGR modified proteins and/or anti-isoDGR autoantibodies.
  • the method comprises providing a biological sample from a patient and detecting the presence of one or more isoDGR-modified proteins and/or anti-isoDGR autoantibodies in the sample.
  • samples obtained from healthy patients and patients with diseases including coronary heart disease, stroke, or vascular dementia are found to contain a number of plasma proteins which are found to be isoDGR- modified to varying degrees and in varying combinations.
  • an aspect includes a method for identifying patients who have, or who are at increased risk of developing, an isoDGR-associated disease, the method comprising: determining the level of one or more isoDGR-modified proteins in a biological sample from a patient; and comparing the level of the one or more isoDGR-modified proteins to a control or reference value, wherein an increased level of one or more isoDGR-modified proteins relative to the control or reference value is indicative that the patient has, or is at increased risk of developing, an isoDGR-associated disease.
  • the method comprises determining the levels of one or more isoDGR-modified proteins selected from: apolipoprotein B100 (ApoB100), apolipoprotein (ApoD), complement component C6 (C6), complement factor B (CFB), complement factor H (CFH), coagulation factor IX (F9), coagulation factor XIII (F13), fibrinogen beta chain (FGB), fibrinogen gamma chain (FGG), plasminogen (PLG) and/or fibronectin (FN1).
  • apolipoprotein B100 ApoB100
  • ApoD complement component C6
  • C6 complement factor B
  • CH complement factor H
  • F9 coagulation factor IX
  • F9 coagulation factor IXIII
  • FGB fibrinogen beta chain
  • FGG fibrinogen gamma chain
  • PLG plasminogen
  • FN1 fibronectin
  • the level of one or more isoDGR-modified proteins in a biological sample is determined using a the method comprising: providing a biological sample suspected of containing isoDGR-modified proteins from a patient; contacting the sample with an anti-isoDGR antibody, optionally an antibody or fragment thereof described herein, under conditions permissive for forming isoDGR:antibody complexes; detecting the presence of any isoDGR:antibody complexes; and determining the level of one or more isoDGR-modified proteins based on the detected isoDGR:antibody complexes.
  • the step of providing a biological sample from a patient may or may not include the step of obtaining the sample from the patient.
  • the method comprises providing a biological sample suspected of containing isoDGR-modified proteins that has been obtained from the patient.
  • stroke is associated with increased levels of isoDGR-modified FGB and FGG, but no significant increase in ApoB100 relative to healthy controls.
  • the isoDGR-associated disease is stroke, and the patient is identified as having, or being at increased risk of developing, stroke, if the levels of isoDGR-modified FGB and/or FGG but not ApoB100 are increased relative to the control or reference value.
  • the patient is identified as having, or being at increased risk of developing, stroke if the levels of isoDGR-modified FGB and/or FGG but not ApoB100 are increased, optionally at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold, relative to a healthy control or reference value.
  • the patient is identified as having, or being at increased risk of developing, stroke if the levels of isoDGR-modified FGB and/or FGG are higher, optionally at least 3-fold, at least 4-fold, at least 5-fold, or at least 6-fold, relative to the levels of isoDGR-modified ApoB100 in the biological sample from the patient.
  • coronary artery disease is associated with increased levels of isoDGR-modified ApoB100, as well as increased isoDGR- modified FGB and FGG.
  • the isoDGR-associated disease is coronary artery disease, and the patient is identified as having, or being at risk of developing, coronary artery disease if the levels of isoDGR-modified ApoB100 (and optionally FGB and/or FGG) are increased relative to the control or reference value.
  • the patient is identified as having, or being at risk of developing, coronary artery disease if the levels of isoDGR-modified ApoB100 (and optionally FGB and/or FGG) are increased, optionally at least 2-fold, at least 3-fold, or at least 4-fold, relative to a healthy control or reference value.
  • the patient is identified as having, or being at risk of developing, coronary artery disease if the levels of isoDGR-modified ApoB100 are higher, optionally at least 2-fold or at least 3-fold, relative to the levels of isoDGR-modified ApoD, C6, CFB, CFH, PLG, and/or FN1 in the biological sample from the patient.
  • vascular dementia is associated with increased levels of isoDGR modified C6, CFB, and CFH, as well as increased isoDGR- modified FGB and FGG.
  • the isoDGR-associated disease is vascular dementia, and the patient is identified as having, or being at risk of developing, vascular dementia if the levels of isoDGR-modified C6, CFB, and/or CFH (and optionally FGB and/or FGG) are increased relative to the control or reference value.
  • the patient is identified as having, or being at risk of developing, vascular dementia if the levels of isoDGR-modified C6, CFB, and/or CFH (and optionally FGB and/or FGG) are increased, optionally at least 2-fold, at least 3-fold, or at least 4-fold, relative to a healthy control or reference value.
  • the patient is identified as having, or being at risk of developing, vascular dementia if the levels of isoDGR-modified C6, CFB, and/or CFH are higher, optionally at least 2-fold or at least 3-fold, relative to the levels of isoDGR-modified ApoB100, ApoD, F13B, PLG, and/or FN1 in the biological sample from the patient.
  • Another aspect provides a method of detecting whether a sample comprises anti-isoDGR autoantibodies.
  • the method comprises: providing a biological sample suspected of containing anti-isoDGR autoantibodies from a patient; contacting the sample with an isoDGR epitope or peptide under conditions permissive for forming isoDGR:autoantibody complexes; and detecting the presence of any isoDGR:autoantibody complexes, wherein the presence of detectable complex is indicative that the sample contains anti-isoDGR autoantibodies.
  • the method further comprises determining the level of anti-isoDGR autoantibodies based on the detected isoDGR:autoantibody complexes, and optionally comparing the level of anti-isoDGR autoantibodies to a control.
