WO1992011859A1 - Peptide-mediated hyposensitization of the human t cell response to dermatophagoides spp. (house dust mite) - Google Patents

Peptide-mediated hyposensitization of the human t cell response to dermatophagoides spp. (house dust mite) Download PDF

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Publication number
WO1992011859A1
WO1992011859A1 PCT/US1992/000205 US9200205W WO9211859A1 WO 1992011859 A1 WO1992011859 A1 WO 1992011859A1 US 9200205 W US9200205 W US 9200205W WO 9211859 A1 WO9211859 A1 WO 9211859A1
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allergen
peptide
molecules
derived
cell
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PCT/US1992/000205
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French (fr)
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Jonathan R. Lamb
Robyn E. O'hehir
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Immulogic Pharmaceutical Corporation
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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43531—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from mites
    • 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
    • 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
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • atopic allergy is applied to a group of allergies, including asthma, hay fever, allergic rhinitis, urticaria (hives), eczema, conjunctivitis and food allergies, all of which are induced by environmental antigens.
  • the mechanisms include the synthesis of specific immunoglobulin E (IgE) with the differentiation and growth of effector cells such as mast cells and eosiniphils. The activity of these effector cells is dependent upon T-helper (CD4+) cells and their products.
  • IgE immunoglobulin E
  • CD4+ T-cells are dependent upon antigen receptors on T-cells recognizing peptide fragments of foreign proteins (allergens) in association with self gene products of the major histocompatibility complex (MHC) expressed on the surface of antigen presenting cells.
  • allergens peptide fragments of foreign proteins
  • MHC major histocompatibility complex
  • Allergic IgE-mediated diseases are currently treated by desensitization procedures that involve the periodic injection of allergen components or extracts. Desensitization treatments may induce an IgG response that competes with IgE for allergen, or they may induce specific suppressor T-cells that block the synthesis of IgE directed against allergen. These procedures are not always effective and pose the risk of provoking an allergic response.
  • a therapeutic treatment that would decrease or eliminate the allergic-immune response to a particular allergen, without altering the immune reactivity to other foreign antigens or triggering an allergic response itself would be of great benefit to allergic individuals.
  • the present invention is a method of reducing T cell response to a selected allergen, in which a non-allergen derived, non-stimulatory peptide is used and interferes with T cell recognition of the aller ⁇ gen, resulting in a decreased T cell response.
  • the present invention is useful in reducing (decreasing or eliminating) hypersensitivity of an individual to an allergen.
  • a non-allergen derived, non-stimulatory peptide is administered to the individual to modulate T cell recognition of the allergen and, as a result, to reduce the individual's immune response to the allergen.
  • the non-allergen derived, non-stimulatory peptide admin- istered is one which binds to selected MHC class II molecules of antigen presenting cells and, as a result, inhibits the specific T-helper cell response.
  • the present invention further relates to peptides and compositions useful in the method of reducing hyper- sensitivity.
  • the present invention relates to a method of reducing hypersensitivity of an individual to Dermatophagoides spp. (house dust mite or HDM) . It also relates to non-HDM derived non-stimulatory peptides, such as non-stimulating peptide analogs of influenza haemagglutinin which bind to DRw52b class II molecules, useful in the present method.
  • non-HDM derived non-stimulatory peptides such as non-stimulating peptide analogs of influenza haemagglutinin which bind to DRw52b class II molecules, useful in the present method.
  • the invention in another embodiment, relates to an in vitro method of determining the capability of a non-allergen derived, non-stimulatory peptide to reduce the immune response to an allergen. This is achieved by determining whether the peptide binds to specific MHC class II molecules of antigen presenting cells and whether binding of the peptide to specific MHC class II molecules modulates the T-cell MHC-restricted recognition of the allergen, thereby reducing the immune response to the allergen.
  • Currently used methods of achieving hyposensi- tization to allergens, including Dermatophagoides spp. depend upon the administration of the components or extracts of allergens. However, these components or extracts are not always successful and can themselves provoke allergic responses.
  • the present invention provides an alternative approach to such therapy and is particularly advantageous because desensitization can be achieved while the risk of triggering an allergic response is reduced.
  • Figure 1 is a graphic representation of HA S-309 induced inhibition of the polyclonal T-cell response of dust mite allergic individuals to specific allergen.
  • Figure 2 is a graphic representation of HA S-309 modulation of polyclonal T-cell responses to Dermatophagoides spp., M. tuberculosis and PHA.
  • Figure 3 is a graphic representation of HA S-309 modulation of monoclonal T-cell responses to insolu- bilized anti-CD3 antibody and Dermatophagoides spp.
  • Figure 4 is a graphic representation of the binding of HA S-309 to DRw52b HLA class II molecules expressed on the surface of murine fibroblasts.
  • the present invention is based on the discovery that a non-allergen derived, non-stimulatory peptide is able to bind to selected MHC class II molecules and inhibit both polyclonal and monoclonal T cell response to an allergen (referred to as an allergen of interest) which causes a T cell response in allergic individuals.
  • an allergen of interest referred to as an allergen of interest
  • a non-stimulatory peptide analogue derived from influenza virus hemagglutinin (HA) binds selected MHC class II molecules and inhibits the reponse of mite- specific CD4+ T cells restricted by the MHC class II molecules.
  • T-cell MHC- restricted recognition is defined as a response of a T-cell resulting from an interaction with a MHC class II molecule of an antigen presenting cell and an allergen derived peptide.
  • the present invention relates to a method of reducing (decreasing or eliminating) a specific allergic response by an individual to an allergen by administering to the individual a peptide which is not derived from the allergen to which the response occurs, is itself non-stimulatory, and binds to MHC class II molecules which also bind the allergen.
  • the invention relates to a method of reducing T cell recognition of HDM through the use of a peptide analogue derived from influenza virus HA.
