WO2011157905A1 - Anticorps polyclonal se liant à la hmgb1 acétylée - Google Patents

Anticorps polyclonal se liant à la hmgb1 acétylée Download PDF

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Publication number
WO2011157905A1
WO2011157905A1 PCT/FI2011/050585 FI2011050585W WO2011157905A1 WO 2011157905 A1 WO2011157905 A1 WO 2011157905A1 FI 2011050585 W FI2011050585 W FI 2011050585W WO 2011157905 A1 WO2011157905 A1 WO 2011157905A1
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WO
WIPO (PCT)
Prior art keywords
acetylated
peptide
antibody
hmgbl
hmgb1
Prior art date
Application number
PCT/FI2011/050585
Other languages
English (en)
Inventor
Heikki Rauvala
Ari Rouhiainen
Original Assignee
Helsingin Yliopisto
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Helsingin Yliopisto filed Critical Helsingin Yliopisto
Publication of WO2011157905A1 publication Critical patent/WO2011157905A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/23—Immunoglobulins specific features characterized by taxonomic origin from birds
    • 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

Definitions

  • the present invention relates to the field of polyclonal antibodies binding to human HMGBl (high- mobility group box 1) protein and diagnostic and therapeutic use of same.
  • the present invention provides an isolated polyclonal antibody binding to hyperacetylated HMGBl but not binding to hypoacetylated HMGBl.
  • the present invention is directed to an isolated polyclonal IgY antibody generated in chicken binding to hyperacetylated HMGBl but not binding to hypoacetylated HMGBl.
  • the invention provides a method for detecting the presence or amount of an acetylated HMGBl in a biological sample.
  • HMGBl is a cytokine-like molecule that is secreted via a poorly characterized mechanisms as it lacks a classical signal peptide. Acetylation has been shown to be a mechanism that may regulate HMGBl secretion (Bonaldi et al., 2003, The EMBO Journal 22(20):5551-5560). The majority of HMGBl is in the hypoacetylated form and only a specific, minor pool of HMGBl that is directed to secretion is hyperacetylated. Acetylated HMGBl is actively secreted from the cells whereas hypoacetylated HMGBl can be released from the dying cells.
  • HMGBl-form having acetylated lysines in the positions 176-187 can be found in plasma during liver injury and in activated immune cells using mass spectrometry (Bonaldi et al., 2003, The EMBO Journal 22(20):5551-5560, Antoine DJ et al., 2009, Toxicol Sci. 112(2):521-531). Therefore, the ability to distinguish between the two forms of HMGBl (acetylated vs. non-acetylated) is critical.
  • An multiply acetylated protein HMGBl is disclosed in US 2006/0111287.
  • the non-histone nuclear protein HMGBl belonging to the B family of high mobility group proteins having acetylated lysines in one or more of the positions in the amino acid sequence of the protein is particularly described. No disclosure of a polyclonal antibody against the acetylated HMGBl is found in the publication.
  • FIG. 1 Anti-acetylated HMGBl IgY (1966) stains cytoplasmic vesicles in lipopolysaccharide (LPS) activated macrophages.
  • Figure 6. A higher magnification of anti-acetylated HMGBl-peptide antibody stained LPS activated macrophages. Arrows indicate some of the cytoplasmic vesicles that are stained with anti acetylated HMGBl peptide antibody.
  • the present invention provides an isolated polyclonal antibody binding to acetylated HMGBl but not binding to non-acetylated HMGBl.
  • said antibody is IgY antibody generated in chicken as shown in the Experimental Section below.
  • polyclonal antibody denotes herein a mixture of different antibody molecules which react with more than one immunogenic determinant of an antigen.
  • the invention also provides an isolated polyclonal antibody obtained by a method comprising the steps of: a) contacting a chicken with an acetylated peptide KAEKSKKKKEEC (SEQ ID NO:l) so that said chicken is immunized with said peptide; and b) collecting the polyclonal antibody from the egg yolk of an egg laid by said chicken.
