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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • 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
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • 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
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
  • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
  • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation

Definitions

• T lymphocytes can mediate and regulate both the specific and non-specific effector mechanisms of immune responses.
• CD4+ T lymphocytes provide help for antibody production and secrete cytokines which modulate the growth of other T cells and the growth and differentiation of other immune cells such as monocytes and granulocytes. Functional and
• MHC major histocompatibility complex
• T cell receptor engagement activates or inhibits the T cell's ability to subsequently respond to the peptide/MHC protein complex may be determined by the presence or absence of costimulatory signals delivered to the T cell by the antigen-presenting cell (Jenkins, M.K., et al. (1990) J. of Immunol. 144:1585-1590; and Jenkins, M.K. and Schwartz, R.H. (1987) J. of Exper. Med.
• HEL hen egg lysozyme
• OVA ovalbumin
• cl lambda repressor
• a peptide derived from Hepatitis B surface antigen (HBsAg amino acid residues 19-33) has recently been shown to stimulate T cell responses in a majority of human subjects who had been immunized with a recombinant hepatitis B vaccine (Schad, V.C. et al., Seminars in Immunol., 3:217-224 (1991)).
• a major mycobacterial antigen 65-kD protein has also been epitope-mapped (Lamb, J.R. et al., EMBO J., 6(5):1245-1249 (1987)). T cell epitopes have been identified in the peptides comprised of amino acid residues 112-132 and 437-459 of the 65-kD protein.
• Myelin basic protein (MBP), an autoantigen which induces experimental autoimmune encephalomyelitis (EAE) and the presumed autoantigen in multiple sclerosis (MS) has also been epitope-mapped in both human (Ota, K. et al., Nature. 346:183-187 (1990)) and rodent (Zamvil et al., Nature 324:258-260(1986)) systems.
• Ota et al. have identified a major T cell epitope recognized by MS patients, MBP amino acid residues 84-102.
• Minor epitopes (MBP amino acid residues 143-168, 61-82, 124-42 and 31-50) recognized by T cells from MS patients were also described.
• Zamvil et al. have shown that MBP amino acid residues 1-11 contain the major T cell e ⁇ itope(s) causing EAE, in susceptible rodent strains.
• T cell epitopes present in allergenic proteins have very recently been described (O'Hehir, R. et al., Ann. Rev. Immunol., 9:67-95 (1991)).
• Several peptides derived from the house dust mite allergen Per p I have been shown to be T cell-reactive
• the present invention is based on the discovery that subcutaneous administration of a peptide to a mammal decreases the T cell response to subsequent challenge with the same peptide, whether a mammal is naive or preexposed to the protein allergen or other antigen from which the peptide is derived.
• This invention also relates to peptides useful for tolerization by subcutaneous or other routes of administration. For such tolerization to be clinically useful, it must be effective upon environmental exposure to whole protein allergen or other protein antigen.
• subcutaneous adminstration of a peptide comprising at least one T cell epitope of a protein allergen or other protein antigen results in T cell tolerization or anergy upon exposure to the allergen or other antigen.
• Such peptide-induced T cell tolerance can be used to treat immune mediated diseases such as allergy.
• the ability to tolerize with subcutaneous injection of a peptide rather than intravenous or intraperitoneal injection provides a more practical and efficient therapeutic approach.
• Peptides useful in methods of tolerizing a mammal, such as a human can be derived from protein allergens. These peptides comprise at least one T cell epitope of an allergen and preferably have minimal immunoglobulin E stimulating activity. In addition, for therapeutic purposes, it is preferred that such peptides bind immunoglobulin E to a mammal.
• immunoglobulin E More preferably, peptides derived from protein allergens do not bind immunoglobulin E specific for the protein allergen in a substantial percentage (at least about 75%) of a population of individuals sensitive to the protein allergen, or if such binding occurs, such binding does not result in mediator release e.g., histamine, from mast cells or basophils.
• the present invention also relates to
• a peptide derived from an autoantigen such as insulin, myelin basic protein and acetylcholine receptors.
• peptides derived from protein allergens or other protein antigens can be designed such that an undesirable property of the native protein (e.g., enzymatic activity) can be eliminated for therapeutic purposes.
• peptides as described herein are preferably administered in soluble form, subcutaneously, to a mammal to tolerize T cells of the mammal to the protein antigen from which the peptide is derived.
• compositions comprising one or more of the following peptides can be administered to an individual
• peptide X (SEQ ID NO: 7); peptide Y (SEQ ID NO: 8); peptide Z (SEQ ID NO: 9); peptide A (SEQ ID NO: 10); peptide B (SEQ ID NO: 11); peptide C (SEQ ID NO: 12); peptide D (SEQ ID NO: 13); and peptide E (SEQ ID NO: 14) of TRFP.
• Methods of treating sensitivity to Felis domesticus in an individual comprising administering to the individual a therapeutically effective amount of one or more therapeutic compositions comprising at least one of such peptides are also within the scope of the invention.
• Figure 1 is the nucleic acid sequence and deduced amino acid sequence of chain 1 of the human T Cell Reactive Feline Protein (TRFP) including leader sequences A (SEQ ID NO: 1 and 2) and B (SEQ ID NO: 3 and 4).
• TRFP T Cell Reactive Feline Protein
• Figure 2 is the nucleic acid sequence and deduced amino acid sequence of chain 2 of TRFP including a leader sequence (SEQ ID NO: 5 and 6).
• Figure 3 is the amino acid sequences of peptide X (SEQ ID NO: 7), peptide Y (SEQ ID NO: 8), peptide Z (SEQ ID NO: 9), peptide A (SEQ ID NO: 10) and peptide B (SEQ ID NO: 11), peptide C (SEQ ID NO: 12), peptide D (SEQ ID NO: 13) and peptide E (SEQ ID NO: 14) of TRFP, each of which contains at least one T cell epitope of TRFP.
