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WO1999038526A1 - Variants de ligands peptidiques induisant selectivement l'apoptose - Google Patents

Variants de ligands peptidiques induisant selectivement l'apoptose Download PDF

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Publication number
WO1999038526A1
WO1999038526A1 PCT/US1999/001912 US9901912W WO9938526A1 WO 1999038526 A1 WO1999038526 A1 WO 1999038526A1 US 9901912 W US9901912 W US 9901912W WO 9938526 A1 WO9938526 A1 WO 9938526A1
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WIPO (PCT)
Prior art keywords
cells
cell
peptide ligand
derived
apoptosis
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PCT/US1999/001912
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English (en)
Inventor
Michael J. Lenardo
Ronald N. Germain
Behazine Combadiere
Caetano Reis E. Sousa
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The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Application filed by The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services filed Critical The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority to AU24813/99A priority Critical patent/AU2481399A/en
Publication of WO1999038526A1 publication Critical patent/WO1999038526A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/41—Porphyrin- or corrin-ring-containing peptides
    • A61K38/415—Cytochromes
    • 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
    • A61P37/02—Immunomodulators
    • 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
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5403—IL-3
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/55—IL-2
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/555—Interferons [IFN]
    • G01N2333/57—IFN-gamma
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to selective modulation of specific T cell responses in a subject. Specifically, the present invention is directed to the identification and characterization of variant peptide ligands for the T cell receptor which act as partial agonists by inducing apoptosis in cycling T cells without concomitant production and release of non-death inducing cytokines.
  • variant ligands can be used to treat or prevent T cell associated disorders such as autoimmune disease, allergic disorders, graft rejection and graft versus host disease by selectively eliminating specific T cell populations.
  • T cell receptor (TCR) engagement can lead to several different responses in mature peripheral T lymphocytes including activation and proliferation, anergy, cytokine elaboration and programmed cell death (apoptosis). These events are controlled by signals resulting from interaction of the TCR and CD4 or CD8 coreceptors with peptide/ major histocompatibility complex (MHC) ligands and contribute to the quality and extent of the T cell response (1-5).
  • MHC major histocompatibility complex
  • the activation state of a T cell also dictates the outcome of interactions between the TCR and the peptide/TVIHC ligand.
  • Agonist ligands stimulate cytokine production and proliferation in resting T cells whereas in cycling cells, they cause cytokine production followed by death via apoptosis (7, 8). The uncoupling of apoptosis and non-death inducing cytokine production in response to a ligand has not been demonstrated. 2
  • the present invention provides variant peptide TCR ligands with selective T cell death-inducing activity without concomitant release of non-death inducing cytokines and methods for the use of these variant peptide ligands in treating or preventing T cell associated disorders such as autoimmune disease, allergic disorders, graft rejection and graft versus host disease in transplantation recipients.
  • the present invention provides a method for treating or preventing a T cell associated disorder in a subject comprising administering to the subject a peptide ligand for the T cell receptor which induces apoptosis of T cells without release of T cell- derived, non-death inducing cytokines by T cells which recognize the peptide ligand.
  • Also provided is a method for enhancing immune tolerance in a transplant recipient comprising administering to the recipient a peptide ligand for the T cell receptor which induces apoptosis of T cells without release of T cell-derived, non-death inducing cytokines by T cells which recognize the peptide ligand.
  • a method for inducing apoptosis of T cells without release of T cell-derived, non-death inducing cytokines comprising contacting the T cells with a peptide ligand for the T cell receptor which induces apoptosis of T cells without release of T cell-derived, non-death inducing cytokines.
  • a method for improving transplantation of bone marrow cells comprising inducing apoptosis of T cells without release of T cell- derived, non-death inducing cytokines, comprising contacting the T cells with a peptide ligand for the T cell receptor which induces apoptosis of T cells without release of T cell-derived, non-death inducing cytokines.
