WO1998021245A1 - Anticorps a haute affinite diriges contre le bdnf humain, procedes permettant sa production, et son utilisation - Google Patents

Anticorps a haute affinite diriges contre le bdnf humain, procedes permettant sa production, et son utilisation Download PDF

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Publication number
WO1998021245A1
WO1998021245A1 PCT/EP1997/005956 EP9705956W WO9821245A1 WO 1998021245 A1 WO1998021245 A1 WO 1998021245A1 EP 9705956 W EP9705956 W EP 9705956W WO 9821245 A1 WO9821245 A1 WO 9821245A1
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WO
WIPO (PCT)
Prior art keywords
bdnf
human bdnf
antibody
antibodies
against human
Prior art date
Application number
PCT/EP1997/005956
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German (de)
English (en)
Inventor
Ilse Bartke
Walter Eberle
Roland Kolbeck
Yves-Alain Barde
Original Assignee
Roche Diagnostics Gmbh
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 Roche Diagnostics Gmbh filed Critical Roche Diagnostics Gmbh
Priority to AU71798/98A priority Critical patent/AU7179898A/en
Publication of WO1998021245A1 publication Critical patent/WO1998021245A1/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

  • the invention relates to high-affinity antibodies against human BDNF (brain-derived neurotrophic factor), processes for their preparation and their use both in diagnostics and for therapy
  • BDNF body fluids
  • MAK monoclonal antibody
  • PAK polyclonal antibody
  • MAK-PAK ELISA sandwich Immunoassay using a monoclonal antibody (MAK) and a polyclonal antibody (PAK) against human BDNF described (MAK-PAK ELISA)
  • MAK-PAK ELISA sandwich Immunoassay using a monoclonal antibody (MAK) and a polyclonal antibody (PAK) against human BDNF described
  • BDNF-specific monoclonal antibodies of the IgGl isotype which are not suitable for an immunoassay (MAK-MAK ELISA) the antibodies described by Radka are unable to block the biological activity of BDNF.
  • the antibodies were obtained after immunization of mice with recombinant BDNF from E. coli. The immunization was carried out according to SF Radka et al, J Immunol 128 (1982) 2804-2806.
  • Radka monoclonal antibody RP 43-01 which is said to have a high affinity, blocks the BDNF immunoassay signal in plasma at a concentration of 10 ⁇ g / ml
  • the object of the present invention is then to provide high-affinity antibodies, preferably monoclonal antibodies, against human BDNF which allow a further increase in the sensitivity and specificity of immunological determinations of human BDNF (in particular in a MAK-MAK ELISA), the biological activity blocking of BDNF (inhibiting antibodies) and in Western blot and also therapeutically usable
  • the object is achieved by an antibody against human BDNF which inhibits the survival of nodosum neurons by 90% and / or more in a concentration of 500 ng / ml.
  • the antibody according to the invention preferably inhibits at a concentration of 250 ng / ml, particularly preferably already at concentrations of 120 ng / ml, 90% and / or more the survival of nodosum neurons.
  • the antibodies according to the invention are even able to increase the survival of nodosum neurons quantitatively (more than 90%) even in concentrations of 80 ng / ml inhibit
  • the antibodies according to the invention can be used Antibodies, a BDNF signal in a sandwich immunoassay with recombinant human BDNF at concentrations below 5 pg / well (50 pg / ml), preferably between 1 and 5 pg / well (10-50 pg / ml)
  • nodosum neurons The survival of nodosum neurons is essentially determined by dissociating nodosum ganglia in individual cells and incubating these cells with human BDNF, with and without antibodies according to the invention, in different concentrations.
  • the stimulation achieved by human BDNF is used as the standard value (0% inhibition).
  • a measured value without the addition of human BDNF is used as the standard value for 100% inhibition
  • Immunization is preferably carried out over a period of several months (6-12 months).