• Figure 4 is a graphic representation of the induction of T cell tolerance in mice by
• Lymph node cells were isolated, cultured in vitro with peptide Y and tested for the presence of IL-2.
• Figure 5 is a graphic representation of the dose response necessary to induce T cell tolerance with subcutaneous administration of peptide Y in mice.
• Figure 6 is a graphic representation of the dose response necessary to induce T cell tolerance with subcutaneous administration of peptide X in mice.
• Figure 7 is a graphic representation of the induction of T cell tolerance in mice by
• Lymph node cells were isolated, cultured in vitro with a combination of peptide X and peptide Y or with peptide X or peptide Y separately and tested for the presence of IL-2.
• Figure 8 is a graphic representation of the response of mice preimmunized with TRFP upon
• Figure 9A is a graphic representation of the induction of T cell tolerance in mice primed with TRFP and tolerized with peptide Y. Lymph node cells were isolated, cultured in vitro with peptide Y and tested for the presence of IL-2.
• Figure 9B is a graphic representation of the induction of IgG tolerance in mice primed with TRFP and tolerized with peptide Y. A sera sample was obtained and assayed for binding to peptide Y in a standard ELISA assay.
• FIGS 10A and 10B are graphic representations of the induction of T cell tolerance in mice primed with TRFP and tolerized with peptide Y. Lymph node cells were isolated, cultured in vitro with peptide Y and tested for the presence of IL-2 (culture with CTLL-3) or IL-4 (culture with CT4S).
• Figure 11 is a graphic representation of the induction of T cell tolerance in mice with
• TRFP peptide Y and peptide X
• peptide Y and peptide X peptides Fel 3-1, peptide Y, peptide C and peptide D.
• mice were challenged with recombinant chain 1 of TRFP. Lymph node cells were isolated, cultured
• Figure 12 is a graphic representation of the
• TRFP TRFP, peptide X and peptide Y.
• Figure 13 is a graphic representation of the
• Figure 14 shows the amino acid sequences of the following peptides: Fel 33, Fel 34, Fel 35, Fel 36,
• Figure 15 A, B and C is a graphic representation depicting the response of T cells from patient #688 primed is
• Figure 16 A, B and C is a graphic representation depicting the response of T cells from patient #730 primed ia
• Figure 17 A, B and C is a graphic representation depicting the response of T cells from patient #738 primed in
• Figure 18 A, B and C is a graphic representation depicting . the response of T cells from patient #807 primed ia
• Figure 19 A, B and C is a graphic representation depicting the response of T cells from patients #688, #730, #738, and #807 primed in vitro to TRFP and analyzed for response to various peptides derived from TRFP as measured by 11-2 production and 11-4 production.
• Figure 20 is a graph depicting the ratio of 11-4 production to 11-2 production by T cell lines from cat allergic patients, #688, #730, #738, and #807 in response to various peptides derived from TRFP.
• Figure 21 is a graph depicting the results of a direct ELISA assay in which various antigens were incubated with human plasma obtained from a cat allergic individual.
• Figure 22 is a graph depicting the results of a depletion ELISA assay in which human plasma obtained from a cat allergic individual was preabsorbed on various antigens followed by incubation on fresh antigen coated plates.
• the present invention is based on the discovery that subcutaneous administration of a peptide
• T cell epitope of a protein allergen or other protein antigen results in T cell tolerance upon subsequent exposure to the allergen or other antigen.
• Such peptides include peptide X (SEQ ID NO: 7), peptide Y (SEQ ID NO: 8), peptide Z (SEQ ID NO: 9), peptide A (SEQ ID NO: 10), peptide B (SEQ ID NO: 11), peptide C (SEQ ID NO: 12), peptide D (SEQ ID NO: 13), and peptide E (SEQ ID NO: 14), each as shown in Figure 3. It is preferred that such peptides be administered in soluble form.
• peptides useful in methods of tolerization have human T cell stimulating activity as determined by standard T cell biology techniques and, thus, comprise at least one T cell epitope.
• precise T cell epitopes can be determined by, for example, fine mapping techniques.
• This technique involves modifying a peptide comprising at least one T cell epitope as defined by standard T cell biology techniques by addition or deletion of amino acid residues at either the amino or carboxy terminus of the peptide. Following modification, the peptide is tested to determine a change in T cell reactivity. If two or more peptides derived from a protein antigen share an area of overlap and are both found to have human T cell stimulating activity (as determined by standard 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 the above fine mapping procedure. As a result of fine mapping, a set of human T cell epitopes for a protein allergen or other antigen comprising amino acid residues essential to T cell recognition can be produced.
• a peptide useful in methods of subcutaneous tolerization may be obtained by reviewing the known protein structure of an allergen or other antigen and theoretically dividing the allergen or antigen into at least two peptide regions of desired lengths. For example, the protein sequence of the allergen or other antigen can be systematically divided into at least two
• non-overlapping peptide regions of desired lengths or at least two overlapping peptide regions of desired lengths.
• This division into peptide regions can be arbitrary, can be made according to an
• the entire protein sequence of the protein allergen or other protein antigen is divided into one or more
• the peptide or peptides can then be produced recombinantly or synthetically and the ability of the peptide(s) to stimulate human T cells can be determined.
• a peptide derived from a protein allergen can be tested to determine whether the peptide binds immunoglobulin E specific for the allergen and result in the release of mediators
• mast cells e.g., histamine
• basophils e.g., histamine
• Those pe ⁇ tide(s) 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 used in methods of the invention.
• Constructing peptides derived from phospholipase A 2 a major allergen from honeybee venom, can be used as an illustrative example when the protein structure of a protein antigen is known, but the T cell
• Phospholipase A 2 is composed of 134 amino acids as defined by cDNA cloning (Kuchler, K. et al. Eur. J. Biochem.
• This amino acid sequence can be divided into regions, each preferably containing 20 to 35 amino acid residues, each region preferably overlapping another region by about 10 amino acids.