  • a method for screening a variant peptide ligand for the T cell receptor for the ability to induce apoptosis of T cells without release of T cell- derived, non-death inducing cytokines by T-cells which recognize the peptide ligand comprising: contacting cycling T-cells which recognize the variant peptide ligand with 3 the variant peptide ligand; detecting the presence or absence of apoptosis of the T cells; and detecting the presence or absence of release of non-death inducing cytokines by the T-cells, whereby the presence of apoptosis of the T cells and the absence of release of non-death inducing cytokines by the T cells indicates a variant peptide ligand having the ability to induce apoptosis of T cells without release of non-death inducing cytokines by T cells which recognize the variant peptide ligand.
  • FIG. 1 A-D (A) Variant ligands induce apoptosis of CD4+ Thl cells without the accompanying production of IL-2, IL-3 or IFN- ⁇ . (B,C,D) Dose response analyses of cycling A.E7 cells stimulated by P13.9 APC incubated with PCC (88-104) WT peptide (B), variant peptide R99 (C) or variant peptide Y99 (D).
  • T cell apoptosis (solid square), IL-2 (diamond), IL-3 (circle) and IFN- ⁇ (triangle) production in cycling A-E7 cells were measured after stimulation for 24 hours with P13.9 APC in the presence of the indicated concentrations of WT peptide or variant peptides. All values were expressed as a percentage of the response obtained with 100 ⁇ M WT peptide.
  • the present invention is based on the surprising discovery of variant peptide ligands which have the partial agonistic effect of inducing apoptosis of T cells without production and release of non-death inducing cytokines by selective T cell populations.
  • the present invention provides a method for treating or preventing a T cell associated disorder in a subject comprising administering to the subject a peptide ligand for the T cell receptor which induces apoptosis of T cells without concomitant release 4 of T cell-derived, non-death inducing cytokines (i.e, cytokines which do not induce cell death) in T cells which recognize the peptide ligand.
  • non-death inducing cytokines By “concomitant” is meant that the production and release of non-death inducing cytokines is associated with the binding of the peptide ligand under normal conditions. In this manner, a specific population of T cells capable of causing a T cell associated disorder can be eliminated while leaving the remainder of the subject's T cells unaffected. In addition, adverse effects on the subject from the release of T cell derived, non-death inducing cytokines are avoided.
  • the peptide ligand of this invention can be a ligand that binds a very specific population of T cells for a very focused effect on the subject's T cells or the peptide ligand can be a ligand that binds the T cell receptor of many T cell types for a broad effect on the subject's T cells.
  • the T cells which recognize the peptide ligand can be cycling T cells in the subject or the T cells which recognize the peptide ligand can be induced to cycle by administering antigen to the subject which is specific for the population of T cells of interest. Whether the T cells of interest are already cycling in the subject can be determined by flow cytometric analysis, allowing for identification of cells expressing antigens characteristic of cycling cells, such as Ki-67 (75) or identification of cells having a cell surface phenotype manifesting DNA replication by the incorporation of labeled nucleotides.
  • the antigen that can be administered to induce cycling of the specific T cell population of interest can be a peptide (e.g., the wild type peptide from which the variant peptide is produced), protein, polysaccharide, antibody, lectin, pharmacological mediator that can be administered to the subject, organic molecule or nucleic acid, as well as any other substance now known or identified in the future to function as an antigen or agonist for activating T cells.
  • activated T cells are T cells which manifest a genetic response to an encounter with an antigen which comprises induction of various genes, including those encoding T cell growth factors such as interleukin-2 (IL-2) or other T cell growth cytokines (e.g., interleukin-4, interleukin-7, interleukin-10, interleukin-12 5 and interleukin-15) and components of the high affinity receptors which bind the growth factors.
  • T cell growth factors such as interleukin-2 (IL-2) or other T cell growth cytokines (e.g., interleukin-4, interleukin-7, interleukin-10, interleukin-12 5 and interleukin-15) and components of the high affinity receptors which bind the growth factors.
  • cycling T cells are T cells that are actively progressing through the cell cycle as manifested by the synthesis of DNA, entry into and completion of mitosis and an increase in cell numbers due to cell division. Cycling occurs after and as a consequence of activation.
