Procedure for detecting and priparing anti-IgE autoantibodies and use of these antibodies as active agents in diagnostic and therapeutic compositions
The present invention concerns a method for detecting anti-Ige autoantibodies in bodyfluids, a method for preparing auto-anti-IgE antibodies and the use of these antibodies for diagnostic and therapeutic purposes.
The existence of antibodies directed against distinct immunoglobulin isotypes is well documented. For example, anti-IgE autoantibodies have been described as rheumatoid factors and have been associated with disease ( Magnusson C.G.M., Vaerman J.P., Int.Arch.Allergy appl. Immunol. 79(1986), 149, Stevens W. J., Bridts CH., J.
Allergy clin. Immunol., 73(1984), 276, Iganäs M.,
Johansson S.G.O., Bennich H. ,Int. Archs. Allergy
Appl. Immunol. 65(1981), 51).
Anti-IgE-autoantibodies were first described in 1972 (Williams R.C., Griffith R.W., Emmons J.D., Field R.C., J. Clin. Invest: 51(1972), 955-1003). Later such specific anti-IgE autoantibodies were reported in
conjunction with different atopic disorders (Nawata Y., Koike T., Hosokawa H., Tomoika H. Yoshida S., J Immunol. 135(1985), 478; Nawata Y. Koike T., Yanagisawa T.,
Iwamoto I. Itaya T., Yoshida S., Tomioka H., clin.Exp. Immunol. 58(1984), 348; Koike T., Nawata Y., Tsutsumi A., Tomoika H., Allergy Today 2(1987), 4; and Gruber B.L., Baeza M.L., Marchese M.J., Agnello V., Kaplan A. P., J, Invest. Dermatol. 90(1988), 213) as well as in non-atopic diseases with elevated serum IgE levels (Stadler B.M., Nakajima K., Yang X., De Week A.L., Int. Arch. Allergy Appl. Immunol. 88(1989) 206-208). However, high levels of anti-IgE-autoantibodies were also encountered in normal induviduals (Stadler B.M., Nakajima K., Yang X., De Week A.L., Int. Arch. Allergy Appl. Immunol. 88(1989);
206-208, Wilson P.B., Fairfield J.E., Beech N., Int.
Archs. Allergy Appl. Immunol. 84(1987) 198).
In in-vitro experiments anti-IgE-autoantibodies have been shown to trigger histamine release from human basophils, tissue mast cells and passively sensitized human bone marrow cells (Marone G., Casolaro V.,
Paganelli R., Quinti I., J. Invest. Dermatol., 93(1989.) 246-252; Quinti I., Brozek C., Wood N., Geha R.S., Leung D.Y.M., J. Allergy Clin. Immunol. 77 (1986),586-594).
However, in other instances, anti-IgE autoantibodies failed to trigger histamine release from blood basophils (Nakajima K., de Week A.L., Stadler B.M., Allergy
44(1989), 187-191; Kemeny D.M., Tomoika H., Tsutsumi A., Koike T., Lessof M.H., Lee T.H., Clin. Exper. Allergy 20(1990), 67-69; Devey M.E., Wilson D.V., Wheeler A.W., Clin. Allergy 6(1976), 227-236). The pathophysiological role of anti-IgE autoantibodies in allergic diseases has remained unclear up to now, essentially because the methods used to detect them, such as ultracentrifugation, are technically difficult and not suitable for large scale investigations. Accordingly, no clear picture has emerged yet from literature about the harmful or possible beneficial role of anti-IgE-autoantibodies in humans.
The object of the present invention is thus to provide a simple and reliable method for a quantitative detection of anti-IgE-autoantibodies.
An object of the present invention is also a method for distinction of functional categories of human anti-IgE-autoantibodies for an assessment of their function in allergic and other diseases associated with anti-IgE-autoantibodies.
Another object of the present invention is a method for the selection of human plasmas comprising
anti-IgE-autoantibodies being useful for preparing therapeutic agents against allergic and other IgE
associated diseases.
