EP0618928A1 - Antikörper gegen allergene, verfahren zu deren reinigung und eine pharmazeutische zusammensetzung, die diese enthält - Google Patents

Antikörper gegen allergene, verfahren zu deren reinigung und eine pharmazeutische zusammensetzung, die diese enthält

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Publication number
EP0618928A1
EP0618928A1 EP93902289A EP93902289A EP0618928A1 EP 0618928 A1 EP0618928 A1 EP 0618928A1 EP 93902289 A EP93902289 A EP 93902289A EP 93902289 A EP93902289 A EP 93902289A EP 0618928 A1 EP0618928 A1 EP 0618928A1
Authority
EP
European Patent Office
Prior art keywords
antibodies
allergens
immunoglobulins
pharmaceutical composition
pollen
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP93902289A
Other languages
English (en)
French (fr)
Inventor
Bertrand Basuyaux
Alain Laroze
Thierry Batard
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
FONDATION NATIONALE DE TRANSFUSION SANGUINE
Original Assignee
FONDATION NATIONALE DE TRANSFUSION SANGUINE
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 FONDATION NATIONALE DE TRANSFUSION SANGUINE filed Critical FONDATION NATIONALE DE TRANSFUSION SANGUINE
Publication of EP0618928A1 publication Critical patent/EP0618928A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to an anti-allergic composition, comprising purified immunoglobulins specifically directed against the constituents of an aqueous crude extract of grass pollen, in particular cocksfoot (dactylis glomerata).
  • the mechanism of allergy involves the interaction of the immune system with the inflammatory system. Upon first contact with the pollen grains, these penetrate the respiratory tract and release their antigens on contact with the mucous membranes, the secretions of which are essentially aqueous. The body will then synthesize class E immunoglobulins (or IgE) directed specifically against some of these antigens, then called allergens. These IgEs will bind to mast cells (tissues) and basophils (circulating) which have membrane receptors for the constant part (or Fc) of class E immunoglobulins. During subsequent contacts of the body with pollen allergens , the latter being recognized by IgE fixed to mast cells and basophils will effect bridging between these immunoglobulins.
  • IgE class E immunoglobulins
  • Immune complexes were injected into patients suffering from the same allergy, formed by the association of grass pollen allergens with purified immunoglobulins specifically directed against these allergens.
  • the immunoglobulins in question were purified from their own plasma (autologous immunoglobulins) after elimination of the class M immunoglobulins (IgM). If this clinical trial has led to a lasting improvement in the disease in patients treated with UC. MACHIELS et al. 1990. Clin. Exp. Allergy 20: 653-660), its application on a large scale seems impractical, for practical reasons (purification of a different batch of immunoglobulins for each patient) and cost (realization of a different purification column for each sick, due to possible contamination).
  • the present invention specifically aims to overcome these drawbacks; it consists in purifying immunoglobulins directed specifically against grass pollen antigens, which are obtained from a pool of plasmas from naturally immunized voluntary donors. More particularly, the subject of the present invention is a pharmaceutical composition rich in specific immunoglobulins intended for the treatment and / or prevention of allergic symptoms linked to one or more allergens, characterized in that said immunoglobulins are a mixture containing primary anti- allergen and secondary anti-isotype IgE secondary antibodies obtained from non-selected human plasmas or sera, purified by affinity chromatography on specific immobilized antigens.
  • Such preparation has many advantages; firstly, such a preparation could be available in a lyophilized form, suitable for long storage.
  • this preparation by intravenous route would allow the injection of significant amounts of active product with a limited loss in the body.
  • a nebulizable form could also be considered, very suitable for local treatment of the respiratory tract, sites of manifestation of symptoms.
  • the present invention precisely makes it possible to implement these objectives. It consists of preparing compositions rich in specific immunoglobulins for therapeutic or prophylactic uses, with a view not only to eliminating allergic symptoms during the pollen season but also to treating the disease itself, by intervening at the level of the system. immune.
  • the implementation of this invention consists in practicing an affinity chroma ⁇ tography which implements an immobilization of the water-soluble dactyl pollen antigens on an immuno-affinity support.
  • the media containing these immunoglobulins are biological liquids which can be of various sources, but more particularly human plasmas or sera. Plasmas or human sera not selected are particularly suitable for implementing the invention.
  • the antigens immobilized on the support come from a water-soluble extract of cocksfoot pollen allergens, but water-soluble extracts of pollen allergens from other grasses (timothy, fescue, ryegrass ...) can perfectly be suitable since major allergens are common to all grasses. ''
