EP0787150A1 - Immunogenes peptidiques synthetiques du domaine d'ancrage de la membrane de l'ige - Google Patents

Immunogenes peptidiques synthetiques du domaine d'ancrage de la membrane de l'ige

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Publication number
EP0787150A1
EP0787150A1 EP95938912A EP95938912A EP0787150A1 EP 0787150 A1 EP0787150 A1 EP 0787150A1 EP 95938912 A EP95938912 A EP 95938912A EP 95938912 A EP95938912 A EP 95938912A EP 0787150 A1 EP0787150 A1 EP 0787150A1
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EP
European Patent Office
Prior art keywords
peptide
seq
gly
leu
val
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EP95938912A
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German (de)
English (en)
Inventor
Chang Yi Wang
Alan M. Walfield
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United Biomedical Inc
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United Biomedical Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6018—Lipids, e.g. in lipopeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
    • C12N2760/18422—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to the use of a composition of a synthetic peptide immunogen comprising a target antigenic site and a helper T cell epitope covalently linked in a linear tandem form. More particularly, the present invention relates to the use of such a composition to elicit the production in healthy mammals, including humans, of high titer antibodies specific to sites on the e heavy chain of B cell-expressed membrane-bound human IgE, i.e., sites on the extracellular segment of the anchor domain of membrane-bound human e- chain and to the use of such a composition as a vaccine to provide an immunotherapy for the treatment of allergy.
  • Immunotherapy for the prevention of IgE-mediated allergic responses such as asthma and hay fever has involved desensitization or hyposensitization by administering a gradually increasing amount of an allergen to a patient to reduce the effects of subsequent exposure to that allergen 1) .
  • Limitations to such an allergen-based immunotherapy include difficulties in identifying the allergen involved and the adverse reactions frequently caused by the use of the identified allergen 0 '.
  • Other treatments for the relief of allergies employ therapeutic compounds to block the cascade of cellular events that is responsible for allergic reactions. These compounds include anti-histamines, decongestants, ⁇ 2 agonists, and corticosteroids.
  • Anti-histamines, decongestants, and ⁇ 2 agonists act on events downstream of IgE in the allergic cascade, making them palliative remedies which address allergic symptoms rather than preventative treatments which must act on events closer to the initiation of IgE- mediated allergic reactions. These palliative remedies provide relief that is short term and partial, frequently accompanied by adverse side effects. For example, anti- histamines may cause restlessness or sedation, and ⁇ 2 agonists are sometimes associated with increased morbidity in asthmatic patients.
  • corticosteroids are powerful immunosuppressants and are highly efficacious for the treatment of allergic symptoms. However, they produce adverse hormonal activities and may cause an undesirably broad immunosuppression.
  • a means of suppression selectively targeted to IgE This may be accomplished by suppressing IgE synthesis directly or indirectly. Indirect suppression can be accomplished by desensitization or by inhibition of IL-4 and other T cell-produced mediators of IgE synthesis 05 .
  • Direct suppression as suggested by Chang et al. (4) , can be accomplished by specifically targeting IgE-producing B cells with selective antibodies.
  • Chang et al. 4 and others (9) have studied human e-chains and corresponding antibodies, as well as the genes and mRNAs by which the e-chains are encoded. They have elucidated the molecular basis for the expression of two types of IgE: the secreted and membrane-bound forms by B cells committed to IgE synthesis.
  • the membrane-bound form of IgE may be distinguished from the secreted form by an additional membrane anchoring domain that extends from the C-terminus of the heavy chains and is contiguous with the CH4 constant domain of IgE.
  • the membrane-bound form is distinctive to the surface of B cells committed to IgE synthesis.
  • IgE-secreting cells By targeting such cells with antibodies specific for the exposed extracellular portion of that anchor domain, such cells may be eliminated or inactivated.
  • the mechanisms for elimination of IgE-secreting cells by such antibodies can be through antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytolysis (6,7) .
  • ADCC antibody-dependent cellular cytotoxicity
  • the reduction of circulating IgE and IgE-expressing cells by anti-IgE antibodies has been demonstrated in vivo in mice (10) , and by the inhibition in vivo of passive cutaneous anaphylaxis in a rat model. It has also been demonstrated in human IgE- secreting cell lines where anti-IgE was shown to lead to reductions in cell growth, decreased IgE accumulation and cytolysis in complement-mediated and ADCC-mediated cytolysis assays (11) .
  • mlgE membrane-bound IgE
  • Another objective is to design optimal peptide immunogens, with specific amino acid sequences taken from the human mlgE heavy chain membrane anchoring domain (SEQ ID NOS:l,2) and attached to peptides containing promiscuous human helper T cell epitopes in specified orientations which, when introduced into mammals, including humans, will stimulate production of efficacious antibodies to the sites on human mlgE anchor domain.