  • cardiovascular disease is correlated with a decreased level of anti-isoDGR autoantibodies compared with healthy controls.
  • an aspect provides a method for identifying patients who have, or who are at increased risk of developing, an isoDGR-associated disease, for example a cardiovascular disease, the method comprising: determining the level of anti-isoDGR autoantibodies in a biological sample from a patient; and comparing the level of the anti-isoDGR autoantibodies to a control or reference value, wherein the patient is identified as having or being at increased risk of developing an isoDGR-associated disease depending on the level of autoantibodies relative to the control or reference value.
  • the step of providing a biological sample suspected of containing anti-isoDGR autoantibodies from a patient may or may not include the step of obtaining the sample from the patient.
  • the method comprises providing a biological sample suspected of containing anti-isoDGR autoantibodies that has been obtained from the patient.
  • sample refers to any material in which the presence or amount of one or more components therein is unknown and can be determined in an assay.
  • the sample may be for example a biological sample obtained from a human or non-human animal, including clinical samples and swabs.
  • the sample may comprise cellular and non-cellular material, including, but not limited to, tissue samples, saliva, sputum, urine, blood, serum, other bodily fluids and/or secretions.
  • Suitable biological samples include, without limitation, a tissue sample, which can be for example a solid tissue biopsy such as a brain biopsy, a lung biopsy, a liver biopsy, a kidney biopsy, a bone marrow biopsy, or a bone biopsy, or a liquid biopsy such as a blood sample, serum sample, plasma sample, or cerebrospinal fluid (CSF).
  • a tissue sample which can be for example a solid tissue biopsy such as a brain biopsy, a lung biopsy, a liver biopsy, a kidney biopsy, a bone marrow biopsy, or a bone biopsy, or a liquid biopsy such as a blood sample, serum sample, plasma sample, or cerebrospinal fluid (CSF).
  • a tissue sample which can be for example a solid tissue biopsy such as a brain biopsy, a lung biopsy, a liver biopsy, a kidney biopsy, a bone marrow biopsy, or a bone biopsy
  • a liquid biopsy such as a blood sample, serum sample, plasma sample, or cerebrospinal fluid (CSF).
  • Biological samples may also include, without limitation, saliva, stool, urine, semen, sputum, mucous, lymph, synovial fluid, ascites, pleural effusion, seroma, nasal swab, mid-turbinate swab, nasopharyngeal swab, nasal sponge, nasal wash, oral swab, oral wash or gargle, buccal swab, throat swab, oropharyngeal swab, skin swab, vaginal swab, meatal swab, urethral swab, rectal swab, or skin scraping.
  • the biological sample is a blood sample, serum sample, or plasma sample.
  • tissue samples include tissue biopsy, fine needle aspiration cytology, fluid cytology, needle biopsy, CT-guided biopsy, ultrasound-guided biopsy, or aspiration biopsy.
  • the sample is obtained from a human patient.
  • the patient is suspected of being at risk of developing an isoDGR- associated disease.
  • the step of obtaining the sample may or may not form part of the method.
  • Various methods and assays may be used to detect the presence of any isoDGR:antibody and/or isoDGR:autoantibody complexes and/or to determine (or measure) the levels of one or more isoDGR-modified proteins and/or anti-isoDGR autoantibodies.
  • the assay is a quantitative assay.
  • the assay is a qualitative assay.
  • the method comprises comparing the level of one or more isoDGR-modified proteins and/or anti- isoDGR autoantibodies to one or more controls or reference values.
  • Controls may include, for example and without limitation, an expression level of the modified protein in a healthy patient or a reference expression level obtained or determined from samples of a group of healthy patients, which can be used to create a "control value".
  • a control value may be obtained from the historical expression data from a pool of healthy patients (e.g. a negative control value) or from a pool of patients with the disease (e.g. a positive control value).
  • Controls may also include an internal control, for example the level of isoDGR-modified protein relative to total protein, or the relative level of isoDGR modifications between two different proteins from the patient sample (e.g. the level of isoDGR-modified protein X relative to the level of isoDGR-modified protein Y).
  • a reference value may include for example a threshold value, above or below which (depending on the specific isoDGR-modified protein and reference value) may indicate the patient has an isoDGR-associated disease or increased risk thereof, or may indicate the patient does not have an isoDGR- associated disease, or has a decreased risk thereof.
  • the method includes an immunoassay, such as an enzyme-linked immunosorbent assay (ELISA), optionally a sandwich ELISA or an indirect ELISA, flow cytometry, a dot blot, a Western blot, or immunoprecipitation followed by a SDS-PAGE immunocytochemistry.
  • ELISA enzyme-linked immunosorbent assay
  • Other immune-based methods may be used, including singleplex and multiplex immunoassay platforms (e.g.
  • Bead- or particle-based immunoassays can be used in which the capture antibody is immobilized on a particle such as a fluorescently dyed bead or paramagnetic bead, and analyte is detected with a second detection antibody.
  • Suitable bead-based multiplex platforms include for example Luminex® from AbCam, FirePlexTM from AbCam, and those available from Quanterix.
  • Bead- or particle-based assays utilizing a fluorescently labeled detection antibody may be followed by single-molecule counting in which the detection antibodies are eluted from the immunocomplex and quantified by detecting and counting individual fluorescent molecules using capillary fluidics and a laser.
  • Suitable technologies include those available from for example Singulex.
  • Planar array platforms can be used in which the capture antibody is immobilized in a micro-arrayed format on a solid surface such as a membrane, a glass surface, or an individual well of for example a 96- or 384-well plate, and analyte is detected with a second detection antibody.
  • spot coordinates can be used to identify detected analyte.
  • Suitable planar array platforms include those available from for example Quaternix or Mesoscale.
