Title:  Peptide immunogens for vaccination against and treatment of allergy

United States Patent:  6,610,297

Issued:  August 26, 2003

Inventors:  Kricek; Franz (Biedermannsdorf, AT); Stadler; Beda (Bern, CH)

Assignee:  Novartis AG (Basel, CH)

Appl. No.:  125641

Filed:  August 21, 1998

PCT Filed:  February 28, 1997

PCT NO:  PCT/EP97/01013

PCT PUB.NO.:  WO97/31948

PCT PUB. Date:  September 4, 1997

Abstract

Disclosed are compositions for treating allergies that include either BSW17 mimotope peptides or antibodies raised against BSW17 mimotope peptides. Also disclosed are methods for the treatment of allergies which involve administration of BSW17 mimotope peptides or the administration of antibodies raised against BSW17 mimotope peptides.

SUMMARY OF THE INVENTION

It has now been found that the problems intrinsically linked to the "classical" vaccine approach are overcome by using BSW17 mimotopes for active immunization, either as chemically synthesized peptides coupled to appropriate carriers, or as recombinant fusion constructs (e.g. with ovalbumin, IgG, etc.).

BSW17 is a monoclonal antibody which recognizes a conformational epitope on Fc.epsilon. with at least part of it residing within C.epsilon.3. The hybridoma cell-line producing monoclonal antibody BSW17 has been deposited on Dec. 19,/1996 with ECACC Salisbury, Wilthuv, SP4 OJE, United Kingdom under the provisions of the Budapest Treaty on the deposit of microorganisms, under deposit number 96121916. BSW17 or BSW17-like antibodies circulating within the vascular system protect from allergic reactions by

a) inhibiting the triggering of mast cells and basophils through competitive inhibition of the IgE/IgERI interaction and

b) lowering serum IgE levels through downregulation of IgE synthesis at the B cell level.

BSW17 "mimotope" peptides have now been identified by random peptide phage display library screening, i.e. peptides which mimic at least part of the complex conformational epitope on the IgE molecule. Chemically synthesized mimotope peptides coupled to an immunogenic carrier protein can be used e.g. as vaccines for the specific generation of antibodies in an allergic host which inhibit mast cell/basophil triggering by blocking IgE/Fc.epsilon.RI.alpha. binding and/or IgE synthesis. As mimotopes of an anti-IgE antibody they induce an immune response which results in the production of BSW17-like antibodies in the host. Since BSW17 has been shown to be non-anaphylactogenic, inhibitory to IgE/Fc.epsilon.RI binding and IgE synthesis on B cells, these antibodies raised against the BSW17 mimotope-based vaccines have analogous protective properties. The immune response is very specific since, in contrast to the "classical vaccine, approach", no IgE-derived protein fragments are present which could generate crosslinking antibodies in the immunized patients.

The invention thus concerns immunogenic molecules comprising

(a) at least one moiety of a BSW17 mimotope peptide and

(b) a moiety capable of eliciting an immune response against that peptide, hereinafter briefly named "the immunogens of the invention".

Component (a) preferably consists of up to five, preferably one or two, especially one moiety of a BSW17 mimotope peptide. Component (b) preferably is a conventional immunogenic carrier as set out hereunder, especially BSA or KLH.

The BSW17 mimotope peptide in component (a) preferably is up to about 15 amino acids altogether, it is e.g. one of the sequences (A) to (Q) (Seq.id.no. 1 to no. 17) hereafter. However, it may appropriately include further components for hapten-carrier binding, e.g. to facilitate coupling to component (b) or further processing. Thus, when the BSW17 mimotope peptide is cyclic, the two ends can e.g. be held together by two additional cystein residues forming a disulfide bridge, or the ends can be chemically crosslinked, e.g. with lysine; or when the BSW17 mimotope peptide is linear, the carboxy terminal amino acid may conveniently be blocked by amidation, and/or the amino terminal amino acid may conveniently be blocked by acetylation. Further, the BSW17 mimotope peptide moieties in component (A), e.g. the preferred moieties (A) to (Q) hereafter, may be flanked in the immunogens of the invention by a few, preferably one or two, additional ancillary groups, such as acetyl, cysteine or lysine, and/or an additional coupling group, such as DC or BSS.

The antibodies elicited by the immunogens of the invention, in contrast to the antibodies produced by hybridoma BSW17, will be endogenous and thus, in a patient, human; they may be used for prophylactic treatment.

They may be prepared by appropriately coupling components (a) and (b) as defined above.

DETAILED EXPLANATION OF THE INVENTION

The immunogens of the invention are e.g. in the form of a polymeric peptide or a recombinant fusion protein, whereby a monomeric component of the polymeric peptide, or one partner of the fusion protein, constitutes a moiety of a BSW17 mimotope peptide (a) and the remainder of the polymeric peptide or fusion protein constitutes the immune response-eliciting moiety (b).

