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WO1996006630A1 - Procedes et compositions permettant de moduler une reponse des cellules t - Google Patents

Procedes et compositions permettant de moduler une reponse des cellules t Download PDF

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Publication number
WO1996006630A1
WO1996006630A1 PCT/US1994/009795 US9409795W WO9606630A1 WO 1996006630 A1 WO1996006630 A1 WO 1996006630A1 US 9409795 W US9409795 W US 9409795W WO 9606630 A1 WO9606630 A1 WO 9606630A1
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Prior art keywords
cell
peptide
cells
peptides
antigen
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PCT/US1994/009795
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English (en)
Inventor
Eli E. Sercarz
Vipin Kumar
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The Regents Of The University Of California
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Priority to PCT/US1994/009795 priority Critical patent/WO1996006630A1/fr
Priority to JP8508689A priority patent/JPH10511541A/ja
Publication of WO1996006630A1 publication Critical patent/WO1996006630A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates generally to the T cell response and specifically to methods and compositions for modulating the Th1 and Th2 response to a specific antigen.
  • the immune response is often seen as beneficial, in certain circum ⁇ stances the immune response to an antigen can actually be harmful to the animal in which the immune response occurs.
  • An example where the immune response creates a condition wherein the host is subject to serious pathologic sequelae is in such autoimmune diseases as lupus erythematosus.
  • lupus erythematosus antibodies are often present which react with determinants in the thyroid, erythrocytes, DNA, and platelets of the host. Consequently, for autoimmune disease it has traditionally been desirable to suppress the immune response.
  • IgE antibodies against allergens cause hay fever, and are involved in the other allergic diseases such as extrinsic asthma.
  • the crucial role of IgE antibodies in the allergic diseases raised the possibility that the regulation and suppres ⁇ sion of the IgE antibody formation against allergens would be one of the fundamental treatments for allergic diseases.
  • the immune system has the potential to make a variety of responses and, as noted above, the response is sometimes deleterious.
  • a correct immune response to an infectious agent is dependent on the activation of an appropri ⁇ ate set of immune effector functions, both cell-mediated and humoral.
  • CD4 T Two parallel T cell subsets, one that bears the CD4 receptor (CD4 T ) and another that bears the CD8 receptor (CD8 " ") have been identified. These subsets are specialized to recognize different antigen presenting MHC molecules, CD4+ T cells recognizing class II MHC molecules and CD8+ T cells recognizing class I MHC molecules.
  • the CD4+ subset can be further subdivided into subpopulations, termed Th1 and Th2, which secrete different patterns of lympho/cytokines and also reciprocally modify each other's effector functions.
  • the cytokines are molecules which affect other cells and are responsible for much of the effector function of the different subpopulations.
  • Th1 cells secrete interferon 7 (IFN-r), interleukin 2 (IL-2) as well as tumor necrosis factor (TNF) and Th2 cells secrete interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin-9 (IL-9), interleukin 10 (IL-10), and interleukin 13 (IL-13).
  • IFN-r interferon 7
  • IL-2 interleukin 2
  • TNF tumor necrosis factor
  • Th2 cells secrete interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin-9 (IL-9), inter
  • ThO A third subset phenotype (ThO), which secretes a combination of the cytokines characteristic of both subsets, has been reported and thought to be present early during an immune response. Recent studies have established the human equivalent for CD4+ T cells with cytokine pattern functions that are comparable to murine Th1 and Th2 cells (Romagnani, S., Ann. Rev. Immunol., 12:227, 1994), as well as a group of cells in both species which appear to secrete both families of cytokines (ThO). The emerging consensus is that the Th1 and Th2 subsets arise as alternative progeny from a common precursor as a result of antigen activation and the presence of cytokines (R ⁇ cken, M., ef al., J.
  • Th2 cells stimulate the production of three important features of allergic diseases: IL-4 induces IgE production, IL-5 is responsible for eosinophilia and the combination of IL-3, IL-4 and IL-10 leads to mast cell production (O'Hehir, R.E., et al., Annu. Rev. Immunol., 9_:67, 1991) (cells which release their content of allergy-causing mediators upon stimulation with an allergen).
  • Th1 cells mediate a very different set of inflammatory immune responses including delayed type hypersensitivity and some autoimmune responses.
  • autoimmune diseases are generally multifactorial in humans, in most experimental models of human autoimmune conditions, for example, experimental allergic encephalomyelitis (EAE) a model for multiple sclerosis, has been shown to be primarily mediated by myelin basic protein (MBP)- specific Th1 cells (Powell, M.B., et al., Int. Immunol., 2:539, 1990; Ruddle, N.H., et al., J. Exp. Med., 172:1193, 1990). There is also evidence of Th1 cell involvement in insulin-dependent diabetes mellitus (IDDM) in humans and animal models (Tisch, R., et al., Nature, 366:72-75, 1993).
  • IDDM insulin-dependent diabetes mellitus
  • Anti-IFN- 7 has been shown to protect against diabetes in non obese diabetic (NOD) mice. In this situation, a predominant Th2 response may be beneficial. Indeed, recently the presence of CD45RC lo (in rat) and CD45RB ,ow (in mouse) CD4+ cells, which predominantly secrete Th2-like cytokines, have been shown to be protective in autoimmunity (Powrie, F., ef al., J. Exp. Med., 179_:589-600, 1994).
  • the present invention fulfills a need to exploit the immune system itself to divert predominant T cell responses away from disease; diverting the response from the dangerous Th2 to the possibly protective or less dangerous Th1 in the case of allergic responses, as well as from the deleterious Th1 to Th2 in autoimmunity.
  • deviations in cytokine production seen after altering ligand-receptor affinities are useful in achieving protective immune responses after vaccination.
  • the present invention shows that rather than trying to prevent an autoimmune or an allergic response, the most successful strategy is instead to elicit another, diversionary and/or protective response.
  • the present invention is based on the discovery that the ratio of subsets of T cells, Th1 to Th2, can be altered by stimulation of a subset, thereby providing a means for eliminating deleterious effects caused by elevated levels of one or the other of these cell populations.
  • Th1 cell responses are often associated with autoimmune disorders, while Th2 responses are associated with allergic responses.
  • the invention provides a method for identifying a T cell modulatory peptide for a specific antigen, wherein the peptide stimulates an IFN- 7 secreting T cell population, comprising preparing an array of variant immunodominant peptides from the antigen and selecting from the array, a peptide that has high MHC binding affinity and that induces proliferation of T cells specific for the antigen.
