WO1994024281A1 - T cell epitopes of the major allergens from dermatophagoides (house dust mite) - Google Patents
T cell epitopes of the major allergens from dermatophagoides (house dust mite) Download PDFInfo
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- WO1994024281A1 WO1994024281A1 PCT/US1993/003471 US9303471W WO9424281A1 WO 1994024281 A1 WO1994024281 A1 WO 1994024281A1 US 9303471 W US9303471 W US 9303471W WO 9424281 A1 WO9424281 A1 WO 9424281A1
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention provides isolated modified peptides of the major protein allergens of the genus Dermatophagoides.
- Peptides within the scope of the invention comprise at least one T cell epitope, preferably at least two T cell epitopes of a protein allergen selected from the allergens Der p I, Der p II, Der f I, or Der f II.
- the invention further provides peptides comprising at least two regions, each region comprising at least one T cell epitope of a mite protein allergen. The regions are derived from the same or from different protein allergens of the genus Dermatophagoides.
- peptides of the invention which have been further modified having similar or enhanced therapeutic properties as the corresponding, naturally-occurring allergen or portion thereof, but having reduced side effects, as well as modified peptides having improved properties such as increased solubility and stability.
- Peptides of the invention are capable of modifying, in a house dust mite-sensitive individual to whom they are administered, the allergic response of the individual to a house dust mite allergen or an allergen immunologically corss-reactive with house dust mite allergen.
- Methods of treatment or of diagnosis of sensitivity to house dust mite in an individual and therapeutic compositions comprising one or more peptides of the invention are also provided.
- Fig. 1 shows the subcloning and expression of Group I and Group II allergens from EX pteronyssinus and IX farinae.
- Fig. 2a shows adaptors used in the expression of Der p I and Der f I and Fig. 2b shows primers for amplification of Der f II and Der p II and a Der f II mutagenesis primer.
- Fig. 3 shows various peptides of desired lengths derived from the Der p I and Der p II protein allergens.
- Fig. 4 shows various peptides of desired lengths derived from the Der f I and Der f II protein allergens.
- Fig. 5 is a graphic representation depicting the responses of T cell lines from 33 patients primed in vitro to either purified native (N) or recombinant (R) Der p I protein and analyzed for response to various overlapping Der p I peptides by percent of responses with a T cell Stimulation Index (S.I.) of at least 2 within the individuals tested, the mean T cell stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
- S.I. T cell Stimulation Index
- Fig. 6 is a graphic representation depicting the responses of T cell lines from 16 patients primed in vitro to the Der f I protein and analyzed for response to various overlapping Der f I peptides by percent of responses with an (S.I.) of at least 2 within the individuals tested, the mean T cell stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
- Fig. 7 is a graphic representation depicting the responses of T cell lines from 14 patients primed in vitro to the Der p I protein and analyzed for response to various overlapping Der p I peptides and substantially matching Der f I peptides by percent of responses with an S.I. of at least 2 within the individuals tested, the mean T cell stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
- Fig. 8 is a graphic representation depicting the responses of T cell lines from 8 patients primed in vitro to the Der f I protein and analyzed for response to various overlapping Der f I peptides and substantially matching Der p I peptides by percent of responses with an S.I. of at least 2 within the individuals tested, the mean T cell stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
- Fig. 9 is a graphic representation depicting the responses of T cell lines from 29 patients primed in vitro to the Der p II protein and analyzed for response to various overlapping Der p II peptides by percent of responses with an S.I. of at least 2 within the individuals tested, the mean T cell stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
- Fig. 10 is a graphic representation depicting the responses of T cell lines from 10 patients primed in vitro to the Der f II protein and analyzed for response to various overlapping Der f II peptides by percent of responses with an S.I. of at least 2 within the individuals tested, the mean T cell stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
- Fig. 11 is a graphic representation depicting the responses of T cell lines from 10 patients primed in vitro to the Der f II protein and analyzed for response to various overlapping Der f II peptides and substantially matching Der p II peptides by percent of responses with an S.I. of at least 2 within the individuals tested, the mean T cell stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
- Fig. 12 is a graphic representation depicting the responses of T cell lines from 26 patients primed in vitro to the Der p II protein and analyzed for response to various overlapping Der f II peptides by percent of responses with an S.I. of at least 2 within the individuals tested, the mean T cell stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
- Fig. 13 is a graphic representation depicting the responses of T cell lines from 33 patients primed in vitro to the Der p I protein and analyzed for response to selected peptides of desired lengths derived from the Der p I protein allergen by percent of responses with an S.I. of at least 2 within the individuals tested, the mean T cell stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
- Fig. 14 is a graphic representation depicting the responses of T cell lines from 9 patients primed in vitro to the Der f I protein and analyzed for response to selected peptides of desired lengths derived from the Der f I protein allergen, by percent of responses with an S.I. of at least 2 within the individuals tested and the mean T cell stimulation index of positive responses for the peptide and the ranked sum of peptide responses.
- Fig. 15a is a graphic representation depicting the responses of T cell lines from 30 matched patients primed in vitro to the Der p I protein and analyzed for response to selected peptides of desired lengths derived from the Der p I and the Der f I protein allergens, by percent of responses with an S.I. of at least 2 within the individuals tested and the mean T cell stimulation index of positive responses for the peptide.
- Fig. 15b is a graphic representation derived from the same data shown in Fig. 15a showing the response of Der p I primed T cells to preferred Der £ I peptides analyzed by percent of response with an S.I. of at least two within the individuals tested (above each bar), the mean T cell stimulation index (above each bar in parenthesis) and the ranked sum of peptide responses.
- Fig. 16a is a graphic representation depicting the responses of T cell lines from 9 patients primed in vitro to the Der f I protein and analyzed for response to selected peptides of desired lengths derived from the Der f I and Der p I protein allergens, by percent of responses with an S.I. of at least 2 within the individuals tested and the mean T cell stimulation index of responses with an S.I. of at least 2 for the peptide.
- Fig. 16b is a graphic representation derived from the same data shown in Fig. 16a showing the response of Der p I primed T cell lines to preferred Der f I and Der p I peptides by percent of responses with an S.I. of at least 2 within the individuals tested and the mean T cell stimulation index of responses with an S.I. of at least 2 for the peptide.
- Fig. 17a is a graphic representation depicting the response of T cells from 29 patients primed in vitro to the Der p II protein and analyzed for response to selected peptides of desired lengths derived from the Der p II protein, by the T cell stimulation index of a response with an S.I. of at least 2 for the peptide.
- Fig. 17b is a graphic representation depicting the response of 30 patients primed in . vitro to the Der p II protein and analyzed for response to selected peptides derived from the Der p II protein by percent of responses with an S.I. of at least 2 within the individuals tested (above each bar), the mean T cell stimulation index (above each bar in parenthesis) and the ranked sum of peptide response (X-axis).
- Fig. 18a is a graphic representation depicting the response of T cells from 10 patients primed in vitro to the Der f II protein and analyzed for response to selected peptides of desired lengths derived from the Der p II and Der f II protein, by the T cell stimulation index of a response with an S.I. of at least 2 for the peptide.
- Fig. 18b is a graphic representation derived from the same data shown in Fig. 18a showing the response of Der f II primed T cell lines to preferred Der p II peptides analyzed by percent responses with an S.I. of at least 2 within the individuals tested (above each bar), the mean T cell stimulation index (above each bar in parenthesis) and the ranked sum of peptide responses (X-axis).
- Fig. 18c is a graphic representation depicting the responses of T cell lines from 4 matched patients primed in vitro with mite group I allergen and analyzed for response to preferred Der p I and Der f I peptides by percent of responses with an S.I. of at least 2 within the individuals tested and the mean T cell stimulation index of positive responses for the peptide.
- Fig. 18d is a graphic representation depicting the responses of T cell lines from 6 matched patients primed in vitro with mite group II allergen and analyzed for response to preferred Der p II peptides by percent of responses with an S.I. of at least 2 within the individuals tested and the mean stimulation index of positive responses for the peptide.
- Fig. 19a- 19b are graphic representations of the results of a direct binding assay of IgE to affinity purified and recombinant Der p I and Der p II proteins and certain Der p I and Der p II overlapping peptides.
- Fig. 20a- 20b are graphic representations of the results of a direct binding assay of IgE to affinity purified and recombinant Der f I and Der f II proteins and certain Der f I and Der f II overlapping peptides.
- Fig. 21a-21h are graphic representations of the results of a direct binding assay of IgE to a mixture of biochemically purified mite allergens (PMA) and various peptides derived from Der p I.Der f I, Der p II and Der f II.
- PMA biochemically purified mite allergens
- Fig. 22 is a composite alignment of the amino acid sequences of five Der p I clones (a)-(e) which illustrates polymorphism in the Der p I protein.
- the numbering refers to the sequence of the Der p 1(a) clone.
- the symbol (-) is used to indicate that the amino acid residue of a Der p I clone is identical to the corresponding amino acid sequences of Der p 1(a) at that position.
- the amino acid sequences of these clones indicate that there may be significant variation in Der p I, with five polymorphic amino acid residues found in the five sequences.
- Fig. 23 is a composite alignment of the amino acid sequences of three Der p II clones (c), (1) and (2) which illustrates polymorphism in the Der p II protein.
- the numbering refers to the sequence of the Der p 11(c) clone.
- the symbol (.) is used to indicate that the amino acid residue of a Der p II clone is identical to the corresponding amino acid residue of Der p II (c) at that position.
- Fig. 24 is a composite alignment of the amino acid sequences of six Der f II clones (i.e., pFLl, pFL2, MT3, MT5, MT18 and MT16) which illustrates polymorphism is the Der f II protein.
- the numbering refers to the sequences of the Der f pLFl clone.