  • all epsilon amino groups of lysine side chains of the antigen peptide are acetylated.
  • an alpha amino group of the amino terminal lysine is preferably acetylated.
  • Said peptide (without the cysteine residue) corresponds to positions 177-187 in the amino acid sequence of human HMGBl (Gene ID:3146; NP_002119.1).
  • the peptide can also be coupled to a carrier such as keyhole limpet hemocyanin (KLH) when used as an antigen.
  • KLH keyhole limpet hemocyanin
  • the peptide is linked to KLH via a cysteine added to the C-terminal of the peptide.
  • the polyclonal IgY obtained by this method can easily be recognized by a skilled person of the art by two simple test: i) to confirm the class and origin of the antibody, the skilled person may use anti-chicken IgY antibody, and ii) to confirm the specificity of the antibody, the skilled person may perform the Western blot described in the Experimental Section below (see, e.g., Figure 4). Further, the invention is directed to a method for detecting the presence or amount of an acetylated HMGB1 in a biological sample comprising contacting the sample with an isolated polyclonal antibody and determining the amount of the acetylated HMGB1 bound to the antibody. Said antibody is preferably labeled with a detectable label.
  • Such label can be a radioisotope, fluorescent compound, chemiluminescent compound, enzyme, or enzyme co-factor, or any other labels known in the art.
  • the antibody that binds to an entity one wishes to measure is not labeled, but is instead detected by binding of a labeled secondary antibody that specifically binds to the primary antibody.
  • Anti acetylated HMGB1 peptide antibody can be used for diagnosis or treatment in the pathological situations belonging to inflammatory, autoimmune, neurodegenerative or malignant
  • diseases/disorders include sepsis, rheumatoid arthritis, septicemia, shock, organ ischemia, reperfusion injury, atherosclerosis, stroke, multiple sclerosis, and cancer.
  • the antibodies of the present invention are useful as diagnostic antibodies to detect specifically the acetylated form of HMGB1 in tissue fluids like plasma, cerebrospinal fluid or synovial fluid. Further, the antibodies of the present invention are useful as diagnostic antibodies to detect specifically the acetylated form of HMGB1 in tissues.
  • the antibodies of the present invention are also useful to block specifically the acetylated form of HMGB1 in patients with inflammatory, autoimmune, neurodegenerative or malignant
  • the present invention is further directed to a peptide comprising or consisting of sequence
  • KAEKSKKKKEEC SEQ ID NO:l
  • lysines of the peptide are partly ot totally acetylated, and its use as an antigen (see Experimental Section below).
  • the peptide of the invention can be synthesized by well-known methods (see, e.g., Atherton, E.; Sheppard, R.C., 1989, Solid Phase peptide synthesis: a practical approach. Oxford, England: IRL Press. ISBN 0199630674).
  • Antibodies were generated in two chickens using keyhole limpet hemocyanin (KLH) coupled acetylated HMGBl-peptide as an antigen (see, e.g, R Schade et al., Chicken Yolk Antibodies, Production and Application, IgY-Technology, Springer-Verlag Berlin Heidelberg, New York (2001) pp. 201, 203, 204).
  • Antigen peptide was synthetized and acetylated using solid phase peptide synthesis.
  • Peptide sequence was (Ac-)(Ac-K)AE(Ac-K)S(Ac-K)(Ac-K)(Ac-K)(Ac-K)EEC(-COOH).
  • Peptide was linked to KLH via C-terminal cysteine. Chickens were immunized with KLH-coupled peptide and adjuvants as described in Figure 1.
  • Lipopolysaccharide (LPS) treated mouse macrophages (RAW 264.7 cells) were stained with anti- acetylated HMGB1 peptide antibody.
  • RAW 264.7 cells were cultured in medium containing 10% foetal calf serum and treated over night with or without 10 micrograms/milliliter of LPS. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100. Cells were treated with 0.12% glycine followed by 50 mM NH 4 CI treatment, and blocked with 5% milk powder in phosphate buffered saline (PBS).
  • PBS phosphate buffered saline