  • the method of the invention is utilized for identifying a peptide which stimulates an IL-4 secreting T cell population and the peptide has low MHC binding affinity.
  • the invention provides a method of modulating a T cell response to a specific antigen in a subject with a T cell disorder, comprising administering to the subject an effective amount of a T cell modulatory peptide for the antigen, wherein the peptide stimulates an IFN- 7 or an IL-4 secreting T cell population.
  • the invention provides T cell modulatory peptides. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIGURE 1 shows profiles of the determinant regions on the PSP molecule in BALB/c and B6 mice.
  • Lymph node cells of four to six pooled BALB/c (A and B) or B6 (C and D) mice previously immunized with 2 ⁇ g of rPSP (A and C) or 2.5 ⁇ g of HPSP (B and D) were cultured in 28 ⁇ M solutions of each of 155 peptides for 72 hours and then pulsed for an additional 18 hours with 1 ⁇ Ci of [ 3 H]thymidine.
  • Peptides were 15mers advancing by three residues and cover the entire PSP molecule.
  • Peptide number refers to the N-terminal residue of the 15mer. Data are expressed as [ 3 H]thymidine incorporation in individual cultures in FIGURES 1 , 2 and 3. To assure reproducibility, experiments were performed two to four times.
  • FIGURE 2 shows fine determinant mapping of PSP determinant regions in BALB/c mice.
  • Lymph node cells of individual BALB/c mice previously immunized with 2.5 ⁇ g of HPSP were cultured along with each peptide (at 14 ⁇ M) within determinant regions with cores 138-147/143-155 (A) and 369-376 (B) under the same conditions used in FIGURE 1.
  • Peptides are 15mers advancing by one residue and cover selected determinate regions represented in FIGURE 1.
  • FIGURE 3 shows fine determinant mapping of PSP determinant regions in B6 mice. Lymph node cells of individual B6 previously immunized with 2.5 ⁇ g of
  • HPSP were cultured along with each peptide (at 14 ⁇ M) within determinant regions with cores 138-147/143-155(A), 194-201 (B), or 456-464 (C) under the same conditions used in FIGURE 1.
  • Peptides are 15mers. advancing by one residue and cover selected determinant regions shown in FIGURE 1.
  • FIGURE 4 shows core sequences for determinant regions defined in FIGURE 2. The cores are defined according to the proliferation results form FIGURE 2 and include shared residues of peptides which induce at least 50% of the maximal proliferation for that determinant region.
  • PSP peptide sequences are indicated by the single-letter amino acid code.
  • FIGURE 5 shows lymphokine profile for the major PSP determinant regions in BALB/c and B6.
  • Lymph node cells (5 x 10 6 ) from individual BALB/c (A. mice 1 and 2) and B6 mice (B mice 1 and 2) were cultured with peptides corre ⁇ sponding to determinant regions with cores 369-376 (BALB/c) and 456-464 (B6) in 1 ml of complete medium.
  • Supernatants were collected after 48 hours centrifuged, stored at -70" C and thawed 10 minutes before assay for IFN- 7 and IL-4 by EL1SA cultures were established as in FIGURE 2 and assayed for proliferation.
  • the results express the lymphokine and proliferation profiles from two individual animals for both BALB/c and B6.
  • FIGURE 6 shows peptides within the determinant region with core 369-376 are able to induce Th2 cells in the presence of anti-IFN- 7
  • BALB/c mice were immunized with 2.5 ⁇ g of HPSP (A and B) and injected i.p. with 500 ⁇ g of XMG 1.2 antibody in PBS (B only) on days 1 and 3.
  • popliteal lymph node cells from three animals were pooled from each group and cultivated for 48 hours with peptides 364-378 (A and B) or 368-382 (A) in the presence or absence of 20 ⁇ g/m of antibody R46A2 (anti-IFN- 7 ) (A and B).
  • Live cells recovered after 48 hours were assayed by ELISPOT as in Table 2.
  • FIGURE 7 shows the frequency of variant peptide antigen-induced IFN-7 (Th1)
  • FIGURE 8 shows proliferation of T cell clones measured by [ 3 H]-thymidine incorporation after stimulation with varying concentrations of peptide variants.
  • FIGURE 9 shows a comparison of the proliferation pattern (A), and lymphokine, IL-4 (B) and IFN- 7 (C) secretion profile by a 9.4 Lys-specific T cell clone, 3C10, in response to 9.4 Lys (•), 9.4 Arg (.) and 9.4 Met (*).
  • the present invention provides a method for identifying a T cell modulatory peptide for a specific antigen, wherein the peptide stimulates an IFN- 7 secreting T cell population or an IL-4 secreting T cell population.
  • the method of the invention entails identifying peptides with low and high affinity binding for MHC and induction of T cell proliferation. Once identified, these T cell modulatory peptides are useful for treatment of subjects with Th1 or Th2 associated T cell disorders, such as allergic or autoimmune disorders.
  • the invention provides a method for identifying a T cell modulatory peptide for a specific antigen, wherein the peptide stimulates an
  • IFN- 7 secreting T cell population comprising preparing an array of variant immunodominant peptides from the antigen and selecting from the array, a peptide that has high MHC binding affinity and that induces proliferation of T cells specific for the antigen.
  • the term 'T cell modulatory peptide refers to a peptide that causes a change in a pre-existing T cell response.
  • the peptide can be used to divert a predominant T cell response away from a disease state by stimulating a protective T cell response.
  • Th1 cells preferably secrete lymphokines such as IFN- 7 , IL-2, and tumor necrosis factor (TNF).
  • the method of the invention operates by eliciting a protective response.
  • the invention provides a method for identifying a T cell modulatory peptide for a specific antigen, wherein the peptide stimulates an IL-
  • Th2 cells preferably secrete lymphokines such as IL-5, IL-6, IL-9, IL-10 and IL-13. Therefore, when the Th1 response is dangerously high in a subject, such as in the case of autoimmune disorders, it is desirable to stimulate a Th2 response.
  • the methods described above for identifying a T-cell modulatory peptide comprise the steps of preparing an array of variant immunodominant peptides from the antigen and selecting from the array a peptide that has either high or low MHC binding affinity, depending on the T cell population one desires to stimulate, and that induces proliferation of T cells specific for the antigen.