• the entire protein sequence of the protein may be divided into regions, however, the total sequence used to determine those peptides useful for
• subcutaneous administration to an individual can be substantially less than the entire protein sequence.
• epitopes in the constructed peptide(s), areas of overlap and length of each region can be designed to maintain the presence of T cell epitopes predicted using algorithms (see e.g., Rothbard, J. and Taylor, W.R. EMBO J. 7:93-100 (1988); Berzofsky, J.A. Philos Trans R. Soc. Lond. 323:535-544 (1989)).
• human T cell epitopes within a protein allergen can be predicted using known HLA class II binding
• T cell epitopes such as glutamic acid decarboxylase (e.g., Samama, J.P., and Mallet, J. Journal of Neurochemistry 54:703-705
• T cell stimulating activity i.e., proliferation, lymphokine secretion and/or induction of T cell anergy/ tolerization
• human T cell stimulating activity can be tested by culturing T cells obtained from an allergen or other antigen
• responses by T cells to peptides useful in methods of the invention can be calculated as the maximum CPM in response to the peptide divided by the medium control CPM.
• a peptide derived from a protein allergen may have a stimulation index of about 2.0.
• a stimulation index of at least 2.0 is generally considered positive for purposes of defining peptides useful as immunotherapeutic agents.
• Preferred peptides have a stimulation index of at least 2.5, more preferably at least 3.5, and most preferably at least 5.0.
• preferred peptides derived from protein allergens do not bind immunoglobulin E (IgE) or bind IgE to a substantially lesser extent than the protein allergen(s) from which the peptide is derived binds IgE.
• IgE immunoglobulin E
• the major complications of standard immunotherapy are systemic responses such as
• 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 chemotactic
• anaphylaxis could be avoided by the use of a peptide which does not bind IgE, or if the peptide binds IgE, such binding does not result in the release of mediators (e.g., histamine, etc.) from mast cells or basophils.
• mediators e.g., histamine, etc.
• 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 whole protein allergen.
• Peptides useful in methods of the invention as well as the preferred peptides derived from TRFP can be produced by recombinant DNA techniques in a host cell transformed with a nucleic acid vector directing expression of a nucleotide sequence coding for such peptide, or by chemical synthesis, or in certain limited situations by chemical cleavage of a protein allergen or other protein antigen.
• host cells transformed with nucleic acid vectors directing expression of a nucleotide sequence coding for a peptide are cultured in a medium
• the peptides may be secreted and harvested from a mixture of cells and cell culture medium. Alternatively, the peptide may be retained cytoplasmically and the cells harvested, lysed and the peptide isolated and purified.
• Peptides can be isolated using techniques known in the art for purifying peptides or proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for the peptide, the protein allergen or other antigen from which the peptide is derived, or a portion thereof. Any of the peptides described herein are isolated such that the peptide is
• peptides derived from a protein allergen or other antigen are administered, preferably subcutaneously to a mammal (such as a human) sensitive to the protein allergen or antigen in a form which results in a decrease in the T cell response of the mammal upon subsequent exposure to the allergen or other antigen.
• a decrease or modification of the T cell response of a mammal sensitive to a protein allergen or other antigen can be defined as non-responsiveness or diminution in symptoms to the allergen or other antigen in the mammal, as
• diminution in symptoms to an allergen includes any reduction in the allergic response of a mammal, such as a human, to the allergen following a treatment regimen with a peptide as described herein. This diminution in symptoms may be determined subjectively in a human (e.g., the patient feels more comfortable upon exposure to the allergen), or clinically, such as with a standard skin test.
• peptides derived from protein allergens or other protein antigens and having T cell stimulating activity i.e., having at least one T cell epitope
• the preferred peptides for use in treating sensitivity to Felis domesticus and derived from TRFP i.e., peptide X, peptide Y, peptide Z, peptide A, peptide B, peptide C, peptide D, peptide E.
• T cell epitopes are believed to be involved in initiation and
• 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, recruitment of additional immune cells to the site, and
• a T cell epitope is the basic element or smallest unit of recognition by a T cell receptor, where the epitope comprises amino acids essential to receptor recognition and may be contiguous and/or non-contiguous in the amino acid sequence of the protein.
• a T cell epitope, as used herein has a stimulation index of at least 2.0, more preferably at least 2.5, even more preferably at least 3.5 and most preferably at least 5.0.
• subcutaneous administration of a peptide as described herein to a mammal, such as a human will tolerize or anergize appropriate T cell subpopulations such that they become unresponsive to the protein allergen or other antigen and do not participate in stimulating an immune response upon such exposure (see
• Exemplification A administration of such a peptide may modify the lymphokine secretion profile as compared with exposure to the
• T cell subpopulations which normally participate in the response to the allergen 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
• Peptides useful for tolerization can comprise as many amino acid residues as desired and preferably comprise at least about 7 , more preferably at least about 15, even more preferably at least about 20 and most preferably at least about 25 amino acid residues of a protein allergen or other protein antigen.
• a peptide length of about 20-40 amino acid residues is preferred as increases in length of a peptide may result in difficulty in peptide synthesis as well as retention of an undesirable property (e.g.,
• the amino acid sequences of one or more peptides can be produced and joined by a linker to increase
• Such linker can be any non-epitope amino acid sequence or other appropriate linking or joining agent.
• the peptide is preferably derived from a protein allergen of the following genus: the genus Dermatophagoides; the genus Felis: the genus Ambrosia: the genus Lolium: the genus Cryptomeria: the genus Alternaria: the genus Alder: the genus
• Betula the genus Ouercus; the genus Olea: the genus Artemisia: the genus Plantago; the genus Parietaria: the genus Canis: the genus Blattella, the genus Apis; and the genus Periplaneta.