Another object of the present invention consists in providing a method for purification of anti-IgE autoantibodies having specific properties.
Another object of the present invention is the in-vivo production of selected anti-IgE-autoantibodies of desired properties by active immunisation by using selected recombinant IgE fragments.
The present invention comprises the subjects according to the definition in the appended claims.
It was found that auto-anti-IgE antibodies can be detected either in free form or in the form of IgE-IgG complexes. Their fine specifity can be located with the use of recombinant IgE fragments
The invention is illustrated by the appended figures mentioned below and described in detail by the following Examples, concerning only special embodiments of the invention.
Fig. 1a shows the principle of a direct assay for detection of anti-IgE antibodies.
Fig. 1b shows the principle of a sandwich assay for the detection of IgE/anti-IgE autoantibody immune complexes.
Fig. 1c shows the principle of a competitive assay for determining the specifity of anti-IgE
autoantibodies.
Fig 2. is a diagram corresponding to Table 4 which shows the intradermal reaction of Rh monkey to anti-IgE antibodies.
Fig. 3 is a diagram corresponding to Table 5 showing the effect of various human sera containing various amounts of anti-IgE antibodies on histamine release induced by Le27 moAb anti-IgE on "stripped" human basophils resensitized by rIgE (CH 1-4).
Fig. 4 is a diagram showing the effect of various sera containing various amounts of anti-IgE antibodies on Rhesus skin reaction to Le 27 moAb anti-IgE.
Fig. 5 is a diagram showing the effect of human anti-IgE antibodies (pool) purified by passage on
immunosorbent columns made of various rlgE (CH 1-4 or CH 3-4) on Le 27 induced skin reaction in Rhesus monkeys.
Fig. 6 is a diagramm showing the effect of human serum after treatment with mixtures of allergen/IgG anti- allergen (postr.) versus pretreatment serum (pretr,) and serum of a classically desensitized patient on histamine release induced by allergen.
Fig. 7 is a diagram showing the effect of serum of Rhesus immunized with rlgE (Immun.) versus
pretreatment (pretr.) serum and serum of Rhesus immunized with allergen on the histamine release induced by
allergen
Example 1
This is a test with a plastic strip coated with nitrocellulose which uses purified IgE myelomas, chimeric IgE antibodies, recombinant IgE fragments and/or IgE synthetic peptides and monoclonal anti-IgE antibodies in
order to assess the presence and fine specifity of auto- anti IgE antibodies in direct assay (Fig. 1a).
In this type of assay, IgE materials are deposited on the solid phase material, usually in the form of a 1 to 2 μl dot, at a concentration varying between 100 to 1000 μg/ml. Following blocking by neutral protein of the adjacent solid phase areas, the strip (consisting preferably from PVC coated with
nitrocellulose) is incubated with the serum sample to be investigated at a dilution of 1:1 to 1:10 for a period of 1 - 18 h. After suitable washings, the strip is
incubated with a horse radish peroxidase (HRP) labelled monoclonal anti-IgG antibody, which is non-crossreactive with IgE and/or any of the IgE materials dotted. When using monoclonal antibodies against IgG subclasses (e.g. IgG1, 2, 3, 4) or other immunoglobulin classes (e.g. A, M) , the various classes of Ig anti-IgE can be determined. This second incubation is usually for a period of 1 to 2 h and is followed by incubation with chromogen. In the case of HRP labelling, the preferred chromogen is a mixture of 2-4 chloronaphtol and hydrogen peroxide. The ensuing blue dots may be measured quantitatively by a suitable refractometer.
An example of direct detection of IgG auto-anti-IgE antibodies in various samples or plasmas is shown in Table 1.
Example 2
This is a nitrocellulose/PVC strip test which uses various monoclonal anti-IgE antibodies to assess the presence of auto-anti-IgE antibodies in the form of immune complexes, in a sandwich assay (Fig 1b).