- the symbol (.) is used to indicate that the amino acid residue of a Der f II clone is identical to the corresponding amino acid residue of Der f II pFLl at that position.
- Fig. 25 shows the amino acid sequences of modified peptides.
- Fig. 26 shows the amino acid sequenced of modified peptides in accordance with the invention.
- the present invention provides isolated modified peptides derived from the major protein allergens of the genus Dermatophagoides.
- a peptide or fragment of a protein refers to an amino acid sequence having fewer amino acid residues than the entire amino acid sequence of the protein.
- a modified peptide refers to a peptide deried from a protein allergen of the genus Dermatophagoides is which the amino acid sequence has been altered by amino acid substitution, deletion or addition.
- Peptides of the invention include odified peptides derived from Der p I (SEQ ID NO: 1 and 2), Der p II (SEQ ID NO: 3 and 4), Der f I (SEQ ID NO: 5 and 6) and Der f II (SEQ ID NO: 7 and 8) which comprise at least one T cell epitope of the allergen.
- Peptides comprising at least two regions, each region comprising at least one T cell epitope of a protein allergen of the genus Dermatophagoides are also within the scope of the invention. Each region of such peptides is derived from the same or from different mite allergens. Isolated peptides or regions of isolated peptides, each comprising at least two T cell epitopes of a mite protein allergen are particularly desirable for increased therapeutic effectiveness. Peptides which are immunologically related (e.g., by antibody or T cell cross-reactivity) to peptides of the present invention are also within the scope of the invention.
- Peptides immunologically related by antibody cross-reactivity are bound by antibodies specific for a peptide of a protein allergen of the genus Dermatophagoides.
- Peptides immunologically related by T cell cross-reactivity are capable of reacting with the same T cells as a peptide of the invention.
- Isolated peptides of the invention can be produced by recombinant DNA techniques in a host cell transformed with a nucleic acid having a sequence encoding such peptide.
- the isolated peptides of the invention can also be produced by chemical synthesis. In certain limited situations, isolated peptides can be produced by chemical cleavage of the protein allergen.
- peptides When a peptide is produced by recombinant techniques, host cells transformed with a nucleic acid having a sequence encoding the peptide or the functional equivalent of the nucleic acid sequence are cultured in a medium suitable for the cells and peptides can be purified from cell culture medium, host cells, or both using techniques known in the art for purifying peptides and proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis or immunopurification with antibodies specific for the peptide, the protein allergen of the genus Dermatophagoides from which the peptide is derived, or a portion thereof.
- Isolated peptides of the invention are substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when synthesized chemically.
- the present invention provides expression vectors and host cells transformed to express the nucleic acid sequences of the invention.
- a nucleic acid sequence coding for a mite allergen, or at least one fragment thereof may be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO).
- bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO).
- Suitable expression vectors, promoters, enhancers, and other expression control elements may be found in Sambrook et al. Molecular Cloning: A Laboratoiy Manual, second edition. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York (1989).
- Other suitable expression vectors, promoters, enhancers, and other expression elements are known to those skilled in the art.
- yeast or insect cells leads to partial or complete glycosylation of the recombinant material and formation of any inter- or intra-chain disulfide bonds.
- Suitable vectors for expression in yeast include YepSecl (Baldari et al. (1987) Embo J. 6: 229-234); pMFa (Kurjan and Herskowitz (1982) Cell ___ ⁇ 933-943); JRY88 (Schultz et al. (1987) Gene 54: 1 13-123) and pYES2 (Invitrogen Corporation, San Diego, CA). These vectors are freely available. Baculovirus and mammalian expression systems are also available.
- baculovirus system is commercially available (PharMingen, San Diego, CA) for expression in insect cells while the pMSG vector is commerically available (Pharmacia, Piscataway, NJ) for expression in mammalian cells.
- suitable expression vectors include, among others, pTRC (Amann et al. (1988) Gene 69: 301-315); pGEX (Amrad Corp., Melbourne, Australia); pMAL (N.E. Biolabs, Beverly, MA); pRIT5 (Pharmacia, Piscataway, NJ); pET-l ld (Novagen, Madison, WI) Jameel et al., (1990) J. Virol. 64:3963-3966; and pSEM (Knapp et al. (1990) BioTechniques 8: 280-281).
- pTRC Amann et al. (1988) Gene 69: 301-315
- pGEX Amrad Corp., Melbourne, Australia
- pMAL N.E. Biolabs, Beverly, MA
- pRIT5 Pharmacia, Piscataway, NJ
- pET-l ld Novagen, Madison, WI
- Jameel et al. (1990) J
- pM AL maltose E binding protein
- pRIT5 protein A
- PSEM protein A
- glutathione S-transferase glutathione S-transferase
- a mite protein allergen or fragment thereof may then be recovered from the fusion protein through enzymatic cleavage at the enzymatic site and biochemical purification using conventional techniques for purification of proteins and peptides.
- Suitable enzymatic cleavage sites include those for blood clotting Factor Xa or thrombin for which the appropriate enzymes and protocols for cleavage are commercially available from, for example, Sigma Chemical Company, St. Louis, MO and N.E. Biolabs, Beverly, MA.
- the different vectors also have different promoter regions allowing constitutive or inducible expression with, for example, IPTG induction (PRTC, Amann et al., (1988) supra: pET-1 Id, Novagen, Madison, WI) or temperature induction (pRIT5, Pharmacia, Piscataway, NJ) .
- IPTG induction PRTC, Amann et al., (1988) supra: pET-1 Id, Novagen, Madison, WI
- temperature induction pRIT5, Pharmacia, Piscataway, NJ
- a mite allergen is divided into non-overlapping peptides of desired length or overlapping peptides of desired lengths as discussed in Example III which can be produced recombinantly, synthetically, or in certain limited situations, by chemical cleavage of the allergen.
- Peptides comprising at least one T cell epitope are capable of eliciting a T cell response, such as stimulation (i.e. proliferation or lymphokine secretion) and/or are capable of inducing T cell anergy (i.e., tolerization).
- isolated peptides are tested by, for example, T cell biology techniques, to determine whether the peptides elicit a T cell response or induce T cell anergy. Those peptides found to elicit a T cell response or induce T cell anergy are defined as having T cell stimulating activity.
- human T cell stimulating activity can be tested by culturing T cells obtained from an individual sensitive to a mite allergen, (i.e., an individual who has an IgE mediated immune response to a mite allergen) with a peptide derived from the allergen and determining whether proliferation of T cells occurs in response to the peptide as measured, e.g., by cellular uptake of tritiated thymidine.
- Stimulation indices for responses by T cells to peptides can be calculated as the maximum CPM in response to a peptide divided by the control CPM.
- a T cell stimulation index (S.I.) equal to or greater than two times the background level is considered "positive.” Positive results are used to calculate the mean stimulation index for each peptide for the group of peptides tested.
- Preferred peptides of this invention comprise at least one T cell epitope and have a mean T cell stimulation index of greater than or equal to 2.0.
- a peptide having a T cell stimulation index of greater than or equal to 2.0 is considered useful as a therapeutic agent.
- Preferred peptides have a mean T cell stimulation index of at least 2.5, more preferably at least 3.5, even more preferably at least 4.0, and most preferably at least 5.0.
- preferred peptides have a positivity index (P.I.) of at least about 100, more preferably at least 150, even more preferably at least about 200 and most preferably at least about 250.
- the positivity index for a peptide is determined by multiplying the mean T cell stimulation index by the percent of individuals, in a population of individuals sensitive to house dust mite (e.g., preferably at least 9 individuals, more preferably at least 16 individuals or more, more preferably at least 29 individuals or more, or even more preferably at least 30 individuals or more), who have T cells that respond to the peptide.
- the positivity index represents both the strength of a T cell response to a peptide (S.I.) and the frequency of a T cell response to a peptide in a population of individuals sensitive to house dust mite.
- a peptide having T cell stimulating activity and thus comprising at least one T cell epitope as determined by T cell biology techniques is modified by addition or deletion of amino acid residues at either the amino or carboxy terminus of the peptide and tested to determine a change in T cell reactivity to the modified peptide. If two or more peptides which share an area of overlap in the native protein sequence are found to have human T cell stimulating activity, as determined by T cell biology techniques, additional peptides can be produced comprising all or a portion of such peptides and these additional peptides can be tested by a similar procedure. Following this technique, peptides are selected and produced recombinantly or synthetically.
- Peptides are selected based on various factors, including the strength of the T cell response to the peptide (e.g., stimulation index), the frequency of the T cell response to the peptide in a population of individuals sensitive to house dust mite, and the potential cross-reactivity of the peptide with other allergens of the genus Dermatophagoides.
- the physical and chemical properties of these selected peptides e.g., solubility, stability
- the ability of the selected peptides or selected modified peptides to stimulate human T cells e.g., induce proliferation, lymphokine secretion is determined.
- preferred peptides of the invention do not bind immunoglobulin E (IgE) or bind IgE to a substantially lesser extent than the protein allergen from which the peptide is derived binds IgE.
- IgE immunoglobulin E
- the major complications of standard immunotherapy are IgE-mediated responses such as anaphylaxis.
- Immunoglobulin E is a mediator of anaphylactic reactions which result from the binding and cross-linking of antigen to IgE on mast cells or basophils and the release of mediators (e.g., histamine, serotonin, eosinophil chemotacic factors).
- anaphylaxis in a substantial percentage of a population of individuals sensitive to house dust mite could be avoided by the use in immunotherapy of a peptide or peptides which do not bind IgE in a substantial percentage (e.g., at least about 75%) of a population of individuals sensitive to a house dust mite allergen, or if the peptide binds IgE, such binding does not result in the release of mediators from mast cells or basophils.
- the risk of anaphylaxis could be reduced by the use in immunotherapy of a peptide or peptides which have reduced IgE binding.