  • T cell growth factors When T cell growth factors are secreted, they interact with their newly expressed high affinity receptors and prompt the cell into cell cycle progression.
  • Cell cycle progression is associated with distinct molecular events including changes in cyclin and cyclin-dependent kinase molecules, DNA synthesis, mitosis and cell division.
  • the peptide ligand for the T cell receptor can be a variant peptide having one or more amino acid substitutions or modifications in the wild type peptide amino acid sequence.
  • the wild type peptide ligand can first be selected as described herein for its ability to bind the TCR of a specific T cell subpopulation in association with a suitable MHC molecule and to activate the T cells.
  • the amino acid sequence of the wild type peptide can be determined either by amino acid sequencing according to standard protocols, by synthesizing a peptide having a specific amino acid sequence or by isolating or synthesizing a nucleic acid encoding a specific amino acid sequence.
  • the wild type amino acid sequence can be substituted or modified as described herein to generate a variant peptide ligand that can be screened for the partial agonist profile described herein.
  • the variant peptide can be produced according to protocols standard in the art, such as peptide synthesis, cleavage of proteins into peptide sequences or expression of nucleic acid encoding the peptide sequence.
  • Amino acid substitutions and/or modifications in the wild type amino acid sequence can be produced by first determining the wild type amino acid sequence and introducing substitutions or modifications into the amino acid sequence of the peptide ligand at the level of synthesizing a peptide or at the level of synthesizing or isolating a nucleic acid encoding the amino acid sequence containing the substitution and/or modification.
  • An example of a substitution in a peptide is replacement of one or more amino acid of the wild type sequence with another amino acid.
  • An example of a modification in a peptide ligand is introduction of one or more amino acids not present 6 in the wild type sequence and/or deletion of one or more amino acids present in the wild type sequence.
  • variant peptide ligand of this invention can comprise non-natural amino acids.
  • substitutions and/or modifications to the wild type ligand amino acid sequence should not eliminate the binding of the peptide ligand to the MHC molecule or completely prevent binding to the TCR, as can be determined according to methods well known in the art.
  • the peptide ligands of the present invention can be administered to any subject having T cells which bind the peptide ligands.
  • the subject of this invention is an animal and most preferably a human.
  • non-human animals to which the peptide ligand of this invention can be administered include, but are not limited to, horses, sheep, rabbits and any other animal having T cells which bind peptide ligands and in which it is desirable to modulate a T cell response.
  • the therapeutic advantage of administering the peptide ligands of the present invention is that a specific T cell population or large numbers of T cells can be eliminated without concomitant production and release of T cell derived, non-death inducing cytokines, many of which have a role in the induction of an inflammatory response which can have an adverse effect on the subject.
  • the production and release, by specific T cells which bind the peptide ligand of this invention, of non- death inducing cytokines such as interleukin-2 (IL-2), interleukin-3 (IL-3) and/or interferon gamma (IFN- ⁇ ), as well as other non-death inducing cytokines identified as having a role in the induction of an inflammatory response, can be inhibited according to the methods described herein.
  • IL-2 interleukin-2
  • IL-3 interleukin-3
  • IFN- ⁇ interferon gamma
  • the inhibition of the production and release of T cell derived, non-death inducing cytokines by T cells by administering the ligand of this invention can be determined according to assays well known in the art, such as enzyme linked immunosorbent assays (ELISA) and ELISPOT (76) for detection and quantification of the production and/or release of specific cytokines, as described herein and in the literature.
  • assays well known in the art, such as enzyme linked immunosorbent assays (ELISA) and ELISPOT (76) for detection and quantification of the production and/or release of specific cytokines, as described herein and in the literature.
  • the methods of the present invention can be employed to treat any T cell associated disorder.
  • a T cell associated disorder is any disease or 7 syndrome which is caused by or exacerbated by a T cell response.
  • the present invention provides a method for modulating a T cell response in a subject.