  • the administration of anti-isoDGR antibodies is shown to decrease the levels of isoDGR- modified proteins in mice.
  • methods for reducing the level of isoDGR-modified proteins for treating or preventing a disease in a patient in need thereof comprising administering an effective amount of an anti- isoDGR antibody, optionally an antibody described herein, to the patient.
  • an anti-isoDGR antibody optionally an antibody described herein, for reducing the level of isoDGR-modified proteins in a patient in need thereof.
  • a further aspect includes a use of an anti-isoDGR antibody, optionally an antibody described herein, in the manufacture of a medicament for reducing the level of isoDGR-modified proteins in a patient in need thereof.
  • Another aspect includes an anti-isoDGR antibody, optionally an antibody described herein, for use in reducing the level of isoDGR-modified proteins in a patient in need thereof.
  • the antibody comprises an Fc domain and/or interacts with or forms a complex with Fc receptors on patient immune cells.
  • the antibody is a neutralizing antibody.
  • kits for inducing immune clearance of isoDGR-modified proteins for treating or preventing a disease in a patient in need thereof comprising administering an effective amount of an anti-isoDGR antibody, optionally an antibody described herein, to the patient.
  • an anti-isoDGR antibody optionally an antibody described herein, for inducing immune clearance of isoDGR-modified proteins in a patient in need thereof.
  • a further aspect includes a use of an anti-isoDGR antibody, optionally an antibody described herein, in the manufacture of a medicament for inducing immune clearance of isoDGR-modified proteins in a patient in need thereof.
  • Another aspect includes an anti-isoDGR antibody, optionally an antibody described herein, for use in inducing immune clearance of isoDGR-modified proteins in a patient in need thereof.
  • the antibody comprises an Fc domain and/or interacts with or forms a complex with Fc receptors on patient immune cells.
  • the administration of anti-isoDGR antibodies is shown to decrease the levels of isoDGR-modified proteins in mice, and prevent the development, or diminish the extent, of tissue damage and chronic inflammation associated with the accumulation of isoDGR-modified proteins in PCMT1-/- mice.
  • anti-isoDGR antibodies are shown to prevent the development, or diminish the extent, of signs of atherosclerotic cardiovascular disease, liver disease, pulmonary disease, clotting disorders, and cognitive impairment, which are associated with the accumulation of isoDGR-modified proteins in PCMT1-/- mice.
  • the administration of anti-isoDGR antibodies is also shown to reduce or prevent atherosclerosis in western diet (WD)-fed wildtype and apoE-/- mice.
  • an anti-isoDGR antibody for treating or preventing an isoDGR-associated disease in a patient in need thereof.
  • a further aspect includes a use of an anti-isoDGR antibody, optionally an antibody described herein, in the manufacture of a medicament for treating or preventing an isoDGR-associated disease in a patient in need thereof.
  • Another aspect includes an anti-isoDGR antibody, optionally an antibody described herein, for use in treating or preventing an isoDGR- associated disease in a patient in need thereof.
  • the antibody comprises an Fc domain and/or interacts with or forms a complex with Fc receptors on patient immune cells.
  • the antibody is a neutralizing antibody.
  • the isoDGR-associated disease is cardiovascular disease.
  • the cardiovascular disease is endothelial dysfunction, atherosclerosis, coronary heart disease (CHD), coronary artery disease (CAD), heart failure, myocardial ischemia, myocardial infarction, hypertrophic cardiomyopathy, or left ventricular hypertrophy.
  • the isoDGR-associated disease is cerebrovascular disease.
  • the cerebrovascular disease is stroke, transient ischemic attack (TIA), carotid artery disease, neurovascular inflammation, blood brain barrier dysfunctions, vascular cognitive impairment, or dementia including vascular dementia and Alzheimer’s disease.
  • TIA transient ischemic attack
  • carotid artery disease neurovascular inflammation
  • blood brain barrier dysfunctions blood brain barrier dysfunctions
  • vascular cognitive impairment or dementia including vascular dementia and Alzheimer’s disease.
  • the isoDGR-associated disease is liver disease.
  • the liver disease is chronic liver inflammation, fatty liver disease, non-alcoholic fatty liver disease, or non-alcoholic steatohepatitis (NASH).
  • NASH non-alcoholic steatohepatitis
  • the isoDGR-associated disease is pulmonary disease.
  • the pulmonary disease is chronic lung inflammation, parenchymal lung disease, emphysema, chronic obstructive pulmonary disease (COPD), asthma, or lung fibrosis.
  • the isoDGR-associated disease is a clotting disorder.
  • the clotting disorder is thrombosis.
  • the isoDGR-associated disease is primary and/or metastatic cancer.
  • the primary and/or metastatic cancer is lung cancer, liver cancer, colon cancer, breast cancer, skin cancer, prostate cancer, ovarian cancer, kidney cancer, or pancreatic cancer.
  • the isoDGR-associated disease is an inflammatory disease.
  • the inflammatory disease is chronic inflammation and inflammaging, vascular inflammation including neurovascular inflammation, or chronic liver inflammation.
  • the administration of anti-isoDGR antibodies is shown to reduce isoDGR-associated chronic inflammation in mice.
  • methods for reducing isoDGR-associated chronic inflammation in a patient in need thereof comprising administering an effective amount of an anti-isoDGR antibody, optionally an antibody described herein, to the patient.
  • an anti-isoDGR antibody for reducing isoDGR-associated chronic inflammation in a patient in need thereof.
  • a further aspect includes a use of an anti- isoDGR antibody, optionally an antibody described herein, in the manufacture of a medicament for reducing isoDGR-associated chronic inflammation in a patient in need thereof.
  • Another aspect includes an anti-isoDGR antibody, optionally an antibody described herein, for use in reducing isoDGR-associated chronic inflammation in a patient in need thereof.