  • the resin used carries carboxyl functions and the antigens are linked to the support by their amine function.
  • the specific anti-pollen activity of the mixture is 1000 to 10,000 times greater than the starting biological fluid, this is due to the fact that each of the classes of immunoglobulins in the mixture exhibits biological activity in vitro, as we demonstrate in the examples.
  • each immunoglobulin subclass has an activity, whereas, in the scientific literature, it was admitted that only IgE and subclass • + of IgG (IgG) had an anti-allergenic effect.
  • IgG IgG
  • the second surprising factor is that, in the different classes of immunoglobulins, both the primary antibodies directed against the allergens and the secondary antibodies directed against the constant Fc part of the IgE are effective, which leads to an inhibition of the degranulation of the basophils by IgE.
  • the anti-pollen antibodies which are the subject of the present invention can be administered to humans, alone or in combination with another compound in a pharmaceutical composition. They are especially useful in preventing or treating symptoms related to grass pollen allergy.
  • compositions can be intended for general administration by systemic route, or local in nebulizable form suitable for the respiratory tracts.
  • the present invention also relates to pharmaceutical compositions containing, as active principle, anti-pollen antibodies according to the invention.
  • FIGURE 1 Inhibition of histamine release by an immunopurified fraction from plasmas rich in IgG --- anti-pollen.
  • FIGURE 2 Inhibition of histamine release by an immunopurified fraction from non-preselected plasmas.
  • FIGURE 3 In vitro activity of the fractions of chromatography on immobilized Anti-IgE.
  • EXAMPLE 1 Purification by Anti-Pollution Antibody by Immuno-Affinity Chromatography
  • cocksfoot pollen (Institut Pasteur, France) are incubated in 15 ml of distilled water with stirring for 1 hour at room temperature. After centrifugation for 5 minutes at 9000 g, the supernatant is recovered and then filtered on a 5 ⁇ m Minisart filter then 0.22 ⁇ m (Sa ⁇ orius): 14 ml at 5 mg of proteins per ml are thus recovered (the concentration is evaluated using the dosage by BCA, PIERCE kit) or 70 mg of proteins.
  • the gel is then drained on sintered glass and then washed successively with 50 ml of 0.1 M glycine citrate buffer pH 2.5 and 50 ml of 0.1 M phosphate buffer pH 7.5.
  • the test was carried out on an IBF 25 column containing 25 ml of the above-mentioned gel.
  • the chromatic system consists of:
  • a peristaltic pump (Gilson, type minipuls 2); a UV detector, DO: 280 nra (Pharmacia, S.P.M. UV - 1), equipped with an HR-10 cell; a recorder (Pharmacia, REC - 482).
  • the source to be purified is 700 ml of a mixture of plasmas diluted to half with 0.1 M phosphate buffer pH 7.5 (ie 1400 ml in total ) .
  • the 1400 ml of diluted plasma (approximately 25 g "l " 1 of protein) were injected into the column at a flow rate of 70 ml per hour, ensuring a residence time of about twenty minutes.
  • specific antibodies are eluted with citrate / glycine buffer 0.1 M pH
  • Glucose-Dextran 3% (w / v) in physiological saline.
  • Tris-albumin buffer, for washing cells 25 mM Tris, 5 mM NaCl, 5 mM KC1, 0.03% human albumin (w / v) in distilled water
  • Tris-albumin buffer - Ca 2+ - Mg 2 + for cell incubations in the histamine release experiment proper: Tris 25 mM / 5 mM NaCl, 5 mM KC1 / CaCl2, 2 H2 0 0.6 mM / Mg Cl2. 6 H2 0 1 mM, human albumin 0.03% (w / U), in distilled water
  • Cocksfoot pollen (dactylis glomerata) allergic patients were sampled for the isolation of human basophils.
  • a mixture of 26 rich plasmas anti-pollen antibodies and a fraction COMPOR t ant IgG, IgM and IgA after normal plasmas were immunopurified using the technique described in Example 1. During 2 hours under stirring and at room t Empera t ure, these immunopurified fractions are preincubated for differen t es concentrations of total immunoglobulins with cocksfoot pollen extract concentrated to 2.5 or 25 ng / ml total protein, according the
  • the histamino-release technique used is that described by May et al.
  • the fluorometric assay of histamine is carried out according to the technique of Shore et al. (1959 J. Pharmac. Exp. Ther. 121: 182-186) on an automatic chain developed by B. Lebel et al. (1983 - Anal. Biochem. 121: 25 16-29).
  • the method comprises a double extraction of histamine with butanol saturated with NaCl in an alkaline medium, then with hydrochloric acid. After alkalization, orthophthaldehyde forms a fluorochrome with histamine.
  • the first fraction tested consists of immunopurified dactyl pollen immunoglobulins from a batch of 26 plasmas preselected by ELISA method for their richness in specific IgG4.
  • the inhibitory effect of this fraction 5 is observed from a concentration 10 " ⁇ g / 1 in total immunoglobulins. This inhibitory effect increases with the concentration of immunoglobulins to be total (100% inhibition) from 10" 2 g / 1- ( Figure 1).
  • the second fraction consists of immunopurified dactyl pollen immunoglobulins from a plasma sub-fraction from a batch of non-preselected plasmas (product IgGAM, BioTransfusion France).