- peptides which have minimal IgE stimulating activity are desirable for therapeutic effectiveness.
- Minimal IgE stimulating activity refers to IgE production that is less than the amount of IgE production and/or IL-4 production stimulated by the native protein allergen (e.g., Der p I).
- a peptide of the invention when administered to a house-dust mite- sensitive individual, is capable of modifying the allergic response of the individual to the allergen.
- peptides of the invention comprising at least one T cell epitope of a mite allergen or at least two regions derived from a mite allergen, each comprising at least one T cell epitope, when administered to an individual sensitive to house dust mite are capable of modifying T cell response of the individual to the allergen.
- modification of the allergic response of an individual to a house dust mite allergen can be defined as non-responsiveness or diminution in symptoms upon exposure to a mite allergen, as determined by standard clinical procedures (see e.g., Varney et al., British Medical Journal 302: 265-269 (1990)) including dimunition in mite protein allergen induced asthmatic symptoms.
- a diminunition in symptoms includes any reduction in allergic response of an individual to the allergen after the individual has completed a treatment regimen with a peptide or protein of the invention. This dimunition may be subjective (i.e. the patient feels more comfortable in the presence of the allergen). Dimunition in symptoms can be deterined clinically as well, using standard skin tests as is known in the art.
- modified peptides derived from mite allergens comprising at least one T cell epitope have been produced.
- T cell epitopes are believed to be involved in initiation and perpetuation of the immune responses to mite allergen(s) which are responsible for the clinical symptoms of mite allergy.
- These T cell epitopes are thought to trigger early events at the level of the T helper cell by binding to an appropriate HLA molecule on the surface of an antigen presenting cell and stimulating the relevant T cell subpopulation. These events lead to T cell proliferation, lymphokine secretion, local inflammatory reactions, the recruitment of additional immune cells to the site, and activation of the B cell cascade leading to production of antibodies.
- T cell epitope is the basic element or smallest unit of recognition by a T cell receptor where the epitope comprises amino acid residues essential to receptor recognition which may be contiguous and/or non-contiguous in the amino acid sequence of the protein.
- Amino acid sequences which mimic those of T cell epitopes and which modify the allergic response to protein allergens of the genus Dermatophagoides are within the scope of this invention.
- Exposure of mite allergic patients to peptides of the present invention may tolerize or anergize appropriate T cell subpopulations such that they become unresponsive to mite allergen(s) and do not participate in mounting an immune response upon such exposure.
- administration of a peptide of the present invention may modify the lymphokine secretion profile as compared with exposure to the naturally-occurring mite protein allergen or portion thereof (e.g., result in a decrease of IL-4 and/or an increase in IL-2).
- T cell subpopulations which normally participate in the response to mite allergen(s) such that these T cells are drawn away from the site(s) of normal exposure to the allergen (e.g., nasal mucosa, skin, and lung) towards the site(s) of therapeutic administration of the peptide.
- This redistribution of T cell subpopulations may ameliorate or reduce the ability of an individual's immune system to mount an immune response at the site of normal exposure to the mite allergen(s), resulting in a diminution in allergic symptoms.
- Isolated peptides of the invention comprise at least one T cell epitope of a protein allergen of the genus Dermatophagoides and accordingly, the peptide comprises at least approximately seven amino acid residues of the protein allergen.
- therapeutic compositions of the invention preferably comprise at least two T cell epitopes of a mite allergen.
- isolated peptides of the invention preferably comprise at least two T cell epitopes and accordingly, the peptide comprises at least approximately eight amino acid residues, and preferably fifteen amino acid residues.
- compositions of the invention preferably comprise a sufficient percentage of the T cell epitopes of the entire protein allergen such that a therapeutic regimen of administration of the composition to an individual sensitive to house dust mite, results in T cells of the individual being tolerized to the protein allergen.
- Synthetically produced peptides of the invention comprising up to approximately forty-five amino acid residues in length, and most preferably up to approximately thirty amino acid residues in length are particularly desirable as increases in length may result in difficulty in peptide synthesis as well as retention of an undesirable property (e.g., immunoglobulin binding or enzymatic activity) due to maintenance of conformational similarity between the peptide and the protein allergen from which it is derived. All of the peptides shown in Fig. 3 and Fig. 4 were found to have human T cell stimulating activity.
- One embodiment of the present invention features a modified peptide of a protein allergen of the genus Dermatophagoides.
- the peptide or portion thereof comprises at least one T cell epitope of the protein allergen and has a formula X n -Y-Z m .
- Y is an amino acid sequence selected from the group consisting of: DP 1-21.7; DP 1-23.10; DP I- 23.11; DP 1-23.12; DP 1-23.5; DP 1-23.6; DP 1-23.7; DP 1-23.8; DP 1-23.9; DP 1-26.2; DP II-20.7; DP II-22.6; DP II-22.3; DP II-22.4; DP II-22.5; DP II- 25.3; DP II-25.4; DP 1-23.13; DP 1-23.14; DP 1-23.15; DP 1-23.16; DP I- 23.17; DP 1-26.3; DP 1-26.4; DP 1-26.5; DP II-22.7; DP II-22.8; DP II-22.9; DP ⁇ -22.10; and DP 11-22.11 all as shown in Fig. 26.
- X n are amino acid residues contiguous to the amino terminus of Y in the amino acid sequence of the protein allergen and Z m are amino acid residues contiguous to the carboxy terminus of Y in the amino acid sequence of the protein allergen.
- n is 0-30 and m is 0-30.
- Another embodiment of the present invention provides peptides comprising at least two regions, each region comprising at least one T cell epitope of a protein allergen of the genus Dermatophagoides and accordingly, each region comprises at least approximately seven amino acid residues).
- These peptides comprising at least two regions can comprise as many amino acid residues as desired and preferably comprise at least about 14, even more preferably about 30, and most preferably at least about 40 amino acid residues of a mite allergen.
- Each region of such peptide preferably comprises up to 45 amino acid residues in length, more preferably up to 40 residues in length and most preferably up to 30 amino acid residues in length as increases in length of a region may result in difficulty in peptide synthesis as well as retention of an undesirable property (e.g., immunoglobulin binding or enzymatic activity) due to maintenance of conformational similarity between the peptide and the protein allergen from which it is derived.
- the amino acid sequences of the regions can be produced and joined by a linker to increase sensitivity to processing by antigen-presenting cells.
- linker can be any non-epitope amino acid sequence or other appropriate linking or joining agent.
- the regions are arranged in a configuration different from a naturally-occurring configuration of the regions in the allergen or a combination of different mite protein allergens.
- the regions containing T cell epitope(s) can be arranged in a noncontiguous configuration and can preferably be derived from the same protein allergen or a combination of protein allergens.
- Noncontiguous is defined as an arrangement of regions containing T cell epitope(s) which is different than that of an amino acid sequence present in the protein allergen from which the regions are derived.
- noncontiguous regions containing T cell epitopes can be arranged in a nonsequential order (e.g., in an order different from the order of the amino acids of the native protein allergen from which the region containing T cell epitope(s) are derived in which amino acids are arranged from an amino terminus to a carboxy terminus).
- a peptide can comprise at least 15%, at least 30%, at least 50% or up to 100% of the T cell epitopes of a mite allergen.
- the individual peptide regions can be produced and tested to determine which regions bind immunoglobulin E specific for a mite allergen and which of such regions would cause the release of mediators (e.g., histamine) from mast cells or basophils.
- mediators e.g., histamine
- Those peptide regions found to bind immunoglobulin E and cause the release of mediators from mast cells or basophils in greater than approximately 10-15% of the allergic sera tested are preferably not included in the peptide regions arranged to form peptides of the invention.
- Preferred peptides of the invention comprise two or more regions derived from the same or from different mite allergens (e.g. Der p I, Der p II, Der f
- one region can be derived from Der p I and one region can be derived from Der p II; one region can be derived from Der p I and one region can be derived from Der f I; one region can be derived from Der p II and one region can be derived from Der f I; one region can be derived from Der p
- one region can be derived from Der f II: one region can be derived from Der rj I and one region can be derived from Der f II; and one region can be derived from Der f I and one region can be derived from Der f II.
- the regions can be derived from the same protein allergen, e.g., Der p I and Der p I, etc.
- Preferred peptides comprise various combinations of two or more regions, each region comprising at least one T cell epitope of a protein allergen of the genus Dermatophagoides, the regions may be derived from the same or different protein allergens of the genus Dermatophagoides, wherein at least one region comprises an amino acid sequence selected from the following group: DP 1-21.1 (SEQ ID NO: 27); DP 1-21.2 (SEQ ID NO: 28); DP 1-22.1 (SEQ ID NO: 29); DP 1-23.1 (SEQ ID NO: 33); DP 1-25.2 (SEQ ID NO: 36); DP 1-26.1 (SEQ ID NO: 37); DP 1-28.1 (SEQ ID NO: 39); DP I- 1 (SEQ ID NO: 9); DF 1-1 (SEQ ID NO: 72); DF 1-21.1 (SEQ ID NO: 90); DF 1-22.1 (SEQ ID NO: 92); DF 1-23.1 (SEQ ID NO: 95); DF 1-25.1 (SEQ ID NO:
- At least one region comprises an amino acid sequence selected from the group consisting of: DP 1-21.7; DP 1-23.10; DP 1-23.11; DP 1-23.12; DP 1-23.5; DP 1-23.6; DP 1-23.7; DP 1-23.8; DP I- 23.9; DP 1-26.2; DP II-20.7; DP II-22.6; DP II-22.3; DP II-22.4; DP II-22.5; DP II-25.3; DP II-25.4; DP 1-23.13; DP 1-23.14; DP 1-23.15; DP 1-23.16; DP 1-23.17; DP 1-26.3; DP 1-26.4; DP 1-26.5; DP II-22.7; DP II-22.8; DP II-22.9; DP ⁇ -22.10; and DP 11-22.11 all as shown in Fig. 26.