  • an individual's sensitivity to an allergen such as HDM can be reduced by administering to the individual a therapeutically effective dose of a non-allergen derived, non-stimulatory peptide, which binds to specific MHC class II molecules of antigen- presenting cells. This binding inhibits the T-cell response to the allergen and thereby, decreases or eliminates the allergic response.
  • the non-allergen derived, non-stimulatory peptide used in the present method is one derived from or having essentially the same sequence as a peptide from a source other than the allergen of interest. It can have the same (unmodified) amino acid sequence as occurs in the peptide from which it is derived or can have an altered amino acid sequence (i.e., is a peptide "analogue" which differs from the amino acid sequence as it occurs naturally by deletion, addition or substitution of at least one amino acid) .
  • the term "derived from” includes both amino acid sequences (peptides) physically obtained from an existing sequence (e.g., by cutting or cleaving using chemical or enzymatic processes) and amino acid sequences which are produced, using known methods such as genetic engineering techniques or synthetic chem ⁇ istry to have substantially the same sequence as that of an existing sequence.
  • the term peptide is intended to include both unmodified and modified amino acid sequences.
  • the non-allergen derived, non-stimulatory peptide can be any peptide that does not provoke a response to the allergen to which desensitization is desired, binds a site on a MHC class II surface molecule of an antigen-presenting cell to which the allergen can also bind, and inhibits the T-cell response to the aller ⁇ gen.
  • a peptide analogue (designated S-309) derived from the natural sequence of the carboxyl terminus of the HA-1 peptide of influenza hae agglutinin (residues 307-319, with the tryosine at position 309 substituted by serine) is used.
  • S-309 binding is restricted to a particular MHC class II molecule, whereas the native peptide is non-restricted in the number of MHC class II molecules it has the capabity to bind.
  • this peptide analogue has been shown to bind class II molecules which also bind HDM and to inhibit the antigen- dependent re ⁇ sponse of cloned HDM specific T cells with the same restriction specificity.
  • the S-309 peptide analogue having the sequence:
  • a peptide analogue useful in the present method differs from the normally-occurring peptide by at least one amino acid (e.g., an addition, deletion or substitution) . Alterations in the amino acid sequence can be made to enhance the ability of the peptide analogue to inhibit T cell response to the allergen.
  • Peptides and peptide analogues can be prepared by a variety of known methods. For example, they can be prepared using Merrifield's procedure of solid-phase peptide synthesis (Merrifield, R.B., J. Am. Chem. Soc. , 86; 304 (1964), hereby incorporated by refer- ence) .
  • the first step in the solid-phase synthesis of amino acid peptide analogues, as well as other pep ⁇ tides, is the formation of a covalent bond between the C-terminal protected amino acid of the chosen peptide sequence and the solid support or resin.
  • the peptide chain is then build up residue by residue by repeti ⁇ tive cycles of deprotection, during which the N- ternamal Boc-protecting (N-tect-bitoxycarboxyl) group is removed by trifluoroacetic acid (TFA) . This is followed by neutralization with disopropylethylamine (DEA) of the amino group left as a salt and coupling of the next amino acid in the sequence. The cycle is repeated until the sequence is completed.
  • DEA disopropylethylamine
  • peptides useful in the present invention can be produced using genetic engineering techniques.
  • DNA encoding the desired amino acid sequence can be incorporated into an appropriate expression vector and introduced into a host cell suitable for expression of the encoded product.
  • Administration of the non-stimulatory peptide to an individual can be by any route by which a therapeu- tically effective quantity of the peptide can be delivered.
  • administration can be via a parenteral route, such as by subcutaneous, intravenous or intramuscular administration, transdermal passage or uptake from the respiratory tract.
  • Administration can also be via the gastrointestinal tract, such as by oral or rectal administration.
  • the form in which the peptide is administered (e.g., capsule, tablet, solution, emulsion) will depend, at least in part, on the route by which it is administered.
  • administration by injection would involve the use of physiologic saline or other physiological compatible carrier.
  • a therapeutically effective amount of a non- stimulatory peptide is that amount which will decrease or eliminate the T-helper cell response to a specific allergen.
  • the therapeutically effective amount will be determined on an individual basis and will be based, at least in part, on consideration of the individual's size, the severity of symptoms to be treated, the result sought, the particular non- stimulatory peptide used, etc.
  • the effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.
  • the non-allergen derived, non-stimulatory peptides used in the method of the present invention bind to ⁇ IHC class II molecules and interfere with the recognition of allergen MHC-molecule complexes by T-helper cells. As a consequence, the T-cell response is inhibited. This inhibition is reflected by a lack of clonal expansion and the secretion of one or more lymphokines which are soluble factors that have multiple effects on B-cells and other T-cells. Lack of or decrease in a T-cell response to an allergen is referred to as a hyposensitization of the T-cells to the allergen.
  • non-allergen non-stimulating peptides of influenza haemagglutinin which is used to modulate the immune response to HDM
  • Identification of other inhibitors of specific allergens can be achieved by 1) identifying the specificity of the restriction (MHC class II) molecules of importance with the specific allergen; 2) searching the database for peptides known to bind to these molecules; 3) synthesizing and testing the above peptides for their ability to inhibit binding of and/or recognition of the allergen in a competition assay; and 4) modifying peptides to produce peptide analogues with enhanced binding and/or biological activity.
  • peptides can be randomly selected and screened for their capacity to bind specific MHC class II molecules and inhibit allergen-dependent T-cell proliferation.
  • Lyophilized extracts of Dermatophagoides farinae (D. farinae) and Dermatophagoides pteronyssinun (D. pteronyssinun) were the general gifts of Drs. Lowenstein and Schou (ALK, Horsholm, Denmark) and Bencard (Brentford, Middlesex, U.K.), respectively.