• peptides useful for subcutaneous tolerization can be derived from known or unkown protein allergens including those of the following species: Dermatophagoides pteronyssinus (e.g.. Per p I, Per p II);
• Dermatophagoides farinae e.g., Per f I, Per f II
• Ambrosia artemisiifolia e.g., Amb a 1.1, Amb a 1.2, Amb a 1.3, Amb a 1.4, Amb a II
• Cryptomeria japonis e.g.. Cry j I, Cry j II
• Lolium perenne e.g. Lol p I, Lol p IX
• Felis domesticus e.g., Fel d I
• Canis familiaris e.g., Can f I, Can f II
• Peptides comprising at least one epitope of protein allergens have been described in the following applications, the contents of which are incorporated herein by reference: U.S.S.N. 866,679 entitled "T Cell
• Particularly preferred peptides for treating sensitivity to Felis domesticus are derived from the genus Felis and include the following: peptide X (SEQ ID NO: 7); peptide Y (SEQ ID NO: 8); peptide Z (SEQ ID NO: 9); peptide A (SEQ ID NO: 10); peptide B (SEQ ID NO: 11), peptide C (SEQ ID NO: 12); peptide D (SEQ ID NO: 13); and peptide E (SEQ ID NO: 14), of TRFP, the amino acid sequences of each peptide as shown in Fig. 3, and modifications thereof. These preferred peptides from TRFP can be produced
• Such peptides can be administered in the form of a therapeutic composition to treat sensitivity to Felis domesticus.
• a therapeutic composition to treat sensitivity to Felis domesticus.
• one or more of peptides X, Y, Z, A, B, C D and E can be combined in a single composition or one or more separate compositions for simultaneous or sequential administration to an individual to be treated for sensitivity to Felis domesticus.
• Such compositions can be adminstered subcutaneously or otherwise, including, but not limited to intravenously and intraperitoneally in a form suitable for tolerization.
• the peptide is administered in soluble form.
• Additional peptides useful for subcutaneous tolerization can be derived from protein antigens other than protein allergens where modification of an antigen specific immune response is desired.
• protein antigens other than protein allergens where modification of an antigen specific immune response is desired.
• peptides having human T cell stimulating activity or peptides having known T cell epitopes can be identified.
• One or more of such peptides can be administered subcutaneously to a mammal, such as a human, afflicted with an autoimmune disease or at risk of developing an autoimmune disease to decrease the antibody response to the autoantigen, to
• peptides of the autoantigen having human T cell stimulating activity can be defined by standard T cell biology techniques, or if desired, precise T cell epitopes can be defined by above-described fine mapping techniques.
• Peptides which stimulate T cells and do not have undesired properties associated with the autoantigen e.g., do not bind autoantibodies in a substantial percentage of individuals sensitive to the
• composition the peptide would be delivered
• autoantigens useful in producing peptides are insulin, glutamic acid decarboxylase (64K), PM-1 (a 69KD autoantigen) and carboxypeptidase in diabetes; myelin basic protein in multiple sclerosis; rh factor in erythroblastosis fetalis; acetylcholine receptors in myasthenia gravis; thyroid receptors in Graves' Disease; basement membrane proteins in Good Pasture's syndrome; and thyroid proteins in thyroiditis.
• known regions of myelin basic protein (MBP) having human T cell stimulating activity include a region comprising all or a portion of amino acid residues 84-106 (preferably amino acid residues
• sensitivity in a mammal such as a human, to a protein allergen (including the preferred peptides X, Y, Z, etc. derived from TRFP) or other protein antigen are in the form of a therapeutic
• compositions include a peptide and a pharmaceutically acceptable carrier or diluent.
• Administration of the therapeutic compositions of the present invention to desensitize or tolerize an individual to a protein allergen or other protein antigen can be carried out using procedures, at dosages and for periods of time effective to reduce sensitivity (i.e., reduce the allergic response) of the individual to a protein allergen or other protein antigen. Effective amounts of the therapeutic compositions will vary according to factors such as the degree of sensitivity of the individual to to the allergen or other antigen, the age, sex, and weight of the individual, and the ability of the peptide(s) to elicit an antigenic response in the individual. Dosage periods may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
• Peptides derived from TRFP used in treating sensitivity to Felis domesticus may be administered in a convenient manner such as by injection (subcutaneous,
• the active compound may be coated with in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.
• the protein or peptide may be administered to an individual in an appropriate diluent or adjuvant, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes.
• Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
• Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon.
• Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.
• Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol.
• Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al., (1984) J. Neuroimmunol. 7:27).
• the therapeutic composition is preferably administered in non-immunogenic form, e.g., one that does not contain adjuvant.
• the active compound may also be administered parenterally or intraperitoneally.
• Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these
• preparations may contain a preservative to prevent the growth of microorganisms.
• compositions suitable for are:
• injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene gloycol, and the like), suitable mixtures thereof, and vegetable oils.
• the proper fluidity can be maintained, for example, by the use of a coating such as licithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, asorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the
• Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions can be prepared by incorporating active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required,
• dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
• sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient (i.e., a peptide of the invention) plus any additional desired ingredient from a previously sterile-filtered
• the peptide may be orally administered, for example, with an inert diluent or an assimilable edible carrier.
• the peptide and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the individual's diet.
• the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
• compositions or preparations should contain at least 1% by weight of active compound.
• the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
• the amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
• Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit contains between from about 10 ⁇ g to about 200 mg of active compound.
• pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for
• Supplementary active compounds can also be incorporated into the compositions.
• Dosage unit form refers to physically
• each unit suited as unitary dosages for the mammalian subjects to be treated; each unit
• compositions of the invention can be administered subcutaneously to mammals, such as humans, sensitive to an allergen or other protein antigen from which the peptide is derived, at dosages and for lengths of time effective to reduce
• compositions comprising at least two peptides (e.g., a physical mixture of at least two peptides), can also be used in methods of
• 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.
• the amino acid residues essential to T cell epitope function can be determined using known techniques (e.g., substitution of each residue and determination of presence or absence of T cell reactivity).