- Preferred peptides comprise a combination of two or more regions (each region having an amino acid sequence as shown in Fig. 3, Fig. 4, and Fig. 25) including: DP 1-21.7, DF 1-22.2, DP 1-23.13, DP 1-26.1, DP II-20.6, DP II-22.3, and DP II-25.2; DP 1-21.2, DF 1-22.2, DP 1-23.10, DP 1-26.2, DP 1-20.6, DP II- 22.4, and DP ⁇ -25.3, DP 1-21.7, DF 1-22.2, DP 1-23.11, DP 1-26.3, DP II-20.7, DP II-22.5, and DP II-25.4; DP 1-21.7, DF 1-22.2, DP 1-23.12, DP 1-26.4, DP II-20.6, DP II-22.6, and DP II-25.2; DP 1-21.7, DF 1-22.2, DP 1-23.5, DP 1-26.5, DP II- 20.7, DP II-22.7
- Additional preferred peptides comprise at least two of the following combination of regions in any arrangement: X,Y,Z,A,B,C,D, wherein X is DP 1-21.2 or DP 1-21.7; Y is DF 1-22.2; Z is DP 1-23.1, DP I- 23.10, DP 1-23.11, DP 1-23.12, DP 1-23.13, DP 1-23.14, DP 1-23.15, DP I- 23.16, DP 1-23.17, DP 1-23.5, DP 1-23.6, DP 1-23.7, DP 1-23.8 or DP 1-23.9.; A is DP 1-26.1, DP 1-26.2, DP 1-26.3, DP 1-26.4, or DP 1-26.5; B is DP 1-20.6 or DP II-20.7; C is DP 11-22, DP II-22.6, DP II-22.7, DP II-22.8, DP II-22.9, DP 11-22.10, DP 11-22.11, DP II-22.3, DP II
- Another preferred peptide comprises a specific sequential arrangement of amino acid sequences, said specific sequential arrangement having the formula:
- ADYCZBX wherein X is DP 1-21.2 or DP 1-21.7; Y is DF 1-22.2; Z is DP 1-23.1, DP I- 23.10, DP 1-23.11, DP 1-23.12, DP 1-23.13, DP 1-23.14, DP 1-23.15, DP I- 23.16, DP 1-23.17, DP 1-23.5, DP 1-23.6, DP 1-23.7, DP 1-23.8 or DP 1-23.9.; A is DP 1-26.1, DP 1-26.2, DP 1-26.3, DP 1-26.4, or DP 1-26.5; B is DP 1-20.6 or DP II-20.7; C is DP 11-22, DP II-22.6, DP II-22.7, DP II-22.8, DP II-22.9, DP 11-22.10, DP II-22.11, DP II-22.3, DP II-22.4, or DP II-22.5; and D is DP II-25.2, DP ⁇ -25.3, or DP
- Another preferred peptide comprises a specific sequenctial arrangement of amino acid sequences, said specific sequential arrangement having the formula: - 13 -
- DYZCAXB wherein X is DP 1-21.2 or DP 1-21.7; Y is DF 1-22.2: Z is DP 1-23.1, DP I- 23.10, DP 1-23.11, DP 1-23.12, DP 1-23.13, DP 1-23.14, DP 1-23.15, DP I- 23.16, DP 1-23.17, DP 1-23.5, DP 1-23.6, DP 1-23.7, DP 1-23.8 or DP 1-23.9.; A is DP 1-26.1, DP 1-26.2, DP 1-26.3, DP 1-26.4, or DP 1-26.5; B is DP 1-20.6 or DP II-20.7; C is DP 11-22, DP II-22.6, DP II-22.7, DP II-22.8, DP II-22.9, DP 11-22.10, DP II-22.1 1, DP II-22.3, DP II-22.4, or DP II-22.5; and D is DP II-25.2, DP II-25.3, or DP II-25
- Yet another preferred peptide has a specific arrangement of amino acid sequences, said specific sequential arrangement having the formula:
- DXZACBY wherein X is DP 1-21.2 or DP 1-21.7; Y is DF 1-22.2; Z is DP 1-23.1, DP I- 23.10, DP 1-23.11, DP 1-23.12, DP 1-23.13, DP 1-23.14, DP 1-23.15, DP I- 23.16, DP 1-23.17, DP 1-23.5, DP 1-23.6, DP 1-23.7, DP 1-23.8 or DP 1-23.9.; A is DP 1-26.1, DP 1-26.2, DP 1-26.3, DP 1-26.4, or DP 1-26.5; B is DP 1-20.6 or DP II-20.7; C is DP 11-22, DP II-22.6, DP II-22.7, DP II-22.8, DP II-22.9, DP 11-22.10, DP II-22.11, DP II-22.3, DP II-22.4, or DP II-22.5; and D is DP II-25.2, DP ⁇ -25.3, or DP
- Peptides of protein allergens of the genus Dermatophagoides within the scope of the invention can be used in methods of treating and preventing allergic reactions to mite allergens.
- one aspect of the present invention provides therapeutic compositions comprising a peptide or a modified peptide of Der p I, Der p II, Der f I, or Der f II including at least one T cell epitope, or preferably at least two T cell epitopes, and a pharmaceutically acceptable carrier or diluent.
- the therapeutic composition comprises a pharmaceutically acceptable carrier or diluent and a peptide comprising at least two regions, each region comprising at least one T cell epitope of a mite allergen and derived from the same or from different mite protein allergens.
- Preferred therapeutic compositions comprise at least one peptide and/or a modified peptide of at least one protein allergen of the genus Dermatophagoides.
- the composition comprises a sufficient percentage of the T cell epitopes of at least one protein allergen such that a therapeutic regimen of administration of the composition to an individual sensitive to a house dust mite allergen resulting in T cells of the individual being tolerized to the protein allergen.
- the composition comprises a sufficient percentage of the T cell epitopes of one or more protein allergens such that at least about 40%, and more preferably at least about 60% of the T cell reactivity of each protein is included in the composition.
- Such compositions can be administered to an individual to treat or prevent sensitivity to house dust mite or to an allergen which is immunologically cross-reactive with house dust mite.
- a peptide derived from a mite allergen comprising at least one T cell epitope can be administered in combination with an appropriate diluent, a carrier, and/or an adjuvant.
- the terapeutic composition is preferably administered in non-immunogenic form, e.g. it does not contain adjuvant.
- Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
- Pharmaceutically acceptable carriers include polyethylene glycol (Wie et al.
- compositions will generally be administered by injection, (subcutaneous, intravenous, etc.) oral administration, (e.g. as in the form of a capsule), inhalation, transdermal application or rectal administration.
- the therapeutic compositions of the invention are administered to individuals sensitive to house dust mite at dosages and for lengths of time effective to reduce sensitivity (i.e., reduce the allergic response) of the individual to a house dust mite allergen.
- a therapeutically effective amount of one or more of the same or of different therapeutic compositions can be administered simultaneously or sequentially to an individual sensitive to house dust mite. Effective amounts of the therapeutic compositions will vary according to factors such as the degree of sensitivity of the individual to house dust mites, the age, sex, and weight of the individual, and the ability of the peptide to stimulate a T cell response in the individual.
- a preferred therapeutic composition of the inventino comprises at least one of the following modified peptides of the invention comprising at least one T cell epitope and a pharmaceutically acceptable carrier or diluent: DP 1-21.7; DP 1-23.10; DP 1-23.11; DP 1-23.12; DP 1-23.5; DP 1-23.6; DP I- 23.7; DP 1-23.8; DP 1-23.9; DP 1-26.2; DP II-20.7; DP II-22.6; DP II-22.3; DP II-22.4; DP II-22.5; DP II-25.3; DP II-25.4; DP 1-23.13; DP 1-23.14; DP 1-23.15; DP 1-23.16; DP 1-23.17; DP 1-26.3; DP 1-26.4; DP 1-26.5; DP II- 22.7; DP II-22.8; DP I ⁇ -22.9; DP 11-22.10; and DP 11-22.1 1 all as shown in Fig. 26.
- a composition comprising at least two peptides (e.g., a physical mixture of at least two peptides), each comprising at least one T cell epitope of a protein allergen of the genus Dermatophagoides.
- the peptides or modified peptides are derived from the same or from different mite allergens.
- Such compositions can be administered in the form of a therapeutic composition with a pharmaceutically acceptable carrier of diluent.
- a therapeutically effective amount of one or more of such compositions can be administered simultaneously or sequentially to an individual sensitive to house dust mite.
- Preferred therapeutic compositions comprising a pharmaceutically acceptable carrier or diluent and at least two peptides each comprising at least one T cell epitope, comprise at least one peptide selected from the group consisting of: DP 1-1 (SEQ ID NO: 9); DP 1-2 (SEQ ID NO: 10); DP 1-3 (SEQ ID NO: 11); DP 1-4 (SEQ ID NO: 12); DP 1-11.1 (SEQ ID NO: 13); DP 1-12.1 (SEQ ID NO: 14); DP 1-5 (SEQ ID NO: 15); DP 1-13 (SEQ ID NO: 17); DP 1-14 (SEQ ID NO: 18); DP 1-15 (SEQ ID NO: 19); DP 1-6.1 (SEQ ID NO: 20); DP 1-7.1 (SEQ ID NO: 21); DP 1-8 (SEQ ID NO: 22); DP 1-9 (SEQ ID NO: 23); DP I- 16 (SEQ ID NO: 24); DP I- 10 (SEQ ID NO: 25); DP 1-17
- At least one region comprises a modified amino acid sequence selected from the following group: DP 1-21.7; DP 1-23.10; DP 1-23.11; DP 1-23.12; DP 1-23.5; DP 1-23.6; DP 1-23.7: DP I- 23.8; DP 1-23.9; DP 1-26.2; DP II-20.7; DP II-22.6; DP II-22.3; DP II-22.4; DP II-22.5; DP ⁇ -25.3; DP II-25.4; DP 1-23.13; DP 1-23.14; DP 1-23.15; DP 1-23.16; DP 1-23.17; DP 1-26.3; DP 1-26.4; DP 1-26.5; DP II-22.7; DP II- 22.8; DP H-22.9; DP 11-22.10; and DP 11-22.11 all as shown in Fig. 26.