  • a soluble extract of Mycobacteriu tuberculosis (MTSE) was generously provided by Dr. A. Rees (M.R.C.
  • HA peptide analogue (residues 307-319: serine at 309) and the keratin peptide (residues 1-9) were synthesized using solid phase techniques (Barany, G. and Merrifield, R. , Solid phase peptide synthesis In: Gross, E. Meinhofer, J. (Eds), The Peptides, New York, Academic Press (1979) hereby incorporated by reference) on an Applied Biosystems Peptide Synthesizer with Pam resins, t-Boc protected amino acids, and commercially available reagents (Applied Biosystems, Foster City, CA) . Peptides were kindly provided by J.
  • the T cell mitogen phytahaemagglutinin (PHA-P) was purchased from Sigma Chemicals Co. (St. Louis, MO) , and the murine monoclonal anti-CD3 antibody (OKT3) purified from the hybridoma cell line purchased from the ATCC (Rockville, MD) .
  • PBMC Peripheral blood mononuclear leucocytes obtained from unmedicated atopic adults with symptomatic HDM allergy (perennial rhinitis) were isolated by centrifugation on a discontinuous gradient of Ficoll-Paque (Pharmacia) . All subjects had positive skin prick tests to Dermaphatogoides spp., and positive HDM radioallergosorbent tests.
  • PBMC were resuspended in complete medium, RPMI-1640 supplemented with A+ or AB+ serum, 2mM L-glutamine and 100 IU/ml of penicillin/streptomycin.
  • HDM specific T cell clones were isolated as described previously (O'Hehir, R.E., et al. ,
  • PBMC 2.5 X 5 10 /ml
  • D farinae 7 days in complete medium.
  • Lymphoblasts enriched on Ficoll-Paque were maintained as a long term line in the presence of irradiated
  • T cell clones Prior to their use in proliferation assays, the T cell clones were rested for 6-8 days after the last addition of filler cells and antigen. A number of T cell clones specific for D. Farinae and others cross-reactive with D. farinae and D. pteronyssiunus were isolated, and the MHC class II restriction specificities of these clones were mapped (O'Hehir, R.E., et al. , Immunology, 64: 627 (1988)).
  • PBMC 2.5 X 10 /ml were cultured with soluble antigen in a total volume of 200 ⁇ l of complete medium in 96-well round bottom plates.
  • Peptides were added at various concentrations to selected wells at the initiation of cultures. After 6 days, tritiated methyl thymidine (l ⁇ Ci [ H] .
  • TdR/well Amersham International, Amersham, U.K.
  • Amersham International Amersham, U.K.
  • Proliferation as correlated with [ H] -TdR incorporation was measured at day 7 by liquid scintillation spectroscopy. The results are expressed as mean counts per minute (cpm) for triplicate cultures.
  • PHA and anti-CD3 induced activation PHA at a concentration of 1 ⁇ g/ml induced marked proliferation in each subject (see Figures 2C i and ii) . No inhibitory effect was observed with the addition of either peptide. Similarly, both peptides failed to inhibit the proliferative response of T cells stimulated directly with immobilized anti-CD3 antibody and IL-2 that mimics the recognition of peptide/MHC class II complexes as illustrated in Figure 3A i.
  • Example I Cloned T cells (5 X 10 /ml) were cultured with soluble antigen in the presence of irradiated
  • D. pteronyssinus (3 ⁇ g/ml) presented by autologous irradiated PBMC as accessory cells.
  • the addition of the HA analogue resulted in marked inhibition of the proliferative response in a dose dependent manner, with 60% inhibition at 100 ⁇ g/ml.
  • coculture using the keratin peptide failed to inhibit the HDM induced proliferation as illustrated in Figure
  • D. farinae specific T cell clones (DE12 and DE47) restricted by the DRAB3 gene product DRw52b and T cell clone DE9 were stimulated with increasing concentra ⁇ tions of stimulating antigen (D. farinae) in the presence of autologous irradiated PBMC as a source of antigen presenting cells.
  • the HA analogue was added at a fixed concentration (100 ⁇ g/ml) and T cell proliferation determined.
  • the HA peptide was able to compete with HDM for presentation to the cloned T cells and effectively inhibit the proliferation (see Figures 3B i-iii) .
  • increasing the concentration of HDM in the assays was associated with a decrease in the inhibition mediated by the HA peptide in each case.
  • Murine fibroblasts transfected with the HLA-D region genes, DRw52b, DR1 and DR2Dw2B5 were examined for their capacity to bind the HA analogue as de ⁇ scribed in Busch, R. , et al. , Int. Immunol., 2 ⁇ 442 (1990) .
  • the fibroblasts were then washed and stained with fluorescein avidin D (Vector Labora ⁇ tories, CA) was added to the cells prior to a further incubation with fluorescein avidin D.
  • HLA class II molecules To control for differential expression of HLA class II molecules on the fibroblasts, cells were incubated with a fluore- ⁇ ceinated monomorphic anti-HLA-DR antibody (L243, Becton Dickinson, CA) as described in Lampson, L.A. and Levy, R. , J. Immunol., 125: 293 (1980) . Stained cells were analyzed by flow cytometry using a FASCcan analyzer (Becton Dickinson) . Only viable cells, identified by their ability to exclude propidium iodide, were analyzed.
  • the appropriate genes were transfected in murine fibroblasts.
  • Murine fibroblasts transfected with the DRw52b gene were able to bind the HA peptide in a dose-dependent manner in contrast to the control fibroblasts expressing DR1 or the untransfected cell line (DAP3) , as determined by the level of fluor ⁇ escence as illustrated in Figure 4.
  • the HA peptide was able to bind in a dose-dependent manner to murine fibroblasts transfected with DR2Dw2B5 genes, which were functionally able to present HDM to clone DE9.