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un anticorps polyclonal isolé se liant à la HMGB hyperacétylée mais ne se liant pas à la HMGB hypoacétylée. En particulier, la présente invention concerne un anticorps polyclonal IgY isolé généré chez le poulet se liant à la HMGB1 hyperacétylée mais ne se liant pas à la HMGB1 hypoacétylée. L'invention a en outre pour objet une méthode de détection de la présence ou de la quantité d'une HMGB acétylée dans un échantillon biologique.
PCT/FI2011/050585 2010-06-18 2011-06-17 Anticorps polyclonal se liant à la hmgb1 acétylée WO2011157905A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FI20105715A FI20105715A0 (fi) 2010-06-18 2010-06-18 Asetyloituun HMGB1:een sitoutuva polyklonaalinen vasta-aine
FI20105715 2010-06-18

Publications (1)

Publication Number Publication Date
WO2011157905A1 true WO2011157905A1 (fr) 2011-12-22

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PCT/FI2011/050585 WO2011157905A1 (fr) 2010-06-18 2011-06-17 Anticorps polyclonal se liant à la hmgb1 acétylée

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FI (1) FI20105715A0 (fr)
WO (1) WO2011157905A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9187780B2 (en) 2011-12-07 2015-11-17 Singapore Volition Pte. Limited Method for detecting nucleosome adducts
WO2015175375A1 (fr) 2014-05-13 2015-11-19 Short Jay M Protéines biologiques conditionnellement actives
WO2016033331A1 (fr) 2014-08-28 2016-03-03 Bioatla, Llc Récepteurs d'antigènes chimères conditionnellement actifs pour cellules t modifiées
WO2016118531A1 (fr) * 2015-01-21 2016-07-28 University Of Hawaii Méthodes et trousses pour l'analyse d'isoformes d'hmgb1
US10900064B2 (en) 2011-09-01 2021-01-26 Belgian Volition Sprl Method for detecting nucleosomes containing nucleotides
US11111288B2 (en) 2014-08-28 2021-09-07 Bioatla, Inc. Conditionally active chimeric antigen receptors for modified t-cells
US11879011B2 (en) 2016-05-13 2024-01-23 Bioatla, Inc. Anti-ROR2 antibodies, antibody fragments, their immunoconjucates and uses thereof
WO2024016944A1 (fr) * 2022-07-18 2024-01-25 华东师范大学 Marqueur pour la prévention et la protection contre les maladies ischémiques cérébrales et l'application d'un dérivé d'aspirine dans la prévention et la protection contre les maladies ischémiques cérébrales

Citations (6)

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WO2000014536A1 (fr) * 1998-09-04 2000-03-16 New England Biolabs, Inc. Production d'anti-corps specifiques d'un motif et independants du contexte au moyen de banques de peptides utilisees comme antigenes
WO2004044001A2 (fr) * 2002-11-11 2004-05-27 Fondazione Centro San Raffaele Del Monte Tabor Proteine acetylee
EP2123298A1 (fr) 2007-02-15 2009-11-25 Fukuoka University Agent servant a supprimer le rejet dans une greffe d'organe comprenant un anticorps anti-hmgb-1
EP2123297A1 (fr) 2007-02-15 2009-11-25 Kumamoto University Agent therapeutique comportant un anticorps capable de se lier specifiquement a la hmgb-1 humaine comme ingredient actif
EP2123299A1 (fr) 2007-02-15 2009-11-25 Kyushu University, National University Corporation Agent therapeutique pour maladie pulmonaire interstitielle comportant un anticorps anti-hmgb-1
US20100061987A1 (en) 2004-10-22 2010-03-11 Medimmune, Llc High Affinity Antibodies Against HMGB1 and Methods Of Use Thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
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WO2000014536A1 (fr) * 1998-09-04 2000-03-16 New England Biolabs, Inc. Production d'anti-corps specifiques d'un motif et independants du contexte au moyen de banques de peptides utilisees comme antigenes
WO2004044001A2 (fr) * 2002-11-11 2004-05-27 Fondazione Centro San Raffaele Del Monte Tabor Proteine acetylee
US20060111287A1 (en) 2002-11-11 2006-05-25 Bianchi Marco E Acetylated protein
US20100061987A1 (en) 2004-10-22 2010-03-11 Medimmune, Llc High Affinity Antibodies Against HMGB1 and Methods Of Use Thereof
EP2123298A1 (fr) 2007-02-15 2009-11-25 Fukuoka University Agent servant a supprimer le rejet dans une greffe d'organe comprenant un anticorps anti-hmgb-1
EP2123297A1 (fr) 2007-02-15 2009-11-25 Kumamoto University Agent therapeutique comportant un anticorps capable de se lier specifiquement a la hmgb-1 humaine comme ingredient actif
EP2123299A1 (fr) 2007-02-15 2009-11-25 Kyushu University, National University Corporation Agent therapeutique pour maladie pulmonaire interstitielle comportant un anticorps anti-hmgb-1