  • the first immunization of a mammal, preferably mouse or rat, with fish BDNF, the subsequent immunizations (at least one) with human BDNF (natural or recombinant) or preferably alternately with human BDNF and fish BDNF A particularly preferred immunization scheme is shown in the examples
  • An antibody according to the invention is to be understood as a protein which consists of one or more polypeptides which are essentially encoded by antibody genes. Such antibody genes contain both genes for constant and variable regions.
  • Antibodies can be in a variety of different forms, for example FvFab and F (ab) 2 as well as single chains (Huston et al, Proc Natl Acad Sei USA 85 (1988) 5879-5883, Bird et al, Science 242 (1988). Both monoclonal and polyclonal antibodies, with monoclonal antibodies and their fragments being preferred
  • the antibodies according to the invention preferably comprise at least two light polypeptide chains and two heavy polypeptide chains.
  • Each of the heavy and light chains contains a variable region (usually denoted by the amino-terminal part of the polypeptide chain (which contains a binding domain that interacts with the antigen (human BDNF)))
  • the light and heavy polypeptide chains also contain a constant region in the polypeptide chain (usually referred to as carboxy terminus) which mediates the binding of the antibody to cells or tissues of the host organism or factors of the immune system, such as phagocytic cells or a first component (Clq) of the complement system
  • the light and heavy chains are complete chains, which consist essentially of a variable and complete constant region.
  • variable regions of the antibody according to the invention can be bound to constant regions of different isotypes
  • a polypeptide that has the variable region of an anti-human BDNF antibody heavy chain of the ⁇ l isotype can be bound to a polynucleotide that encodes the constant region of a heavy chain of another class or subclass
  • Antibody structures are described, for example, in Creighton, TE , Proteins Structures and Molecular Properties, Publ Freeman, New York (1984), Branden, C, Tooze, J, Introduction to Protein Structure, Garland Publishing, New York (1991), Fornton et al, Nature 354 (1991) 105 Die invent Antibodies according to the invention can be both monoclonal and polyclonal antibodies, fragments thereof, chimeric or humanized antibodies, as long as the characteristic properties of the high-affinity binding to human BDNF are retained. Shortened antibody fragments which, for example, only contain the CDR regions or parts thereof, are also suitable. which are able to produce high affinity for human BDNF tie.
  • the antibodies are preferably labeled (eg radioactive or enzymatic) and immobilized or derivatized so that they can be immobilized in the course of the immunological determination.
  • Antibodies of the IgGl isotype are preferred.
  • the antibodies according to the invention are preferably used for the diagnosis and determination of BDNF in body fluids such as serum or plasma or on solid tissue samples such as tissue sections.
  • the immunological determination is carried out in a manner familiar to the person skilled in the art, the antibodies being able to be used in labeled and / or immobilized form.
  • a change in a measurement signal which is based on the binding of at least one antibody according to the invention to BDNF and this is followed Binding can be assigned to a signal change.
  • signals are, for example, color reactions due to enzymatic reactions or radioactivity after separation of bound and unbound antibody to BDNF
  • the antibodies according to the invention are also for the therapeutic treatment of diseases in which a reduction in the BDNF concentration in body cells or in body fluids is advantageous for therapy.
  • An anti-human BDNF antibody for neutralizing the BDNF is particularly preferred.
  • Activity and prevention of axonal sprouting used The prevention of sprouting leads to the reduction or healing of epilepsy
  • FIG. 1 shows the cross-reaction of anti-human BDNF antibodies according to the invention with BDNF and other neurotrophins (ELISA 10 ng neurotrophin / well)