  • This fraction shows an inhibitory effect from the concentration of 10 "-5 g / 1 in total immunoglobulins. This inhibition is dose-dependent and almost total for an immunoglobulin concentration of 0.4 g / 1 ( Figure 2).
  • control tests were carried out, on one side with non-immunopurified immunoglobulin fractions and without specific specificity, on the other side with a sample of the batch of 26 plasmas rich in specific IgG4, not immunopurified.
  • Non-specific immunoglobulin fractions have no inhibitory effect on histamine release for total immunoglobulin concentrations ranging from 10 " ⁇ to 1 g / 1, while the sample of the batch of specific plasmas shows slight inhibition for a concentration of 1 g 1 of immunoglobulins (and more).
  • the test was carried out on an IBF 11 column containing 5.5 ml of the above-mentioned gel.
  • the chromatographic system is identical to that described in Example 1.
  • the source to be purified is a mixture of eluates containing the anti-pollen antibodies purified according to Example 1, which has been concentrated (8 ml at a rate of 1.05 g / 1 of IgG / 0.45 g 1 of IgM / 0.13 g / 1 IgA and 10 mg / 1 IgE) and dialysis 16 hours at + 4 ° C against 0.1 M phosphate buffer pH 7.5.
  • the source was injected into the column at a rate of 14 ml per hour, ensuring a residence time of about twenty minutes.
  • the retained antibodies were eluted with 0.1 M citrate / glycine buffer pH 2.5 (see Example 1).
  • the eluate is neutralized extemporaneously with solid Tris.
  • the IgG, IgM and IgA present in the eluate with the IgE are secondary antibodies or autoantibodies. This was confirmed when immunoblotting was carried out with a fraction of this eluate heated for 2 hours at 56 ° C. (destruction of Fc from IgE): the secondary antibodies no longer recognize the immobilized allergens.
  • the IgG, IgM and IgA present in the filtrate are on the other hand primary antibodies, really directed against the allergens of the pollen of da ⁇ yle (these are well revealed in immuno-imprints).
  • EXAMPLE 4 Biological Activity in Vitro of Primary Anti-Pollen Antibodies and Secondary Antibodies
  • Example 3 The biological activity of the primary and secondary anti-pollen antibodies whose preparation is described in Example 3 was evaluated in the same manner as in Example 2.
  • the fraction purified by chromatography on immobilized allergen (so-called starting fraction); - the filtrate, that is to say fraction not retained after passage through the column comprising immobilized anti-human IgE antibodies, of the starting fraction; of the eluate, that is to say the fractions retained after passage through the column comprising immobilized human anti-IgE antibodies, of the starting fraction.
  • the fraction "primary antibodies” is the fraction not retained (filtrate). It is considerably depleted in IgE, as indicated in Example 3.
  • FIG. 3 indicates an in vitro biological effect of this fraction, equivalent to that of the starting fraction (despite an absence of activity at 10 " 4 g / 1).
  • the “secondary antibody” fraction which contains the cocksfoot anti-pollen IgE is the retained fraction (eluate).
  • Figure 3 also indicates that the biological effect of this fraction is little different from that of the starting fraction
  • This activity is provided by all classes of immunoglobulins, namely IgG, IgM, IgA and IgE.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Botany (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
EP93902289A 1991-12-03 1992-12-03 Antikörper gegen allergene, verfahren zu deren reinigung und eine pharmazeutische zusammensetzung, die diese enthält Withdrawn EP0618928A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FR9114954 1991-12-03
FR9114954A FR2684384B1 (fr) 1991-12-03 1991-12-03 Anticorps anti-allergenes, procede de purification de ces anticorps et composition pharmaceutique les contenant.
PCT/FR1992/001139 WO1993011160A1 (fr) 1991-12-03 1992-12-03 Composition pharmaceutique contenant des anticorps anti-allergenes, procede de purification de ces anticorps et anticorps ainsi obtenus

Publications (1)

Publication Number Publication Date
EP0618928A1 true EP0618928A1 (de) 1994-10-12

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EP93902289A Withdrawn EP0618928A1 (de) 1991-12-03 1992-12-03 Antikörper gegen allergene, verfahren zu deren reinigung und eine pharmazeutische zusammensetzung, die diese enthält

Country Status (3)

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EP (1) EP0618928A1 (de)
FR (1) FR2684384B1 (de)
WO (1) WO1993011160A1 (de)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08501799A (ja) * 1992-12-21 1996-02-27 タノックス バイオシステムズ インコーポレイテッド アレルゲン特異的ＩｇＡモノクローナル抗体及びアレルギー治療のための関連物質

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1336818C (en) * 1987-04-16 1995-08-29 International Institute Of Cellular And Molecular Pathology Treatment of allergy and composition thereof
JPH0325366A (ja) * 1989-06-22 1991-02-04 Dai Ichi Pure Chem Co Ltd 特異抗体の測定法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9311160A1 *

Also Published As

Publication number Publication date
WO1993011160A1 (fr) 1993-06-10
FR2684384A1 (fr) 1993-06-04
FR2684384B1 (fr) 1995-05-12

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