  • These antibodies may result in the reduction in IgE- producing B lymphocytes and thereby attenuate allergen- induced IgE production, constrain mast cell activation by IgE-allergen complexes, reduce the consequent release of chemical mediators such as histamines responsible for allergic symptoms and depress IgE- ediated passive cutaneous anaphylaxis (PCA) .
  • PCA passive cutaneous anaphylaxis
  • Another objective is to develop an effective mlgE e-chain peptide-based vaccine, employing compositions containing such linear peptide immunogens, so as to provide immunotherapy for the treatment of allergic reactions.
  • a series of linearly arranged synthetic peptides which contain either of two peptide sequences corresponding to sites on the exposed portion of the membrane anchoring domain of human mlgE (SEQ ID NOS:l,2) or their immunogenic analogs thereof together with a portion of a helper T-cell epitope (Th epitope) are made by solid phase synthesis.
  • Compositions of the invention are used to immunize healthy mammals, e.g. rats and humans for the production of high titer antisera that is specific for the mlgE anchor membrane sites (SEQ ID N0S:1,2) and free of irrelevant antibodies.
  • vaccines containing the synthetic peptide compositions as the key immunogen may also be prepared with an immunologically effective amount of linear synthetic peptide in the presence of a proper adjuvant and/or delivery vehicle. It is expected that such vaccine compositions will elicit a more focused anti-IgE peptide response than those of the peptide-carrier protein conjugates currently used by Chang et al. (4,6*8) , thus providing better immunotherapy for the treatment of allergy.
  • This invention is directed to the use of a novel group of peptide-based immunogens for the generation of high titer antibodies to mlgE anchor membrane sites (SEQ ID NOS:l,2) on human IgE e heavy chain in healthy mammals, including humans, ultimately for the treatment of IgE- mediated allergic diseases.
  • Table I shows the arrangement and the amino acid sequence of the Membrane Anchor Domain of the e heavy chain of human membrane-bound IgE (mlgE) , as deduced from the nucleic acid sequence of the predominant species of mRNA that codes for membrane-bound e chain* 7,8 '.
  • the regions on the e chain sequence of the peptides used as the target immunogens of the invention are underlined: a single underline for SEQ ID NO:l, and a double underline for SEQ ID NO:2.
  • the allergen/antibody binding crosslinks the bivalent surface-bound IgE and induces conformational changes in the distal Fc region of IgE, the region of IgE in direct contact with a high affinity Fc receptor on the mast/basophil cell surface as well as receptor site(s) on the cell surface.
  • the conformational changes activate the cell-IgE-allergen complex with the resultant release from the cell of chemical mediators, including histamine, inducing allergic symptoms and the further secretion of IgE.
  • the secretory IgE which mediates the allergic reaction is produced by terminally differentiated B cells in response to allergen.
  • B cells committed to IgE synthesis also display membrane-bound IgE (mlgE) on their surface.
  • mlgE membrane-bound IgE
  • the mlgE molecules are allergen receptors and are believed to play regulatory roles in the maturation of the B cells, and in activation of the B cells by allergen-specific T cells.
  • the mlgE is distinguishable from the secreted IgE by a membrane- anchoring segment which extends from the C-terminus of the heavy chains which serves to attach the mlgE to the cell membrane.
  • amino acid sequences for two immunogenic sites on the extracellular portion of the anchor domain were deduced (SEQ ID NOS:l,2). These sites are present on alternative forms of mlgE that result from different mRNA splicing events. Both sites are present on the predominant species of membrane-bound e-chain, in the orientation shown in Table I. The presence and specificity of these sites as well as their accessibility to antibodies were confirmed with specific antibodies' 4,8 '.
  • anti-IgE antibodies directed against such specific mlgE sites can lead to the reduction in the numbers of IgE-producing B cells and a concomitant reduction in circulating IgE, possibly through the lytic removal of the IgE-expressing cells 00 - 11 '.
  • it is desirable to target anti-IgE antibodies to the membrane-anchor domain because the domain is a surface marker specific to IgE- expressing cells. This target site is not available on secreted IgE.
  • anti-mlgE cannot bind and crosslink IgE bound to receptors on mast cells and basophils, and cannot by itself induce histamine release.
  • the removal of IgE-expressing cells in hosts suffering from allergic reactions may result in the down-regulation of IgE production and have a therapeutic outcome.
  • Such interventions employed in the treatment of allergy through the use of specific anti-IgE antibodies can be achieved either passively, through prophylactic treatment with specific "site-directed" antibodies to IgE, or, more preferably, actively, by providing the host with a vaccine comprised of site-directed peptide immunogens, to elicit the production by the host of its own site-directed anti-IgE antibodies. It is believed that active immunization will provide a more effective and longer lasting form of protection.
  • the sites on the extracellular segment of the membrane anchor domain (SEQ ID NOS:l,2), arranged on membrane-bound IgE as shown in Table I, have been confirmed as immunogenic sites that are acessible to antibodies through the cross-reactivities of the surface of IgE-secreting cells to antibodies that had been generated by immunizing animals with mlgE anchor membrane peptides coupled to a carrier protein, keyhole limpet hemocyanin (KLH) (4,8) .