- compositions comprising a pharmaceutically acceptable carrier or diluent and at least two peptides each comprising at least one T cell epitope, comprise at least one peptide selected from the group consisting of: DP 1-21.1 (SEQ ID NO: 27): DP 1-21.2 (SEQ ID NO: 28); DP 1-22.1 (SEQ ID NO: 29); DP 1-22.2 (SEQ ID NO: 30); DP I- 22.3 (SEQ ID NO: 31); DP 1-22.4 (SEQ ID NO: 32); DP 1-23.1 (SEQ ID NO: 33); DP 1-23.2 (SEQ ID NO: 34); DP 1-25.1 (SEQ ID NO: 35); DP 1-25.2 (SEQ ID NO: 36); DP 1-26.1 (SEQ ID NO: 37); DP 1-27.1 (SEQ ID NO: 38); DP 1-28.1 (SEQ ID NO: 39); DP 1-28.2 (SEQ ID NO: 40): DP 1-1 (SEQ ID NO: 9); DF
- compositions of the invention comprise the following combination of peptides and/or modified peptides and a pharmaceutically acceptable carrier or diluent: DP 1-21.7, DF 1-22.2, DP 1-23.13. DP 1-26.1. DP II-
- a composition comprising a pharmaceutically acceptable carrier or diluent and at least two, preferably at least four, more preferably at least six, even more preferably at least seven of the following combination of peptides: X, Y, Z, A, B, C, D, wherein X is DP 1-21.2 or DP 1-21.7; Y is DF 1-22.2; Z is DP 1-23.1, DP I- 23.10, DP 1-23.11, DP 1-23.12, DP 1-23.13, DP 1-23.14, DP 1-23.15, DP I- 23.16, DP 1-23.17, DP 1-23.5, DP 1-23.6, DP 1-23.7, DP 1-23.8 or DP 1-23.9.; A is DP 1-26.1, DP 1-26.2, DP 1-26.3, DP 1-26.4, or DP 1-26.5; B is DP 1-20.6 or DP II-20.7; C is DP 11-22, DP II-22.6,
- the present invention also provides methods of detecting sensitivity in individuals to house dust mite allergens comprising combining a blood sample obtained from the individual with a peptide of the present invention, under conditions appropriate for binding of blood components with the peptide and determining the extent to which such binding occurs.
- the extent to which binding occurs is determined by assessing T cell function, T cell proliferation or a combination thereof.
- a modified peptide can be produced in which the amino acid sequence has been altered, such as by amino acid substitution, deletion, or addition, to modify immunogenicity and/or reduce allergenicity, or to which a component has been added for the same purpose.
- a peptide can be modified so that it maintains the ability to induce T cell anergy and bind MHC proteins without the ability to induce a strong proliferative response or possibly, any proliferative response when administered in immunogenic form.
- critical binding residues for the T cell receptor can be determined using known techniques (e.g., substitution of each residue and determination of the presence or absence of T cell reactivity).
- Those residues shown to be essential to interact with the T cell receptor can be modified by replacing the essential amino acid with another, preferably similar amino acid residue (a conservative substitution) whose presence is shown to enhance, diminish but not eliminate, or not effect T cell reactivity.
- those amino acid residues which are not essential for T cell receptor interaction can be modified by being replaced by another amino acid whose incorporation may enhance, diminish or not effect T cell reactivity but does not eliminate binding to relevant MHC.
- peptides of the invention can be modified by replacing an amino acid shown to be essential to interact with the MHC protein complex with another, preferably similar amino acid residue (conservative substitution) whose presence is shown to enhance, diminish but not eliminate or not effect T cell activity.
- amino acid residues which are not essential for interaction with the MHC protein complex but which still bind the MHC protein complex can be modified by being replaced by another amino acid whose incorporation may enhance, not effect, or diminish but not eliminate T cell reactivity.
- Preferred amino acid substitutions for non-essential amino acids include, but are not limited to substitutions with alanine, glutamic acid, or a methyl amino acid.
- Another example of a modification of peptides is substitution of cysteine residues preferably with serine, threonine, leucine or glutamic acid to minimize dimerization via disulfide linkages.
- the stability of a peptide of Der p II or Der f II which includes the first ten amino acid residues of either allergen can be enhanced by replacing the cysteine located at the 8th amino acid residue, preferably with alanine, or glutamic acid, or alternatively with serine or threonine.
- peptides can also be modified to incorporate one or more polymorphisms in the amino acid sequence of a protein allergen resulting from natural allelic variation.
- D-amino acids, non-natural amino acids or non-amino acid analogues can be substituted or added to produce a modified peptide within the scope of this invention.
- peptides of the present invention can be modified using the polyethylene glycol (PEG) method of A. Sehon and co-workers (Wie et al. supra ) to produce a peptide conjugated with PEG.
- PEG polyethylene glycol
- Modifications of peptides or portions thereof can also include reduction/alkylation (Tarr in: Methods of Protein Microcharacterization. J.E. Silver ed. Humana Press, Clifton, NJ, pp 155-194 (1986)); acylation (Tarr, supra); esterification (Tarr, supra): chemical coupling to an appropriate carrier (Mishell and Shiigi, eds, Selected Methods in Cellular Immunology. WH Freeman, San Francisco, CA (1980); U.S. Patent 4,939,239); or mild formalin treatment (Marsh International Archives of Allergy and Applied Immunology 41 : 199-215 (1971)).
- peptides within an allergen group can be modified to enhance T cell reactivity.
- a peptide of one group allergen which may be less reactive than a peptide of another group allergen corresponding in amino acid position can have one or more amino acids substituted with one or more amino acids from the corresponding peptide.
- peptides can be modified to incorporate a polymorphism in the amino acid sequence of a protein allergen resulting from natural allelic variation. Modification of peptides to include one or more of these polymorphisms may result in enhanced stability and/or reactivity.
- reporter group(s) to the peptide backbone.
- poly-histidine can be added to a peptide to purify the peptide on immobilized metal ion affinity chromatography (Hochuli, E. et al., Bio/Technology. :1321-1325 (1988)).
- specific endoprotease cleavage sites can be introduced, if desired, between a reporter group and amino acid sequences of a peptide to facilitate isolation of peptides free of irrelevant sequences.
- canonical protease sensitive sites can be recombinantly or synthetically engineered between regions, each comprising at least one T cell epitope.
- charged amino acid pairs such as KK or RR
- the resulting peptide can be rendered sensitive to cathepsin and/or other trypsin-like enzymes cleavage to generate portions of the peptide containing one or more T cell epitopes.
- such charged amino acid residues can result in an increase in solubility of a peptide.
- Site-directed mutagenesis of DNA encoding a peptide of the invention can be used to modify the structure of the peptide. Such methods may involve PCR (Ho et al., Gene. 77:51-59 (1989)) or total synthesis of mutated genes (Hostomskv. Z.. et al.. Biochem. Biophvs. Res. Comm.. 161: 1056-1063 (1989)). To enhance bacterial expression, the aforementioned methods can be used in conjunction with other procedures to change the eucaryoti ⁇ codons in DNA constructs encoding peptides of the invention to ones preferentially used in _____, coli.
- modifications to peptides 23.1 include the addition of charged amino acids (DP 1-23.7) charged amino acid pairs (DP 1-23.5) to increase solubility and replacement of a cysteine residue with serine (DP 1-23.6) or glutamic acid to increase solubility.
- Changes to peptide DP 11-22 include the addition of charged amino acid pairs (22.6 and 22.3) to increase solubility as shown in Fig. 25.
- the present invention also provides nucleic acids having sequences encoding peptides of the invention.
- Nucleic acid sequences used in any embodiment of this invention can be cDNA as described herein, or alternatively, can be any oligodeoxynucleotide sequence having all or a portion of a sequence represented herein, or their functional equivalents. Such oligodeoxynucleotide sequences can be produced chemically or mechanically, using known techniques.
- Monoclonal antibodies 4C1 and 6D6 (University of Virginia, NC) were used for immunoaffinity purification of group I or group II mite allergens respectively (Chapman et al., J. Allergy Clin. Immunol. 80: 1479-1484 (1987); Hevmann et al.. J. Allergy Clin. Immunol. 83: 1055-1067 (1989)).
- the 4Cl monoclonal antibody reacts with an epitope shared by both the Der f I and Der p I proteins; similarly, the 6D6 monoclonal antibody reacts with both the Der f II and Der p II proteins.
- ascites fluid was cut in 50% ammonium sulfate and the antibodies coupled to CNBr-activated Sepharose 4B (Pharmacia) in 100 mM NaHCO ⁇ . 500 mM NaCl, pH 8.3, overnight at 4°C.
- Monoclonal antibody columns were equilibrated in PBS and the filtered extracts loaded at 15 ml per hour. The column was then washed in 20 volumes of PBS, after which a more stringent wash of 20 column volumes was carried out with PBS supplemented with 500 mM NaCl. Proteins were eluted in 500 mM NaCl, 100 mM glycine pH 11.0, and fractions evaluated for protein content by spectrophotometric absorbance at 280 nm. Peak fractions were pooled and dialyzed extensively against PBS, concentrated with a negative pressure dialysis device (obtained from Amicon, Beverly MA) and were used in T-cell epitope mapping studies of the mite group I and group II allergens. Recovered proteins were obtained at purities ranging from 80% (group II) to 90% (group I).