  • antigen refers to a substance which elicits an immune response.
  • the antigens of the invention which are sources for a T cell modulatory peptide may or may not be exogenously derived relative to the host.
  • the method of the invention may be used to identify a T cell modulatory peptide that is an "autoantigen".
  • An autoantigen is a normal constituent of the body that reacts with an autoantibody or activates T cells.
  • the invention also includes inducing tolerance to an "alloantigen". Alloantigen refers to an antigen found only in some members of a species, for example the blood group substances. Xenoantigens are substances which cause an immune reaction due to differences between different species.
  • an immunodominant peptide is a peptide derived from a whole antigen that is well-processed and presented to the T cell and is competitively favored among all of the potential determinants. The more poorly processed determinants from an antigen are termed “subdominant” and "cryptic” in descending hierarchical order of antigen presentation.
  • an immunodominant peptide is typically identified by immunizing an animal, such as a mouse, at a local site, for example intracutan- eously in a footpad, removing the draining lymph nodes from the animal and preparing cell suspensions approximately 9-11 days post injection.
  • the cells are distributed onto a solid support, such as the wells of a 96-well culture plate, to which, successively one of an overlapping set of peptides is added.
  • the overlapping peptides are approximately 10-20-mers, and most preferably 15-mers, with overlaps of from 10-14 amino acids.
  • Antigen-induced T cell proliferation can be determined by standard methods known to those of skill in the art (See, Coligan, et al., Current Protocols in Immunology, Wiley Interscience, 1991 , Unit 3).
  • proliferation is assessed by measuring the incorporation of tritiated thymidine ([ 3 H]) to indicate DNA synthesis.
  • an immunodominant peptide is identified, preferably the core of the determinant plus about 3 residues on each side of the core is used as the test immunogen.
  • An array of variants at each amino acid residue within the core is produced, with about three variants at each residue.
  • a "variant" refers to a peptide which is a mutagenized form of an immuno ⁇ dominant or core peptide, or one produced through recombination or peptide synthesis, for example.
  • substitution of amino acid residues such as any of the known methods for accomplishing random or site-directed mutagenesis of the DNA encoding the peptide.
  • the three variants at each position of the wild-type sequence to be screened for the MHC binding affinity and induction of a T cell proliferative response are: (1 ) a conservative change; (2) a non-conservative change; and (3) a change to alanine variant.
  • the wild-type residue is alanine
  • one of the residue changes will be to serine or a single small residue.
  • Amino acid changes which affect MHC binding can be evaluated directly or indirectly. For example, binding of a variant peptide can be determined directly by an assay using purified MHC molecules. Binding can be measured indirectly by a cellular competition assay. The peptides can be assayed for MHC binding in liquid phase or bound to a solid phase carrier.
  • peptides in these assays can be detectably labeled in various ways.
  • types of assays which can utilize the peptides of the invention are competitive and non-competitive assays in either a direct or indirect format. Those of skill in the art will know, or can readily discern, other assay formats without undue experimentation.
  • the peptides of the invention can be bound to many different carriers and used to detect the binding of the peptide to MHC.
  • Examples of well-known earners include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite.
  • the nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding peptides, or will be able to ascertain such, using routine experimentation.
  • a high affinity peptide variant has an MHC binding affinity at least 1 ,000 times, preferably at least 5,000 times, greater than the corresponding wild-type peptide.
  • a low affinity peptide variant of the invention has an MHC binding affinity at least 1 ,000 times, preferably at least 5,000 times, less than the corresponding wild-type peptide.
  • T cells specific for the wild type peptide are stimulated with these variants and the lymphokine secretion pattern is determined.
  • Amino acid changes which stimulate a T cell proliferative response can be assayed directly or indirectly.
  • the method described above utilizing tritiated thymidine incorporation is an example of a direct assay. Induction of proliferation of the appropriate T cell population can be determined indirectly by measuring lymphokine production from a T cell population after stimulation with a variant immunodominat peptide.
  • induction of stimulation of proliferation of Th1 cells can be determined by assay for IFN- 7 , IL-2, and/or tumor necrosis factor (TNF) and induction of stimulation of proliferation of Th2 cells can be determinined by assay for IL-5, IL-6, IL-9, IL-10 and IL-13.
  • the lymphokines are measured by standard immunoassay techniques, however, one of skill in the art will know of other assays for such detection.
  • cloned T cells as described below, are incubated with an increasing concentration of peptide variants in the presence of syngeneic antigen presenting cells (APCs).
  • APCs syngeneic antigen presenting cells
  • irradiated splenic cells are preferably used as APCs. Supernatants are then assayed, for example, by immunoassay or bioassay.
  • the method of the invention for identifying T cell modulatory peptides may further include the step of retesting the variants for their T cell specificity both in vitro and in vivo.
  • T cell clones from primed lymph nodes are prepared which react with the wild type immunodominant peptide. These clones are prepared by methods known to those of skill in the art such as the limiting dilution method described by Kimoto and Fathman (J. Exp. Med., 1 ⁇ 2:759, 1980). These clones are then stimulated with each of the variant peptides in vitro.
  • mice Animals, such as mice, are challenged with both the wild type peptide as well as the variant peptide and approximately 10 days later, lymph node proliferative responses are recalled with either peptide.
  • Cross- reactivity in proliferative responsiveness indicates a similarity in T cell specificity observable with the response induced by variants and the wild type peptide.
  • the invention also provides a synthetic T cell modulatory peptide with the amino acid sequences ASQXRPSQR and YSDGSCTQRASEAHASLLPFN (SEQ ID NO:
  • conservative variations denotes the replacement of an amino acid residue by another, biologically similar residue.
  • conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.
  • SEQ ID NO:1 represents amino acids 1 to 9 of myelin basic protein (MBP) polypeptide, wherein X is preferably glutamic acid, glutamine, alanine, valine, tyrosine or methionine.
  • SEQ ID NO:2 represents amino acids 361 to 382 of promastigote surface protease, PSP (gp63) of Leishmania major.
  • the core of SEQ ID NO:2 is amino acids RASEAHAS.
  • synthetic peptide denotes a peptide which does not comprise an entire naturally occuring protein molecule.
  • the peptide is "synthetic” in that it may be produced by human intervention using such techniques as chemical synthesis, recombinant genetic techniques, or fragmentation of whole antigen or the like.