  • the antibody comprises an Fc domain and/or interacts with or forms a complex with Fc receptors on patient immune cells.
  • the antibody is a neutralizing antibody.
  • isoDGR epitope can be used as an immunogen or vaccine to induce an adaptive immune response in a patient, leading to the production of anti-isoDGR autoantibodies, thereby inducing immune clearance of isoDGR-modified proteins, reducing isoDGR-modified protein levels in the patient, and/or treating or preventing isoDGR-associated diseases.
  • a further aspect includes a method of inducing the production of anti-isoDGR autoantibodies in a patient, and/or inducing immune clearance of isoDGR-modified proteins in the patient, and/or reducing isoDGR-modified protein levels in the patient, and/or treating or preventing isoDGR-associated diseases, the method comprising administering to the patient an effective amount of an isoDGR immunogen or composition comprising an isoDGR immunogen as described herein.
  • an isoDGR immunogen or composition comprising an isoDGR immunogen described herein, for inducing the production of anti-isoDGR autoantibodies in a patient, and/or inducing immune clearance of isoDGR-modified proteins in the patient, and/or reducing isoDGR-modified protein levels in the patient, and/or treating or preventing isoDGR-associated diseases.
  • a further aspect includes a use of an isoDGR immunogen or composition comprising an isoDGR immunogen described herein, in the manufacture of a medicament for inducing the production of anti-isoDGR autoantibodies in a patient, and/or inducing immune clearance of isoDGR-modified proteins in the patient, and/or reducing isoDGR-modified protein levels in the patient, and/or treating or preventing isoDGR-associated diseases.
  • Another aspect includes an isoDGR immunogen or composition comprising an isoDGR immunogen described herein, for use in inducing the production of anti- isoDGR autoantibodies in a patient, and/or inducing immune clearance of isoDGR- modified proteins in the patient, and/or reducing isoDGR-modified protein levels in the patient, and/or treating or preventing isoDGR-associated diseases.
  • the isoDGR-associated disease is cardiovascular disease, optionally endothelial dysfunction, atherosclerosis, coronary heart disease (CHD), coronary artery disease (CAD), heart failure, myocardial ischemia, myocardial infarction, hypertrophic cardiomyopathy, or left ventricular hypertrophy.
  • the isoDGR- associated disease is a cerebrovascular disease, optionally stroke, transient ischemic attack (TIA), carotid artery disease, neurovascular inflammation, blood brain barrier dysfunctions, vascular cognitive impairment, or dementia including vascular dementia and Alzheimer’s disease.
  • the isoDGR- associated disease is liver disease, optionally chronic liver inflammation, fatty liver disease, non-alcoholic fatty liver disease, or non-alcoholic steatohepatitis (NASH).
  • the isoDGR-associated disease is pulmonary disease, optionally chronic lung inflammation, parenchymal lung disease, emphysema, chronic obstructive pulmonary disease (COPD), asthma, or lung fibrosis.
  • the isoDGR-associated disease is a clotting disorder, optionally thrombosis.
  • the isoDGR- associated disease is primary and/or metastatic cancer, optionally lung cancer, liver cancer, colon cancer, breast cancer, skin cancer, prostate cancer, ovarian cancer, kidney cancer, or pancreatic cancer.
  • the isoDGR-associated disease is an inflammatory disease, optionally chronic inflammation and inflammaging, vascular inflammation including neurovascular inflammation, or chronic liver inflammation.
  • patient in need thereof refers to a patient that could benefit from the method(s) or treatment(s) described herein, and optionally refers to a patient identified as having increased levels of one or more isoDGR-modified proteins and/or having an increased risk of developing an isoDGR-associated disease.
  • treating or “treatment” as used herein and as is well understood in the art means an approach for obtaining beneficial or desired results, including clinical results.
  • Beneficial or desired clinical results include, but are not limited to alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable.
  • Treating” and “treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Treatment methods comprise administering to a patient a therapeutically effective amount of one or more antibodies described herein and optionally consist of a single administration, or alternatively comprise a series of administrations.
  • “Palliating” a disease, disorder or condition means that the extent and/or undesirable clinical manifestations of a disease, disorder or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the disorder.
  • prevention or “prophylaxis”, or synonym thereto, as used herein refers to a reduction in the risk or probability of a patient becoming afflicted with a disease, disorder or condition or manifesting a symptom associated with a disease, disorder or condition, for example an isoDGR-associated disease.
  • a patient identified as being at risk of developing an isoDGR-associated disease can be treated with one or more immunogens or antibodies described herein to prevent an isoDGR-associated disease from developing, or to reduce the severity or extent of an isoDGR-associated disease compared to expected severity or extent if not receiving preventative or prophylactic treatment.
  • Prevention methods comprise administering to a patient a therapeutically effective amount of one or more immunogens or antibodies described herein and optionally consist of a single administration, or alternatively comprise a series of administrations.
  • the term “disease, disorder or condition” as used herein refers to a disease, disorder or condition associated with increased levels of isoDGR-modified proteins (i.e. an isoDGR-associated disease).
  • the term “administered” or “administering” as used herein means administration of a therapeutically effective amount of a compound or composition of the disclosure to a patient.
  • the immunogens or antibodies described herein may be administered using a variety of routes of administration.
  • the immunogens or antibodies described herein may be administered by parenteral administration.
  • the immunogens or antibodies described herein may be administered by intravenous, intra-arterial, intraperitoneal, subcutaneous, or intramuscular administration.
  • the administration may be by continuous infusion over a selected period of time
  • coadministration or “combination therapy” shall mean that at least two compounds or compositions are administered to the patient at the same time, such that effective amounts or concentrations of each of the two or more compounds may be found in the patient at a given point in time.