• Those residues shown to be essential can be modified (e.g., replaced by another amino acid whose presence is shown to enhance T cell reactivity), as can those which are not required for T cell reactivity (e.g., by being replaced by another amino acid whose
• incorporation enhances T cell reactivity but does not diminish binding to relevant MHC).
• Another example of a modification of peptides is substitution of cysteine residues preferably with alanine, or
• glutamic acid or alternatively with serine or threonine to minimize dimerization via disulfide linkages.
• 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 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. Modifications of peptides 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
• 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. 6:1321-1235 (1988)).
• specific affinity chromatography Hochuli, E. et al., Bio/Technology. 6:1321-1235 (1988)
• 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.
• 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.
• solubility of a peptide by adding functional groups to the peptide or by not including hydrophobic T cell epitopes or regions containing hydrophobic epitopes in the peptide.
• 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 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 (Hostomsky, Z., et al., Biochem. Biophys. Res. Comm. 161:1056-1063 (1989)). To enhance bacterial expression, the aforementioned methods can be used in conjunction with other procedures to change the eucaryotic codons in DNA constructs encoding peptides to ones preferentially used in E. coli.
• Another aspect of the invention provides
• a therapeutic composition can comprise one or more of such peptides.
• a preferred composition comprises at least one peptide selected from the group consisting of peptide X (amino acid residues 7-30 from chain 1 of TRFP; also referred to as Fel 8-3 and shown in Figure 3); peptide Y (amino acid residues 29-55 from chain 1 of TRFP; also referred to as Fel 30-4 and shown in Figure 3); peptide Z (amino acid residues 14-39 from chain 2 of TRFP; also referred to as Fel 31-2 and shown in Figure 3); peptide A (amino acid residues 51-69 from chain 1 of TRFP and shown in Figure 3); peptide B (amino acid residues 74-92 from chain 2 of TRFP; also referred to as Fel 29 and shown in Figure 3); peptide C (amino acid residues 51-66 from chain 1 of TRFP, also referred to as Fel 23 and shown in Figure 3); peptide D (amino acid residues 56-69 from chain 1 of TRFP; a modified form of Fel 21 and shown
• mice subcutaneous or intravenous administration of peptide X or peptide Y separately or in combination.
• the tolerization of the mice was evidenced by decreases in peptide specific IL-2 production, IL-4 production, and antibody production. Decreases in the T cell activities that are expected to be associated with IgE production, T cell help for B cells and IL-4 production have been shown.
• T cells specific for recombinant chain 1 of TRFP were
• T cell tolerance was induced to myelin basic protein (MBP) in mice by (1) administering one or a combination of two
• EAE Encephalomelitis
• the invention further encompasses at least one therapeutic composition useful in treating a disease which involves an immune response to protein antigen (e.g., an allergen, an autoantigen, etc.) comprising at least one peptide having a sufficient percentage of the T-cell epitopes of the protein antigen such that in a substantial percentage of a population of individuals sensitive to the protein antigen, the response of such individuals to the protein antigen is substantially diminished, with the provisio that the at least one peptide does not comprise the entire protein antigen.
• protein antigen e.g., an allergen, an autoantigen, etc.
• TRFP Human T Cell Reactive Feline Protein
• TRFP Human T Cell Reactive Feline Protein
• the peptides were purified by reverse phase HPLC and examined by amino acid analysis.
• the lyophilized peptides were resuspended in PBS and sterilized by gamma irradiation (10,000 rads) or by passage through a 0.2 ⁇ filter.
• Native TRFP protein was purified from an extract of house dust as described by Chapman et al. (J.
• the TRFP was eluted from the column with 100 mM glycine pH 2.5 and was neutralized.
• TRFP chain 1 was expressed and purified as described in Rogers et al. (Rogers, B.L. et al., International Symposium - Workshop, Molecular Biology and Immunology of Allergens (1992), CRC-Press, Inc. (in Press)). TRFP chain 1 was approximately 96% pure and contains the amino acids glycine and serine from a thrombin cleavage recognition site and the
• Subcutaneous peptide inhibits the response to
• mice (H-2 bxa ), Jackson Lbas, Bar Harbor, ME.) 6-10 week old female mice) were treated with subcutaneous
• mice were chosen because of their strong T cell response to peptide Y.
• the mice were then challenged with peptide Y in CFA.
• a peptide specific T cell response was measured by peptide Y-specific IL-2, IL-4, and IFN- ⁇ production 10 days after challenge.
• peptide Y specific production of all of the examined cytokines by lymph node or spleen cells was decreased in cells from the peptide-tolerized animals. Note that in each of the cytokine assays there were similar low backgrounds using cells from both groups of animals in cultures with no peptide Y.
• PBS lymph node 1 50 49.86 (6.46) ⁇ 2.82 (0.29) 1.20 (0.02)
• IPC-2 lymph node 150 6.82 (0.64) 1.04 (0.08) 0.26 (0.11)
• mice Five naive B6CBAF 1 mice were tolerized by subcutaneously administering either IPC-2 peptide in PBS or PBS alone. They were then challenged with IPC-2.
• IPC-2 as shown. Supemaiants were analyzed for cytokine production.
• mice Females, 6-8 weeks of age mice (females, 6-8 weeks of age), were injected either subcutaneously in a single dorsal site between the forelimbs or intravenously through one of the tail veins.
• BDF1 C57BL/6J ⁇ DBA/2J mice
• PBS Phosphate Buffered Saline
• each animal was challenged with 100 ⁇ g peptide Y in 200 ⁇ l Complete Freuds adjuvant (CFA) in four sites, two subcutaneous sites at the base of the tail and two subcutaneous sites on the thigh.
• CFA Complete Freuds adjuvant
• the animals were sacrificed by cervical dislocation on day 20 and inguinal and popliteal nodes were removed and placed in rinsing buffer, cold RPMI 1640 containing 1% Fetal Bovine Serum (FBS).
• the nodes were rinsed with rinsing buffer and forced through a fine stainless steel mesh, using a glass pestal to suspend them in rinsing buffer.