  • the peptides of the invention include "functional fragments" of the peptides, as long as the activity of the peptide as noted above remains. Such peptides can be readily identified by those of skill using the routine screening methods described herein without resorting to undue experimentation. These peptides can be as few as 5, preferably as few as 10 amino acids in length.
  • Minor modifications of the primary amino acid sequence of the peptides of the invention may result in peptides which have substantially equivalent activity as compared to the specific peptides described herein or those identified by the method of the invention. Such modifications may be deliberate, as by site- directed mutagenesis, or may be spontaneous. All of the peptides produced by these modifications are included herein as long as the biological activity of the original variant peptide still exists.
  • the first 6 amino acids of the peptide of SEQ ID NO:1 may stimulate Th1 cells to the same extent as the first 8 or 9 amino acids. Such determinations are routine and can be done without undue experimentation.
  • deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its biological activity. This can lead to the development of a smaller active molecule which would also have utility.
  • one of skill in the art can use standard techniques to remove amino or carboxy terminal amino acids from SEQ ID NO:1 , or delete amino acids from SEQ ID NO:2, as long as the amino acids are not required for biological activity of the particular peptide.
  • Peptides of the invention can be synthesized by such commonly used methods as t-BOC or FMOC protection of alpha-amino groups. Both methods involve stepwise syntheses whereby a single amino acid is added at each step starting from the C terminus of the peptide (See, Coligan, et al., Current Protocols in Immunology, Wiley Interscience, 1991 , Unit 9). Peptides of the invention can also be synthesized by the well known solid phase peptide synthesis methods described by Merrifield, (J. Am. Chem.
  • This can normally be purified by such techniques as gel filtration on Sephadex G-15 using 5% acetic acid as a solvent. Lyophilization of appropriate fractions of the column will yield the homogeneous peptide or peptide derivatives, which can then be characterized by such standard techniques as amino acid analysis, thin layer chromatogra ⁇ phy, high performance liquid chromatography, ultraviolet absorption spectros- copy, molar rotation, solubility, and quantitated by the solid phase Edman degradation.
  • the peptides of the invention can be used singularly, in mixtures, or as multimers such as aggregates, polymers, and the like.
  • the invention embraces synthetic peptides which comprise one or more of the same, or different, peptides of the invention to produce a homogeneous or heteroge ⁇ neous polymer with respect to the particular peptides of the invention which are contained therein.
  • Appropriate techniques for producing various mixtures, aggregates, multimers and the like will be known to those of skill in the art.
  • polynucleotide refers to a polymer of deoxyribo- nucleotides or ribonucleotides, in the form of a separate fragment or as a component of a larger construct.
  • DNA encoding a peptide of the invention can be assembled from cDNA fragments or from oligonucleotides which provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit.
  • Polynucleotide sequences of the invention include DNA, RNA and cDNA sequences. A polynucleotide sequence can be deduced from the genetic code; however, the degeneracy of the code must be taken into account.
  • Polynucleotides of the invention include sequences which are degenerate as a result of the genetic code.
  • the polynucleotide encoding the peptides of the invention includes a polynucleotide that encodes SEQ ID NO:1 or 2, as well as complementary nucleic acid sequences.
  • a complementary sequence may include an antisense nucleotide.
  • the sequence is RNA
  • the deoxynucleotides A, G, C, and T of SEQ ID NO:1 or 2 are replaced by ribonucleotides A, G, C, and U. respectively.
  • fragments of the above- described nucleic acid sequences that are at least 15 bases in length, which is sufficient to permit the fragment to selectively hybridize to DNA that encodes the peptides of the invention under physiological conditions.
  • Polynucleotide sequences encoding the peptides of the invention can be expressed in either prokaryotes or eukaryotes.
  • Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art.
  • Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences of the invention.
  • the present invention also provides a method of modulating a T cell response to a specific antigen in a subject with a T cell disorder, comprising administer- ing to the subject an effective amount of a T cell modulatory peptide for the antigen, wherein the peptide stimulates an IFN- 7 or an IL-4 secreting T cell population.
  • a subject has a T cell disorder associated with elevated levels of Th2
  • the disorder is typically an allergic disorder or a helminth infection.
  • the disorder is typically an autoimmune disorder, or a disorder having a bacterial, viral or protozoan etiology.
  • the subject is a human.
  • the term "effective amount” as used herein refers to the amount of T cell modulatory peptide administered is in sufficient quantity to decrease the subject's response to the antigen, for example, decrease the symptoms of an allergy.
  • the amount of peptide that is considered effective can be determined by monitoring the antigen-specific Th1 :Th2 ratio of a subject. Preferably, this is determined by ELISA measurements of lymphokines secreted by peripheral blood lymphocyte samples. Other methods will be known to those of skill in the art.
  • the dosage ranges for the administration of the peptide of the invention are those large enough to produce the desired effect. Generally, the dosage will vary with the age, condition, sex, and extent of the infection with bacteria or other agent as described above, in the patient and can be determined by one skilled in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. In any event, the preadminstration and post administration ratio of Th1 :Th2 should correlate with recovery of the patient.
  • allergic disorders include allergic rhinitis, asthma, atopic dermatitis, and food allergies.
  • autoimmune disorders where the immune system attacks the host's own tissues, include, but are not limited to, type 1 insulin-dependent diabetes mellitus, adult respiratory distress syndrome, inflammatory bowel disease, dermatitis, meningitis, thrombotic thrombocy- topenic purpura, Sj ⁇ gren's syndrome, encephalitis, uveitic, leukocyte adhesion deficiency, rheumatoid arthritis, rheumatic fever, Reiter's syndrome, psoriatic arthritis, progressive systemic sclerosis, primary biliary cirrhosis, pemphigus, pemphigoid, necrotizing vasculitis, myasthenia gravis, multiple sclerosis, lupus erythematosus, polymyositis, sarcoidosis, granulomatosis, vasculitis, pernicious
  • T cells can be separated from other PBLs by techniques known in the art.
  • Procedures for separation of cells may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, for example, complement and cytotoxins, and "panning" with antibody attached to a solid matrix, for example, plate, or other convenient technique.
  • Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, for example, a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
  • the T cell modulatory peptide is administered by any suitable means, including parenteral, subcutaneous, intrapulmonary, oral, and intranasal administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, or intraperitoneal administration.
  • the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or prolonged.
  • peptides of the invention are administered in a physiologically acceptable solution.