- Reverse Phase HPLC chromatography was applied to further purify the immunoaffinity purified group II mite protein allergen according to the conditions in Heymann et al., J. Allergy Clin. Immunol. 8 : 1055-1067 (1989). Briefly, immunoaffinity purified protein was applied to a 5 ⁇ m 300 A C-8 column (Applied Biosystems Inc.) in 0.1% vol./vol. TFA/H2O. The flow rate for the column was 1 ml/min. with a gradient of 0-60% acetonitrile/0.1% TFA over 60 minutes. Group ⁇ proteins eluted around 45% acetonitrile/0.1% TFA. Fractions were analyzed for purity by SDS-polyacrylamide gel electrophoresis followed by densitometric scanning. Those fractions with group II protein of purity greater than 90% were subsequently utilized for human T-cell mapping of the allergen.
- the cDNAs encoding the group I allergens from E pteronyssinus and P. farinae were obtained from Dr. Wayne Thomas in plasmid form as subclones from ⁇ gtl 1 (Chua et al, J. Exp. Med. 167: 175-182 (1988); Dilworth et al., Clin. Exp. Allergy 21: 25-32 (1991)).
- the Der p I cDNA had been subcloned from an M13 RF plasmid as an EcoRI fragment into pUC18 by Dr. Roland Buelow at ImmuLogic (Palo Alto), while the Der f I cDNA was manipulated directly from the M13mpl9 RF plasmid Df 1(1).
- the leader sequence was used as a purification aid as described above, while the RRS was added to enhance recombinant message stability and thereby increase recombinant protein yield (Skoglund et al., Gene 88: 1-5 (1990)).
- an insert in this case an Amb a 1.1 cDNA encoding the major allergen of the short ragweed (Rafnar et al.. J. Biol. Chem. 226: 1229-1236 (1991)) was subcloned in frame with the Hg leader as an EcoRI to PstI fragment.
- the Amb a 1.1 cDNA was excised by EcoRI/Hind III digestion and replaced by adaptors with ⁇ EcoRI/Hind in overhangs (composed of a pair of complimentary oligonucleotides).
- These adaptors encoded the first 5 amino acids, up to the Pst I site, of the mature Der p I NH2 terminus, and the first 10 amino acids, up to the Hpal site, of the mature Der f I NH2 terminus.
- the EcoRI site in the vector was destroyed, leaving the EcoRI site encoded internal to the adaptor as the sole EcoRI site in the intermediate constructs pTrc99A Hisg 5' pi RRS and pTrc99A Hiss 5' fl RRS.
- the Der p I cDNA was inserted as a Pstl/EcoRI fragment into Pstl/EcoRI digested pTrc99A Hisg 5' pi RRS, while the Der f I cDNA was inserted as a Hpal/EcoRI fragment into Hpal/EcoRI digested pTrc99A Hisg 5' fl RRS.
- BL21 [DE3] contains a recombinant phage ⁇ lysogen, DE 3, with a phage T7 RNA polymerase gene under the transcriptional control of the lac UV5 promoter.
- T7 RNA polymerase gene expression is induced by the addition of IPTG (Isopropyl-B-D-thiogalactopyrano- side), which in turn leads to high level expression of the recombinant gene subcloned 3' of the pET vector's T7 gn 10 promoter.
- the cDNAs encoding the group II allergens from EX pteronyssinus and EX farinae were also obtained from Dr. Wayne Thomas in plasmid form as subclones from ⁇ gtl l (Chua et al.. Int Arch. AppI. Immun. 91 : 118-123 (1990); Trudinger et al., Clin. Exp. Allergy 21: 33-37 (1991)).
- the original Der p II cDNA was subcloned from an M13RF plasmid into the pCA and pGEX vectors by Dr. Roland Buelow at ImmuLogic (Palo Alto). Dr.
- Thomas' group had supplied the Der f II cDNA as a subclone in pGEX.
- Both pGEX plasmids harboring group II cDNAs were used as templates for PCR amplification with the same 5' sense/3' antisense primer pair (Fig. 2b).
- the primers were designed to fuse an EcoRI site (GAATTC encoding the amino acids EF) in frame with the NH2 terminus of the mature group II proteins and place a PstI site 3' of the group H coding region.
- An MJ Research Thermal Controller with a program of 30 X (94°C 1 min./55°l min.
- the Der f II cDNA possessed a sequence polymorphism at position 54 (i.e., threonine residue in place of isoleucine).
- site directed mutagenesis of the T54 residue in the Der f II cDNA was performed using a Muta-Gene kit from Bio-Rad Laboratories, based on the method of Kunkel, Proc. Natl. Acad. Sci. USA 82: 488-492 (1985).
- the original M13mpl9RF with the T54 Der_f II cDNA was transformed into CJ236 bacteria, which tolerate the incorporation of uracil in place of thymidine during DNA replication, and single stranded phage DNA prepared as template.
- a mutagenic 17 base pair primer (Fig. 2b) was annealed to the phage template DNA.
- T4 DNA polymerase was used to copy the template DNA primed by the mutagenic oligonucleotide, and T4 DNA ligase served to seal gaps in the DNA strand.
- the reaction was transformed into MV1190 bacteria, which are wild type for the editing of uracil residues from DNA and therefore selectively replicate the mutagenized (non-uracil containing) strand, and single stranded phage DNA prepared from recombinant (white) plaques.
- the two mite group II cDNAs were subcloned as Hg fusion proteins into T7 vector.
- pTrc ppA Hisg f II RRS and pTrc99A Hisg p II RRS were Nco I Nhel digested, the cDNA inserts isolated, and subcloned into Ncol Nhel digested pETl Id Hiss P (Fig. 1). Recombinant plasmids were transformed into BL 21 [DE3] bacteria for expression.
- BL21 DE3 host bacteria harboring the pETl Id mite allergen expression constructs were freshly streaked onto a BHI agar plate (3.7% wt./vol. Difco Brain Heart Infusion; 1.5% wt./vol. Difco agar) supplemented with 200 ⁇ g/ml ampicillin and incubated overnight at 37°C.
- a single colony was innoculated into a 2 ml of 200 ⁇ g/ml ampicillin/BHl media (3.7% wt./vol. Difco Brain Heart Infusion) and shaken at 300 rpm at 37°C until turbid but not saturated.
- the 2 ml culture was then added to 100 ml of 200 ⁇ g/ml ampicillin/BHl media, shaken at 300 rpm at 37°C until turbid but not saturated, at which point the culture was divided into 18 X 500 ml (9 litres) of 200 ⁇ g/ml ampicillin/BHl media and shaken at 300 rpm at 37°C.
- the OD595 of the culture reached 1.0, expression of the recombinant molecules was induced by the addition of IPTG to 400 ⁇ M, and allowed to continue for two hours.
- Bacteria were harvested by centrifugation at 10,000 X g for 15 minutes, and resuspended in l/20 m volume of lysis buffer (0.2 mg/ml lysozyme, 100 mM NaP ⁇ 4 pH 8.0, 50 mM NaCl), incubated on ice for 30 minutes, and frozen at -70°C.
- the frozen bacteria were fractured by a quick thaw at 37 °C and then sonicated 5 times for 20 seconds at 30 second intervals.
- the sonicated samples were centrifuged at 15,000 X g to separate soluble and paniculate bacterial proteins.
- the soluble proteins were poured off, and the pelleted protein resuspended in 6 M guanidine HC1, 100 mM 2- mercaptoethanol, 100 mM NaP ⁇ 4, 10 mM Tris pH 8.0.
- This suspension was subjected to centrifugation at 15,000 X g, and the supernatant removed, adjusted to pH 8.0 with 10 N NaOH, and applied to an NTA agarose column that had been equilibrated in 6 M guanidine HC1, 100 mM NaP ⁇ 4, 10 mM Tris pH 8.0.
- the column was washed in 6 M guanidine HC1, 100 mM NaPO4, 10 mM Tris pH 8.0 until the OD28O of the effluent reached background.
- the column buffer was then switched to 8 M urea, 100 mM NaPO4, 10 mM Tris pH 8.0. After equilibration, a more stringent wash was performed in 8 M urea, 100 mM NaOAc, 10 mM Tris pH 6.3 until the OD28O of the effluent reached background.
- Recombinant mite protein (as an H fusion) was then eluted in 8 M urea, 100 mM NaOAc, 10 mM Tris pH 4.5 and collected in aliquots whose OD28O profile was monitored.
- the protein peak was dialyzed 3 times into 500 volumes of PBS for human T-cell analysis. Yield ranged from 10 to 70 mg of recombinant protein per liter with purity (as determined by densitometric scanning) ranging from 80 (Der f II) to 95%.
- Der p I, Der f I, Der p II, and Der f II overlapping peptides as shown in Figs. 3 and 4 were synthesized using standard Fmoc/tBoc synthetic chemistry and purified by dialysis or Reverse Phase HPLC.
- the amino acid residues of the synthesized peptides are in brackets by the peptide name and the amino acid sequence (in single letter code) is next to the peptide name.
- the peptide names are consistent throughout the Figures.
- the Der p I, Der f I, Der p II and Der f II proteins were divided into overlapping peptides in such a way that the overlapping peptides of Der p I and Der f I as well as for Der p ⁇ and Der f II have corresponding amino acid residue numbers, e.g. DP 1-1 and DF 1-1 both contain amino acid residues 1-20 of the Der p I and Der f I allergens, respectively.
- PBMC Peripheral blood mononuclear cells
- PBMC Peripheral blood mononuclear cells
- 5 X 10 ⁇ PBMC were cultured in triplicate microwells for 7 days at 37°C in the presence of various concentrations of antigen in 200 ⁇ l RPMI-1640 containing 5% human AB serum. Each well then received 1 ⁇ Ci tritiated thymidine for 16 hours.