  • KLH keyhole limpet hemocyanin
  • the peptide immunogens of the current invention while being substantially incapable of mediating non- cytolytic histamine release, are capable of eliciting antibodies with serological cross-reactivity with the target amino acid sequences of the extracellular region of the human mlgE membrane anchor domain (SEQ ID NOS:l,2) .
  • the initial dose e.g. 0.2-2.5 mg; preferably 1 mg, of immunogen is to be administered by injection, preferably intramuscularly, followed by repeat (booster) doses. Dosage will depend on the age, weight and general health of the patient as is well known in the vaccine and therapeutic arts.
  • immunogen relates to a synthetic peptide which is capable of inducing antibodies against the mlgE membrane anchor domain (SEQ ID NOS:l,2), leading to suppression of IgE-mediated basophil and mast cell degranulation.
  • the immunogen of the present invention includes linear synthetic peptides which contain promiscuous helper T cell epitopes (Th epitopes) .
  • the Th peptides are covalently attached to the mlgE membrane anchor domain peptide (SEQ ID NOS:l,2), with a spacer, so as to be adjacent to either the N- or C-terminus of the membrane anchor peptides, in order to evoke efficient antibody responses.
  • the immunogen may also comprise an immune stimulatory amino acid sequence corresponding to a domain of an invasin protein from the bacteria Yersinia spp 05 '. The invasin domain is attached through a spacer to a Th peptide.
  • the immunogen of the present invention minimizes the generation of irrelevant antibodies to elicit a more focused immune response to the "target sequences", the desired reactivity to mlgE membrane anchor sites (SEQ ID N0S:1,2), without producing undesirable side effects which may complicate the immunotherapy process for the treatment of allergy.
  • the desired target sequence is short, such as the 26 and 17 amino acid mlgE peptides (SEQ ID NOS:l,2) of the present invention
  • T cell-dependent antigens i.e. the presence of a T helper epitope is required to render such antigens immunogenic.
  • A is an amino acid, ⁇ -NH 2 , a fatty acid or a derivative thereof, or an invasin domain;
  • B is an amino acid
  • Th is a helper T cell epitope or an immune enhancing analog or segment thereof;
  • mlgE peptide is:
  • n is from 1 to about 10
  • m is from 1 to about 4
  • o is from 0 to about 10.
  • the peptide immunogen of the present invention comprises from about 20 to about 100 amino acid residues, preferably from about 20 to about 50 amino acid residues and more preferably from about 20 to about 35 amino acid residues.
  • A is an amino acid
  • it can be any non- naturally occurring or any naturally occurring amino acid.
  • Non-naturally occurring amino acids include, but are not limited to, ⁇ -alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, ⁇ -amino butyric acid, homoserine, citrulline and the like.
  • Naturally-occurring amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.
  • m is greater than one, and two or more of the A groups are amino acids, then each amino acid may be independently the same or different.
  • A is a fatty acid, such as stearic acid, palmitic acid or a fatty acid derivative, such as tripalmitoyl cysteine (Pam 3 Cys) , it acts as an adjuvant by enhancing the immune stimulating properties of the Th epitope 06 '.
  • the fatty acid moiety or its derivative is preferably located at the amino terminus of the mlgE peptide.
  • the peptide im uogen comprises 2 or 3 additional A moieties which are amino acids.
  • the fatty acid is selected from the group with a hydrocarbon chain of 8 to 24 carbon atoms. The hydrocarbon chain can be saturated or unsaturated.
  • A When A is an invasin domain, it is an immune stimulatory epitope from the invasin protein of a Yersinia species. This immune stimulatory property results from the capability of this invasin domain to interact with the ⁇ l integrin molecules present on T cells, particularly activated immune or memory T cells.
  • the specific sequence for an invasin domain found to interact with the ⁇ l integrins has been decribed by Brett et al ⁇ 15) , incorporated herein by reference.
  • the invasin domain (Inv) for linkage to a promiscuous Th epitope has the sequence:
  • Thr-Tyr-Gln-Phe (SEQ ID NO:3) or is an immune stimulatory ar.alog thereof from the corresponding region in another Yersinia species invasin protein.
  • Such analogs thus may contain substitutions, deletions or insertions of amino acid residues to accommodate strain to strain variation, provided that the analogs retain immune stimulatory properties.
  • n is 1 and A is ⁇ -NH 2 . In another embodiment, n is 4 and A is ⁇ -NH 2 , an invasin domain (Inv) , glycine and glycine, in that order.
  • Inv invasin domain
  • B is a spacer and is an amino acid which can be naturally occurring or the non-naturally occurring amino acids as described above.
  • Each B is independently the same or different.
  • the amino acids of B can form a flexible hinge, or spacer, to enhance the immune response to the Th epitope and mlgE peptides and their immunogenic analogs thereof.