  • FIG. 2 shows the inhibition of the survival of nodosum neurons, depending on the concentration of the antibodies 4 D3 3A3 and 4 B3 9D3 (m-BDNF 1 ng) according to the invention.
  • BALB / c mice were alternately immunized intraperitoneally with fish BDNF and recombinant, human BDNF (produced in E. coli).
  • the initial immunization was carried out in complete Freund's adjuvants (CFA) - all further immunizations were carried out in incomplete Freund's adjuvants (IFA)
  • CFA complete Freund's adjuvants
  • IFA incomplete Freund's adjuvants
  • the dose was between 60 - 100 ⁇ g.
  • the immunization was carried out every 4 weeks (Table 1).
  • the total immunization period was 9 months.
  • the last three immunizations were carried out intravenously (iv) at intervals of one day.
  • the immortalization took place the spleen cells of the immunized animals with the myeloma cell line P3X63 Ag8 653
  • the fusion of the spleen cells with the myeloma cell line is carried out according to the standard method according to Goding, JW, J of Imm Meth, 39 (1980) 285-308.
  • the fusion ratio of spleen cells to myeloma cells is 1 1.
  • the fusion products are placed on 24-well culture dishes (Nunc) with HFCS (Boehringer Mannheim GmbH, cat. No.
  • the Hyb ⁇ dom cell clones thus obtained are expanded in vivo.
  • 5 ⁇ 10 ⁇ Hyb ⁇ dom cells are inoculated intraperitoneally in mice pretreated with P ⁇ stan (Sigma Chemical Company, St Louis, USA). After 10-21 days, 2-3 per mouse ml of ascites are taken and the monoclonal antibody is obtained by conventional methods.
  • the yield is about 3-10 mg IgG / ml ascites
  • An enzyme-linked immunosorbent assay (ELISA) is used to determine the specificity of the antibodies in the culture supernatant of the hybridoma cells.
  • 96-well microtiter plates (Nunc) are coated with 50 ⁇ l human BDNF, fish BDNF and chicken BDNF (100 ng / ml) in carbonate buffer (Boehringer Mannheim GmbH, cat. No. 726559), with 50 ⁇ l culture supernatant for 2 hours Incubated 37 ° C and washed with 3 x 250 ul PBS / 0.05% Tween 20. Then it is incubated with ß-Gal labeled sheep anti-mouse IgG (Amersham) for 60 minutes at room temperature, washed with 3 ⁇ 250 ⁇ l / 0.05% Tween and the Detection reaction with 50 ⁇ l methyl umbiliferyl galactoside.
  • carbonate buffer Boehringer Mannheim GmbH, cat. No. 726559
  • 20 nodosum ganglia are prepared from chicken embryos (day 8) and dissociated into single cells by trypsin treatment. After a pre-plating step, 2000 cells are pipetted into the well of a 48-well plate. The cells are stimulated overnight with human BDNF or fish BDNF, i.e. the cells survive and differentiate. Without the addition of BDNF, the cells die after 24-48 h. By adding antibodies + BDNF (1 ng) there is a strong inhibition of cell survival (Fig. 2).
  • BDNF (Peprotech / 5 - 100 ng) is separated using 10 - 20% SDS - PAGE under reducing conditions.
  • the electrophoretically separated BDNF is transferred to a PVDF membrane (BM ID no. 1722026) by blotting.
  • the non-specific binding sites are blocked with 1% RSA / PBS (30 min / Rt.), which is followed by a washing step.
  • Incubation of the primary antibody ⁇ BDNF> 4.F11.1A1 1 ⁇ g / ml
  • the secondary antibody anti-mouse IgG-POD, Fab fragments (BM Cat. No. 1500686) is incubated 1: 1000 for 1 h at 37 ° C.
  • the excess secondary antibody is removed by washing, the detection is carried out with the BM Chemiluminescence Blotting Substrate (POD) / (BM Cat.No. 1500708).
  • POD BM Chemiluminescence Blotting Substrate
  • Immunohistochemical staining is carried out according to Sternberger et al, Immunocytochemistry, Willey - New York (1986).
  • the sections are washed several times in TPS (0.05M Tris 0.1M sodium phosphate, 0.1M NaCl) and then overnight with monoclonal anti-BDNF antibody (20 ⁇ g / ml in TPS and 0.3% Triton X-100 and 1% sheep serum). After washing several times, the sections are incubated for one hour with sheep anti-mouse IgG, Fab fragments (BM 1 500 686) and then with peroxidase anti-peroxidase for one hour (BM 1 092 626). After further washing steps, the reaction is initiated with diaminobenzidine (Sigma).