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Abstract

A method of reducing immune response to an allergen in which a non-allergen derived, non-stimulatory peptide which binds to specific MHC class II molecules of antigen presenting cells is used, as well as non-allergen derived, non-stimulatory peptides useful in the method.

Description

PEPTIDE-MEDIATED HYPOSENSITIZATION OF THE HUMAN T CELL RESPONSE TO DERMATOPHAGOIDES SPP. (HOUSE DUST MITE)
Background of the Invention The term "atopic allergy" is applied to a group of allergies, including asthma, hay fever, allergic rhinitis, urticaria (hives), eczema, conjunctivitis and food allergies, all of which are induced by environmental antigens. The mechanisms include the synthesis of specific immunoglobulin E (IgE) with the differentiation and growth of effector cells such as mast cells and eosiniphils. The activity of these effector cells is dependent upon T-helper (CD4+) cells and their products. In turn, the activation of CD4+ T-cells is dependent upon antigen receptors on T-cells recognizing peptide fragments of foreign proteins (allergens) in association with self gene products of the major histocompatibility complex (MHC) expressed on the surface of antigen presenting cells. Allergic IgE-mediated diseases are currently treated by desensitization procedures that involve the periodic injection of allergen components or extracts. Desensitization treatments may induce an IgG response that competes with IgE for allergen, or they may induce specific suppressor T-cells that block the synthesis of IgE directed against allergen. These procedures are not always effective and pose the risk of provoking an allergic response. A therapeutic treatment that would decrease or eliminate the allergic-immune response to a particular allergen, without altering the immune reactivity to other foreign antigens or triggering an allergic response itself would be of great benefit to allergic individuals.
Summary of the Invention
The present invention is a method of reducing T cell response to a selected allergen, in which a non-allergen derived, non-stimulatory peptide is used and interferes with T cell recognition of the aller¬ gen, resulting in a decreased T cell response. The present invention is useful in reducing (decreasing or eliminating) hypersensitivity of an individual to an allergen. A non-allergen derived, non-stimulatory peptide is administered to the individual to modulate T cell recognition of the allergen and, as a result, to reduce the individual's immune response to the allergen. In the method of the present invention, the non-allergen derived, non-stimulatory peptide admin- istered is one which binds to selected MHC class II molecules of antigen presenting cells and, as a result, inhibits the specific T-helper cell response. The present invention further relates to peptides and compositions useful in the method of reducing hyper- sensitivity.
In particular, the present invention relates to a method of reducing hypersensitivity of an individual to Dermatophagoides spp. (house dust mite or HDM) . It also relates to non-HDM derived non-stimulatory peptides, such as non-stimulating peptide analogs of influenza haemagglutinin which bind to DRw52b class II molecules, useful in the present method.
In another embodiment, the invention relates to an in vitro method of determining the capability of a non-allergen derived, non-stimulatory peptide to reduce the immune response to an allergen. This is achieved by determining whether the peptide binds to specific MHC class II molecules of antigen presenting cells and whether binding of the peptide to specific MHC class II molecules modulates the T-cell MHC-restricted recognition of the allergen, thereby reducing the immune response to the allergen. Currently used methods of achieving hyposensi- tization to allergens, including Dermatophagoides spp., depend upon the administration of the components or extracts of allergens. However, these components or extracts are not always successful and can themselves provoke allergic responses. The present invention provides an alternative approach to such therapy and is particularly advantageous because desensitization can be achieved while the risk of triggering an allergic response is reduced.
Brief Description of the Drawings
Figure 1 is a graphic representation of HA S-309 induced inhibition of the polyclonal T-cell response of dust mite allergic individuals to specific allergen.
Figure 2 is a graphic representation of HA S-309 modulation of polyclonal T-cell responses to Dermatophagoides spp., M. tuberculosis and PHA.
Figure 3 is a graphic representation of HA S-309 modulation of monoclonal T-cell responses to insolu- bilized anti-CD3 antibody and Dermatophagoides spp.
Figure 4 is a graphic representation of the binding of HA S-309 to DRw52b HLA class II molecules expressed on the surface of murine fibroblasts.
Detailed Description of the Invention
The present invention is based on the discovery that a non-allergen derived, non-stimulatory peptide is able to bind to selected MHC class II molecules and inhibit both polyclonal and monoclonal T cell response to an allergen (referred to as an allergen of interest) which causes a T cell response in allergic individuals. As described herein, it has been shown that a non-stimulatory peptide analogue derived from influenza virus hemagglutinin (HA) binds selected MHC class II molecules and inhibits the reponse of mite- specific CD4+ T cells restricted by the MHC class II molecules. As is also described herein, although polyclonal T cell responses were negatively modulated (decreased) by the peptide, recognition of common recall antigens remained intact. T-cell MHC- restricted recognition is defined as a response of a T-cell resulting from an interaction with a MHC class II molecule of an antigen presenting cell and an allergen derived peptide.
The present invention relates to a method of reducing (decreasing or eliminating) a specific allergic response by an individual to an allergen by administering to the individual a peptide which is not derived from the allergen to which the response occurs, is itself non-stimulatory, and binds to MHC class II molecules which also bind the allergen. In particular, the invention relates to a method of reducing T cell recognition of HDM through the use of a peptide analogue derived from influenza virus HA. As a result, an individual's sensitivity to an allergen such as HDM can be reduced by administering to the individual a therapeutically effective dose of a non-allergen derived, non-stimulatory peptide, which binds to specific MHC class II molecules of antigen- presenting cells. This binding inhibits the T-cell response to the allergen and thereby, decreases or eliminates the allergic response.