  • Examples of sequences encoding flexible hinges are found in the immunoglobulin heavy chain hinge region. Flexible hinge sequences are often proline rich.
  • One particularly useful flexible hinge is provided by the sequence Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID N0:4) , where Xaa is any amino acid, and preferably aspartic acid.
  • Immunogenicity can also be improved through the addition of spacer residues (e.g. Gly-Gly) between the promiscuous Th epitope and the mlgE membrane anchor peptides (SEQ ID NOS:l,2) and immunogenic analogs thereof.
  • spacer residues e.g. Gly-Gly
  • the spacer glycine residues can disrupt any artifactual secondary structures created by the joining of the Th epitope with the the mlgE membrane anchor peptides (SEQ ID NOS:l,2) and immunogenic analogs thereof and thereby eliminate interference between the T and/or B cell responses.
  • the conformational separation between the helper cell and the antibody eliciting domains thus permits more efficient interactions between the presented immunogen and the appropriate Th and B cells.
  • Th is a sequence of amino acids (natural or non- natural amino acids) that comprises a Th epitope.
  • a Th epitope can consist of a continuous or discontinuous epitope. Hence not every amino acid of Th is necessarily part of the epitope. Accordingly, Th epitopes, including analogs and segments of Th epitopes, are capable of enhancing or stimulating an immune response to the mlgE membrane anchor peptides (SEQ ID NOS:l,2) and immunological analogs thereof. Th epitopes that are immunodominant and promiscuous are highly and broadly reactive in animal and human populations with widely divergent MHC types 07 ' 19 '.
  • the Th domain of the subject peptides has from about 10 to about 50 amino acids and preferably from about 10 to about 30 amino acids. When multiple Th epitopes are present (i.e. m ⁇ 2), then each Th epitope is independently the same or different.
  • Th epitope analogs include substitutions, additions, deletions and insertions of from one to about 10 amino acid residues in the Th epitope.
  • Th segments are contiguous portions of a Th epitope that are sufficient to enhance or stimulate an immune response to the mlgE membrane anchor peptides (SEQ ID NOS:l,2) and immunological analogs thereof.
  • Th epitopes of the present invention include hepatitis B surface and core antigen helper T cell epitopes (HB,Th and HB c Th) , pertussis toxin helper T cell epitopes (PT Th) , tetanus toxin helper T cell epitopes (TT Th) , measles virus F protein helper T cell epitopes (MV F Th) , Chla ydia trachomatis major outer membrane protein helper T ceil epitopes (CT Th) , diphtheria toxin helper T cell epitopes (DT Th) , Plasmodi ⁇ m falciparu circumsporozoite helper T cell epitopes (PF Th) , Schistosoma mansoni triose phosphate isomerase helper T cell epitopes (SM Th) , Escherichia coli TraT helper T cell epitopes (TraT Th) and immune-enhancing analogs and segments of any of these
  • Ser-Leu-Asp SEQ ID NO: 5
  • PT 1A Th Tyr-Met-Ser-Gly-Leu-Ala-Val-Arg-Val-His-Val-Ser- Lys-Glu-Glu (SEQ ID NO: 9)
  • MV 'pF2 Th: Gly-Ile-Leu-Glu-Ser-Arg-Gly-Ile-Lys-Ala-Arg-Ile- Thr-His-Val-Asp-Thr-Glu-Ser-Tyr (SEQ ID NO: 14) TT 4 Th: Trp-Val-Arg-Asp-Ile-Ile-Asp-Asp-Phe-Thr-Asn-Glu-
  • CT Th Ala-Leu-Asn-Ile-Trp-Asp-Arg-Phe-Asp-Val-Phe-Cys-
  • TraT 3 Th Ser-Thr-Glu-Thr-Gly-Asn-Gln-His-His-Tyr-Gln-Thr- Arg-Val-Val-Ser-Asn-Ala-Asn-Lys (SEQ ID NO:24)
  • the linear synthetic peptides of this invention as described by the formulas (A) n - (Th) m - (B) 0 - (mlgE peptide) or (mlgE peptide) - (B) 0 - (Th) m - (A) n , have the Th epitope covalently attached through spacer B to the N terminus of either of the the mlgE membrane anchor peptides (SEQ ID NOS:l,2) and immunogenic analogs thereof.
  • the Th epitope is HB S Th, PT, Th, PT 2 Th, TT, Th, TT 3 Th, or MV F , Th.
  • the sequence of the mlgE membrane anchor peptide comprises:
  • Immunogenic peptide analogs of the mlgE anchor peptides may further comprise substitutions, additions, deletions, or insertions of from one to about four amino acid residues provided that the peptide analogs are capable of eliciting immune responses crossreactive with the mlgE membrane anchor peptides (SEQ ID NOS:l,2).
  • the substitutions, additions, and insertions can be accomplished with natural or non-natural amino acids as defined herein.