They especially are in the form of a conjugate of at least one BSW17 mimotope peptide moiety (a) and an immunogenic carrier moiety (b).

Preferred BSW17 mimotope peptide moieties, i.e. component (a), of the immunogens of the invention essentially consist of or contain an amino acid sequence selected from

More preferred are (A), (D) and (G) above, especially (A) and (D).

The invention also concerns pharmaceutical compositions, especially vaccines, comprising immunogen molecules as defined above and an adjuvant.

It also concerns ligands, i.e. antibodies or fragments derived therefrom directed against BSW17 mimotope peptides used in "passive immunization" (see below), whereby the antibody or antibody fragments also recognize the natural epitope for BSW17 on human IgE; namely, it concerns ligands comprising an antibody domain specific for a moiety of a BSW17 mimotope peptide as defined above, whereby the antibody domain is reactive also with the sequence of amino acids on the heavy chain of IgE which comprises the natural epitope recognized by BSW17. Such ligands can be generated in mammals as polyclonal or monoclonal antibodies; they preferably are in the form of monoclonal antibodies, preferably in the form of an Fab' fragment or an F(ab')₂ fragment thereof.

It further concerns a process for the preparation of an immunogen of the invention, comprising covalently coupling

a) at least one moiety of a BSW17 mimotope peptide with

b) a moiety capable of eliciting an immune response against that peptide.

It also concerns immunogenic molecules as defined above, for use as a pharmaceutical, e.g. in the treatment of IgE-mediated diseases such as allergy and atopic dermatitis.

It further concerns the use of immunogenic molecules as defined above in the preparation of pharmaceutical compositions, especially vaccines, against IgE-mediated diseases, in particular allergy and atopic dermatitis.

It further concerns a method of prophylactic or curative immunization against IgE-mediated diseases such as allergies and atopic dermatitis comprising the administration of a therapeutically effective amount of immunogenic molecules as defined above to a patient in need of such treatment.

The immunogens of the invention, while being substantially incapable of mediating non-cytolytic histamine release, are capable of eliciting antibodies with strong serological cross-reactivity with the target amino acid sequences of the Fc region of IgE. They are thus useful in, or as, vaccines.

The initial dose of immunogen (e.g. from about 0.2 mg to about 5 mg, especially about 1 mg) is for example administered intramuscularly, followed by repeat (booster) doses of the same 14 to 28 days later. Doses will of course depend to some extent on the age, weight and general health of the patient. Immunization may be "active" or "passive".

In "active" immunization the patient receives immunogen of the invention and thereby an anti-hIgE response is actively induced by the patient's immune system.

"Passive" immunization is achieved by administering anti-BSW17 mimotope antibodies, either polyclonal or monoclonal, to a patient suffering from IgE-mediated disease, preferably by injection.

Polyclonal anti-BSW17 mimotope antibodies can be prepared by administering immunogen of the invention, preferably using an adjuvant, to a non-human mammal and collecting the resultant antiserum. Improved titres can be obtained by repeated injections over a period of time. There is no particular limitation to the species of mammals which may be used for eliciting antibodies; it is generally preferred to use rabbits or guinea pigs, but horses, cats, dogs, goats, pigs, rats, cows, sheep, etc., can also be used. In the production of antibodies, a definite amount of immunogen of the invention is e.g. diluted with physiological saline solution to a suitable concentration and the resulting diluted solution is mixed with complete Freund's adjuvant to prepare a suspension. The suspension is administered to mammals, e.g. intraperitoneally, e.g. to a rabbit, using from about 50 .mu.g to about 2500 .mu.g immunogen of the invention per administration. The suspension is preferably administered about every two weeks over a period of up to about 2-3 months, preferably about 1 month, to effect immunization. Antibody is recovered by collecting blood from the immunized animal after the passage of 1 to 2 weeks subsequently to the last administration, centrifuging the blood and isolating serum from the blood.

Monoclonal anti-BSW17 mimotope antibodies may e.g. be human or murine. Preferably, the patient will be treated with an Fab' fragment preparation from murine monoclonal antibody or a chimeric human-mouse antibody (comprising human Fc region and mouse Fab' region) so as to minimize any adverse reaction to the foreign animal immunoglobulin. Murine monoclonal antibodies may be prepared by the method of Kohler and Milstein (Kohler, G. and Milstein, C., Nature 256 [1975] 495), e.g. by fusion of spleen cells of hyperimmnunized mice with an appropriate mouse myeloma cell line. Numerous methods may be utilized to raise human monoclonal antibodies, including production by:

(1) Epstein-Barr virus (EBV)--transformed B-cells;

(2) cell line for B lymphocyte hybridization;

(3) human-murine hybridomas;

(4) human-human hybridomas;

(5 ) human x human-mouse heterohybridomas; and

(6) repertoire cloning (phage display).