• the suspended cells were rinsed two times by centrifugation at 1500 rpm for 10 minutes and resuspended with rinsing buffer. An aliquot of suspended cells from each animal was counted on a Coulter Counter Model ZB.
• the cells (4 ⁇ 10 6 /ml) were incubated in culture media (RPMI media containing 10% FBS, 2 mM
• CTLL-2 an IL-2 dependent T cell clone (ATCC TIB#214).
• CTLL-2 in log phase growth were rinsed 2 times by centrifugation at 1000 rpm for 10 minutes, aspiration of the media, and resuspension of the pellet with culture media.
• CTLL were added to the warmed culture supernatants (5 ⁇ 10 3 CTLL cells/well) and the IL-2 assay was incubated at 37°C and 5% CO 2 . After 24 hours, 1 ⁇ Ci/ml
• 3H-thymidine was added in 50 ⁇ l/well and the CTLL cells were incubated an additional 4-6 hours, then they were harvested with a Tom-Tec 96-well cell harvester. The 3 H-incorporation in each well was counted by a Betaplate Model 1205 scintillation counter. Background counts were not subtracted.
• Each bar on Figure 4 represents the arithmetic mean of triplicate in vivo cultures from one mouse.
• draining lymph node cells isolated from mice who were administered peptide Y intravenously and subcutaneously exhibited a
• mice were tolerized with various doses of peptide X and peptide Y prior to challenge with the peptide in order to determine the lowest dose necessary for the induction of tolerance. Seven groups of mice, each group
• B6CBAF 1 mice (C57BL/6J ⁇ CBA/J) were tolerized and challenged with a mixture of the two peptides.
• B6CBAF1 mice respond well to both peptide X and peptide Y, as measured by peptide-specific IL-2 production from lymph node cells isolated from mice who were challenged with either peptide.
• one group of five B6CBAF 1 mice was injected with 300 ⁇ g/dose each of peptide X and of peptide Y. The animals were injected at a single dorsal site under the skin between the forelimbs on days 0 and 5.
• the animals received a challenge dose of 100 mg each peptide X and peptide Y in 270 ⁇ l total CFA in above location.
• the animals were sacrificed and draining lymph nodes were removed as described above.
• the lymph node cells were isolated, suspended, rinsed, and cultured as above with or without 150 ⁇ g/ml of peptide Y, peptide X, or a combination of peptide X and peptide Y.
• the supernatants from the cultures were assayed for IL-2 as described above.
• Each bar in Figure 7 represents the arithmetic average of cpm values of triplicate cultures of pooled draining lymph nodes of five animals.
• preimmunized with TRFP is tolerized by peptide Y when administered subcutaneously
• mice were immunized with whole protein, TRFP, so that there would be T cell response to peptide Y derived from the TRFP protein. Peptide Y in the native protein structure is obscured by the protein conformation. Thus, following TRFP administration, the mice do not produce antibody specific for peptide Y.
• mice were each immunized with 100 ⁇ g TRFP in 200 ⁇ l IFA (Incomplete Freund's Adjuvant) in a single site intraperitoneally. After four months, the mice were bled and grouped according to their anti-TRFP IgG antibody levels as follows. The mice were warmed with an infra-red lamp and bled through one of the tail veins by nicking the vein with a razor blade (Gilette Platinum Plus). The blood was allowed to drip into a serum separating tube
• TRFP-specific IgG by ELISA.
• immunoaffinity purified TRFP was coated onto Nunc Polysorp 96-well plates by incubation of 50 ⁇ l/well of 2 ⁇ g/ml TRFP in PBS. Unless mentioned, each incubation step in this protocol was performed for 1 hour at 37°C. After rinsing with PBS, the wells were incubated with 0.5% gelatin in PBS to block excess binding to the plate. The plates were rinsed with PBS containing 0.05% Tween 20. Sera were diluted 1/500 in PBS and incubated in triplicate on the plates. After rinsing, the bound mouse antibody was detected by incubation with biotinylated goat
• TRFP primed mice respond to peptide Y
• three TRFP primed, anti-TRFP antibody positive mice were sacrificed by cervical dislocation and the spleens were removed and placed in rinsing buffer. Spleens were used because intraperitoneal injection leads to strong T cell responses in the spleen. The spleens were suspended and rinsed as detailed above for lymph node cells. The cells were cultured as above with 150 ⁇ g/ml peptide Y or with media alone. After 24 hours the supernatants were removed and tested for the presence of IL-2 as previously
• TRFP IgG matched groups of mice were tolerized with peptide Y. The tolerance of these animals to peptide Y was then shown by their failure to specifically secrete IL-2 in vitro
• the IgG matched groups were tolerized and challenged with peptide Y as follows. One of the two IgG matched groups was injected subcutaneously on days 0 and 5 (day 109 and day 114 after TRFP priming) with 0.2 ml PBS containing 300 ⁇ g peptide Y. The other group was injected with PBS only. Both groups were injected at a single dorsal site between the forelimbs. On day 10 (day 119 after TRFP priming) both groups of animals were challenged with 100 ⁇ g peptide Y as described above. On day 20 (day 129 after TRFP priming) the animals were sacrificed and the draining lymph nodes were removed as described above. The lymph nodes were suspended, rinsed, and cultured as described above with 150 ⁇ g/ml peptide Y ( Figure 9, left panel) or with media alone. After 24 hours, the supernatants of the cultures were tested for IL-2 as described above.
• T cells specific for peptide Y can be tolerized in animals which have been preimmunized with TRFP and demonstate a peptide Y specific T cell response at the time of tolerization.