  • preferred peptide immunogens of this invention are the linear synthetic peptides containing the mlgE membrane anchor peptides (SEQ ID NOS:l,2) or immunological analogs thereof and Th.
  • the more preferred peptide immunogens are those linear constructs containing the mlgE membrane anchor peptides (SEQ ID NOS:l,2) or immunogenic analogs thereof; a spacer (e.g Gly-Gly) ; a Th epitope selected from the group consisting of HB S Th, PT, Th, PT 2 Th, TT, Th, TT 3 Th, and MV ⁇ Th (SEQ ID NOS:5,6,11,7,10,12, respectively); and, optionally, an Inv domain (SEQ ID NO:3) or analog thereof.
  • peptide immunogens of this invention can be made by chemical synthesis methods which are well known to the ordinarily skilled artisan. See, for example, Grant, ed. Synthetic Peptides 00 '. Hence, peptides can be synthesized using the automated Merrifield techniques of solid phase synthesis with the ⁇ -NH 2 protected by either t- Boc or F-moc chemistry using side chain protected amino acids on, for example, an Applied Biosystems Peptide Synthesizer Model 430A or 431.
  • A is a fatty acid, it can be easily added to the N-terminal amino group of the synthesized peptide by the well known dicyclohexyl-carbodiimide coupling method.
  • Pam 3 Cys, lipoamino acid S-[2,3- Bis(palmitoyloxy) - (2R) -propyl-N-palmitoyl- (R) -cysteine may also be synthesized by chemical methods.
  • Pam 3 Cys can be coupled to, for example, the N terminus of the mlgE peptide by solid-phase synthesis using Pam 3 Cys-OH in the final coupling step to link the lipoamino acid to a resin- bound mlgE peptide chain.
  • the solid-phase peptide can be elongated with additional serine and lysine residues at the N-terminus.
  • the resin After complete assembly of the desired immunogen, the resin is treated according to standard procedures to cleave the peptide from the resin and deblock the functional groups on the amino acid side chains.
  • the free peptide is purified by HPLC and characterized biochemically, for example, by amino acid analysis or by sequencing. Purification and characterization methods for peptides are well known to one of ordinary skill in the art.
  • a cysteine can be added to the C terminus of a Th-containing peptideand the thiol group of cysteine may be used to form a covalent bond to an electrophilic group such as an N° chloroacetyl-modified or a maleimide-derivatized cu- or e-NH 2 group of a lysine residue attached to the N-terminus of an mlgE membrane anchor peptide (SEQ ID N0S:1 or 2) or immunogenic analogs thereof.
  • an electrophilic group such as an N° chloroacetyl-modified or a maleimide-derivatized cu- or e-NH 2 group of a lysine residue attached to the N-terminus of an mlgE membrane anchor peptide (SEQ ID N0S:1 or 2) or immunogenic analogs thereof.
  • the subject immunogen may also be polymerized. Polymerization can be accomplished for example by reaction between glutaraldehyde and the -NH 2 groups of the lysine residues using routine methodology.
  • the linear "A-Th-spacer- (mlgE peptide) " or " (mlgE peptide) -spacer- (Th) m - (A) n " immunogen can be polymerized or co-polymerized by utilization of an additional cysteine added to the N-terminus of the linear "A-Th-spacer- (mlgE peptide) or "(mlgE peptide) -spacer- (Th) m - (A) n " immunogen.
  • the thiol group of the N-terminal cysteine can be used for the formation of a "thioether" bond with haloacetyl- modified amino acid or a maleimide-derivatized or- or e-NH 2 group of a lysine residue that is attached to the N- terminus of a branched poly-lysyl core molecule (e.g., ⁇ 2 ⁇ , K ⁇ K ⁇ or K-g ⁇ i ⁇ K ) .
  • a branched poly-lysyl core molecule e.g., ⁇ 2 ⁇ , K ⁇ K ⁇ or K-g ⁇ i ⁇ K
  • the longer linear peptide immunogens can be synthesized by well known recombinant DNA techniques. Any standard manual on DNA technology provides detailed protocols to produce the peptides of the invention.
  • a gene encoding a peptide of this invention the amino acid sequence is reverse translated into a nucleic acid sequence, and preferably using optimized codon usage for the organism in which the gene will be expressed.
  • a synthetic gene is made, typically by synthesizing overlapping oligonucleotides which encode the peptide and any regulatory elements, if necessary.
  • the synthetic gene is inserted in a suitable cloning vector and recombinants are obtained and characterized.
  • the peptide is then expressed under suitable conditions appropriate for the selected expression system and host.
  • the peptide is purified and characterized by standard methods.
  • the efficacy of the immunogen of the present invention can be established by injecting an animal, for example, rats, followed by monitoring the humoral immune response to the mlgE membrane anchor peptides (SEQ ID NOS:l,2) and immunogenic analogs thereof, as detailed in the Examples.