In that case, the materials dotted are well defined monoclonal anti-IgE antibodies specific for
various epitopes on the IgE molecule. Following
incubation with serum samples to be investigated, whereby free IgE and IgE contained in immune complexes will be captured, a second incubation with HRP-labelled
monoclonal anti-IgG will detect IgE/IgG anti-IgE immune complexes. Timing and quantitative evaluation of
reactions are essentially like in the Example 1 above.
An example of detection of such IgG anti-IgE immune complexes in serum samples and plasma pools is shown in Table 1.
Example 3
This is a nitrocellulose/PVC strip test, to detect the fine specifity of anti-IgE antibodies, in free or complexed form, using IgE myelomas, IgE recombinant peptides and anti-IgE monoclonal antibodies in
competitive assays (Fig 1c).
In this procedure, IgE myelomas and/or
recombinant IgE peptides are first dotted on the solid phase. Following incubation with the serum sample to be investigated for 2 to 18 h and suitable washings, a second incubation occurs with selected HRP-labelled Anti-IgE monoclonal antibodies. If the serum sample comprises IgE antibodies of the same epitope specifity as the HRP labelled anti-IgE antibody, the reaction will be
inhibited. At present, at least five different epitopes have been identified on the IgE molecule in this way.
An example of detection of auto-anti-IgE
antibodies with variable specifities in human serum is given in Table 2. This procedure enables identification of patterns of specificities, which may be linked to some specific IgE functions and form the basis of selection for immunoglobulin preparations of therapeutic use. Such identification procedures allow the preparation of anti-
allergic human auto-anti-IgE antibodies being active in blocking allergic diseases.
Besides their immunochemical detection and fine specifity determination by the above-described
techniques, auto-anti-IgE antibodies can also be
investigated by functional assays, such as the release of histamine and/or sulfido leukotrienes from blood
basophils, the effect on the binding of IgE to
lymphocytes, the killing effect on IgE bearing B
lymphocytes and the effect on IgE synthesis.
In doing so it was discovered that the auto-anti- IgE antibodies have widely different functional
properties and that these functions are linked to the fine specifity, to the avidity and possibly also to the class or subclass of auto-anti-IgE antibodies produced. The conjunction of these various assays has demonstrated indeed that some auto-anti-IgE antibodies play an
important pathological role, while other appears on the contrary to be beneficial. The above-described immunochemical assays provide a rational and efficient basis for different therapeutic approaches and development of corresponding therapeutic products, as illustrated below.
Depending on their content of auto-anti-IgE antibodies, appropriate plasmas can be selected being suitable for preparing anti-allergic immunoglobulin preparations. In such a procedure, plasmas obtained by blood donation or plasmapheresis are screened for their content in anti-IgE free or complexed antibodies by the above-described immunochemical tests: According to their content in such antibodies and their specifity, they are pooled and tested also for some functional properties (e.g. histamine release). The selected plasmas are then
processed for preparing of immunoglobulin fractions by classical techniques in the art (e.g. alcohol fraction or ion exchange chromatography).
Example 4 Human plasmas obtained by plasmapheresis are submitted to the Immunodot test for detection of anti-IgE antibodies (Table 3) and/or sulfido leukotrienes. The plasmas are also investigated for their capacity to induce histamine release from human basophils (Table 3). The plasmas possessing no, low or high levels of anti- IgE-antibodies and no or high histamine releasing
activity are pooled and immunoglobulin fractions prepared by ion exchange chromatography. When tested on Rhesus monkeys (Table 4 and the corresponding Fig. 2 ) by intradermal injection, it can be seen that human
immunoglobulin preparations devoid of auto-anti-IgE antibodies do not elicit allergic reactions. On the contrary, auto-anti-IgE antibodies with anaphylactogenic properties do so (Table 4). Some of the sera containing anaphylactogenic and non anaphylactogenic anti-IgE antibodies can inhibit the skin reaction induced in
Rhesus monkeys by murine monoclonal anti-IgE-ab Le 27 (Fig. 4).