  • Preparations of peptide for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, and polyethylene glycol.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • a peptide of the invention can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms.
  • These include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric and the like. Salts also include those formed from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like.
  • mice Female BALB/cJ, C57BIJ6, BALB.B, CBA/J, and 510.D2 mice (8-12 weeks old) were either purchased from Jackson Laboratories (Bar Harbor, ME) or bred in our own facilities.
  • HL-1 serum-free medium Ventrex, Portland, ME
  • 2 x 10 '5 M 2-merca- ptoethanol, 20 mM glutamine, and 25 ⁇ g/ml gentamicin was used for pepscan experiments (FIGURES 1A and B and 2A and B).
  • RPMI 1640 Sigma. St. Louis, MO
  • FCS Heat inactivated FCS
  • 2 x 10 "5 M mercaptoe- thanol. 20 mM glutamine, and 25 ⁇ g/ml gentamicin was used.
  • gp63 Recombinant gp63 (rPSP) was a generous gift of Dr. Robert McMaster (University of British Columbia); native gp63 (PSP) in either hydrophilic (HPSP) or amphiphilic (APSP) forms were kindly donated by Dr. Robert Etges (Universite de Lausanne). Soluble L. major (LV 39) 'antigen' was prepared by three cycles of freezing and thawing, followed by centrifugation at 10,000 g for 10 minutes.
  • GK 1.5(anti- mouse CD4 + ), 34-5-3S (anti 1-A C ), and 2.43 (anti-mouse CD ⁇ " ”) cells were purchased form ATCC and the antibodies purified in this laboratory.
  • Anti- mouse Thy-1 " ⁇ " ascites and rabbit Low-tox complement were purchased from Accurate Chemical and Scientific (Westbury, NY);
  • rHulL-2 was a gift of Dr. Ray Apple (Roche Molecular Systems, Alameda, CA);
  • r-mouse IL-4 was a generous gift of Dr. William Paul (NIH)
  • r-mouse IL-5 and IL-10 were purchased from Pharmingen;
  • r-mouse IFN- 7 was purchased from Genzyme (Cambridge, MA).
  • the Fmoc protecting group was then removed and additional Fmoc-protected amino acids were added onto the i -amino group of the lysine by repetitive cycles of Fmoc deprotection and amino acid couplings.
  • the terminal amino group of each peptide was acetylated.
  • cleavage from the pins was performed by exposure to water at neutral pH, under which conditions the C-terminal lysine-proline residues formed a diketopiperazine derivative.
  • Peptide yield was estimated as described (Maeji, N.J., et al., supra). 6. Immunizations and culture conditions
  • mice were immunized s.c. in a footpad with 2 ⁇ g of rPSP or 2.5 ⁇ g of either HPSP or APSP, or 40 ⁇ g of synthetic peptides. All immunogens were emulsified in 50% complete Freund's adjuvant (Difco. Detroit, Ml). Mice were killed 9-10 days later and popliteal lymph nodes were pooled or prepared from individual mice and processed as single cell suspensions. Cells (5 x 10 5 /well) were seeded in 96-well plates (Costar, Cambridge, MA) along with various concentrations of peptide or protein, incubated for 3 days and then pulsed for 18 hours with 1 ⁇ Ci of [ 3 H]thymidine (ICN, Costa Mesa, CA). Cultures were harvested with a Skatron cell harvester (Skatron, Sterline, VA) and [ '
  • lymph node cells were placed in 24-well plates (Costar) at 2-5 x 10 6 cells/ml in 1 ml of medium along with antigen and incubated at 37 °C. Supernatants were harvested after 48, 72 or 96 hours of culture, centrifuged, and stored at -70" C.
  • T cell lines were incubated in 24-well plates at 1 x 10 5 cells/ml along with 2 x 10 6 mitomycin C-treated spleen cells and antigen, in 1 ml of medium. Super ⁇ natants were harvested after 24-48 hours and prepared as above.
  • ELISPOT plates at various concentrations. Alternatively, lymph node cell suspensions were directly seeded in ELISPOT plates along with antigen and processed as described below.
  • 96-well ELISA plates (Nunc Maxisorp, Kamnstrup, Denmark) were coated with 2 ⁇ g/ml of capture antibody in 0.05 M Tris, pH 8.5, and incubated overnight at 4°C. The plates were then washed twice and blocked with 25 mM Tris- buffered saline - 100% FCS (blocking buffer) for 2 hours, washed four times, and incubated with serial dilutions of samples in blocking buffer at room temperature for 90 minutes.
  • Second antibodies were added at 2 ⁇ g/ml in blocking buffer, and the plates were then incubated for 1 hour at room temperatures, washed six times, and incubated with a 1 :4000 dilution of avidi ⁇ D-peroxidase (Vector, Burlingame, CA) for 30 minutes at room temperature. Finally, the plates were washed eight times and the substrate, 2.2'-azino-bis-3 ethylbenzthiazoline-6-sulfonic acid (ABTS: Sigma) added at 100 M g/ml along with H 2 0 2 (1 :2000 of a 30% solution). Optical density was measured using a Titertek Multiskan ELISA reader (Flow Laboratories, Huntsville, AL) at 405 nm. The values were converted to U/ml using recombi ⁇ nant protein as a standard.
  • ABTS 2.2'-azino-bis-3 ethylbenzthiazoline-6-sulfonic acid
  • Cells were added to duplicate wells in concentrations ranging from 10 4 to 2.5 x 10 5 cells/well in 100 ⁇ l of medium and incubated for 20-60 hours at 37 °C (see figure legends for details). Following incubation, plates were first washed extensively with ice-cold PBS with 0.01 M EDTA to remove adherent cells and then with PBS-0.05% Tween 20.
  • Lymph node cells were prepared as described above and incubated in 24-well plates at a concentration of 5-10 x 10 6 cells/ml in 1 ml of complete medium and antigen. Cells were harvested after 6 days and reincubated with fresh mitomycin C-treated syngeneic spleen cells [antigen presenting cells (APC)] and antigen. Seven days later, viable cells were recovered through flotation on Ficoll (Sigma) and incubated in 50 U/ml of rlL-2. From this point on, cells were stimulated with antigen/APC or IL-2 on alternate weeks.
  • APC antigen presenting cells
  • FIGURE 1 shows profiles of the determinant regions on the PSP molecule in BALB/c and B6 mice.