  • Another aspect of this invention provides a vaccine composition
  • a vaccine composition comprising an immunologically effective amount of one or more of the immunogens of this invention in a pharmaceutically acceptable delivery system.
  • Such vaccine compositions are used for prevention of atopic allergic reactions including allergic rhinitis, those of food allergies, asthma, anaphylaxis, and other IgE-mediated hypersensitive reactions such as virally- induced asthma.
  • the subject peptides can be formulated as a vaccine composition using adjuvants, pharmaceutically-acceptable carriers or other ingredients routinely provided in vaccine compositions.
  • Such formulations are readily determined by one of ordinary skill in the art and include formulations for immediate release and/or for sustained release, and for induction of systemic immunity and/or induction of localized mucosal immunity, which may be accomplished by, for example, immunogen entrapment by microparticles.
  • the present vaccines can be administered by any convenient route including subcutaneous, oral, intramuscular, or other parenteral or enteral route. Similarly the vaccines can be administered as a single dose or multiple doses. Immunization schedules are readily determined by the ordinarily skilled artisan.
  • Adjuvants or emulsifiers that can be used in this invention include alum, incomplete Freund's adjuvant, liposyn, saponin, squalene, L121, emulsigen and ISA 720 as well as the other efficacious adjuvants and emulsifiers.
  • the vaccine composition of the instant invention contain an effective amount of one or more of the immunogens of the present invention and a pharmaceutically acceptable carrier.
  • a composition in a suitable dosage unit form generally contains about 0.5 ⁇ g to about 1 mg of the immunogen per kg body weight. When delivered in multiple doses, it may be conveniently divided into an appropriate amount per dosage unit form.
  • Vaccines which contain cocktails of the subject immunogens with two or more of the Th epitopes may enhance immunoefficacy in a broader population and thus provide an improved immune response to the mlgE membrane anchor peptide (SEQ ID NOS:l,2).
  • a cocktail of Peptide Nos. 1-4 of Example 1 and 7-10 of Example 4 is useful.
  • Other immune stimulatory synthetic peptide-based mlgE anchor peptide immunogens are arrived at through modification into lipopeptides, such as Pam 3 Cys, so as to provide a built-in adjuvant for a potent vaccine.
  • the immune response to synthetic mlgE anchor peptide- containing immunogens can be improved by delivery through entrapment in or on biodegradable microparticles of the type described by O'Hagan et al (2i) .
  • the immunogens can be encapsulated with or without an adjuvant, including covalently attached Pam 3 Cys, and such microparticles can carry an immune stimulatory adjuvant such as Freund's Incomplete Adjuvant or alum.
  • the microparticles function to potentiate immune responses to an immunogen, including localized mucosal immunity which may be especially applicable to mucosally localized allergic reactions, and to provide time-controlled release for sustained or periodic responses, for oral administration, and for topical administration 01,22 '.
  • peptide immunogens and compositions are provided in the following examples to illustrate the invention.
  • the peptide immunogen of the invention can be useful for the amelioration of IgE-mediated allergic disorders.
  • Immunogens 1-4 (Table III) were synthesized by solid phase synthesis using F-moc chemistry on an Applied Biosystems Peptide Synthesizer Model 430A or 431 according to manufacturer's instructions. After complete assembly of the peptide, the resin was treated according to standard procedures to cleave the peptide from the resin and deprotect the functional groups on amino acid side chains. The free peptide was then purified by HPLC and characterized biochemically for amino acid content and sequence.
  • Peptide immunogens Nos. 1-4 from the amino terminus to the carboxyl terminus, are symbolized as A-Th-B- (mlgE peptide) or (mlgE peptide) -B- Th-A, where "A” is NH 2 - , "B” is a Gly-Gly spacer, "Th” is the measles virus Fl helper T cell epitope MV F , Th (SEQ ID NO:12) , and "(mlgE peptide)” is either mlgE peptide of SEQ ID NO:l or mlgE peptide of SEQ ID NO:2.
  • Adjuvant Freund's Complete/Incomplete
  • Assay ELISAs for anti-peptide activity. Blood is collected and processed into serum, and stored prior to titering by ELISA with the target peptides (SEQ ID NOS:1,2) .
  • Anti-peptide antibody activities are determined by ELISA (enzyme-linked immunosorbent assay) using 96-well flat bottom microtiter plates which are coated with the corresponding target mlgE peptide epitope as the immunosorbent, either Peptide 5 for mlgE anchor peptide site described by SEQ ID NO:l or Peptide No. 6, described by SEQ ID N0:2. Aliquots (100 ⁇ L) of a peptide immunogen solution at a concentration of 5 ⁇ g/mL are incubated for 1 hour at 37°C. The plates are blocked by another incubation at 37°C for 1 hour with a 3% gelatin/PBS solution. The blocked plates are then dried and used for the assay.
  • ELISA enzyme-linked immunosorbent assay
  • the plates are washed six times with 0.05% PBS/Tween ® buffer.