Human x human-mouse heterohybridomas are the most preferred, and involve combining favourable characteristics of both human and murine parental cell types. Human-mouse heterohybridoma cell lines have been rendered suitable for B cell fusion (Teng, N. N. M. et al., Proc. Natl. Acad. Sci. USA 80 [1983] 7308).

When used for immunization, antibody can be introduced into the host most conveniently by intramuscular injection. Any conventional liquid or solid vehicle may be employed which is acceptable to the host and does not have adverse side effects on the host nor detrimental effects on the vaccine. Phosphate-buffered saline (PBS), at a physiological pH, e.g. about pH 6.8 to 7.2, preferably about pH 7.0, may be used as a vehicle, alone or with a suitable adjuvant, such as an aluminium hydroxide-based adjuvant. The concentration of immunogenic antigen may vary from about 50 .mu.g to about 500 .mu.g, preferably from about 200 .mu.g to about 300 .mu.g per injection, in a volume of solvent generally of from about 0.25 ml to about 1 ml, preferably about 0.5 ml. Multiple injections will be required after the initial injection and may be given e.g. at annual intervals.

As regards "active" immunization, this is preferred for human use, but other mammalian species may be treated similarly, using analogous mimotopes corresponding to the IgE of these specie, as e.g. in the dog. The term "immunogenic carrier" herein includes those materials which have the property of independently eliciting an immunogenic response in a host animal and which can be covalently coupled to polypeptide either directly via formation of peptide or ester bonds between free carboxyl, amino or hydroxyl groups in the polypeptide and corresponding groups on the immunogenic carrier material, or alternatively by bonding through a conventional bifunctional linking group, or as a fusion protein.

Examples of such carriers include: albumins, such as BSA; globulins; thyroglobulins; hemoglobins; hemocyanins (particularly Keyhole Limpet Hemocyanin [KLH]); proteins extracted from ascaris, e.g. ascaris extracts such as those described in J. Immun. 111 [1973] 260-268, J. Immun. 122 [1979] 302-308, J. Immun. 98 [1967] 893-900, a Am. J. Physiol. 199 [1960] 575-578 or purified products thereof; polylysine; polyglutamic acid; lysine-glutamic acid copolymers; copolymers containing lysine or ornithine; etc. Recently, vaccines have been produced using diphteria toxoid or tetanus toxoid as immunogenic carrier material (Lepow M. L. et al., J. of Infectious Diseases 150 [1984] 402-406; Coen Beuvery, E. et al., Infection and Immunity 40 [1983] 39-45) and these toxoid materials can also be used in the present invention. The purified protein derivative of tuberculin (PPD) is particularly preferred for utilization in the "active" immunization scheme since (I) it does not induce a T-cell response itself (i.e. it is in effect a "T-cell hapten"), and yet behaves as a fully processed antigen and is recognized by T-cells as such; (2) it is known to be one of the most powerful hapten "carriers" in the linked recognition mode; and (3) it can be used in humans without further testing.

As hapten-carrier binding agents, those conventionally employed in the preparation of antigens can be employed, e.g. those set out above, or in the Examples hereunder.

The process of the invention for covalently coupling component (a) to moiety (b) can be effected in known manner. Thus, for example, for direct covalent coupling it is preferred to utilize bis-N-succinimidyl derivatives, more preferably bis(sulfosuccinimidyl)suberate (BSS) as coupling agent. Glutaraldehyde or carbodiimide, more preferably dicyclohexyl-carbodiimide (DC) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide may also be used for covalent coupling of peptide (a) to immunogenic carrier material (b).

The amounts of hapten and hapten-carrier binding agent [i.e. component (a)] and carrier [i.e. component (b)] can be readily ascertained in conventional manner. It is preferred that the carrier be employed in an amount of about 1 to about 6 times, preferably about 1 to about 5 times the weight of the hapten, and the hapten-carrier binding agent be employed in an amount of about 5 to about 10 times the molar equivalent of the hapten. After reaction, the carrier is bound to the hapten via the hapten-carrier binding agent to obtain the desired antigen composed of a peptide-carrier complex. The resultant immunogen of the invention can be readily isolated in conventional manner, e.g. by dialysis, gel filtration, fractionation precipitation, etc.

The preparation of the starting materials may be effected in conventional manner. Appropriate peptides for use as component (a) may e.g. be identified by screening of random peptide phage display libraries, and readily synthesized e.g. by conventional solid phase procedures, e.g., for cyclic peptides, by the solid phase procedure employing the well-known "F-moc" procedure, or may alternatively be identified using a peptidomimetic strategy by screening of randomly synthesized peptides.

Claim 1 of 16 Claims

What is claimed is:

1. An immunogenic molecule comprising

(a) at least one mimotope peptide-comprising moiety which comprises a mimotope peptide of up to 15 amino acids, said peptide being a mimotope of the monoclonal antibody produced by the hybridoma cell line deposited under European Collection of Cell Cultures deposit number 96121916, wherein the mimotope peptide is not Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe (SEQ ID NO:28); and

(b) an immunogenic carrier moiety.

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