  • Peptide number refers to the N-terminal residue of the 15mer. Data are expressed as [ 3 H]thymidine incorporation in individual cultures in FIGURES 1 , 2 and 3. To assure reproducibility, experiments were performed two to four times.
  • T cell determinant regions are readily apparent in BALB/c mice after HPSP immunization (FIGURE 1A).
  • the most dominant determinant region was one that can be recalled with peptides p361 ('p361 ' is the 15mer starting at residue 361) through p367 followed by a group of subdominant determinants recalled by peptides (in order to importance) p403 through p409, p136 through p151.
  • p106 through p121.
  • the determinants are referred to by a single number representing the amino terminus of the 15mer peptide giving the peak response.
  • FIGURE 2 shows fine determinant mapping of PSP determinant regions in BALB/c mice.
  • Lymph node cells of individual BALB/c mice previously immunized with 2.5 ⁇ g of HPSP were cultured along with each peptide (at 14 ⁇ M) within determinant regions with cores 138-147/143-155 (A) and 369-376 (B) under the same conditions used in FIGURE 1.
  • Peptides are 15mers advancing by one residue and cover selected determinate regions represented in FIGURE 1.
  • FIGURE 3 shows fine determinant mapping of PSP determinant regions in B6 mice.
  • Lymph node cells of individual B6 previously immunized with 2.5 ⁇ g of HPSP were cultured along with each peptide (at 14 ⁇ M) within determinant regions with cores 138-147/143-155(A), 194-201 (B), or 456-464 (C) under the same conditions used in FIGURE 1.
  • Peptides are 15mers. advancing by one residue and cover selected determinant regions shown in FIGURE 1.
  • FIGURE 4 shows core sequences for determinant regions defined in FIGURE 2.
  • the cores are defined according to the proliferation results form FIGURE
  • PSP peptide sequences are indicated by the single-letter amino acid code. Recall by the 15mer overlapping peptide series (pepscan) after HPSP immunization allowed the core regions, which are here arbitrarily defined as those residues contained in all peptides giving 50% or more of the maximal proliferation for the determinant region, of each determinant to be mapped.
  • FIGURE 2(A and B) shows the pepscan for the regions surrounding peptides p136 through p151 and p361 through p367 respectively in BALB/c mice
  • FIGURE 3(a-c) shows regions that include peptides p136 through p148, p187 through p193, and p451 through p460 respectively in the B6 mice Core regions for each determinant are illustrated in FIGURE 4
  • determinant regions 136-151 and 361 -367 gave clearly similar cores for BALB/c and B6 mice
  • the determinant region 136-151 clearly contains two overlapping determinants in both BALB/c and B6 In BALB/c the cores are 138-148 and 144-155, and in the B6, 138-1 7 and 143-155
  • the determinant region p361- p367 gave exactly the same core, 369-376, for BALB/c and B6 strains.
  • FIGURE 5 shows lymphokine profile for the major PSP determinant regions in BALB/c and
  • Lymph node cells (5 x 10 6 ) from individual BALB/c (A. mice 1 and 2) and B6 mice (B mice 1 and 2) were cultured with peptides corresponding to determinant regions with cores 369-376 (BALB/c) and 456-464 (B6) in 1 ml of complete medium. Supernatants were collected after 48 hours centrifuged, stored at -70 ⁇ C and thawed 10 minutes before assay for IFN- 7 and IL-4 by
  • ELISA cultures were established as in FIGURE 2 and assayed for proliferation. The results express the lymphokine and proliferation profiles from two individual animals for both BALB/c and B6.
  • mice were immunized with 40 ⁇ g of peptide 364-378 or 452-466 respectively.
  • popliteal lymph node cells were pooled form three animals and assayed for proliferation and lymphokine production in response to a frozen and thawed extract of L. major (1 x 10 6 /ml). Proliferation was measured as described in Example 2 and lymphokine production as in Example 5 (c.p.m. values for medium alone were 4-6 x 10 '3 ).
  • a central peptide such as 364-378 almost exclusively stimulated a population of cells with a typical Th1 phenotype while peptide 368-382 induced a frequency of IL-4 producing cells that was up to three times higher than that of IFN- ⁇ producing cells; a similar pattern was induced by the other boundary peptide, 361-375.
  • mice Balb/c, B6 or CBA mice were immunized with p364-378 and pooled popliteal lymph node cells from three animals were assayed for their ability to secrete lymphokines.
  • Cells were cultivated at 10 7 cells/ml for 48 hours with either 10 ⁇ g/ml of p364-378 or 3 ⁇ g/ml of rPSP and then dispersed, washed, and seeded in ELISPOT plates at concentrations from 10 4 to 2.5 x 10 5 cells/well in duplicate. The plates were cultivated for an additional 20 hours and then processed for spot development as described. TABLE 3
  • Th1 and Th2 cells are mutually inhibitory events (Hsieh, C.S.. et al., Science, 2SQ:547, 1993). and that the initial concentration of Th1/Th2 lymphokines is critical, it is possible that the total dominance of IFN- 7 in most of the determi ⁇ nant regions measured is a result of the inability of those peptides to prime a sufficient number of Th2 cells or to induce secretion of enough IL-4 by primary T cells to permit the development of Th2 cells.
  • FIGURE 6 shows peptides within the determinant region with core 369-376 are able to induce Th2 cells in the presence of anti-IFN- 7 .
  • BALB/c mice were immunized with 2.5 ⁇ g of HPSP (A and B) and injected i.p. with 500 ⁇ g of XMG 1.2 antibody in PBS (B only) on days 1 and 3.
  • FIGURE 6(A) ELISPOT results present in FIGURE 6(A) show that the apparent dominance of IFN-7 producing cells in response to peptide 364-378 can be altered markedly with a large increase in IL-5-producing cells. This suggests that peptide 364-378 induces not only IFN- 7 from the former population. A similar increase in Th2 was observed in vivo when mice were injected with anti-IFN- 7 (FIGURE 6B). As can be seen in FIGURE 6(A) when peptide 368-382 was used for recall, there was a clear decrease in the number of IFN- 7 secreting T cells relative to the number of IL-5 secreting T cells. The effect is not as great as that in Table 3 which may be attributable to a number of differences in the two experiments including a difference in immunogens (rPSP versus HPSP) and a difference in the methodology for measure single T cells.