  • 100 ⁇ L of horseradish peroxidase labelled goat-anti-rat antibody is added at a dilution of 1:1,000 in conjugate dilution buffer (Phosphate buffer containing 0.5M NaCl, and normal goat serum) .
  • the plates are incubated for 1 hour at 37°C before being washed as above. Aliquots (100 ⁇ L) of o-phenylenediamine substrate solution are then added.
  • the color is allowed to develop for 5-15 minutes before the enzymatic color reaction is stopped by the addition of 50 ⁇ L 2N H 2 S0 4 .
  • the A 492nm of the contents of each well is read in a plate reader.
  • the peptide immunogen compositions of this invention evoke antibodies that target IgE-secreting human B cells and inhibit IgE production. Unlike most antibodies to IgE, the mlgE peptide-elicited antibodies do not bind to cells bearing only the secretory form of IgE bound to receptors and are therefore incapable, by themselves, of triggering the release of the chemical mediators of allergic response from IgE-sensitized mast cells and basophils. These biological activities are of relevance to immunotherapy for allergy and can be observed in the rat antisera to Peptide immunogens Nos. 1-4 by assaying for the following functions:
  • a human IgE- producing B cell line for example the myeloma cell line SKO-007 (ATCC, Rockville MD) or an EBV-transformed B cell line 8866, is incubated with serial dilutions of the rat anti-mlgE and antibody binding is detected using FITC- labeled ant-rat IgG and quantitated by fluorescence flow cytometry* 4 '.
  • the extent of binding by the rat anti-mlgE antibodies to normal human basophils, prepared from peripheral blood and loaded or sensitized with secreted IgE, is evaluated in a similar fashion (5) .
  • IgE-secretin ⁇ cells Reduction in the I ⁇ E accumulation of IgE-secretin ⁇ cells.
  • IgE accumulates in the culture medium of myeloma cell line SKO-007 and in like IgE-secreting cell lines.
  • Peptide immunogens Nos. 1-4 results in a decrease of IgE secreted into the medium
  • the cells are treated with a range of antibody concentrations, and IgE levels in the medium are monitored over time by IgE-specific ELISA 01 '. Efficacious antibodies result in a dose-related reduction in accumulated IgE.
  • ADCC antibody dependent cell-mediated cytotoxicity
  • Th-spacer- (mlgE peptide) and (mlgE peptide) -spacer-Th peptides wherein there are more than one Th epitope for recognition by diverse MHC types can serve to broaden immune responsiveness in a genetically diverse human population.
  • Promiscuous Th epitopes useful for this purpose are selected from Table II.
  • the Th epitopes of Table II that are useful for such peptide cocktails include but are not limited to the MV F , Th peptide (SEQ ID NO:12) used in Peptide Nos. 1-4 (SEQ ID NOS:25-28) and the HB S Th peptide (SEQ ID NO:5) .
  • Peptides containing either of the two anchor membrane mlgE peptide sequences (SEQ ID NOS:l or 2) and the HB, Th peptide are described in Table IV as Peptide immunogens Nos. 7-10 (SEQ ID NOS: 29-32).
  • Peptide immunogens Nos. 7-10 are synthesized as described in Example 1 and combined with each other and with Peptide immunogens Nos. 1-4, in equal molar ratios, to formulate into a peptide cocktail.
  • a composition of the invention formulated as a cocktail is evaluated for immunopotency in rats by the protocol described below.
  • Alum Alluminum hydroxide
  • CFA/IFA groups receive CFA week 0, IFA weeks 2 and 4.
  • Alum groups receive Alum formulations for all 3 doses
  • This study is designed to demonstrate improved immunogenicity for this embodiment of the peptide invention, and to demonstrate efficacy for a composition of the invention formulated with a pharmaceutically acceptable adjuvant, Alum.
  • Th peptide sequences from measles virus F and hepatitis B surface antigen are promiscuous for multiple human HLA DR antigens, so as to provide maximum immunogenicity in a genetically diverse human population.
  • these Th peptides are derived from children's vaccines, childhood vaccinations are a potential source of Th memory in an immunized human population.
  • children's vaccines have the potential to afford enhanced immunopotency to anti- allergy vaccines comprised of mixtures of such Th peptides.
  • compositions of the invention formulated as a mixture of such linear "Th-Spacer- (mlgE Peptide) " and " (mlgE Peptide) -spacer-Th” peptide constructs, in a widely acceptable adjuvant, Alum.
  • Efficacy and safety of the clinical composition are evaluated by comparisons of serological tests, skin test reaction, by recording patient usage of hay fever medication, by physical examination for allergic symptoms and adverse reactions, and by interviews to obtain subjective patient assessments of the product.
  • Serological evaluations include the aforementioned ELISAs for antipeptide titers, and a standard automated spectrofluorimetric assay to determine reduction in histamine levels 03 ' as well as to ascertain that the products do not trigger histamine release.
