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WO2008002572A2 - Procédé de mesure de facteur neurotrophique dérivé de cerveau mature - Google Patents

Procédé de mesure de facteur neurotrophique dérivé de cerveau mature Download PDF

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Publication number
WO2008002572A2
WO2008002572A2 PCT/US2007/014828 US2007014828W WO2008002572A2 WO 2008002572 A2 WO2008002572 A2 WO 2008002572A2 US 2007014828 W US2007014828 W US 2007014828W WO 2008002572 A2 WO2008002572 A2 WO 2008002572A2
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WO
WIPO (PCT)
Prior art keywords
mbdnf
antibody
bdnf
probdnf
peptide
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PCT/US2007/014828
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English (en)
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WO2008002572A3 (fr
Inventor
Bai Lu
Guhan Nagappan
Eugene Zaitsev
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The Government Of The United States Of America As Represented By The Secretary Of Health And Human Services
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Application filed by The Government Of The United States Of America As Represented By The Secretary Of Health And Human Services filed Critical The Government Of The United States Of America As Represented By The Secretary Of Health And Human Services
Publication of WO2008002572A2 publication Critical patent/WO2008002572A2/fr
Publication of WO2008002572A3 publication Critical patent/WO2008002572A3/fr

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    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators

Definitions

  • BDNF Brain derived neurotrophic factor
  • proBDNF precursor protein
  • mBDNF mature BDNF
  • proBDNF unglycosylated proBDNF having an empirical molecular weight of about 27.8 kDa and mBDNF of about 13.5 kDa [1, 2, 3, 4].
  • proBDNF enters the endoplasmic reticulum (ER) lumen through its signal peptide and processed subsequently by the signal peptidase and N-glycosylated [1, 5].
  • ER endoplasmic reticulum
  • ProBDNF can be cleaved to form mBDNF by intracellular proteases including the serine protease furin in the trans-Golgi network and the prohormone convertases (PCl/3) in the secretory granules [1, 5].
  • proteases including the serine protease furin in the trans-Golgi network and the prohormone convertases (PCl/3) in the secretory granules [1, 5].
  • MMP3 or MMP7 matrix metalloproteinases
  • serine protease plasmin 6, 7
  • BDNF can be sorted into one of the two secretory pathways: constitutive (passive) or regulated (induced or active) [2, 8, 9]. While ProBDNF can be measured by antibodies specific for the pro-domain of proBDNF, tools to detect mBDNF alone are lacking. Therefore, the ratio of proBDNF/mBDNF that is secreted through the constitutive or regulated pathway remains unknown. Until a few years ago, only mBDNF was considered to be biologically active, and the pro-domain was suggested to play a passive role in folding and the maturation process [10, 11,12]. However, proBDNF can be detected in the extracellular milieu.
  • BDNF central nervous system
  • E-LTP early phase long-term potentiation
  • tPA by activating plasminogen, converts proBDNF to mBDNF extracellularly in the hippocampus, and such a conversion is critical for L-LTP expression [24].
  • This work provided a mechanistic link between tPA and BDNF in L-LTP, and revealed a physiological role for extracellular cleavage of proBDNF.
  • proBDNF NMDA receptor-dependent long term depression
  • BDNF protein in various pathological conditions.
  • CSF cerebrospinal fluid
  • serum or postmortem brain tissues are altered under several neurodegenerative disorders, and pathophysiological conditions.
  • BDNF levels are lowered in the serum and CSF of patients with relapsing remitting Multiple Sclerosis [26], Huntington's disease [27], and schizophrenia [28, 29], whereas the levels increase in CSF of children suffering from asphyxia [30].
  • reduced levels of BDNF were found in the plasma of patients with acute coronary syndromes [31, 32], BDNF levels are also reduced in the brain of patients suffering from Alzheimer's [33][34][35].
  • pro- and mature BDNF elicit very different and sometimes opposite biological effects, it is important to have a method that distinguishes mBDNF from proBDNF. This method should allow measurement of the relative amounts of proBDNF and mBDNF synthesized in neurons, and detection of their secretion through either constitutive or regulated pathway.
  • the method comprises one or more of the following steps: i) the use of the N-terminal amino acid sequence of mature BDNF to generate antibody; ii) affinity purification procedure to obtain a substantially purified antibody, and iii) immuno-depletion of the substantially purified antibody using a peptide that spans the cleavage site in proBDNF.
  • the resulting antibody is highly specific for mBDNF because the portion that reacts with proBDNF has been removed.