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ATHERTON, E., SHEPPARD, R.C.: "Solid Phase peptide synthesis: a practical approach.", 1989, IRL PRESS
BONALDI ET AL., THE EMBO JOURNAL, vol. 22, no. 20, 2003, pages 5551 - 5560
BONALDI TIZIANA ET AL: "Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion.", THE EMBO JOURNAL 15 OCT 2003 LNKD- PUBMED:14532127, vol. 22, no. 20, 15 October 2003 (2003-10-15), pages 5551 - 5560, XP002658473, ISSN: 0261-4189 *
KIM SUNG CHAN ET AL: "Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.", MOLECULAR CELL AUG 2006 LNKD- PUBMED:16916647, vol. 23, no. 4, August 2006 (2006-08-01), pages 607 - 618, XP002658474, ISSN: 1097-2765 *
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PARKKINEN J ET AL: "Amphoterin, the 30-kDa protein in a family of HMG1-type polypeptides. Enhanced expression in transformed cells, leading edge localization, and interactions with plasminogen activation.", THE JOURNAL OF BIOLOGICAL CHEMISTRY 15 SEP 1993 LNKD- PUBMED:8366113, vol. 268, no. 26, 15 September 1993 (1993-09-15), pages 19726 - 19738, XP002658475, ISSN: 0021-9258 *
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10900064B2 (en) 2011-09-01 2021-01-26 Belgian Volition Sprl Method for detecting nucleosomes containing nucleotides
US9187780B2 (en) 2011-12-07 2015-11-17 Singapore Volition Pte. Limited Method for detecting nucleosome adducts
US11193939B2 (en) 2011-12-07 2021-12-07 Belgian Volition Sprl Method for detecting nucleosome adducts
US9709569B2 (en) 2011-12-07 2017-07-18 Singapore Volition Pte. Limited Method for detecting nucleosome adducts
WO2015175375A1 (fr) 2014-05-13 2015-11-19 Short Jay M Protéines biologiques conditionnellement actives
US10329556B2 (en) 2014-05-13 2019-06-25 Bioatla, Llc Conditionally active biological proteins
EP4074735A1 (fr) 2014-08-28 2022-10-19 BioAtla, Inc. Récepteurs d'antigène chimérique conditionnellement actifs pour cellules t modifiées
US11111288B2 (en) 2014-08-28 2021-09-07 Bioatla, Inc. Conditionally active chimeric antigen receptors for modified t-cells
WO2016033331A1 (fr) 2014-08-28 2016-03-03 Bioatla, Llc Récepteurs d'antigènes chimères conditionnellement actifs pour cellules t modifiées
US11584927B2 (en) 2014-08-28 2023-02-21 Bioatla, Inc. Conditionally active chimeric antigen receptors for modified T-cells
WO2016118531A1 (fr) * 2015-01-21 2016-07-28 University Of Hawaii Méthodes et trousses pour l'analyse d'isoformes d'hmgb1
US11879011B2 (en) 2016-05-13 2024-01-23 Bioatla, Inc. Anti-ROR2 antibodies, antibody fragments, their immunoconjucates and uses thereof
WO2024016944A1 (fr) * 2022-07-18 2024-01-25 华东师范大学 Marqueur pour la prévention et la protection contre les maladies ischémiques cérébrales et l'application d'un dérivé d'aspirine dans la prévention et la protection contre les maladies ischémiques cérébrales

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