  • the skin test is an intradermal test in which a standardized solution of allergens is injected into the upper layers of the skin. Reactions to the allergens are quantitated in the skin test by determining the area of the typical "wheal and flare" produced in response to the allergens.
  • the expected results include significant improvement in allergic symptoms at the endpoint of the study, and no evidence of histamine release. This experiment demonstrates the clinical efficacy and safety of a pharmaceutically acceptable composition of the invention.
  • ADDRESSEE Maria C.H. Lin
  • B STREET: 345 Park Avenue
  • Lys Lys Lys Leu Arg Arg Leu Leu Tyr Met lie Tyr Met Ser 1 5 10
  • Lys Lys Gin Tyr lie Lys Ala Asn Ser Lys Phe lie Gly 1 5 10 lie Thr Glu Leu 15
  • Trp Val Arg Asp lie lie Asp Asp Phe Thr Asn Glu Ser 1 5 10

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Abstract

La présente invention se rapporte à un procédé destiné à déclencher la production, chez des animaux en bonne santé, y compris des êtres humains, d'anticorps à titrage élevé spécifiques des sites se trouvant sur le segment extracellulaire du domaine d'ancrage de la chaîne lourde ε liée à une membrane de l'IgE humaine exprimée dans les cellules, à l'aide d'une composition comprenant un immunogène peptidique synthétique contenant des sites d'ancrage de la membrane extracellulaire, ceci afin de réduire la production des leucocytes B sécrétant l'IgE et celle de l'IgE induite par des allergènes. L'invention se rapporte également à l'utilisation d'immunogènes comme composants clés, conçus de manière optimale, liés à la chaîne ε de l'IgE, dépourvus de protéines porteuses, dans un vaccin synthétique afin d'assurer une immunothérapie s'appliquant au traitement des allergies. Les peptides de l'invention contiennent des séquences stimulatrices immunes, y compris un épitope des lymphocytes T auxiliaires liés en tandem, afin de faciliter la stimulation de la réponse immune vers le domaine d'ancrage de la membrane de mIgE.
EP95938912A 1994-10-25 1995-10-25 Immunogenes peptidiques synthetiques du domaine d'ancrage de la membrane de l'ige Withdrawn EP0787150A1 (fr)

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DE69718150T3 (de) 1996-03-01 2009-03-05 Novartis Ag Peptid-immunogene zur schutzimpfung gegen und behandlung von allergien
US6913749B2 (en) 1998-11-02 2005-07-05 Resistentia Pharmaceuticals Ab Immunogenic polypeptides for inducing anti-self IgE responses
US7265208B2 (en) 2001-05-01 2007-09-04 The Regents Of The University Of California Fusion molecules and treatment of IgE-mediated allergic diseases
WO2003015716A2 (fr) 2001-08-13 2003-02-27 Ige Therapeutics, Inc. Vaccins d'immunoglobuline e et leurs procedes d'utilisation
WO2004000217A2 (fr) * 2002-06-20 2003-12-31 The Trustees Of The University Of Pennsylvania Vaccins permettant la suppression de maladies allergiques liees a l'immunoglobuline e (ige) et leurs procedes d'utilisation
DE102004035337A1 (de) * 2004-07-21 2006-03-16 Merck Patent Gmbh Varianten der Gruppe 1-Allergene aus Poaceae mit reduzierter Allergenität und erhaltener T-Zellreaktivität
JP5389442B2 (ja) * 2005-09-29 2014-01-15 メディミューン，エルエルシー 膜Ｉｇ特異的抗体を同定する方法および免疫グロブリンを生成する前駆体細胞を標的化するための使用
AU2007247965A1 (en) * 2006-05-03 2007-11-15 Guthrie Foundation For Education & Research Immunoglobulin associated cell-surface determinants in the treatment of B-cell disorders
JP5723594B2 (ja) 2007-03-22 2015-05-27 ジェネンテック， インコーポレイテッド アポトーシス性抗ＩｇＥ抗体
AU2009305406A1 (en) * 2008-10-15 2010-04-22 Alk-Abello A/S Pharmaceutical product for up-dosing of allergy vaccine
EP2401300B1 (fr) * 2009-02-25 2018-01-10 Academia Sinica ANTICORPS ANTI-C[epsilon]MX CAPABLES DE SE LIER AUX mIgE HUMAINS SUR DES LYMPHOCYTES B
DE102009034779A1 (de) 2009-07-25 2011-02-03 Emc Microcollections Gmbh Synthetische Analoga bakterieller Lipopeptide und ihre Anwendung zur Therapie und Prophylaxe allergischer Erkrankungen
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JP5918851B2 (ja) * 2012-06-18 2016-05-18 日本全薬工業株式会社 ＩｇＥペプチドワクチン
CN107849119A (zh) 2015-07-07 2018-03-27 阿费里斯股份公司 用于治疗和预防IgE介导的疾病的疫苗
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