- the counts incorporated were collected onto glass fiber filters and processed for liquid scintillation counting. Table I shows the results of this assay. The CPM +/- standard deviation are shown.
- the stimulation index of each response is the ratio of the -1H-thymidine CPM incorporated by the cells in response to antigen divided by the ⁇ H-thymidine CPM incorporated by cells in medium only.
- S.I. The stimulation index of each response
- the results indicate that this patient responds with an S.I. of at least 2.0 to peptides DP I-l, DP 1-3, DP 1-8, DP I- 10, DP 1-5.2, DP II-4 and DP II-9.
- these peptides contain Der p I or Der p II T cell epitopes recognized by T cells from this particular allergic patient.
- PBMC Peripheral blood mononuclear cells
- 10 7 PBMC from individual 543 were cultured in 10 ml RPMI- 1640 containing 5% pooled human AB serum and supplemented vith glutamine, penicillin, streptomycin and HEPES buffer in the presence of 20 ⁇ g/ml purified native Der p I/ml at 37°C for 7 days. Viable cells were then purified by Ficoll-Hypaque centrifugation and cultured for 2 additional weeks in RPMI- 1640/5% AB serum containing 5 units recombinant human IL-2/ml and 5 units recombinant human IL-4/ml. The resting T cells were then tested in a secondary proliferation assay to assess T cell responses to various house dust mite proteins and peptides.
- 2 X 10 ⁇ resting T cells were cultured in 200 ⁇ l of RPMI- 1640/5% AB serum for 3 days at 37 °C in the presence of 2 X 10 ⁇ autologous Epstein-Barr virus transformed B cells (20,000 Rads) as antigen presenting cells with various concentrations of purified native Der p I or synthetic Der p I peptides. Each well then received 1 ⁇ Ci tritiated thymidine for 16 hours. The counts incorporated were collected onto glass fiber filters and processed for liquid scintillation counting. Medium alone, acting as negative control, contained no allergen or peptide. The results of this experiment indicate that this particular patient responds with an S.I.
- DP I- 11 SEQ ID NO: 117
- DP 1-12 SEQ ID NO: 118
- DP II-3 SEQ ID NO: 119
- Three additional peptides DP I-l 1.1 (SEQ ID NO: 13), DP 1-12.1 (SEQ ID NO: 14) and DP II-3.1 (SEQ ID NO: 43) were synthesized with no changes from the native sequence.
- T cell stimulation index is calculated by:
- the bar represents the cumulative rank of the peptide response in the group of patients. To determine the cumulative rank, the 5 peptides with the highest S.I. in each individual were determined and assigned a numerical rank in descending order, with 5 representing the strongest response. The ranks for each peptide were then summed in the group of patients to -33 -
- the positivity index (P.I.) for each peptide can be calculated.
- the P.I. for each individual is determined by multiplying the mean S.I. by the percent of individuals, in a population of individuals sensitive to house dust mite (e.g. preferably at least 15 individuals, more preferably at least 30 individuals or more) who responded with an S.I. of at least 2.0 to that peptide (e.g., for DP I-l in Fig. 5, the P.I. would be about 343 (73% x 4.7).
- Fig. 5 demonstrates that peptides DP I-l, DP I- 2, DP 1-3, DP 1-4, DP 1-5, DP 1-6.1, DP 1-7.1, DP 1-8, DP 1-9, DP 1-16, and DP 1-10 contain significant regions of T cell reactivity in this panel of patients.
- Example V Experiments similar to those of Example V were performed to determine the T cell-reactive areas of the Der f I protein.
- PBMC from house dust mite-allergic patient 783 were isolated as described in Example V and were stimulated in vitro with recombinant purified Der f I at 20 ⁇ g/ml.
- the results of the proliferation assay with Der f I peptides using x- irradiated (3500 Rads) autologous PBMC as antigen presenting cells indicate that T cells from this patient respond to the peptides DF 1-8.1, DF 1-9, DF 1-6, DF 1-10, DF 1-2.1, DF 1-3, DF Ml, DF 1-5, DF I-l, and DF 1-17 (data not shown).
- T cell lines were primed with affinity purified Der f I at 20 ⁇ g/ml or at 10 ⁇ g/ml and x-irradiated (25,000 Rads) autologous Epstein- Barr virus transformed B cells were used as antigen presenting cells.
- affinity purified Der f I at 20 ⁇ g/ml or at 10 ⁇ g/ml
- x-irradiated (25,000 Rads) autologous Epstein- Barr virus transformed B cells were used as antigen presenting cells.
- Fig. 6 The data was analyzed as described in Example V.
- the Der p I and Der f I proteins are very homologous (81 % identity).
- T cell lines were primed in vitro as described in Example V and tested for response to a set of substantially matching Der p I and Der f I peptides (e.g., DP I-l (amino acid residues 1-20 of Der p I) or DF I-l (amino acid residues 1-20 of Der f I).
- the data was analyzed as described in Example V except the highest and lowest S.I.
- the Der f I peptide was a more potent stimulator of Der p I-primed T cells than the corresponding Der p I peptide.
- Fig. 8 shows the results of inverse experiments in which T cells from a number of patients were primed in vitro to the Der f I protein and analyzed for response to various Der p I peptides and a set of substantially matching Der f I peptides. The results of 8 positive experiments indicate that Der f I primed T cells respond significantly to peptides DP I-l, DP 1-3, DP 1-4, DP M l/11.1, DP 1-14, DP 1-5, DP M5, and DP 1-8.
- Example V Experiments similar to those of Example V were performed to determine the T cell-reactive areas of the Der p II protein.
- PBMC from house dust mite-allergic patient 348 were isolated as described in Example V and were stimulated in vitro with 20 ⁇ g/ml purified native Der p ⁇ .
- the results of a proliferation assay using x-irradiated (25,000 Rads) Epstein-Barr virus transformed autologous B cells as antigen-presenting cells with various Der p II peptides demonstrate that this particular patient responds well to peptides DP IM, DP II-7, DP II-8, DP II-2, and DP II-9 (data not shown).
- T cell lines were primed with recombinant Der p II at 20 ⁇ g/ml or at 3 ⁇ g/ml and x-irradiated (3500 Rads) autologous PBMC were used as antigen-presenting cells.
- Fig. 9 A summary of the results of 26 positive experiments is shown in Fig. 9.
- the data was analyzed as described in Example V, except the ranked sum of peptide responses was analyzed assigning a value of 3, 2 or 1 to the three highest S. I. responses. Areas of significant T cell reactivity within the Der p II protein for this panel of patients are found in peptides DP II- 1, DP II-2, DP II-3, DP II-4, DP II-7, DP II-8 and DP II-9.
- T cell-reactive areas of the Der f II protein were performed. For example, PBMC from house dust mite- allergic patient 384 were stimulated in vitro with purified recombinant Der f II and the T cell line was then challenged in the presence of x-irradiated (25,000 Rads) autologous Epstein-Barr virus transformed B cells as antigen presenting cells with various overlapping Der f ⁇ peptides. The results of this proliferation assay indicate that T cells from this particular patient respond well to peptides DF IM, DF II-2, DF II-3.1, DF ⁇ -4.5, DF IM5, DF 11-16, and DF II-19.1 (data not shown).
- T cell lines were primed by stimulating the patient PBMC with 20 ⁇ g ml or 3 ⁇ g/ml purified native Der f II, and were assayed in the presence of x-irradiated (3500 Rads) autologous PBMC as antigen-presenting cells.
- x-irradiated 3500 Rads
- Fig. 10 The data was analyzed as detailed in Example IX, except the highest and lowest S.I. values of the positive responses to each peptide were not omitted from the calculations.
- T cells primed with the Der f II protein were challenged with various Der f II peptides and a set of substantially matching Der p II peptides.
- a summary of the results of 10 positive experiments is shown in Fig. 11. The results indicate that Der f II primed T cells respond significantly to peptides DP II- 1 , DP ⁇ -3/3.1, DP II-4, and DP ⁇ -7.
- Fig. 12 shows the results of inverse experiments in which T cells from a number of patients were primed in vitro to the Der p II protein and analyzed for response to various Der f II peptides. The results of 26 positive experiments indicate that Der p II primed T cells respond significantly to peptides DF IM, DF II-4.5, DF 11-15, DF 11-17, DF II-18 and DF IM9.1.
- Region 1 amino acid residues 1-28 of the Der p I and Der f I proteins; Region 2, amino acid residues 36-68 of the Der p I and Der f I proteins; Region 3, amino acid residues 74-109 of the Der p I and Der f I proteins; Region 4, amino acid residues 118-139 of the Der p I and Der f I proteins; Region 5, amino acid residues 141-166 of the Der p I and Der f I proteins; and Region 6a, amino acid residues 161-185 of the Der p I and Der f I proteins and Region 6b, amino acid residues 173-201 of the Der p I and Der f I proteins.
- Region 7 amino acid residues 1-26 of the Der p H and Der f II proteins
- Region 8 amino acid residues 33-67 of the Der p H and Der f II proteins
- Region 9 amino acid residues 79-104 of the Per p II and Der f II proteins
- Region 10 amino acid residues 107-129 of the Der p II and Der f II proteins.
- peptides derived from Der p I, Der f I, Der p II, and Der f II were selected and modified by addition or deletion of amino acid residues at either the 5' or 3' end of the peptide.
- various factors were considered, including the ranked sum of the overlapping peptides, the percentage of responses with an S.I. of at least 2.0 to the peptides, the potential cross-reactivity of the peptides, the difficulty of manufacture of the peptides, etc.
- T cell studies similar to those described in Examples V-X were performed using these selected peptides to more precisely define the major areas of T cell reactivity within Regions l-6a and 6b of the Der p I and Der f I protein and Regions 7- 10 of the Der p ⁇ and Der f II protein.