• mice The ability of mice to mount an antibody
• peptide Y specific T helper cell activity As shown in Example 5, immunization of BDFI mice with TRFP generates antibodies specific for whole TRFP, with no detectable antibodies specific for peptide Y. The two matched groups of mice described above were bled at the time of sacrifice (ten days after peptide Y/CFA challenge). The serum was separated as
• mouse antibody was detected by incubation with biotinylated goat anti-mouse IgG (Southern Biotechnology Associates). Streptavidin conjugated to horse radish peroxidase (Southern
• the sera were assayed for the presence of peptide Y specific IgM, IgE, and IgG isotypes.
• the ELISA used to detect antigen specific binding of IgGl, IgG2a, IgG2b, IgG3 and IgM isotypes were similar to the IgG assay described above with the only difference being the biotinylated
• Antigens bound IgE was detected similarly, but using biotinylated EM95, a rat
• Biotinylated goat anti-rat Ig was used as an added signal amplification step in the IgE ELISA. No binding above background was detected in assays for peptide Y specific IgG2a, IgG3, or IgE.
• IgG1 was the major isotype seen in the animals which were injected with saline only, with a titer of 1/2700 for all three animals. Of the peptide Y tolerzied animals, two had an IgG1 titer of 1/300 and one had no binding above background. The titers for IgM and IgG2b were similarly effected. The sera from all of the
• the sc peptide injected in saline decreased the antibody response in these primed animals without changing the isotype distribution of the anti-peptide antibody.
• Example 6 The animals described in Example 6 which were tolerized to peptide Y had a preexisting antibody response to whole TRFP protein.
• tolerization injection i.e., peptide Y in PBS vs. PBS alone
• challenge injection i.e., peptide Y in CFA
• the anti-TRFP antibody assay was followed as detailed above.
• the mouse sera was diluted from 1/500 to 1/13,500 for this assay.
• the data from the 1/4500 sera dilution is shown in Table 2. Each sera was assayed in duplicate wells and the values were averaged. The background in this assay was about 0.1. The data show that there was no signifigant change in the quantity of antibody specific for TRFP in any of these mice in this time frame.
• tolerization of the T cells specific for peptide Y had no effect on the anti-TRFP antibody in the 20 days following tolerization.
• Table 2
• T helper cells There are several phenotypic classes of T helper cells which can be differentiated by their production of lymphokines. Th 1 cells produce IL-2 and
• Th 2 cells produce IL-4 and other lymphokines.
• IL-2 production is easily measured from primary in vitro lymph node cultures through the IL-2 dependent CTLL-2 cell line.
• IL-4 can be measured with cultures of the IL-4 dependent CT4S cells (provided by W. Paul). However, production of IL-4 from primary in vitro culture is low and not
• T helper cells we pooled lymph node cells from the two groups of mice (described previously) that were tolerized, challenged, and assayed for T cell
• the cells (2 ⁇ 10 7 total) were cultured in 5 ml culture media containing 30 ⁇ g/ml peptide Y. After 21 days incubation at 37°C and 5% CO2, the cells were rinsed two times with culture media and replated at 4 ⁇ 10 6 cells/ml in triplicate 0.2ml wells with or without 150 ⁇ g/ml peptide Y for both the IL-2 production assay and the IL-4 production assay. Samples (50 ⁇ l each) of the supernatants for the IL-2 assay were removed at 24 hours and assayed as detailed above. Samples for the IL-4 assay were removed at 48 hours and stored at 4°C until use (2 days minimum).
• the IL-4 content was assayed by the ability of the supernatants to support the growth of the CT4S line.
• CT4S cells (1 ⁇ 10 4 /well) were added to the warmed culture supernatants and incubated 40 hours at 37°C and 5% CO 2 .
• 3 H-thymidine (1 ⁇ Ci/50 ml culture media/well) was added for an additional 8 hours of culture to quantitate CT4S growth through incorporation of the thymidine into DNA.
• the cells were harvested after freezing the plates to dislodge the slightly adherent CT4S cells.
• the plates were harvested and counted identically to the IL-2 assays detailed above.
• mice Four groups of B6CBAF 1 mice were "tolerized" by subcutaneous injection with either PBS alone or PBS containing recombinant chain 1 of TRFP; PBS
• mice were injected with 150 ⁇ g/dose of either the TRFP protein or each of the peptides (days 0 and 5).
• the PBS alone and the PBS/peptides were injected in 300 ⁇ l/dose.
• the PBS/chain 1 of the TRFP protein was injected in 100 ⁇ l/dose. All tolerizing doses were at a single site under the dorsal skin between the forelimbs. On day 10 all of the animals received a challenge injection of 100 ⁇ g recombinant chain 1 of TRFP in 200 ⁇ l CFA as described above.
• lymph node cells were suspended, rinsed, and cultured as above with or without 150 ⁇ g/ml recombinant chain 1 of TRFP.
• Each bar in Figure 11 represents the average of IL-2 produced by triplicate cultures of lymph node cells from five individual mice. IL-2 assays from cultures of these lymph node cells without peptide was approximately 500 cpm. The results show that tolerization with peptide X and peptide Y and
• tolerization with chain 1 of TRFP is effective in decreasing IL-2 production specific for chain 1 of TRFP after in vivo challenge with the chain 1 protein TRFP.
• the results indicate that tolerization with two peptides from chain 1 (peptide X and peptide Y) are as effective as tolerization with four peptides from chain 1 (Fel 3-1, peptide Y, peptide C and peptide D) or as effective as tolerization with TRFP chain 1 itself.
• the basic format of these ELISA assays was a direct binding assay wherein the antigen was coated onto the wells of a 96 well microtitre dish and then antibodies in solution are added and assayed for the binding capacity.
• the protocol for these assays was as follows:
• Corning assay plates (#25882-96) were coated with 10 ⁇ g/ml of each coating antigen; affinity purified TRFP, peptide X, or peptide Y, peptide X and peptide Y mixed, peptide X/HSA, or peptide Y/HSA in PBS (phosphate buffered saline), at 50 ⁇ g/well and incubated overnight at 4°C.
• the peptides were conjugated to HSA (human serum albumin) by two different methods based on their chemical structure.