  • substantially purified is intended to mean a compound or substance (e.g. antibody) that is isolated from its natural surroundings, for example, having been removed from whole blood or serum by a process that may include affinity purification, or the like.
  • the N-terminal amino acid sequence (peptide) used in the initial step can be any fragment from the N-terminal of mBDNF of suitable length to effectively elicit antibody formation (e.g. an N-terminal fragment of SEQ ID NO:1).
  • a seven amino acid sequence corresponding to the amino terminus of the cleaved mBDNF is used (HSDPARR, SEQ ID NO:2), preferably with a cysteine residue at the C-terminus as a linker (i.e. HSDPARRC, SEQ ID NO:4)
  • Suitable peptides will generally be from 6 to 50 amino acid residues, more usually 6-25 or 7-15 residues in length.
  • peptides with any integral number of amino acids between 4 and 50, inclusive can be used for this purpose.
  • Peptides that correspond to the amino terminus of cleaved mBDNF, such as SEQ ID NO:2, are referred to herein as B-peptides. Methods for generating peptides are well known in the art.
  • the peptide can be administered to any animal suitable for the production of antibodies (e.g. a rabbit) using methods that are known in the art [37] the serum collected after an interval sufficient for antibody production, and the resulting antibody isolated by affinity purification.
  • the antibody can be purified on a column conjugated with SEQ ID NO:2.
  • the antibody is then further purified by contacting it with a peptide that spans the cleavage point in proBDNF, for example, CSMRVRRHSDPARR (SEQ ID NO:3)
  • Suitable peptides for this purpose will generally be from 12-50, more generally 12-25, and in particular 12-16 amino acid residues in length.
  • peptides with any integral number of amino acids between 8 and 50, inclusive can be used for this purpose. Such peptides are referred to herein as A+B peptides.
  • the resulting antibody has a high specificity for mBDNF. It is also an object of the invention to provide antibodies obtained using the methods of the invention.
  • the antibodies of the invention can be used alone or with other agents in suitable compositions for the detection of mBDNF, for diagnostic or investigative (e.g. research) purposes.
  • the invention includes antibodies made by the methods outlined above, and compositions comprising the antibodies.
  • the antibody is one that has been generated by the method described herein, i.e. an antibody that has been raised against a fragment of the N-terminal of mBDNF, and purified by contacting it with a peptide spanning the cleavage point of proBDNF to mBDNF.
  • the antibody may be labeled, for example with a fluorescent label, to aid in detection of antibody antigen complexes.
  • Many suitable means of labeling antibodies are known in the art (see, e.g.
  • labels may be directly linked through a covalent bond or covalently through a linking molecule which typically bears reactive sites capable of forming covalent bonds with the label and the antibody respectively.
  • Other techniques familiar in the art for example, radiolabels, enzymatic labels, biotin-avidin labels, chemi luminescent labels, quantum dot, may also be used.
  • BDNF "proBDNF” and "mBDNF”, as used herein, mean any isoform of a mammalian BDNF, proBDNF or mBDNF, in particular a human BDNF.
  • BDNF sequences for rodents for rodents (rat and mouse) and other species are known in the art. Information as to the sequences and properties of these forms can be found on the NCBI website and in other resources known to persons of skill in the art [39].
  • the compositions and methods of the present invention are expected to be useful for research purposes and for clinical studies. As mentioned above, most studies to date in measuring BDNF protein levels have used antibodies raised against the mature domain of BDNF, and therefore have measured both proBDNF and mBDNF. A few investigators have used proBDNF specific antibody, but to the inventors' knowledge, no one has a method to detect specifically mBDNF.
  • mBDNF specific antibody generated using the method described in this application will be used to revisit the expression and regulation of mBDNF protein. We therefore anticipate a large commercial usage of reagents based on the mBDNF specific antibody (Western blot, immunoprecipitations, immunocytochemistry, ELISA, etc). Moreover, we expect a wide range of application of the mBDNF-specific detection methods be used in clinical investigations. Further, an ELISA developed using the antibody of the present invention is expected to be useful for differentiating between the two forms of BDNF in these and other clinical disorders. Measurements can be made, for example, on samples of blood or cerebrospinal fluid from such patients.
  • FIG. 1 Western blot detection of purified mBDNF.