When used on blood basophils in histamine release assay, some sera or Ig preparations devoid of
anaphylactogenic anti-IgE antibodies will block the effect of anaphylactogenic anti-IgE antibodies (Table 5 and the corresponding Fig. 3 ). A similar inhibiting effect of Ig preparations can be observed on the
immediate wheal and erythema skin reaction induced by anaphylactogenic anti-IgE antibodies, demonstrating thereby their therapeutic potential.
Sheep anti-IgE antibodies directed against human IgE have also the property to kill IgE-bearing cells which express the IgE receptor (CD 23). Similarly, some human anti-IgE antibodies possess similar potential in vitro, presumably exerting thereby an inhibiting effect on IgE synthesis in vivo.
Since the desired therapeutic properties of some anti-IgE-antibodies are associated with their fine specifity, which can be assessed by interaction with IgE recombinant fragments and anti-IgE monoclonal antibodies, as described above, it was attempted to obtain purified immunoglobulin preparations of desired anti-IgE
specificity by passing selected plasmas over affinity chromatography columns prepared with relevant IgE
recombinant fragments.
Example 5
Plasmas containing anti-IgE antibodies selected by the Immunodot test described above are passed over chromatography columns prepared with recombinant IgE-fragments. The Ig preparations are then processed according to techniques well known in the art.
Such Ig preparations show in functional tests in humans and monkeys (Fig. 5) the desired protective properties, in preventing allergic reactions. Indeed, the preparation of purified auto-anti-IgE antibodies obtained from selected plasmapheresis and passed over appropriate IgE recombinant peptide columns is able to block histamine release from leukocytes of allergic patients challenged by allergen or anti-IgE. It is also capable of blocking the histamine release from leukocytes first "stripped" of their own IgE by acid treatment, reloaded with with recombinant IgE fragment and
challenged with anaphylactogenic anti-IgE monoclonal
antibodies (Table 5). It can inhibit in Rhesus monkeys the skin reactions induced by murine monoclonal anti-IgE Le 27 antibodies (Fig. 5).
Classically, IgG antibodies specific for allergen, such as encountered spontaneously in some highly allergic patients or raised by repeated injections of allergen during hyposensitization therapy are
considered to be beneficial and to function in-vivo as blocking antibodies (Devey M.E., Wilson D.V., Wheeler A.W., Clin. Allergy 6(1976), 227-236; Djurup R., Mailing H.J., Soendergaard I., Weeke B., J. Allergy Clin.
Immunol. 76(1985), 46-55). It must be emphasized, however, that the beneficial role of such antibodies is disputed and that little correlation exists between the level of IgG anti-allergen antibodies reached during hyposensitization and the clinical benefit of that treatment (Golden D.B.K., Meyers D.A., Kagey-Sobotka A., Valentine M.D., Lichtenstein L.M., J. Allergy clin.
Immunol. 69(1982), 489-493). This has induced some authors to attempt another approach, namely the use of anti-idiotypic immunization (Saint-Remy J.M.R., Lebecque S.J., Lebrun P.M.,
Jacquemin M.G., Eur. J. Immunol. 18(1988), 1009-1014). This consists in raising anti-allergen IgG antibodies in patients, purifying such antibodies on allergen affinity chromatography columns and reinjecting to the patient with his own antibodies complexed in vitro with allergen (US-A-4 740 371). This procedure has been claimed to provide clinical benefit to various categories of
allergic patients suffering from IgE-associated diseases.
It was surprisingly found that in fact the majority of apparently allergen-specific IgG antibodies isolated by such procedure as passing through an allergen affinity chromatography column are not allergen-specific
IgG, as hitherto believed, but allergen-specific IgE bound to IgG auto-anti-IgE.
In other words, immunization with IgG anti-IgE complexed to allergen-specific IgE and allergen induces in allergic patients the beneficial immunological changes associated with immunotherapy. Since, as seen above, the functional effects of IgG auto anti-IgE may be very different according to their fine specifity, it became imperative to evaluate these therapeutic methods in terms of specific anti-IgE-antibodies.
