EP0618928A1

EP0618928A1 - Pharmaceutical composition containing anti-allergenic antibodies, method of purifying said antibodies and antibodies thus obtained

Info

Publication number: EP0618928A1
Application number: EP93902289A
Authority: EP
Inventors: Bertrand Basuyaux; Alain Laroze; Thierry Batard
Original assignee: FONDATION NATIONALE DE TRANSFUSION SANGUINE
Current assignee: FONDATION NATIONALE DE TRANSFUSION SANGUINE
Priority date: 1991-12-03
Filing date: 1992-12-03
Publication date: 1994-10-12
Also published as: FR2684384B1; WO1993011160A1; FR2684384A1

Abstract

La présente invention concerne une compositon pharmaceutique riche en immunoglobulines spécifiques destinée au traitement et à la prévention des symptomes allergiques liés à un ou plusieurs allergènes, caractérisée en ce que lesdites immunoglobulines sont un mélange contenant des anticorps primaires anti-allergène et des anticorps secondaires principalement anti-isotype d'IgE obtenus à partir de plasmas ou sérums humains, non sélectionnés, purifiés par chromatographie d'affinité sur des antigènes spécifiques immobilisés. Elle concerne également un procédé de purification de ces anticorps et les anticorps ainsi obtenus.The present invention relates to a pharmaceutical composition rich in specific immunoglobulins intended for the treatment and prevention of allergic symptoms linked to one or more allergens, characterized in that the said immunoglobulins are a mixture containing primary anti-allergen antibodies and mainly secondary anti-allergen antibodies. - IgE isotype obtained from unselected human plasma or serum, purified by affinity chromatography on immobilized specific antigens. It also relates to a process for purifying these antibodies and the antibodies thus obtained.

Description

PHARMACEUTICAL COMPOSITION CONTAINING ANTI-ALLERGENIC ANTIBODIES, METHOD FOR PURIFYING THESE ANTIBODIES AND ANTIBODIES THUS OBTAINED.

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).

In Western Europe, around 15% of the population have type I allergy (or immediate hypersensitivity), half of whom suffer from an allergy to grass pollen. The grass species involved are mainly herbaceous (as opposed to cereals) of which cocksfoot (dactylis glomerata), in France, is the most allergenic. Symptoms range from seasonal allergic rhinitis (hay fever) to bronchial asthma, to anaphylactic shock (which can be fatal).

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. These bypasses induce the release of chemical mediators contained by mast cells and basophils, mainly histamine, following a cascade of complex biochemical reactions. Histamine then causes vascular hyperpermeability followed by extravasation of blood constituents (case of allergic rhinitis), or even hypersecretion of mucus in the lungs with bronchoconstriction (case of bronchial asthma).

To control the symptoms of allergy, certain treatments which act on the inflammatory system or on the respiratory system are effective; these are antihistamines and corticosteroids (action on the inflammatory system), or bronchodilators (action on the respiratory system). But these treatments, if they have a demonstrated effect on the symptoms, do not cure the allergic disease itself. In this way, a treatment at the level of the immune system seems more promising. 5 Furthermore, desensitization treatments by intramuscular injections of allergens have not reported any serious efficacy; however, they have made it possible to highlight the role of immunoglobulins of the subclass G. (or Ig G ^) in a possible anti-allergic power.

Specifically, the injection of immunoglobulins seems to be a solution for the future. The injection of immunoglobulins without specific specificity in patients allergic to grass pollen, before and at the very beginning of the pollen season, results in a reduction in allergic symptoms and in the taking of antihistamines during the pollen season, by report to a control group (3.P. GIRARD et al. 1988. Clin. Allergy JJ8: 393-400).

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. For each of these patients, 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.

The administration of 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. Preferably, 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. ''

Preferably, the resin used carries carboxyl functions and the antigens are linked to the support by their amine function.

A technique for coupling pollen antigens to a support has certainly already been described (3.3. MACHIELS et al. See above). However, it does not concern the water-soluble constituents of cocksfoot pollen, it includes the elimination of IgM contained in the medium to be purified (these IgM can be the support of a possible anti-allergic power) before the immunopurification itself , and shows a very low capacity, hence its character unsuitable for large-scale production of immunoglobulins.

The originality of the technique used according to the present invention and described in more detail below makes it possible to obtain efficiently and in a single step a mixture of IgG, IgM, IgA and IgE, directed against the antigens water-soluble pollen.