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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)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Selon l'invention, un anticorps dirigé contre le BDNF humain, qui inhibe, en une concentration de 500 ng/ml, la survie de neurones afférents de ganglions à 90 %, convient particulièrement à une utilisation à des fins diagnostiques et thérapeutiques.
PCT/EP1997/005956 1996-11-08 1997-10-29 Anticorps a haute affinite diriges contre le bdnf humain, procedes permettant sa production, et son utilisation WO1998021245A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU71798/98A AU7179898A (en) 1996-11-08 1997-10-29 Highly affine antibody against human BDNF, method for the production and use thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP96117941 1996-11-08
EP96117941.3 1996-11-08
EP97100665 1997-01-17
EP97100665.5 1997-01-17

Publications (1)

Publication Number Publication Date
WO1998021245A1 true WO1998021245A1 (fr) 1998-05-22

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1548435A1 (fr) * 2002-09-24 2005-06-29 Masaomi Iyo Agent diagnostique et procede d'examen pour trouble de l'alimentation
WO2016034968A1 (fr) * 2014-09-02 2016-03-10 Pfizer Inc. Anticorps thérapeutique

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993009798A1 (fr) * 1991-11-12 1993-05-27 Regeneron Pharmaceuticals, Inc. Methodes therapeutiques et diagnostiques basees sur l'expression de nt-3 specifique de tissus et sur la liaison de recepteurs

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993009798A1 (fr) * 1991-11-12 1993-05-27 Regeneron Pharmaceuticals, Inc. Methodes therapeutiques et diagnostiques basees sur l'expression de nt-3 specifique de tissus et sur la liaison de recepteurs

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
A. GHOSH ET AL.: "Requirement for BDNF in activity-dependent survival of cortical neurons.", SCIENCE, vol. 263, no. 5153, 18 March 1994 (1994-03-18), WASHINGTON, DC, VSA, pages 1618 - 1623, XP002029406 *
K. MATSUMOTO ET AL.: "Expression of brain-derived neurotrophic factor and p145TrkB affects survival, differentiation, and invasiveness of human neuroblastoma cells.", CANCER RESEARCH, vol. 55, no. 8, 15 April 1995 (1995-04-15), BALTIMORE, MD, VSA, pages 1798 - 1806, XP002029405 *
R. GÖTZ ET AL.: "Brain-derived neurotrophic factor is more highly conserved in structure and function than nerve growth factor during vertebrate evolution.", JOURNAL OF NEUROCHEMISTRY, vol. 59, no. 2, August 1992 (1992-08-01), NEW YORK, NY, VSA, pages 432 - 442, XP000670104 *
R. GÖTZ ET AL.: "Neurotrophin-6 is a new member of the nerve growth factor family.", NATURE, vol. 372, no. 6503, 17 November 1994 (1994-11-17), LONDON, GB, pages 266 - 269, XP002029408 *
S. COHEN-CORY ET AL.: "Effects of brain-derived neurotrophic factor on optic axon branching and remodelling in vivo.", NATURE, vol. 378, no. 6553, 9 November 1995 (1995-11-09), LONDON, GB, pages 192 - 196, XP002029407 *
S. RADKA ET AL.: "Presence of brain-derived neurotrophic factor in brain and human and rat but not mouse serum detected by a sensitive and specific immunoassay.", BRAIN RESEARCH, vol. 709, no. 1, 12 February 1996 (1996-02-12), AMSTERDAM, NL, pages 122 - 130, XP000670771 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1548435A1 (fr) * 2002-09-24 2005-06-29 Masaomi Iyo Agent diagnostique et procede d'examen pour trouble de l'alimentation
EP1548435A4 (fr) * 2002-09-24 2007-12-05 Masaomi Iyo Agent diagnostique et procede d'examen pour trouble de l'alimentation
US7754434B2 (en) 2002-09-24 2010-07-13 Masaomi Iyo Diagnostic and examination method for eating disorder
WO2016034968A1 (fr) * 2014-09-02 2016-03-10 Pfizer Inc. Anticorps thérapeutique

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