  • the N-terminal peptide fragment of myelin basic protein, acetylated 1-9 (abbreviated here as 9.4Lys), served as the desired ligand because single amino acid substitutions at position 4 allowed the generation of variants with an enormous range of MHC-binding affinities coupled with unchanged T cell specificity (Wraith, D.C., ef al., Cell, 59:247-255, 1989; Hood, L.E., ef al., Cold Spring Harbor Symp. Quant. Biol., 54:859-874, 1989).
  • MHC-binding affinities were determined using a direct MHC-binding assay using purified class II molecules.
  • the final detergent concentration in the incubation mixture was 2.6% digitonin.
  • the amount of MHC required for 10-20% binding was approximately 20-100 nM. All subsequent assays were then performed using this class II concentration.
  • peptide competitors were typically tested at concentrations ranging from 1.2 mg/ml to 0.12 ng/ml. The data were then plotted and the dose yielding 50% inhibition was calculated. Each peptide was tested in 2-4 completely independent experiments.
  • Peptides were synthesized by Drs. S. Horvath (Caltech, Pasadena, CA) or C. Miles (Macromolecular Resources, Fort Collins, CO) using a solid phase technique on a peptide synthesizer (Applied Biosystems Inc., Foster City, CA) and were purified on a reversed phase column by HPLC (Clark-Lewis, I., et ai, Science, 231:134, 1986).
  • I-A u molecules were purified from a l-A u expressing hybrid cell line JK 91.7 that was produced by fusion of the BALB/c B cell lymphoma line A20.2 JA (l-A°, l-E d ) to spleen cells of a PL ⁇ J mouse (l-A u , l-E u ).
  • Variant peptides with higher T cell stimulatory capacities bind more effectively than 9.4Lys as suggested earlier (Wraith, D.C., et al., supra; Hood, L.E., ef al., supra).
  • Peptide variant 9.4Arg is the poorest binder among all variants whereas peptides 9.4Met and 9.4Tyr, which bind almost 10,000 times better than 9.4Lys, are the best binders.
  • the wild type peptide 9.4Lys is only slightly better than 9.4Arg.
  • Peptide variant 9.4Ala showed a 400 fold better binding affinity than 9.4Lys, consistent with earlier observations (Wraith, D.C., ef al., supra; Hood, L.E., ef al., supra).
  • the longer N- terminal peptide of MBP, Ac1-20 binds almost as effectively as 9.4Ala with l-A u .
  • mice challenged with 9.4Met were 4.1 or 5 fold higher than in mice immunized with 9.4Lys/9.4Arg. In contrast, frequencies of IL-5- producing cells were comparable in each group of mice.
  • IFN- 7 producing cells were about 5 times higher in mice challenged with the high affinity peptide, 9.4Met (1170 cells per 10 6 cells) in comparison with mice immunized with the low affinity peptide, 9.4Arg or 9.4Lys (250 cells per 10 6 cells) (FIGURE 7A).
  • the frequency of IL-5 producing cells was comparable in all three groups of mice (70-80 cells per 10 6 cells) and did not increase proportionately in response to the highest affinity ligand (FIGURE 7B).
  • the frequency of IL-4 secreting cells was comparable in each group.
  • T cell proliferation and lymphokine secretion pattern was measured.
  • 9.4Lys-spec ⁇ f ⁇ c T cell clones (Bhardwaj, V., et al., J. Immunol., 151 :5000- 5011 , 1993; Urban, J., ef al., Cell, 54:577-592, 1988) were studied to determine whether they were capable of recognizing variants with low as well as high MHC-binding affinities.
  • a typical proliferation profile of a CD4 + T cell clone (3C10) specific for 9.4Lys, which secreted interleukin 4 (IL-4) but not interferon- 7 is shown in FIGURE 8. Briefly.
  • T cells (5 x 10 4 -1 x 10 5 cells per well) (T cell clone, 3C10, was derived from a long term T cell line, generated from B10.PL mice immunized with MBP, by two sequential limiting dilutions at 0.3 cells per well) were incubated with irradiated syngeneic spleen cells (APC) (1 x 10 5 -5 x 10 5 cells per well) in the absence or presence of peptides at different concentrations (1 nM to 14 ⁇ M). Proliferation of T cell clones was measured by [ 3 H] -thymidine incorporation for the last 18 hours of a 3-day culture.
  • APC irradiated syngeneic spleen cells
  • Peptide variants with position 4 residues possessing hydrophobic side chains smaller than lysine or a negative charge (Glu) did not significantly affect stimulation, whereas amino acid substitutions with either neutral or uncharged polar side chains (gin) were more effective in T cell activation.
  • position 4 variants of 9.4Lys with nonpolar or hydrophobic side chains (ala, val, met) showed a strong heteroclitic proliferative response in that they were able to efficiently stimulate T cells at a 10 3 to 10 5 -fold lower antigen concentration (FIGURE 8).
  • FIG. 9 The relative capability of variants to induce proliferation and IL-4-secretion was compared.
  • IFN- 7 and IL-4 were measured by ELISA as described (Curry, R.C., et al., J. Immunol. Methods, 104:137-145, 1987; Mosmann, T., ef al., J. Immunol. 145:2938-2945, 1990). Briefly, 96-well ELISA plates (NUNC Maxisorp, Kamnstrup, Denmark) were coated with 2 ⁇ g/ml of capture antibody in 0.05 M Tris, pH 8.5, and incubated overnight at 4°C.
  • the plates were then washed 2 times and blocked with 25 mM tris-buffered saline-10% FCS (blocking buffer) for 2 hours, washed 4 times and incubated with serial dilutions of samples in blocking buffer at room temperature for 90 minutes. After 4 washes, second antibodies were added at 2 ⁇ g/ml in blocking buffer, the plates were incubated for 1 hour at room temperature, washed 6 times, and then re-incubated with a 1 :4000 dilution of avidin D-peroxidase (Vector, Burlingame, CA) for 30 minutes at room temperature.
  • Vector Burlingame, CA
  • Murine IFN- 7 -specific monoclonal antibodies R4-6A2 and XMG 1.2 and anti-mouse IL-4 monoclonals, BVD4-1 D11 and BVD6-2A62 were purchased from Pharmingen, San Diego, CA.
  • Recombi ⁇ nant mouse IL-4 was a generous gift of Dr. William Paul (NIH) and r-IFN- 7 was purchased from Ge ⁇ zyme Co., Cambridge, MA.
  • 9.4Met induced significant levels of IFN-7 secretion in 2/4 T cell clones although at a relatively high peptide concentration (FIGURE 9C).