The non-allergen derived, non-stimulatory peptide used in the present method is one derived from or having essentially the same sequence as a peptide from a source other than the allergen of interest. It can have the same (unmodified) amino acid sequence as occurs in the peptide from which it is derived or can have an altered amino acid sequence (i.e., is a peptide "analogue" which differs from the amino acid sequence as it occurs naturally by deletion, addition or substitution of at least one amino acid) . As used herein, the term "derived from" includes both amino acid sequences (peptides) physically obtained from an existing sequence (e.g., by cutting or cleaving using chemical or enzymatic processes) and amino acid sequences which are produced, using known methods such as genetic engineering techniques or synthetic chem¬ istry to have substantially the same sequence as that of an existing sequence. As used herein, the term peptide is intended to include both unmodified and modified amino acid sequences.
The non-allergen derived, non-stimulatory peptide can be any peptide that does not provoke a response to the allergen to which desensitization is desired, binds a site on a MHC class II surface molecule of an antigen-presenting cell to which the allergen can also bind, and inhibits the T-cell response to the aller¬ gen. In one embodiment of the invention a peptide analogue (designated S-309) derived from the natural sequence of the carboxyl terminus of the HA-1 peptide of influenza hae agglutinin (residues 307-319, with the tryosine at position 309 substituted by serine) is used. An advantage of S-309 is that its binding is restricted to a particular MHC class II molecule, whereas the native peptide is non-restricted in the number of MHC class II molecules it has the capabity to bind. As described herein, this peptide analogue has been shown to bind class II molecules which also bind HDM and to inhibit the antigen- dependent re¬ sponse of cloned HDM specific T cells with the same restriction specificity. As described in the Exam¬ ples, the S-309 peptide analogue, having the sequence:
Pro-Lys-Ser-Val-Lys-Gln-Asn-Thr-Leu-Lys-Leu-Ala-Thr, 307 319
has been shown to inhibit polyclonal T cell responses to stimulation induced by D. pteronyssinus and mono¬ clonal T cell responses to stimulation induced by D. pteronyssinus and D. farinae. This suggests that the binding of the peptide to MHC surface molecules prevents activation of the majority of HDM reactive T cells. Possible mechanisms by which the activation of helper T-cells is inhibited include 1) competitive binding of the HA-1 peptide and the T-cell receptor for the MHC surface molecule, and 2) interference with the presentation of processed antigens of the allergen complexed to the MHC surface molecules.
A peptide analogue useful in the present method differs from the normally-occurring peptide by at least one amino acid (e.g., an addition, deletion or substitution) . Alterations in the amino acid sequence can be made to enhance the ability of the peptide analogue to inhibit T cell response to the allergen. Peptides and peptide analogues can be prepared by a variety of known methods. For example, they can be prepared using Merrifield's procedure of solid-phase peptide synthesis (Merrifield, R.B., J. Am. Chem. Soc. , 86; 304 (1964), hereby incorporated by refer- ence) . The first step in the solid-phase synthesis of amino acid peptide analogues, as well as other pep¬ tides, is the formation of a covalent bond between the C-terminal protected amino acid of the chosen peptide sequence and the solid support or resin. The peptide chain is then build up residue by residue by repeti¬ tive cycles of deprotection, during which the N- ternamal Boc-protecting (N-tect-bitoxycarboxyl) group is removed by trifluoroacetic acid (TFA) . This is followed by neutralization with disopropylethylamine (DEA) of the amino group left as a salt and coupling of the next amino acid in the sequence. The cycle is repeated until the sequence is completed. After assembly is completed, the peptide is cleaved from the resin and purified. Alternatively, peptides useful in the present invention can be produced using genetic engineering techniques. For example, DNA encoding the desired amino acid sequence can be incorporated into an appropriate expression vector and introduced into a host cell suitable for expression of the encoded product. Administration of the non-stimulatory peptide to an individual can be by any route by which a therapeu- tically effective quantity of the peptide can be delivered. For example, administration can be via a parenteral route, such as by subcutaneous, intravenous or intramuscular administration, transdermal passage or uptake from the respiratory tract. Administration can also be via the gastrointestinal tract, such as by oral or rectal administration. The form in which the peptide is administered (e.g., capsule, tablet, solution, emulsion) will depend, at least in part, on the route by which it is administered. For example, administration by injection would involve the use of physiologic saline or other physiological compatible carrier.
A therapeutically effective amount of a non- stimulatory peptide is that amount which will decrease or eliminate the T-helper cell response to a specific allergen. The therapeutically effective amount will be determined on an individual basis and will be based, at least in part, on consideration of the individual's size, the severity of symptoms to be treated, the result sought, the particular non- stimulatory peptide used, etc. The effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.
The non-allergen derived, non-stimulatory peptides used in the method of the present invention bind to ΪIHC class II molecules and interfere with the recognition of allergen MHC-molecule complexes by T-helper cells. As a consequence, the T-cell response is inhibited. This inhibition is reflected by a lack of clonal expansion and the secretion of one or more lymphokines which are soluble factors that have multiple effects on B-cells and other T-cells. Lack of or decrease in a T-cell response to an allergen is referred to as a hyposensitization of the T-cells to the allergen.
Although the present invention is described in terms of non-allergen, non-stimulating peptides of influenza haemagglutinin which is used to modulate the immune response to HDM, it is also possible to identify other non-allergen, non-stimulatory peptides useful for the same purpose or for modulating immune responses to other allergens. Identification of other inhibitors of specific allergens can be achieved by 1) identifying the specificity of the restriction (MHC class II) molecules of importance with the specific allergen; 2) searching the database for peptides known to bind to these molecules; 3) synthesizing and testing the above peptides for their ability to inhibit binding of and/or recognition of the allergen in a competition assay; and 4) modifying peptides to produce peptide analogues with enhanced binding and/or biological activity. Alternatively, peptides can be randomly selected and screened for their capacity to bind specific MHC class II molecules and inhibit allergen-dependent T-cell proliferation.