  • compounds according to the present disclosure may be co-administered to a patient at the same time, the term embraces both administration of two or more agents at the same time or at different times, provided that effective concentrations of all coadministered compounds or compositions are found in the patient at a given time.
  • an effective amount means an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • an effective amount is an amount that, for example, prevents the occurrence of an isoDGR-associated disease, or reduces the severity or extent of the isoDGR-associated disease compared to the response obtained without administration of the compound. Effective amounts may vary according to factors such as the disease state, age, sex and weight of the animal.
  • Suitable administration schedules may include, without limitation, at least once a week, from about one time per two weeks, three weeks or one month, about one time per week to about once daily, 2, 3, 4, 5 or 6 times daily.
  • the length of the treatment period may depend on a variety of factors, such as the severity of the disease, disorder or condition, the age of the patient, the concentration and/or the activity of the immunogen, antibody, composition, etc. described herein and/or a combination thereof.
  • the effective dosage of the immunogen, antibody, composition, etc. described herein used for the treatment may increase or decrease over the course of a particular treatment regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration is required.
  • the immunogen, antibody, composition, etc. described herein is administered to the patient in an amount and for duration sufficient to treat the patient.
  • the immunogen, antibody, composition, etc. described herein may be either used alone or in combination with other known agents useful for treating diseases, disorders or conditions. When used in combination with other agents useful in treating such diseases, disorders or conditions, the immunogen, antibody, composition, etc. described herein may be administered contemporaneously with those agents.
  • “contemporaneous administration” of two substances to a patient means providing each of the two substances so that they are both active in the individual at the same time.
  • the exact details of the administration will depend on the pharmacokinetics of the two substances in the presence of each other, and can include administering the two substances within a few hours of each other, or even administering one substance within 24 hours of administration of the other, if the pharmacokinetics are suitable. Design of suitable dosing regimens is routine for one skilled in the art.
  • two substances will be administered substantially simultaneously, i.e., within minutes of each other, or in a single composition that contains both substances.
  • the combination of agents is administered to a patient in a non-contemporaneous fashion.
  • the immunogen, antibody, composition, etc. described herein is administered with another therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. Accordingly, the present description provides a single unit dosage form comprising the immunogen, antibody, composition, etc. described herein, an additional therapeutic agent, and a pharmaceutically acceptable carrier.
  • the immunogens described herein are suitably administered in combination with an adjuvant. [00183] The dosage of the immunogen, antibody, composition, etc.
  • Example 1 The following non-limiting examples are illustrative of the present disclosure: Example 1.
  • mice deficient in deamidation repair enzyme Pcmt1 were previously generated by Clarke and co-worker 38 .
  • Pcmt1 +/ ⁇ C57BL/6 background mice were obtained from the Jackson Laboratory.
  • Pcmt1 +/ ⁇ male and female mice were bred, yielding litters comprising Pcmt1 +/+ , Pcmt1 +/ ⁇ , and Pcmt1 ⁇ / ⁇ offspring in the expected Mendelian ratios.
  • mice were maintained on normal chow diets and housed with regular light/dark cycles.
  • isoDGR immunogens and immunization of mice Synthetic isoDGR peptides (Ac-GC(isoD)GRCGK; SEQ ID NO: 25); GC(isoD)GRCGG-(CH2-CH2-NH2) (SEQ ID NO: 12); GC(isoD)GRCGK (SEQ ID NO: 38); Ac-C(isoD)GRCGGK (SEQ ID NO: 39); or Ac-GC(isoD)GRCGGK (SEQ ID NO: 40) were conjugated to carrier proteins (Keyhole Limpet Hemocyanin (KLH) and/or Bovine Serum Albumin (BSA)), using glutaraldehyde.
  • KLH Keyhole Limpet Hemocyanin
  • BSA Bovine Serum Albumin
  • Anti-isoDGR immunotherapy Pcmt1 -/- pups were i.p. injected with 1mg/kg/week isoDGR-specific mAb 14G7 or 6E11 6 starting from 1-week-old until use in the study. Age matched control animals were injected with an equal volume of 1X PBS only. All mice were fed normal chow diet from weaning onwards, body weight was measured weekly, and all animals were included in the survival analyses.
  • Hind-limb clasping test Mice were suspended by their tails and the extent of hindlimb clasping was observed for 30 s. If both hind-limbs were splayed outward away from the abdomen with spread toes, a score of 0 was given. If one hind- limb was fully retracted or both hind-limbs partially retracted without touching the abdomen and with toes spread, a score of 1 was assigned. If both hind-limbs were partially retracted and in contact with the abdomen but without touching each other, a score of 2 was given.
  • Ledge test The ledge test for evaluating balance and coordination was carried out as previously described, with a minor modification. Mice were placed on a ledge (90 cm long, 0.5 cm wide, 20 cm high), and paw placement and forward movement were observed. Time taken to cross the ledge and number of paw slips were also recorded. Mice that left the ledge were excluded.
  • IsoDGR-antigen uptake / ADCP assays ADCP assays were performed by fluorescence microscopy and FACS.
  • a total of 5 x 10 5 murine RAW macrophages were seeded into 24-well plates and treated with 5 ⁇ g/ml FITC-labelled isoDGR- fibronectin (isoDGR-FN-FITC), FITC-labelled isoDGR fibrinogen (isoDGR-FG-FITC), or native FN-FITC / FG-FITC control for 24h. 28 After 45 min incubation with varying doses of isoDGR-mAb (0 to 5 ⁇ g/ml), excess fluorescence was quenched by adding trypan blue and incubating for 10min. The cells were then washed 3 times and re- suspended in PBS.