  • the maximum amount of IFN- 7 produced by the T cell clone 3C10 was 47.1 u/ml at 100 ⁇ M of 9.4Met.
  • other variants which demonstrated more than 1000-fold higher MHC-binding than 9.4Lys, for example, 9.4Val also induced IFN- 7 .
  • the 9.4Lys and 9.4Arg peptides with very low MHC-binding were not able to induce 7 -IFN, even at very high peptide concentrations (up to 1 mM).
  • purified T cells (3C10) were used to measure IFN- 7 production by the ELISA spot technique.
  • Cytokine production by clone 3C10 at similar levels of the different variants on the surface of the antigen-presenting cell was determined. In the absence of any direct measure of surface antigen/MHC display, this was assessed indirectly, using the 9.4Lys-reactive T cell hybridoma (HY.33). Peptide concentrations of the variants were determined for which they have apparently triggered similar numbers of TCR as measured by proliferation of an indicator
  • T cell line (Table 5).
  • the 9.4Lys-specific T cell hybridoma (Hy.33) cells (1x10 5 cells/well), were stimulated in the presence of syngeneic irradiated spleen cells (5x10 5 ) and various concentrations of the peptides.
  • IL-2 secretion was determined by measuring 3 H-thymidine uptake by the IL-2- dependent cell line, HT-2, in the presence of 100 ⁇ l of 24 hours culture supernatant as before (Bhardwaj, V.. ef a/., J. Immunol., 121:5000-5001 , 1993).
  • the control (APC and T cells without antigen) CPM were 1488+ 138.
  • the data shown are the arithmetic means of triplicate wells. Cytokine production by T cell clone 3C10 was measured by ELISA as in Example 9. Proliferation as well as lymphokine production is compared at a linear range for each of the peptides. As expected, the concentrations required for the same level of TCR-signalling were different for different variants, for example, 14 ⁇ M, 0.7 ⁇ M and 0.0007 ⁇ M for 9.4Arg, 9.4Lys and 9.4Met, respectively. At these peptide concentrations, the T cell clone, 3C10, did not produce detectable levels of IFN- 7 (Table 5). Interestingly, a significant amount of IL-4 was produced in the presence of 9.4Arg or 9.4Lys. This further confirms that lower affinity variants preferentially trigger IL-4 production.

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Abstract

Cette invention concerne des peptides modulant les cellules T, des procédés d'identification de ces peptides ainsi que des procédés d'utilisation associés. Dans une forme de l'invention, on identifie des peptides modulant les cellules T destinés à un antigène spécifique qui stimulent les cellules Th1 sécrétant des interféron-gamma et qui présentent une forte affinité de liaison pour le complexe majeur d'histocompatibilité (CMH); on peut administrer ces peptides modulateurs à un patient pour moduler les pathologies allergiques par exemple. Dans une autre forme de l'invention on identifie des peptides modulant les cellules T destinés à un antigène spécifique qui stimulent les cellules Th2 sécrétant de l'IL-4 et qui présentent une faible affinité de liaison pour le CMH; on peut administrer ces peptides modulateurs à un patient pour moduler les pathologies auto-immunes par exemple.
PCT/US1994/009795 1994-08-26 1994-08-26 Procedes et compositions permettant de moduler une reponse des cellules t WO1996006630A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997002052A1 (fr) * 1995-07-05 1997-01-23 Yeda Research And Development Co. Ltd. Procede de detection ou de surveillance concernant l'efficacite d'un traitement de maladies mediees par des lymphocytes t
WO1998023959A3 (fr) * 1996-11-29 1998-08-06 Univ California Procedes perfectionnes pour le diagnostic et le traitement du diabete sucre insulino-dependant
WO2004058295A1 (fr) * 2002-12-26 2004-07-15 Daiichi Suntory Pharma Co., Ltd. Medicament permettant de traiter la pemphigoide
WO2014115102A1 (fr) * 2013-01-23 2014-07-31 Institut National De La Sante Et De La Recherche Medicale Nombre de lymphocytes t sécrétant de l'il4 et/ou de l'il3 utilisé en tant que biomarqueur pour maladies allergiques
US8835603B2 (en) 2008-11-30 2014-09-16 Immusant, Inc. Agents for the treatment of celiac disease
US10370718B2 (en) 2014-09-29 2019-08-06 Immusant, Inc. Use of HLA genetic status to assess or select treatment of celiac disease
US10449228B2 (en) 2013-09-10 2019-10-22 Immusant, Inc. Dosage of a gluten peptide composition

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997002052A1 (fr) * 1995-07-05 1997-01-23 Yeda Research And Development Co. Ltd. Procede de detection ou de surveillance concernant l'efficacite d'un traitement de maladies mediees par des lymphocytes t
WO1998023959A3 (fr) * 1996-11-29 1998-08-06 Univ California Procedes perfectionnes pour le diagnostic et le traitement du diabete sucre insulino-dependant
US6022697A (en) * 1996-11-29 2000-02-08 The Regents Of The University Of California Methods for the diagnosis and treatment of insulin-dependent diabetes mellitus
US6207159B1 (en) 1996-11-29 2001-03-27 The Regents Of The University Of California Methods for the diagnosis and treatment of insulin-dependent diabetes mellitus
WO2004058295A1 (fr) * 2002-12-26 2004-07-15 Daiichi Suntory Pharma Co., Ltd. Medicament permettant de traiter la pemphigoide
US7449177B2 (en) 2002-12-26 2008-11-11 Asubio Pharma Co., Ltd. Remedy for pemphigoid
US8835603B2 (en) 2008-11-30 2014-09-16 Immusant, Inc. Agents for the treatment of celiac disease
US9464120B2 (en) 2008-11-30 2016-10-11 Immusant, Inc. Compositions for treatment of celiac disease
WO2014115102A1 (fr) * 2013-01-23 2014-07-31 Institut National De La Sante Et De La Recherche Medicale Nombre de lymphocytes t sécrétant de l'il4 et/ou de l'il3 utilisé en tant que biomarqueur pour maladies allergiques
US10449228B2 (en) 2013-09-10 2019-10-22 Immusant, Inc. Dosage of a gluten peptide composition
US10370718B2 (en) 2014-09-29 2019-08-06 Immusant, Inc. Use of HLA genetic status to assess or select treatment of celiac disease

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