• the conjugation reaction with peptide X used the reagents and protocol of the Pierce Imject Immungen EDC conjugation kit (Pierce, Rockland, IL).
• Peptide Y was conjugated to HSA through its cysteine residue using the sulfo-SMCC reagent (Pierce). Both
• the antibody solution was a pool of plasma samples from 20 patients that were skin test positive for commercial cat extract which was
• Membrane Peroxidase Substrate system (Kirkegaard & Perry Laboratories) was freshly mixed, and added at 100 ⁇ l/well. The color was allowed to develop for 2-5 minutes. The reaction was stopped by the addition of 100 ⁇ l/well of 1 M phosphoric acid. Plates are read on a Microplate EL 310 Autoreader (Biotek
• Figure 12 is the graphic representation of ELISA assays for TRFP, peptide X and peptide Y IgE
• the negative control for these binding assays is the level of binding to wells which have had no antigen coated on them.
• For the positive control for the presence of peptide coated on the wells was an ELISA using anti-peptide antisera against either peptide X or peptide Y was used.
• Table 3 shows the percent positive binding as summarized data from different numbers of cat
• the dot blot assay is similar to ELISA in that is a direct binding assay with the antigen bound to a solid phase matrix.
• the matrix is nitrocellulose paper and results are based on autoradiography with 125 l-Streptavidin binding to a biotin labeled second antibody.
• Dot blot protocol Antigen; TRFP, peptide X and peptide Y, were coated on 0.1 ⁇ m nitrocellulose sheet using 96-well dot blot manifold (Biorad, Richmond, CA) for 45 min. at room temperature in 100 ⁇ l sample volumes. TRFP was coated at 2,0.2 and 0.02 ⁇ g/dot and the peptides at 10, 1 and 0.1 ⁇ g/dot. Wells were then washed with 200 ⁇ l PBS.
• the objective of the histamine release analysis was to directly assay the effects of peptides or TRFP in an in vitro allergic response system.
• the release of histamine through IgE recognition and IgE receptor crosslinking on live cells directly assays the allergic potential of a protein antigen.
• the aim of these studies was to compare this allergenic
• histamine release assay used for these studies is based on the detection of an acylated derivative of histamine using a specific monoclonal antibody (Morel, A.M. and Delaage, M.A.; 1988, J. Allergy Clin. Immunol. 82:646-654.) This assay was performed as two separate protocols: 1) the release of histamine from basophils present in heparinized whole blood in the presence of different
• acylation reaction i.e., 100 ⁇ l of each standard and 50 ⁇ l of acylation buffer in each acylation reaction tube.
• 50 ⁇ l of each acylation reaction was added to the bottom of a monoclonal antibody coated 12 ⁇ 75 mM plastic tube. This monoclonal antibody recognizes the acylated histamine derivative as its epitope.
• 500 ⁇ is of 123 I labelled tracer was added to each tube on ice and the binding reaction was carried out
• the standard curve is generated from the histamine standard counts and plotted on semi-log paper.
• This type of analysis for histamine is a very sensitive, accurate method of measurement.
• Figure 13 shows the summarized graphed results of eight individual cat allergic patients comparing histamine release following culture with various concentrations of affinity purified TRFP, peptide X and peptide Y.
• the graph shows the concentration of the various antigens in nMoles versus the percent total histamine released for each patient.
• the graph depicts the average percent release for each
• the results are represented as percent total histamine released because of the great variability between patients in overall histamine levels.
• the source of this variability relates to the number of basophils per unit blood from patient to patient and the variation in quantity of histamine per basophil.
• concentration points for the TRFP peptide antigens emphasizing the higher end of the concentration curve.
• the TRFP concentrations are eight five-fold dilutions with three lower concentrations used for TRFP to get the full response range.
• the reassociated TRFP may be useful as a diagnostic reagent.
• the final volume was 5 mLs and was heated to 55°C for 3 minutes with shaking and then 5 mLs H 2 O was added and the mix was allowed to cool to room temp. This was followed by extensive dialysis against IX PBS at room temp using a membrane with a molecular weight cutoff of 3,500.
• the reassociated TRFP was analyzed by a direct ELISA followed by a depletion ELISA.
• the protocol for the ELISA is as follows: Microtiter plates were coated with 5.0 ⁇ g/mL of coating antigen (Fel d I, recombinant TRFP (rTRFP) chain 1, rTRFP chain 2, a mix of rTRFP chain 1+2, or reassociated rTRFP chain 1+2) in PBS at 100 ⁇ L/well and incubated overnight at 4°C. The plates were washed three times between each step with PBS-T (Phosphate buffered saline + 0.05% Tween 20). The unbound antigen was removed and the plate blocked with 300 ⁇ L/well of 1% bovine serum albumin (BSA) in PBS-T for one hour at room
• BSA bovine serum albumin
• pre-absorption on the reassociated chains exhibited significantly greater reduction of Fel d I specific IgE (second lowest binding curve) than pre-absorption on a mix of the chains or the individual chain. It appeared that the reassociated recombinant chains either have more epitopes for IgE binding (than a mixture of rchain 1 and rchain 2) and/or that the affinity of binding to the epitopes has been increased.
• interleukin 2 IL-2
• interleukin 4 IL-4
• SI 2 Average of the cpm of T cell and antigen
• N 3 Number of patients tested.
• S.I. stimulation index
• Two ⁇ 10 6 rested T cells were cultured in 1 ml of medium containing 20 ⁇ g/ml of test antigesn with 2 ⁇ 10 6 ⁇ -irradiated autologous Epstein-Barr Virus transformed cells as antigen presenting cells.
• IL-2 and IL-4 production are suitable methods for defining peptides which have T cell stimulating activity.
• Figures 15-20 also show that interleukin production favors IL-4 as opposed to IL-2 when T cell lines from cat allergic individuals were cultured by TRFP.
• NAME Mandragouras, Amy E.
• MOLECULE TYPE protein

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