  • A Schematic representation of proBDNF, mBDNF, and the epitopes that the proBDNF and mBDNF antibodies are directed towards. The mBDNF antibody was raised against the "B” peptide, affinity purified using the "B" peptide conjugated column, and depleted against the "A+B” peptide.
  • B Detection of purified mBDNF. Equal amounts of purified proteins (GST fused to Pro-domain of BDNF, proBDNF fused to EGFP and mBDNF) were resolved on 4 - 12% NuPAGE, transferred to PVDF membrane, and probed using the affinity-purified anti-mBDNF antibody at 1 :500 dilution.
  • FIG. 1 Western blot detection of mBDNF in transfected cells.
  • A Schematic representation of the BDNF construct containing the N-terminal tags HA, FLAG, pro domain, mature domain, and the C-terminal tags EGFP and V5. The molecular weights of some fragments shown in C are also indicated.
  • B Western analysis using the anti-mBDNF antibody. AtT20 cells stably transfected with or without the epitope-tagged BDNF were lysed and processed for Western blot.
  • C Control blot showing the existence of various fragments. The same blot was stripped and re-probed using anti-GFP antibody. While three bands of GFP containing proteins (as indicated) could be visualized using anti-GFP antibody (C), only the mBDNF was detectable in cell lysates using the mBDNF-specific antibody (B).
  • FIG. 3 Western blot detection of endogenous mBDNF in brain tissues in vivo. Hippocampal and cortical tissues were dissected from the adult rat brain, and homogenized. The proteins were separated on NuPAGE gel and transferred to the PVDF membrane. The blot was probed with anti-mBDNF antibody. Note that this antibody detects only mBDNF, but not proBDNF.
  • FIG. 4 Immunofluorescence staining of cultured hippocampal neurons expressing epitope tagged BDNF under membrane permeable conditions. Neurons transfected with HA-tagged BDNF construct (shown in Fig. 2A) were grown at low density for more than 14 days in vitro (>14DIV), fixed, permeabilized using 0.2% Triton X-100, and processed for immunocytochemistry using anti-HA (red) and anti-mBDNF (green) specific antibodies. Note the non-overlapping areas. Arrowheadss indicate mBDNF only spots, whereas arrows indicate proBDNF only spots.
  • FIG. 2A Cell surface staining of proBDNF and mBDNF secreted under the constitutive and regulated conditions.
  • Neurons transfected with HA-tagged BDNF construct (shown in Fig. 2A) were grown at low density for >14DIV, fixed under the non-permeable conditions, and processed for immunocytochemistry.
  • A top, constitutive
  • the neuronal activity was completely blocked by TAC (Tetrodotoxin, 2-APV and CNQX) for 12 hours, and cells were immunostained with either a monoclonal antibody against HA for proBDNF (red, 1 :500) or anti-mBDNF antibody (green, 1 :250).
  • Antibodies used in this study (a) Mouse anti-GFP at 1/500 (Covance Research Products, Inc, 800 University Avenue, Berkeley, CA 94710, USA); (b) Rabbit anti-GFP at 1/500 (Abcam- Abcam Inc, One Kendall Square, Bldg.
  • DNA constructs DNA sequences corresponding to the signal peptide followed by haemagglutinin (HA) tag, FLAG tag and the pro-peptide of BDNF were synthesized with Xba I (5') and Nde I (3') ends and ligated upstream between Xba I — Nde I sites in the parent BDNF-EGFP construct in pET21d(+). Subsequently, the entire open reading frame was amplified by polymerase chain reaction using primers with CACC sequence at the 5' and 3' ends to clone into a lentiviral vector using the pLenti6/V5 directional topo-cloning kit (Invitrogen).
  • HA haemagglutinin
  • hippocampus or cortex was removed into HBSS and subjected to trypsin (0.05%) and DNase I digestion (0.1 ⁇ g/ml) for 15 min at 37 0 C.
  • the tissues were then washed three times with Dulbecco's Modified Eagles Medium containing 10% heat inactivated Fetal Bovine serum (DMEM-FBS - Invitrogen) followed by trituration using a blunt ended transfer pipette. Cells were then clarified using a 40/xm cell strainer and on a 4% Bovine serum albumin fraction V (Invitrogen) diluted in DMEM-FBS.
  • 5 X 10 6 cells were mixed with a cocktail of 5/ig endotoxin-free DNA (prepared using Qiagen columns - 27220 Turnberry Lane .Suite 200, Valencia, CA 91355) in the Amaxa reagent mix, and nucleofected using the protocols specified for rat or mouse neurons provided by the manufacturer (Amaxa, 205 Perry Parkway, Suite 7, Gaithersburg, MD 20877, USA).
  • Cells were immediately diluted in DMEM-FBS and seeded according to the required dilutions (20000 — 30000 cells on a 18mm diameter No.l Carolina glass cover slips of 0.17mm thickness coated with Poly-D-Lysine in 0.1 M Borate Buffer followed by Laminin I (ATCC - P.O. Box 1549, Manassas, VA 20108, USA) at lO ⁇ g/ml for low-density seeding or 0.6X10 6 cells/well in a 6 well plate for high-density seeding) and maintained at 37°C in the presence of 5% CO 2 .
  • Laminin I ATCC - P.O. Box 1549, Manassas, VA 20108, USA