The invention is further illustrated by the following specific examples, which are not intended to be limiting in any way. EXAMPLE 1 Peptide Inhibition of Polyclonal T Cell
Responses Antigens
Lyophilized extracts of Dermatophagoides farinae (D. farinae) and Dermatophagoides pteronyssinun (D. pteronyssinun) were the general gifts of Drs. Lowenstein and Schou (ALK, Horsholm, Denmark) and Bencard (Brentford, Middlesex, U.K.), respectively. A soluble extract of Mycobacteriu tuberculosis (MTSE) was generously provided by Dr. A. Rees (M.R.C.
Tuberculosis Unit, Hammersmith Hospital, U.K.). The HA peptide analogue (residues 307-319: serine at 309) and the keratin peptide (residues 1-9) were synthesized using solid phase techniques (Barany, G. and Merrifield, R. , Solid phase peptide synthesis In: Gross, E. Meinhofer, J. (Eds), The Peptides, New York, Academic Press (1979) hereby incorporated by reference) on an Applied Biosystems Peptide Synthesizer with Pam resins, t-Boc protected amino acids, and commercially available reagents (Applied Biosystems, Foster City, CA) . Peptides were kindly provided by J. Rothbard (ImmuLogic Pharmaceutical Corporation, Palo Alto, CA) . The T cell mitogen phytahaemagglutinin (PHA-P) was purchased from Sigma Chemicals Co. (St. Louis, MO) , and the murine monoclonal anti-CD3 antibody (OKT3) purified from the hybridoma cell line purchased from the ATCC (Rockville, MD) .
Preparation of Lymphocytes and Donor Characterization Peripheral blood mononuclear leucocytes (PBMC) obtained from unmedicated atopic adults with symptomatic HDM allergy (perennial rhinitis) were isolated by centrifugation on a discontinuous gradient of Ficoll-Paque (Pharmacia) . All subjects had positive skin prick tests to Dermaphatogoides spp., and positive HDM radioallergosorbent tests. PBMC were resuspended in complete medium, RPMI-1640 supplemented with A+ or AB+ serum, 2mM L-glutamine and 100 IU/ml of penicillin/streptomycin.
Isolation of Antigen Reactive T cell Clones
HDM specific T cell clones were isolated as described previously (O'Hehir, R.E., et al. ,
Immunobiology, 62: 635 (1987)). Briefly, PBMC (2.5 X 5 10 /ml) were stimulated with an optimal concentration of D farinae for 7 days in complete medium.
Lymphoblasts enriched on Ficoll-Paque were maintained as a long term line in the presence of irradiated
(2500 Rads) autologous PBMC, D farinae and interleukin 2 (IL-2, 10% v/v; Lymphocult-T, Biotest Folex,
Frankfurt, FRG) and then cloned by limiting dilution from the T cell line. For cloning, viable cells (0.3 cells/well) were plated in Microtest II trays together with irradiated autologout PBMC (5 X 10 /ml) , D. farinae and IL-2. After 7 days, growing clones were transferred to flat bottom 96-well microtitre trays and, subsequently, to 24 well trays. At each transfer, the clones received filler cells, antigen and IL-2. The clones were maintained and expanded by the addition of IL-2 every 3-4 days, and antigen together with filler cells every 7 days. Prior to their use in proliferation assays, the T cell clones were rested for 6-8 days after the last addition of filler cells and antigen. A number of T cell clones specific for D. Farinae and others cross-reactive with D. farinae and D. pteronyssiunus were isolated, and the MHC class II restriction specificities of these clones were mapped (O'Hehir, R.E., et al. , Immunology, 64: 627 (1988)).
Proliferation Assays
5 Polyclonal Responses: PBMC (2.5 X 10 /ml) were cultured with soluble antigen in a total volume of 200 μl of complete medium in 96-well round bottom plates.
Peptides were added at various concentrations to selected wells at the initiation of cultures. After 6 days, tritiated methyl thymidine (lμCi [ H] .
TdR/well; Amersham International, Amersham, U.K.) was added to the cultures for 8-16 hours and then the cells were harvested onto glass fibre filters. Proliferation as correlated with [ H] -TdR incorporation was measured at day 7 by liquid scintillation spectroscopy. The results are expressed as mean counts per minute (cpm) for triplicate cultures.
Peptide Inhibition of Polyclonal T Cell Responses Stimulation induced with D. pteronyssinus:
Marked proliferation was induced in all cases when PBMC isolated from 5 atopic individuals with symptomatic perennial rhinitis were stimulated with D. pteronyssinus at an optimal concentration (range 3-12 μug/ l) . The addition of the HA peptide analogue. with the tyrosine at position 309 substituted by serine, to the cultures resulted in marked inhibition of the proliferative response over a concentration range (0.5-100 μg/ml) . In the presence of the HA peptide, the percentage proliferation as compared to the maximal HDM response achieved varied from 10 to 34%. As illustrated in Figure 1 (striped bar) , PBMC stimulated with HDM in the presence of peptide analogue; open bar: PBMC stimulated with HDM in the absence of peptide analogue) . In contrast, coculture of the keratin peptide and D. pteronyssinus over the same concentration range failed to decrease the observed response as illustrated in Figure 2A. In all cases, PBMC failed to proliferate to either of the peptides alone.
Stimulation induced with M. tuberculosis soluble extract: Polyclonal T cell proliferation observed in response to stimulation with MTSE (3 μg/ml) was only minimally inhibited by the HA analogue. The maximal inhibitory effect obtained was only 25% (BS) and 40% (MR) at 100 μg/ml, with 13% (BS) and 28% (MR) inhibition at 10 μg/ml. No inhibition of the anti-mycobacterial T cell response was observed in the presence of the control keratin peptide as illustrated in Figure 2B.