- Example VI the procedure described in Example VI was followed with T cell lines from 9 patients primed in vitro to the Der f I protein then analyzed for response to selected peptides derived from the protein. The data was analyzed as described in Example VI. The results of the 9 patients shown in Fig. 14 demonstrate T cell reactivity to selected peptides from Der f I.
- the T cell lines from a number of patients were primed in vitro to the Der p I protein and analyzed for response to selected peptides derived from the Der p I protein and a set of substantially matching peptides derived from the Der f I protein.
- the data from 30 positive experiments was analyzed as described in Example V.
- the Der p I primed T cells respond significantly to peptides DF I- 21.1, DF 1-21.2, DF 1-23.1, DF 1-22.2, DF 1-22.3. DF 1-22.4, DF 1-23.2, DF 1-25.1. DF 1-26.1 and DF 1-27.1.
- Fig. 15b is a subset of the data shown in Fig 15a and shows the response of Der p I primed T cells to peptides analyzed by ranked sum.
- Fig. 15b shows that DPI-23.1 has the highest ranked sum of this group of peptides in this study.
- Fig. 16a shows the results of the inverse experiment in which T cells from 9 patients were primed in vitro to the Der f I protein and challenged with selected Der f I peptides and a set of substantially matching Der p I peptides. The results indicate that Der f I primed T cells from 9 patients respond to selected peptides from Der p I.
- FIG. 16a shows that DF 1-22.1 and DF 1-25.1 have the highest stimulation indexes of this group of peptides.
- Fig. 16b is a subset of the same data as shown in Fig. 16a and shows the response of preferred Der f I and Der p I peptides is shown.
- Fig 16b shows that DF1-22.2 has the highest stimulation index of this group of preferred peptides in this experiment.
- FIG. 17a shows that Der p II primed T cells from one patient respond to selected peptides from Der p II.
- Fig. 17b shows the results from an experiment similar to that shown in Fig. 17a with a set of 30 patients and with the high and low omitted from the mean.
- Fig. 17b shows that DP ⁇ - 20.6 has the highest ranked sum of this group of preferred peptides in this study.
- FIG. 18a shows the inverse experiment in which T cells from 1 patient were primed in vitro to the Der f II protein and challenged with selected peptides derived from the Der p II sequence.
- Fig. 18a shows that Der f H primed T cells from 10 patients respond to selected peptides from Der p II.
- DP 11-25 has the highest stimulation index.
- Fig. 18b is a subset of the same data shown in Fig. 18a and shows the response of native Der f II primed T cell lines to preferred Der p II peptides analyzed by ranked sum.
- DPH-25.2 has the highest ranked sum of the preferred peptides in this study.
- Example VII A study similar to that described in Example VII was carried out with T cells from 4 matched patients primed in vitro with Group I proteins from Der f and Der p then analyzed for response to selected preferred peptides from Der p I and Der f I.
- the results in Fig. 18c demonstrate that T cell reactivity to a number of the selected preferred Der p I peptides was essentially equivalent for their Der f I counterpart and vice-versa.
- Fig. 18d shows the results of a similar study with T cells from 6 matched patients primed in vitro with Group II proteins from Der p and Per f, then analyzed for response to selected preferred Der p H and Der f II peptides. Similar to the results in Fig. 18c, T cell reactivity to a number of the selected preferred peptides of Der p II are essentially equivalent to their Der f II counterparts and vice-versa.
- Corning assay plates (#25882-96) were coated with 5 ⁇ g/ml of each coating antigen listed in Figs. 19, 20 and 21 at 50 ⁇ l/lOOml/well and incubated overnight at 4°C. The coating antigens were removed and the wells are blocked with 0.5% gelatin in PBS, 300 ⁇ l/well for 2 hours at room temperature. Pooled human plasma (a mix of plasma samples from 20 patients that were skin test positive for commerical mite extract) was serially diluted with PBS-Tween 20 (PBS with 0.05% nonionic detergent Tween-20 Sigma, St. Louis, MO) and 100 ⁇ l/well a ⁇ was added and incubated overnight at 4°C (plasma dilutions were tested in duplicate).
- PBS-Tween 20 PBS with 0.05% nonionic detergent Tween-20 Sigma, St. Louis, MO
- the second antibody (biotinylated goat anti-Human IgE, 1:1000, Kirkegaard & Perry Laboratories Inc, Gaithersburg, MD), was added at 100 ⁇ l/well for one hour at room temperature. This solution was removed and streptavidin-HRPO. 1: 10000, (Southern Biotechnology Associates, Inc., Birmingham, AL) was then added at 100 ⁇ l/well for one hour at room temperature (all wells are washed three times with PBS-Tween between each incubation step). TMB Membrane Peroxidase Substrate system (Kirkegaard & Perry Laboratories) was freshly miced, and added at lOOml/well. The color was allowed to develop for 2-5 minutes.
- the reaction was stopped by the addition of lOOml/well of IM phosphoric acid. Plates were read on a Microplate IL310 Autoreader (Biotech Instruments, Winooski, VT) with a 450 nm filter. The absorbance levels of duplicate wells were averaged. The graphed results (log of the dilution versus absorbance) of the ELISA assays are shown in Figs. 19a-b, 20a-b and 21a-h. The order of coating antigens listed vertically in these figure legends corresponds in order from left to right to the coating antigens listed for each histogram.
- the results of the ELISA assay shown in Fig. 19b demonstrate good binding of both biochemically purified Der p II and recombinant Der p II ( " rDer p II) with human IgE and no detectable binding to the Der p II peptides.
- the IgE binding to the Der p set of peptides and proteins shows the same pattern of reactivity as the Der f set. That is, no detectable binding to Der f I or Der f_II peptides or recombinant Der f I with binding to only biochemically purified Der f I and recombinant and biochemically purified Der f II.
- the antigen preparation that was used as a positive control was a mixture of the four major biochemically purified mite allergens (term PMA for Purified Mite Allergen); Der f I, Der f ⁇ , Der p I and Der p. II.
- the stock was generated at a concentration of 100 ⁇ gs of each protein per millimeter or 400 ⁇ gs total protein/ml. This preparation was used on each coated ELISA plate. The results from these ELISA assays are shown in Fig. 21a-h. There is clear binding to either the purified or recombinant protein or the PMA antigen preparation on each plate indicating good IgE reactivity.
- the PMA antigen preparation does exhibit a high degree of non-specific reactivity shown in the background dilution where no first antibody solution was added. This non-specific reactivity occurs between the PMA antigen and the biotinyulated second antibody and does not compromise the finding of specific IgE reactivity to the antigen.
- Trp lie Val Arg Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr 210 215 220
- Trp He Val Arg Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr 210 215 220
- MOLECULE TYPE peptide
- FRAGMENT TYPE N-terminal
Abstract
Description
Claims
Priority Applications (13)
Application Number | Priority Date | Filing Date | Title |
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JP6523074A JPH09501043A (en) | 1993-04-14 | 1993-04-14 | T cell epitopes of major allergens derived from DERMATOPHAGOIDES |
KR1019950704526A KR960701995A (en) | 1993-04-14 | 1993-04-14 | T.CELLS EPITOPES OF THE MAJOR ALLERGENS FROM DERMATOPHAGOIDS (HOUSE DUST MITE) from Dermatopoids (House Dust Mite) |
DE69333979T DE69333979T2 (en) | 1993-04-14 | 1993-04-14 | T CELL EPITOPES OF THE MAIN ALLERGIES OF DERMATOPHAGOIDES (HOUSE DUST MILK) |
PCT/US1993/003471 WO1994024281A1 (en) | 1993-04-14 | 1993-04-14 | T cell epitopes of the major allergens from dermatophagoides (house dust mite) |
EP93910592A EP0694067B1 (en) | 1993-04-14 | 1993-04-14 | T cell epitopes of the major allergens from dermatophagoides (house dust mite) |
CA002160121A CA2160121C (en) | 1993-04-14 | 1993-04-14 | T cell epitopes of the major allergens from dermatophagoides (house dust mite) |
AU41026/93A AU680820B2 (en) | 1993-04-14 | 1993-04-14 | T cell epitopes of the major allergens from dermatophagoides(house dust mite) |
ZA932677A ZA932677B (en) | 1993-04-14 | 1993-04-16 | T cell epitopes of the major allergens of the major allergens of dermatophagoides (house dust mite) |
US08/445,307 US7288256B1 (en) | 1991-10-16 | 1995-05-19 | T cell epitopes of the major allergens from dermatophagoides (house dust mite) |
US08/484,296 US6268491B1 (en) | 1991-10-16 | 1995-06-07 | T cell epitopes of the major allergens from dermatophagoides (house dust mite) |
NO19954095A NO316922B1 (en) | 1993-04-14 | 1995-10-13 | Isolated peptide and nucleic acid of protein allergens from Dermatophagoides (household dust mite) as well as its use and therapeutic composition, and methods for detecting sensitivity to household dust |
FI954895A FI121670B (en) | 1993-04-14 | 1995-10-13 | T-cell epitopes of major allergens from Dermatophagoides (house dust mites) |
FI963331A FI104906B (en) | 1993-04-14 | 1996-08-27 | Process for testing a person's sensitivity to household dust mites |
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PCT/US1993/003471 WO1994024281A1 (en) | 1993-04-14 | 1993-04-14 | T cell epitopes of the major allergens from dermatophagoides (house dust mite) |
ZA932677A ZA932677B (en) | 1993-04-14 | 1993-04-16 | T cell epitopes of the major allergens of the major allergens of dermatophagoides (house dust mite) |
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US88139692A Continuation-In-Part | 1991-10-16 | 1992-05-08 |
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US22777294A Continuation-In-Part | 1991-10-16 | 1994-04-14 |
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