  • the mAb dose-dependent uptake of isoDGR-antigen by RAW cells was determined by FACS using FlowJo software to quantify phagocytic cells. To confirm the FACS results, cells were also examined by fluorescent imaging using a Zeiss LSM710 confocal microscope. The fluorescence intensity of each cell was calculated using Image J software. [00191] Histological assessment and immunostaining of aorta and liver tissues: Tissues from WT, Pcmt1 +/- , Pcmt1 -/- and mAb-treated Pcmt1 -/- mice were collected and fixed with 4% PFA at 4 °C for 24 h.
  • Tissue was embedded in OCT compound with dry ice and cut into 10 ⁇ m sections using A Leica CM3060S Cryostat. The sections were mounted onto Fisherbrand Superfrost plus microscope slides and kept in warm PBS for 20min to remove OCT prior to staining with haematoxylin and eosin.
  • slides were permeabilized with 0.5% PBST for 2-3h and then incubated with blocking buffer (2.5% normal goat serum, 1% BSA in 0.5% PBST) for 1h at RT prior to addition of primary antibodies (isoDGR [1:200] and CD68 [1:200, Abcam, ab283654]) overnight at 4 °C. Slides were next washed with PBS (3X) for 5 min then incubated with secondary antibodies conjugated to AlexaFluor 488 and 594 (1:500) for 1h at RT. The slides were again washed with PBS (3X) for 5 min, then incubated with DAPI for 15 min to visualize cell nuclei.
  • blocking buffer 2.5% normal goat serum, 1% BSA in 0.5% PBST
  • primary antibodies isoDGR [1:200] and CD68 [1:200, Abcam, ab283654]
  • Cytokine multiplex bead assay The LEGENDplex TM mouse inflammation panel (Biolegend, San Diego, CA) was used to measure 13 different cytokines (IL-23, IL-1 ⁇ , IL-1 ⁇ , IL-6, IL-10, IL-12p70, IL-17A, IL-23, IL-27, MCP-1, IFN- ⁇ , IFN- ⁇ , TNF- ⁇ , and GM-CSF) in blood plasma from WT, Pcmt1 +/- , Pcmt1 -/- and mAb- treated Pcmt1 -/- animals (assessed by LSRII flow cytometer according to the manufacturer’s protocol).
  • cytokines IL-23, IL-1 ⁇ , IL-1 ⁇ , IL-6, IL-10, IL-12p70, IL-17A, IL-23, IL-27, MCP-1, IFN- ⁇ , IFN- ⁇ , TNF- ⁇ , and GM-CSF
  • RNA isolated from tissues was treated with Dnase and reverse-transcribed using a first-strand DNA synthesis kit from Invitrogen. The PCR was performed on an ABI Fast 7500 System (Applied Biosystems, Foster City, CA). TaqMan probes for the respective genes were custom-generated by Applied Biosystems based on the sequences in the Illumina array and used as per the manufacturer’s instructions. Expression levels of target genes were determined in triplicate from the standard curve and normalized to GAPDH mRNA level. [00194] Western blot analysis: Western blot analysis was performed using standard methods.
  • Anti-isoDGR immunotherapy doubles the lifespan of prematurely aging Pcmt1 -/- mice
  • Global deletion of repair enzyme Pcmt1 leads to tissue accumulation of isoaspartate residues and premature death of Pcmt1 -/- mice 21,24 , but the mechanistic basis of this pathology is not fully understood. It was previously shown that isoDGR- modified fibronectin is a critical mediator of vascular inflammation in Pcmt1 +/- mice via interaction with resident macrophages in an atherosclerotic CVD model.
  • isoDGR-modified proteins were previously shown to co-localize with CD68+ monocyte-macrophage cells in both intima and adventitial layers of the aorta, with isoDGR engagement of macrophage integrin receptors playing a key role in triggering inflammatory cytokine release and progression of atherosclerotic lesions. 28 As shown herein, and in order to fully elucidate the role of isoDGR motifs in age-linked disease, global Pcmt1 -/- mice were generated by crossing Pcmt1 +/- parents (25% of pups born were Pcmt1 -/- consistent with the expected Mendelian ratio).
  • Genotypes were confirmed by PCR of tail genomic DNA and western blot analysis of liver protein extracts (Fig.1A).
  • Pcmt1 -/- neonates were viable but displayed significantly reduced body weights and died prematurely compared to PCMT1 +/+ mice. It was hypothesized that isoDGR-induced tissue inflammation is partly responsible for the pathology observed in Pcmt1 -/- mice. It was therefore tested if anti-isoDGR immunotherapy using specific mAb could be used to reduce levels of damaged proteins in body tissues from Pcmt1 -/- pups.
  • Pcmt1 -/- mice exhibited severe hind-limb clasping and high dysfunction scores that were improved upon treatment with isoDGR-specific mAb 14G7 (Fig.1F).
  • Ledge tests also revealed impaired motor coordination in the Pcmt1 -/- mice, as evidenced by difficulty in paw placement and multiple limb slips during forward movement. Consequently, KO mice took longer to traverse the ledge than did PCMT1 +/+ and Pcmt1 +/- mice.
  • administration of isoDGR-specific mAb was sufficient to improve paw placement and reduced limb slip frequency during forward movement of Pcmt1 -/- mice (Fig.1G).
  • the levels of isoDGR-modified protein were therefore assessed by western blot analysis of whole brain and liver lysates from 6-week-old Pcmt1-KO that had been treated or not with anti-isoDGR mAb.
  • This analysis indicated that Pcmt1-KO mice accumulated large quantities of isoDGR-damaged proteins in both liver and brain relative to PCMT1 +/+ and Pcmt1 +/- mice (Fig. 2A and 2B), but motif levels were significantly reduced by specific mAb treatment (Fig. 2C and 2D). While the blood-brain-barrier can limit antibody penetration into brain tissue, a substantial reduction of isoDGR-modified proteins was nonetheless observed in mice treated with motif-specific mAb (Fig.2C).