PHA and anti-CD3 induced activation: PHA at a concentration of 1 μg/ml induced marked proliferation in each subject (see Figures 2C i and ii) . No inhibitory effect was observed with the addition of either peptide. Similarly, both peptides failed to inhibit the proliferative response of T cells stimulated directly with immobilized anti-CD3 antibody and IL-2 that mimics the recognition of peptide/MHC class II complexes as illustrated in Figure 3A i.
EXAMPLE II Peptide Inhibition of Monoclonal T Cell
Responses Proliferation Assays Monoclonal Responses: Antigens and antigen reactive T cell clones were obtained as described in
4 Example I. Cloned T cells (5 X 10 /ml) were cultured with soluble antigen in the presence of irradiated
5 autologous PBMC (1.25 X 10 /ml) in a total volume of 200 μl of complete medium in 96-well round bottom plates. Peptides were added to selected wells as described for polyclonal responses. After 60 hours incubation, [ H] -TdR was added to the cultures for
8-16 hours and then they were harvested as described for the polyclonal T cell proliferation assays.
Cloned T cells (DE9; cross-reactive for both species of Dermatophagoides spp., restricted by products of the Bl gene locus DRwl2 and degenerate in its restriction specificity for DR2 subtypes and DR8 (O'Hehir, R.E. , supra (1988), proliferated markedly to
D. pteronyssinus (3 μg/ml) presented by autologous irradiated PBMC as accessory cells. The addition of the HA analogue resulted in marked inhibition of the proliferative response in a dose dependent manner, with 60% inhibition at 100 μg/ml. In contrast, coculture using the keratin peptide failed to inhibit the HDM induced proliferation as illustrated in Figure
3A i.
D. farinae specific T cell clones (DE12 and DE47) restricted by the DRAB3 gene product DRw52b and T cell clone DE9 were stimulated with increasing concentra¬ tions of stimulating antigen (D. farinae) in the presence of autologous irradiated PBMC as a source of antigen presenting cells. To these cultures the HA analogue was added at a fixed concentration (100 μg/ml) and T cell proliferation determined. The HA peptide was able to compete with HDM for presentation to the cloned T cells and effectively inhibit the proliferation (see Figures 3B i-iii) . However, increasing the concentration of HDM in the assays was associated with a decrease in the inhibition mediated by the HA peptide in each case.
EXAMPLE III Binding of HA Peptide to Antigen
Presenting Cells Expressing DRw52b MHC Class II Molecules
The cloning of the DRIDwl, DR2Dw2B5 and DRw52b genes and their co-transfection with the DRA gene into the Ltk fibroblast cell line (DAP3) have been de¬ scribed in Rothbard, J.B., et al., Cell, 52: 515 (1988); Rabourdin-Combe, C. and Mach, B., Nature, 303: 670 (1983); Borsh, J. , et al. , J. Exp. Med., 162: 105 (1985); Wilkinson, D., et al. , J. Exp. Med., 167: 1442 (1988) . The transfected fibroblasts were the generous gifts of Drs. R. Leckler, J. Trowsdale and B. Mach. Murine fibroblasts transfected with the HLA-D region genes, DRw52b, DR1 and DR2Dw2B5 were examined for their capacity to bind the HA analogue as de¬ scribed in Busch, R. , et al. , Int. Immunol., 2} 442 (1990) . After incubation with peptide, containing long chain biotin at the amino terminus, over a concentration range, the fibroblasts were then washed and stained with fluorescein avidin D (Vector Labora¬ tories, CA) was added to the cells prior to a further incubation with fluorescein avidin D. To control for differential expression of HLA class II molecules on the fibroblasts, cells were incubated with a fluore- εceinated monomorphic anti-HLA-DR antibody (L243, Becton Dickinson, CA) as described in Lampson, L.A. and Levy, R. , J. Immunol., 125: 293 (1980) . Stained cells were analyzed by flow cytometry using a FASCcan analyzer (Becton Dickinson) . Only viable cells, identified by their ability to exclude propidium iodide, were analyzed.
To examine the binding of the HA peptide to DRw252b in isolation from other HLA class II mole¬ cules, the appropriate genes were transfected in murine fibroblasts. Murine fibroblasts transfected with the DRw52b gene were able to bind the HA peptide in a dose-dependent manner in contrast to the control fibroblasts expressing DR1 or the untransfected cell line (DAP3) , as determined by the level of fluor¬ escence as illustrated in Figure 4. Similarly, the HA peptide was able to bind in a dose-dependent manner to murine fibroblasts transfected with DR2Dw2B5 genes, which were functionally able to present HDM to clone DE9.

Claims

1. A method of reducing T cell response to a selected allergen, comprising contacting T cells with a non-allergen derived, non-stimulatory peptide in the presence of the selected allergen and antigen-presenting cells, under conditions appropriate for interference by the non-allergen derived, non-stimulatory peptide with T cell response to the selected allergen.
2. The method of Claim 1 wherein the selected allergen is a house dust mite allergen and the non-allergen derived, non-stimulatory peptide is an influenza virus HA peptide.
3. The method of Claim 2 wherein the influenza virus HA peptide has the following sequence:
Pro-Lys-Ser-Val-Lys-Gln-Asn-Thr-Leu-Lys-Leu-Ala— Thr.
4. A method of inhibiting polyclonal T cell response to stimulation by a selected allergen, comprising combining T cells stimulated by the selected allergen with a non-allergen derived, non-stimulatory peptide which interferes with T cell recognition of the selected allergen.
5. The method of Claim 4 wherein the selected allergen is a house dust mite allergen and the non-allergen derived, non-stimulatory peptide is an influenza virus HA peptide.