After the chromatography, 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.

The surprising nature of this important anti-pollen activity is due to two factors:

The first is that 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. 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.

Given their protein purity, which can be greater than or equal to 50%, 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.

These 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.

The examples and figures given below without implied limitation, in particular for the support which may be any solid phase which may have carboxyl functions suitable for being grafted with the allergens described, make it possible to demonstrate the characteristics of the product and to highlight other features and advantages of the present invention.

In these examples, reference is made to the following figures: 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

1) MATERIAL AND METHODS

1.1 Preparation of the support

a) Extraction of allergens from pollen

753 mg of 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 μm Minisart filter then 0.22 μm (Saπorius): 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.

b) Preparation of the gel

In parallel, 6 g of dry BrCN-Sepharose gel (Pharmacia) are suspended for 15 minutes in 1 liter of an HCl solution l (h ³ M.

Then the gel is wrung on sintered glass. The gel is then washed with 50 ml of coupling buffer (NaH CO30.1 M buffer, 0.5 M NaCl, pH = 8.3) and then wrung on sintered glass.

c) Coupling of allergens

In a 50 ml test tube allowing a hermetic closure, the gel then the 14 ml of allergens and finally 14 ml of coupling buffer are successively introduced. The test piece is sealed and then stirred (rotary shaker) .; the coupling reaction is carried out for 16 hours at + 4 ° C.

The gel is then drained and washed on sintered glass with 20 and 60 ml of coupling buffer. Each filtrate is carefully collected: their volume is specified and their protein concentration measured using the BCA assay (PIERCE kit). Thus, the coupling yield can be determined = 48% (i.e. 2.8 mg of proteins per ml of gel). Then the gel is introduced into a new test of 50 ml and 25 ml of a 1 M ethanolamine solution pH8 are added. The residual active sites not coupled to the allergens are thus saturated; the reaction is carried out for 2 hours at room temperature. The gel is spun on sintered glass and undergoes 5 successive series of washing; each series is carried out with 50 ml of two buffers: sodium acetate buffer 0; I M. 0.5 M NaCl, pH 8.

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.

1.2 Immunopurification assay

a) C rcmatQgraphis

The test was carried out on an IBF 25 column containing 25 ml of the above-mentioned gel. In addition to the column, 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. After washing with phosphate buffer 0.1 M pH 7.5, specific antibodies are eluted with citrate / glycine buffer 0.1 M pH

2.5 (21 g citric acid of pM 210.14 + 6 g of glycine of PM 75, qs 1 1 of PPI water). The eluate is neutralized extemporaneously with solid Tris. b) Analytical monitoring

Assays of the immunoglobulin classes G, M and A in the eluate. They are performed by nephelometry. An immunochemical analyzer (Beckman ISC II immuno-chemistry) is used. Ig E assays in the eluate. They are made using an EIA kit (Pharmacia). Electrophoresis on polyacrylamide gel 4/30 (Pharmacia) in the presence of SDS.

2) RESULTS

Table 1 presented below reports the results obtained:

Knowing that the total volume of the eluate (after neutralization) is 58 ml, we thus purified from 700 ml of a plasma mixture:

EXAMPLE 2 In Vitro Biological Activity of Anti-Pollen Antibodies

The biological activity of the anti-pollen antibodies purified from plasmas selected or not selected by the method described in Example 1. was evaluated in vitro using the histamine release test by the basophils of allergic patients brought into contact of allergens, the ability of antibodies to inhibit this release of histamine was evaluated on this model. 1) MATERIAL AND METHODS

a) Preparation of solutions and buffers

125 mM EDTA pH 7.4 in distilled water.

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

(pH 7.6).

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

(pH = 7.6).

b) Patients

Cocksfoot pollen (dactylis glomerata) allergic patients were sampled for the isolation of human basophils.

c) Pollen extract

50 mg of cocksfoot pollen supplied by the Institut Pasteur (Paris, France) are incubated with shaking with 450 μl of distilled water for 1 h at room temperature, then centrifuged at 9,000 g. The supernatant constitutes the pollen extract. d) Immunoglobulin fractions

A mixture of 26 rich plasmas anti-pollen antibodies and a fraction COMPOR _t ant IgG, IgM and IgA after normal plasmas (product marketed by IgGAM Biotransfusion, France) 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

1 0 sensi _b ili _ty basophils tested (previously determined). The dilutions are made in Tris-albumin-Ca -Mg. e) Histamine release experience

The histamino-release technique used is that described by May et al.