  • IsoDGR mAb reduces monocyte-macrophage infiltration and inflammation of Pcmt1-KO liver
  • IsoDGR mAb reduces monocyte-macrophage infiltration and inflammation of Pcmt1-KO liver
  • isoDGR-macrophage interactions triggered secretion of several pro-inflammatory cytokines / chemokines including CCL-2 and TNF ⁇ , thereby promoting vascular inflammation.
  • immunofluorescent imaging was used to detect isoDGR- modified proteins and CD68+ macrophage distribution in liver (Fig.3), spleen (Fig.4), and thymus tissue (Fig. 5).
  • qRT-PCR was performed using total RNA from liver of 5-week-old Pcmt1 +/- , Pcmt1 -/- , mAb-treated Pcmt1 -/- , and PCMT1 +/+ mice.
  • cytokine levels was assessed in blood plasma from Pcmt1 +/+ , Pcmt1 +/-, Pcmt1 -/- , and mAb-treated Pcmt1 -/- mice.
  • Multiplex bead array confirmed that elevated concentrations of CCL2 and IL-23 in plasma from Pcmt1 -/- mice could be reversed by anti-isoDGR immunotherapy.
  • Individual mice with more extensive isoDGR accumulation (Fig. 2, 3, 4 and 5) also displayed corresponding higher levels of circulating pro-inflammatory cytokines (Fig 6).
  • mice injected with deamidated plasma proteins also displayed increased levels of isoDGR-modified proteins in blood vessel walls that were co-localized with CD68+ monocyte-macrophage infiltration (Fig.7G).
  • synthetic isoDGR peptide Ac-GC(isoD)GRCGK; SEQ ID NO: 25
  • PBS control was injected into WT mice and blood plasma was collected for assessment of pro- inflammatory cytokine levels 24h later. Again, a significant elevation of plasma CCL2, TNF ⁇ , and IL-1 ⁇ concentrations was observed in mice injected with isoDGR-peptide (Fig.8).
  • ADCP antibody-dependent cellular phagocytosis
  • Effector functions mediated by Fc ⁇ Rs include antibody-dependent cellular toxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) which exert a critical influence on the therapeutic efficacy of antibody drugs. 49 It was hypothesised that accumulation of isoDGR-modified proteins in Pcmt1 -/- mice is a key mediator of tissue pathology and premature aging, consistent with the observation that weekly isoDGR-specific mAb therapy was sufficient to double average lifespan.
  • FITC positive phagocytic cells were then analysed and quantified by FACS. This analysis revealed a clear mAb dose-dependent increase in phagocytic activity that was not observed with native FN-FITC control (data not shown) or in the absence of mAb (Fig.11A), thus strongly indicating immune clearance of isoDGR-antigens by ADCP.
  • Accumulation of isoDGR-damaged fibrinogen (isoDGR-FBG) has been identified in both atherosclerotic tissues and plasma from CVD patients, 6,28 and a similar mAb dose-dependent clearance was observed when testing isoDGR-FBG antigen (Fig. 11B). These data indicate that the immune clearance observed was directed against isoDGR rather than being determined by protein identity.
  • WD standard chow or western diet
  • mice fed with chow diet and injected with PBS or mAb are normal and have similar body weight, indicating the mAb did not induce any adverse effect.
  • Oil Red O staining of lipid distribution revealed several en face atherosclerotic lesions in animals that received WD (Fig. 15A), but not in those that were additionally treated with isoDGR-specific mAb 14G4 (Fig.15B).
  • the PBS control group developed fatty livers that were pale, enlarged, and with marked fat accumulation in hepatocytes, whereas livers from the 14G7 mAb-treated group were healthy in appearance (Fig.16A and B).
  • LDL levels, liver size, and overall body weight was also much lower in the mAb-treated groups compared with the PBS group, despite comparable dietary intake (Fig.16C- E).
  • Mice fed with WD have high accumulation of isoDGR motif and macrophages in the liver but 14G7 mAb-treated mice have normal histology as mice fed with chow diet (Fig. 16F). Similar patterns were observed for plasma CRP level, indicating that treated mice have the immune clearance of isoDGR modified proteins, and reduction of inflammation and fat accumulation in liver of high fat diet fed mice.
  • Chronic pulmonary disease due to aging-induced lung pathology is the third leading cause of morbidity and mortality around the world.
  • the aging lung is characterized by loss of elasticity, airway enlargement, reduced strength in the respiratory muscles, decreased in pulmonary function, destruction of vessel and loss of structural integrity 50 .
  • Unique ECM environments are required to support the proper functions of the lungs.
  • PCMT-/- mice exhibited accumulation of damaged isoDGR-modified ECM proteins that can induce chronic inflammation in the lungs, as demonstrated by colocalization of CD68+ macrophages, and the alveoli were damaged by creating larger air space and reducing the surface area available for gas exchange (Fig.18).
  • the compromised oxygen exchange leads to systemic hypoxemia as shown in Fig 13.
  • Fig.19 the aging-induced lung morphological changes and dysfunctions observed in PCMT-/- mice (Fig.19) are the key characteristics of various age-related lung disorders such as emphysema, chronic obstructive pulmonary disease (COPD), asthma, lung cancer and lung fibrosis 51-54 .
  • COPD chronic obstructive pulmonary disease
  • the injection of isoDGR-specific mAb into PCMT1-/- mice reduced isoDGR accumulation and damage to the lung tissues, and can reduce isoDGR-induced chronic pulmonary diseases.
  • Example 8 the injection of isoDGR-specific mAb into PCMT1-/- mice reduced isoDGR accumulation and damage to the lung tissues, and can reduce isoDGR-induced chronic pulmonary diseases.