6. The method of Claim 5 wherein the influenza virus HA peptide has the following sequence: Pro-Lys-Ser-Val-Lys-Gln-Asn-Thr-Leu-Lys-Leu-Ala— Thr.
7. A non-allergen derived, non-stimulatory peptide which inhibits T cell response to a selected allergen.
8. The non-allergen derived, non-stimulatory peptide of Claim 7 which is an influenza virus HA peptide.
9. The non-allergen derived, non-stimulatory peptide of Claim 8 which has the following sequence: Pro-Lys-Ser-Val-Lys-Gln-Asn-Thr-Leu-Lys-Leu-Ala— Thr.
10. A method of reducing, in an individual, immune response to an allergen, comprising administering to the individual a therapeutically effective amount of a non-allergen derived, non-stimulatory peptide which binds to specific MHC class II molecules of antigen-presenting cells, wherein binding of the non-allergen derived, non-stimulatory peptide to specific MHC class II molecules modulates T cell MHC-restricted recognition of the allergen, thereby reducing the immune response of the individual to the allergen.
11. A method of reducing, in an individual, immune response to an allergen of house dust mite, comprising administering to the individual a therapeutically effective amount of a non- allergen derived, non-stimulatory peptide which binds to specific MHC class II molecules of antigen-presenting cells, wherein binding of the non-allergen derived, non-stimulatory peptide to specific MHC class II molecules modulates T cell MHC-restricted recognition of the allergen of house dust mite, thereby reducing the immune response of the individual to the house dust mite allergen.
12. The method of Claim 11 wherein the non-allergen, non-stimulatory peptide is an influenza virus HA peptide.
13. The method of Claim 12 wherein the influenza virus HA peptide has the amino acid sequence: Pro-Lys-Ser-Val-Lys-Gln-Asn-Thr-Leu-Lys-Leu-Ala— Thr.
14. The method of Claim 11 wherein said MHC class II molecules are products of HLA-DRAB3 class II genes, DRw52 class II molecules.
15. A method of Claim 11 wherein the MHC class II molecules are products of the DRAB1 genes, DRwl2 class II molecules.
16. A method of reducing, in an individual, immune response to an allergen, comprising administering to the individual a therapeutically effective amount of a non-allergen derived, non-stimulatory peptide of haemagglutinin which binds to DRw52 class II molecules of antigen-presenting cells, wherein binding of the non-allergen derived, non-stimulatory peptide to DRw52 MHC class II molecules modulates T cell DRw52-restricted response to house dust mites, thereby reducing the immune response of the individual to house dust mites.
17. A method of determining the capability of a non-allergen derived, non-stimulatory peptide to reduce immmune response to an allergen, comprising determining whether the peptide binds to specific MHC class II molecules of antigen presenting cells and whether binding of the peptide to the specific MHC class II molecules modulates T cell MHC-restricted recognition of the allergen, thereby reducing the immune response to the allergen.
18. A method of determining the capability of a non-house dust mite derived, non-stimulatory peptide to reduce the immune response to a house dust mite allergen, comprising determining whether the peptide binds to specific MHC class II molecules of antigen presenting cells and whether binding of the peptide to the specific MHC class II molecules modulates T cell MHC-restricted recognition of house dust mite, thereby reducing immune response to house dust mite.
PCT/US1992/000205 1991-01-08 1992-01-08 Peptide-mediated hyposensitization of the human t cell response to dermatophagoides spp. (house dust mite) WO1992011859A1 (en)

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WO1995026980A2 (en) * 1994-04-01 1995-10-12 Immulogic Pharmaceutical Corporation Haptenated peptides and uses thereof
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WO1999034826A1 (en) * 1998-01-09 1999-07-15 Circassia Limited Methods and compositions for desensitisation
US6737406B1 (en) 1996-03-21 2004-05-18 Circassia, Ltd. Cryptic peptides and method for their identification
US7485708B2 (en) 1996-09-23 2009-02-03 University Of Arkansas Nucleic acids encoding ara h 3 polypeptides
US7879977B2 (en) 1998-01-31 2011-02-01 University Of Arkansas Methods and reagents for decreasing clinical reaction to allergy
US8246945B2 (en) 2000-04-06 2012-08-21 University Of Arkansas Methods and reagents for decreasing clinical reaction to allergy

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Title
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IMMUNOLOGY, Vol. 64, issued 1988, R.E. O'HEHIR et al., "MHC class II restriction specificity of cloned human T lymphocytes reactive with Dermatophagoides farinae (house dust mite)", pages 627-631. *
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5460977A (en) * 1992-08-14 1995-10-24 Torii & Co. Peptide having allergenicity
WO1995026980A2 (en) * 1994-04-01 1995-10-12 Immulogic Pharmaceutical Corporation Haptenated peptides and uses thereof
WO1995026980A3 (en) * 1994-04-01 1995-12-07 Immulogic Pharma Corp Haptenated peptides and uses thereof
US6737406B1 (en) 1996-03-21 2004-05-18 Circassia, Ltd. Cryptic peptides and method for their identification
US7485708B2 (en) 1996-09-23 2009-02-03 University Of Arkansas Nucleic acids encoding ara h 3 polypeptides
WO1999034826A1 (en) * 1998-01-09 1999-07-15 Circassia Limited Methods and compositions for desensitisation
GB2348808A (en) * 1998-01-09 2000-10-18 Circassia Ltd Methods and compositions for desensitisation
GB2348808B (en) * 1998-01-09 2003-03-19 Circassia Ltd Methods and compositions for desensitisation
US7879977B2 (en) 1998-01-31 2011-02-01 University Of Arkansas Methods and reagents for decreasing clinical reaction to allergy
US8246945B2 (en) 2000-04-06 2012-08-21 University Of Arkansas Methods and reagents for decreasing clinical reaction to allergy

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