  • Neurons grown on cover slips were washed three times with phosphate buffered saline (PBS) and then fixed with 4% paraformaldehyde in PBS containing 100 mM sucrose for 3-5min on ice. All the procedures were performed on ice. The cells were then washed three times and incubated with 100 mM glycine for lOmin. Blocking was done using 3% BSA (Pierce - Pierce Biotechnology, Inc., P.O. Box 117, Rockford, IL. 61105 U.S.A) in PBS for 60 min, followed by incubation for 2 hours with primary antibody diluted in blocking buffer.
  • BSA Pierce - Pierce Biotechnology, Inc., P.O. Box 117, Rockford, IL. 61105 U.S.A
  • mBDNF antibody A seven amino acid synthetic peptide sequence corresponding to the amino-terminus of the cleaved mature BDNF (or B-peptide: HSDPARRC) was used for immunizing New Zealand White Rabbits after conjugating the peptide to Keyhole limpet hemocyanin (KLH) through the thiol (-SH) group of Cysteine at the C-terminus.
  • KLH Keyhole limpet hemocyanin
  • -SH thiol
  • the antibodies were diluted 1 :1 with 0.01M Tris buffer pH7.2 and ImM EDTA and allowed to bind to the first affinity column (also called B peptide column).
  • This column was prepared by mixing B-peptide with thiosepharose 6B beads at the ratio of 6:1 for 2 hours at room temperature, and then washing the beads with Phosphate buffered saline (PBS) several times until the free peptide is completely removed from the column [38].
  • PBS Phosphate buffered saline
  • the rabbit serum was collected and affinity purified on the column conjugated with B-peptide. Briefly, the antibody solution was loaded onto the B-peptide column, and excess proteins from the serum were washed with PBS until no protein was detected in the flow through.
  • the B-peptide column was then eluted using 0.1 mM Glycine buffer pH 2.5 to collect fractions with all antibodies against the B-peptide. The fractions were pooled and dialyzed against PBS. The antibodies against the B-peptide were then passed through the second affinity column (also called A+B peptide column, see Fig. 2A, as described above), which was generated the same way as the B peptide column except A+B peptide (CSMR VRRHSDP ARR) (SEQ ID NO: 3) was used instead of B peptide.
  • the flow through in this procedure contains the antibody which specifically recognizes the cleaved end of the mBDNF, but not the uncleaved proBDNF were used in this study.
  • proBDNF and mBDNF elicit different and often opposite biological effects [7], it is essential to distinguish whether secreted BDNF is in pro- or mature isoform.
  • Western blot can distinguish proBDNF and mBDNF based on their molecular weights
  • ELISA and immunocytochemistry are better methods because they are much more sensitive and can be used when very small number of cells/tissues are available. Therefore, we developed a method that allows specific and reliable detection of mBDNF, with high sensitivity.
  • the method is based on the premise that cleavage of proBDNF will generate a previously unexposed epitope, which can be used to generate a specific antibody.
  • An antibody was raised against the synthetic peptide corresponding to the amino termini of the cleaved mBDNF (peptide B, Figl A), affinity-purified using the B peptide column, and depleted against the peptide spanning the cleavage sequence (peptide A+B, Fig. IA).
  • tissue homogenates are prepared from specific subregions of the brain, such as cortex and hippocampus. Briefly, the cortex and the hippocampus were removed from the adult rat brain and immediately homogenized in the presence of protease inhibitors in RIPA buffer containing 1% SDS. The homogenates were then sonicated repeatedly and then centrifuged. The protein concentration of the supernatant was estimated and 30 ⁇ g of total protein from cortex or hippocampus were resolved on a 4-12% NuPAGE, transferred to PVDF membrane and probed using the anti- mBDNF (B-peptide) antibody. Even in brain tissue, the B-peptide antibody specifically detects only the mature BDNF, but not proBDNF or any other proteins (Fig. 3).
  • the methods of the invention are effective in generating useful antibodies for measuring levels of mBDNF, and should accordingly be useful for generating antibodies that can be used both in research and clinical settings. It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
  • Sortilin controls intracellular sorting of brain-derived neurotrophic factor to the regulated secretory pathway. J Neurosci., 2005. 25(26): p. 6156-66.
  • Rattenholl, A., et al. The pro-sequence facilitates folding of human nerve growth factor from Escherichia coli inclusion bodies. Eur J Biochem., 2001.
  • BDNF is a target-derived survival factor for arterial baroreceptor and chemoafferent primary sensory neurons. J Neurosci, 1999. 19(6): p. 2131-42. 14. Balkowiec, A. and D.M. Katz, Brain-derived neurotrophic factor is required for normal development of the central respiratory rhythm in mice. J Physiol,
  • Tan, Y.L., et al. Decreased BDNF in serum of patients with chronic schizophrenia on long-term treatment with antipsychotics.
  • Korhonen, L., et al. Brain derived neurotrophic factor is increased in cerebrospinal fluid of children suffering from asphyxia. Neurosci Lett., 1998. 240(3): p. 151-4.