  • isoDGR-proteins [00208] Using indirect ELISA assays, an accumulation of isoDGR-motifs was detected in plasma from vascular disease patients and mice lacking the isoDGR repair enzyme PCMT1 6 . Therefore, isoDGR-modified tryptic peptides were immunoprecipitated from the plasma of patients with coronary artery disease (CAD), stroke, vascular dementia (VaD), and healthy controls for mass spectrometry analysis. Eleven candidate isoDGR-biomarkers were identified that were associated with lipid metabolism (ApoB100, ApoD), inflammation (C6, CFB, CFH), coagulopathy (F9, F13, FGB, FGG and PLG) and vascular disease (FN1).
  • CAD coronary artery disease
  • VaD vascular dementia
  • FN1 vascular disease
  • Example 10 Active immunization using isoDGR immunogens stimulates production of isoDGR-specific antibodies and extends the lifespan of Pcmt-/- mice [00210] Pcmt1 -/- mice were vaccinated with a synthetic isoDGR immunogen at birth and followed with two booster injections at weeks 2 and 3. Another group of Pcmt1 -/- mice was weekly injected with 1mg/kg isoDGR mAb 14G7, starting from at birth.
  • isoDGR-modified proteins in the tumour microenvironment were significantly reduced in the mAb-treated mice compared to PBS controls, suggesting isoDGR-mAb induced phagocytic clearance of isoDGR-proteins and reduced inflammation in the tumour microenvironment to inhibit tumour development.
  • Example 12. Inhibition of platelet aggregation using isoDGR antibodies [00215] The NGR-motif is highly represented in the proteins involved in the coagulation pathway.
  • Fibrinogen is a protein that plays a crucial role in platelet activation and aggregation. It binds to platelet integrin alphaIIbbeta3 ( ⁇ IIb ⁇ 3), which triggers signaling pathways that lead to platelet activation and spreading. This binding causes the platelets to aggregate and form a platelet plug. The platelet plug is then reinforced by fibrin, which is generated by the coagulation cascade. The formation of a platelet plug is critical in hemostasis that stops bleeding. However, if the platelet plug formation is not regulated properly, it can lead to pathological conditions such as thrombosis or embolism.
  • LC-MS/MS detected two isoDGR motifs in fibrinogen beta or fibrinogen gamma chains, and these motifs are located in the D-domain of fibrinogen as shown in Fig.24A.
  • the D-domain of fibrinogen is particularly important for the process of platelet aggregation, as it contains specific binding sites for the ⁇ IIb ⁇ 3 integrin as shown in Fig.24B.
  • the isoDGR motif which is a damaged form of the NGR motif, can enhance this interaction, leading to increased platelet activation and clot formation.
  • platelet spreading and aggregation assays were used to compare platelet response to isoDGR-modified fibrinogen vs native fibrinogen. Briefly, platelets were isolated from healthy donor blood and washed three times with PBS buffer. Three conditions were tested in vitro to determine the effect of isoDGR-modified fibrinogen on platelet aggregation: (A) Platelet adhesion to a plastic plate (without any coating), (B) Platelet spreading on native fibrinogen coated on a plastic plate, and (C) Platelet aggregation on isoDGR-modified fibrinogen.
  • FN or isoDGR-FN were coated onto 96- well plates (2 ⁇ g protein in 50 ⁇ l PBS, pH 6.8) via overnight incubation at 37°C. Non- adhered proteins were then removed and the wells washed twice with PBS. Then 2x10 6 platelets in 100 ⁇ l PBS buffer containing 2 ⁇ M ADP and 3 ⁇ M CaCl2 were added to each well and incubation at 37°C for 30 min. Images of the platelet morphology were captured using a microscope, as shown in Fig.25.
  • the results show that platelets from male PCMT1 KO mice with isoDGR accumulation in plasma displayed 60-fold increased platelet aggregation in the presence of 2uM ADP compared to male WT mice under the same conditions. Additionally, platelets from female PCMT1 KO mice with isoDGR accumulation in plasma displayed a 16-fold increase in platelet aggregation compared to female WT mice under the same conditions.
  • WT wild-type
  • PCMT1+/- mice with isoDGR accumulation in plasma
  • PCMT1+/- mice injected with 3 mg of isoDGR-specific 14G7 mAb 10 minutes before the experiment.
  • mice were cut 0.7 cm from the tip, and then immersed in a 50-mL Falcon tube containing isotonic saline that was pre-warmed to 37°C. The bleeding times were determined from the time of tail cutting until the bleeding stopped. The hemoglobin concentrations in the collected blood samples were measured using a microplate spectrophotometer at a wavelength of 550 nm. (Fig.27).
  • the results demonstrate that isoDGR-modified plasma proteins in PCMT1 KO mice effectively enhance coagulation and thrombosis, as evidenced by the significantly shorter tail vein bleeding time and higher amount of hemoglobin in the collected blood.

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Abstract

La divulgation concerne des peptides et des immunogènes isoDGR, et des anticorps qui se lient à l'isoDGR, et des procédés d'utilisation desdits peptides, immunogènes et anticorps. La divulgation concerne des immunogènes isoDGR et des anticorps ayant des CDR spécifiques identifiées dans la description, comprenant des variants fonctionnels de domaines variables spécifiques ayant les séquences CDR spécifiées, et des immunoconjugués desdits anticorps et leurs utilisations. L'invention concerne également des compositions et des kits comprenant lesdits peptides, immunogènes et anticorps, et des procédés et des utilisations de ceux-ci. L'invention concerne également des procédés et des utilisations desdits peptides, immunogènes et anticorps pour le diagnostic, le traitement et/ou la prévention de maladies associées à l'isoDGR comprenant une maladie cardiovasculaire.
EP24762836.5A 2023-03-02 2024-01-31 Procédés et agents pour des interventions de maladies induites par des lésions protéiques Pending EP4673464A1 (fr)

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