Example 6
Plasmas from hypersensitized patients are selected on the basis of their apparent IgG specifity for allergens (e.g. such as grass pollen), on the basis of allergen-specific IgG tests and analyzed for the presence of auto-anti-IgE antibodies.
Plasma pools rich or devoid of auto-anti-IgE antibodies are used as source of immunoglobulin
preparations, which are then injected complexed with allergen intradermally. In such a case, the preparation of complexes between allegen-specific IgG in a plasma pool and increasing doses of allergen follows an
empirical schedule dictated by the patient's sensitivity to the corresponding allergen. For a grass pollen mixture, a preferred schedule is given in Table 7. The effect in allergenic individuals is assessed by
intradermal provocation and histamine release (Fig. 6). As can be seen from this Figure, the effect of such a treatment is to raise antibodies which will diminish the reactivity of the patients and of their cells to
allergens.
This technique can in principle be used also for fostering other immune responses which may rest upon IgE
activities, such as the immune defense against some parasites. The presence of blocking antibodies for IgE has been for example described in filiarosis.
Therefore active immunization with IgE
recombinant fragments, in order to raise beneficial anti- IgE antibodies in allergic patients (IgE-vaccine) becomes possible.
Since it was found, as described above, that some of the naturally occuring anti-IgE-antibodies are
beneficial in allergic patients, and that the benefit is associated with fine specifity for some IgE epitopes, it can be attempted to raise similar anti-IgE antibodies actively, by immunization with appropriate IgE fragments, which would contain only the epitopes associated with with a beneficial anti-IgE response.
Accordingly, allergen complexed IgE, recombinant IgE or fragments of IgE prepared by recombinant
techniques and selected for their association with beneficial anti-IgE are brought in suitable form for immunization and injected into experimental animals.
Anti-IgE antibodies of the desired specifity having the desired blocking anti-allergic activity can be produced actively.
Example 7 Recombinant IgE peptides of various sizes and encompassing various domains of the IgE heavy chain are produced according to combinations of procedures known in the art.
These fragments can be used as well for
diagnostic purposes, in establishing Immunodot assays for detection of auto-anti-IgE antibodies, as in purifying these antibodies by affinity chromatography columns.
In the third form of application described here, selected fragments containing the selected domains CHl-4 are used for immunization of rabbits and Rhesus monkeys.
As shown in Table 6, the antibodies raised have the functional properties required, based on the previous analysis of similar but naturally occurring human auto- anti-IgE antibodies. In particular, these antibodies are able to recognize IgE and IgE fragments. These antibodies have also the capacity to block histamine release induced by anaphylactogenic anti-IgE monoclonal antibodies or allergen (Fig. 7).
Using not entirely purified recombinant IgE peptides, a secondary effect of immunization with such peptides is to increase the natural level of IgG
antibodies against E.coli. This may not as such be undesirable, but can be avoided by using purer "IgE vaccine".
Table 7: Schedule and amounts of allergen and antibody to be used as complexes for hyposensitization therapy
Week Antibody* Allergen
(meg) (ng)
0 0.02 - 0.06 4 - 16
2 0.04 - 0.16 - 32
4 0.08 - 0.32 16 - 64
6 0.16 - 0.64 32 - 128
8 0.32 - 1.28 64 - 256
10 0.64 - 2.56 128 - 512
12 1.28 - 5.12 256 - 1024
to be continued as maintenance dose every 2nd week for one year. This schedule is only an example which may be modified according to antibody and allergen strenghts. In principle, enough anti-body must be added to the allergen in order to neutralize it for an in vitro allergen challenge of sensitized basophils and prevent mediator release under such conditions.
As antibody are used allergen-specific "IgG antibodies", which contain as well antiallergen IgE complexed with IgG anti-IgE autoantibodies.