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Abstract

La présente invention concerne un procédé d'obtention d'anticorps spécifique du facteur neurotrophique dérivé du cerveau mature (mBDNF), et ses utilisations.
PCT/US2007/014828 2006-06-27 2007-06-27 Procédé de mesure de facteur neurotrophique dérivé de cerveau mature WO2008002572A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104774264A (zh) * 2014-01-15 2015-07-15 上海易乐生物技术有限公司 抗人proBDNF单克隆抗体及其在疼痛中的作用
EP4164747A4 (fr) * 2020-06-10 2024-07-10 Crystal Bioscience Inc. Anticorps anti-bdnf et leurs procédés d'utilisation

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5235043A (en) * 1990-04-06 1993-08-10 Synergen, Inc. Production of biologically active, recombinant members of the ngf/bdnf family of neurotrophic proteins
WO1996008562A1 (fr) * 1994-09-12 1996-03-21 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin Molecules biologiquement actives derivees de neurotrophines
GB9616105D0 (en) * 1996-07-31 1996-09-11 Univ Kingston TrkA binding site of NGF
JP2009514904A (ja) * 2005-11-07 2009-04-09 ユニバーシティ オブ コペンハーゲン ニューロトロフィン由来ペプチド配列

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104774264A (zh) * 2014-01-15 2015-07-15 上海易乐生物技术有限公司 抗人proBDNF单克隆抗体及其在疼痛中的作用
WO2015106641A1 (fr) * 2014-01-15 2015-07-23 上海易乐生物技术有限公司 Anticorps monoclonal probdnf antihumain et ses utilisations pour des douleurs
US9969799B2 (en) 2014-01-15 2018-05-15 Shanghai Yile Biotechnology Limited Anti-human proBDNF monoclonal antibody, and uses thereof in pains
CN104774264B (zh) * 2014-01-15 2018-09-14 上海易乐生物技术有限公司 抗人proBDNF单克隆抗体及其在疼痛中的作用
EP4164747A4 (fr) * 2020-06-10 2024-07-10 Crystal Bioscience Inc. Anticorps anti-bdnf et leurs procédés d'utilisation

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