(1970 Allergy 4 £: 73-89).

In this example, all the fractions are tested on basophils from the same allergic patient.

20 f) Determination of histamine

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.

302) RESULTS

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).

In addition, 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).

This study shows that the immunopurification method described makes it possible to isolate and concentrate immunoglobulins having an inhibiting power on the release of histamine from human basophils brought into contact with allergens.

In addition, it shows that the selection of specific plasmas does not increase the inhibitory effect of the immunoglobulins purified by the method of the invention.

EXAMPLE 3 Separation of "Primary" Anti-Pollen and "Secondary" Antibodies (Anti-Pollen IgE from a Fraction of Immunopurified Anti-Pollen Antibody Resulting from a Mixture of Plasmas Rich in Specific Antibodies

1) MATERIAL AND METHODS

1.1 Preparation of the support

a) Anri-IgE antibodies

They are goat immunoglobulins directed against the Fc of human IgE (Nordic Immunology).

20 mg of these antibodies (under 8 ml) are dialyzed against 2 liters of 0.1 M NaH CO3 buffer, 0.5 M NaCl pH 8.3, 16 hours at + 4 ° C. with stirring. b) Preparation of the gel and coupling of the antibodies

The methodology is that described in Example 1.

Chromatography

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. After washing with 0.1 M phosphate buffer pH 7.5, 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 filtrate and the eluate are collected carefully. The assays of the different classes of immunoglobulins are carried out as in Example 1.

2) RESULTS

Table 2 presented below reports the results obtained.

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 daαyle (these are well revealed in immuno-imprints). EXAMPLE 4 Biological Activity in Vitro of Primary Anti-Pollen Antibodies and Secondary Antibodies

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.

In Figure 3, the in vitro biological activities are compared

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

These results indicate that the inhibitory power of the release of histamine, of the purified fraction according to Example 1, is carried both by directed primary immunoglobulins, against pollen allergens and by secondary immunoglobulins or anti-autoantibodies. -IgE, and this, in an equivalent way. CONCLUSION

All the results presented in the examples above, indicate that it is possible to purify, from human plasmas, selected or not, in one step, a mixture of immunoglobulins specific for allergens and having anti activity. - o significant allergen.

This activity is provided by all classes of immunoglobulins, namely IgG, IgM, IgA and IgE.

5 It is also made up of primary anti-allergen antibodies and secondary antibodies or auto-antibodies against IgE.

The use of such a mixture of immunoglobulins purified from normal plasmas is therefore particularly promising for the prevention and treatment of o allergy.

Claims

1. Pharmaceutical composition rich in specific immunoglobulins intended for the treatment and prevention of allergic symptoms linked to one or more allergens, characterized in that said immunoglobulins are a mixture containing primary anti-aliergene antibodies and secondary antibodies mainly anti-isotype IgE obtained from unselected human plasmas or sera, purified by affinity chromatography on specific immobilized antigens.

2. Pharmaceutical composition according to claim I, characterized in that the antibodies are directed against the water-soluble fraction of grass pollen allergens.

I- ⁵ 3. Pharmaceutical composition according to one of claims

1 and 2, characterized in that it contains a mixture of IgG, IgM, IgA and

ι *. Pharmaceutical composition according to one of claims 1 to 3, characterized in that it contains a mixture of primary anti-pollen antibodies 0, and secondary antibodies, mainly anti-isotype of IgE, or autoantibodies.

5. Pharmaceutical composition according to one of claims l to *, characterized in that the antibodies have a protein purity greater than or equal to 50%. 5 6. Method for purifying antibodies, characterized in that it comprises an immuno-affinity chromatography step using allergens common to grass pollens, immobilized on a support.

.7. Purification process according to claim 6, characterized 0 in that the allergens used preferably come from dactylis glomerata.

8. Purification method according to one of claims 6 and 7, characterized in that the allergens are immobilized on a support so as to leave their epitopes accessible to antibodies. 9. Purification process according to one of claims 6 to 8, characterized in that the support is a resin which has carboxyl functions and that the antigens are linked to the support by their amino functions.

10. Antibodies obtainable by the method according to one of claims 6 to 9, characterized in that they are used for the prevention or treatment of allergies.