  • the T cell disorder to be treated or prevented by the methods of this invention can be, but is not limited to, autoimmune disease, allergic disease, graft vs host disease, atopic disorder, transplantation rejection, viral infection, human immunodeficiency virus (HIV) associated disorder, T cell leukemia and T cell lymphoma.
  • HIV human immunodeficiency virus
  • an autoimmune disease generally describes a disease state or syndrome whereby a subject's body produces a dysfunctional immune response against the subject's own body components, with adverse effects. This may include production of T cells bearing receptors recognizing self components and producing cytokines that cause inflammation.
  • an allergic disease or disorder describes a disease state or syndrome whereby the body produces a dysfunctional immune response to environmental antigens comprising immunoglobulin E (IgE) antibodies which evoke allergic symptoms.
  • allergic diseases and disorders which can be treated or prevented by the methods of this invention include, but are not limited to, drug hypersensitivity, allergic rhinitis, bronchial asthma, ragweed pollen hayfever, anaphylactic syndrome, urticaria, angioedema, atopic dermatitis, erythema nodosum, erythema multiforme, Stevens-Johnson Syndrome, cutaneous necrotizing venulitis, bullous skin diseases, allergy to food substances and insect venom-induced allergic reactions (54,55,60), as well as any other allergic disease or disorder now known or identified in the future.
  • the methods of the present invention can additionally be employed to treat or prevent transplantation rejection in a transplant recipient, which is a disease state or syndrome whereby the transplant recipient's body produces an immune response against the engrafted tissue, resulting in rejection.
  • Transplantation rejection can occur, for example, with kidney, heart, lung or liver transplants as well as with any other transplanted tissue (58,62,63).
  • the present invention further provides a method for enhancing immune tolerance in a transplant recipient comprising administering to the recipient a peptide ligand for the T cell receptor which induces apoptosis of T cells without release of T cell-derived, non-death inducing cytokines by T cells which recognize the peptide ligand.
  • a peptide ligand for the T cell receptor which induces apoptosis of T cells without release of T cell-derived, non-death inducing cytokines by T cells which recognize the peptide ligand.
  • the variant peptide ligand may be employed in any suitable form.
  • the variant peptide ligand may be used alone or as a peptide/MHC complex and may be used in association with other agents.
  • the variant peptide ligand may be introduced by contacting with cells, expression in cells, presentation on the surface of antigen presenting cells, or introduction by any other appropriate means or combination of means.
  • GvH disease describes a disease state or syndrome whereby an immune response is initiated by engrafted cells and is directed against the recipient's body with adverse effects.
  • GvH disease include, but are not limited to, acute and chronic GvH disease following bone marrow and other organ transplants.
  • a method for inducing apoptosis of T cells without release of T cell-derived, non-death inducing cytokines comprising contacting the T cells with a peptide ligand for the T cell receptor which induces apoptosis of T cells without release of T cell-derived, non-death inducing cytokines.
  • the T cells which recognize the peptide ligand can be cycling T cells or the T cells can be resting T cells which have been induced to cycle by contact with an antigen.
  • the present invention also provides a method for improving transplantation of bone marrow cells, comprising inducing apoptosis of T cells without release of T cell- derived, non-death inducing cytokines, comprising contacting the T cells with a peptide ligand for the T cell receptor which induces apoptosis of T cells without release of T cell-derived, non-death inducing cytokines.
  • the T cells of this method can be a component of transplanted bone marrow cells in a transplantation recipient and/or can be the recipient's own T cells.
  • the T cells can be a component of bone marrow cells that are still in the bone marrow donor or that have been removed from the donor and are contacted with the peptide ligand in vitro or ex vivo prior to 10 transplantation into a recipient.
  • the transplantation of bone marrow cells is improved by eliminating T cells included within the population of transplanted bone marrow cells that are capable of inducing GvH disease and/or by eliminating T cells in the recipient that are capable of causing rejection of the transplanted bone marrow cells.
  • the source of the wild type peptide ligand for the T cell receptor of the present invention can be, for example, residues 84-102 and/or 143-168 of myelin basic protein (MBP) (for multiple sclerosis) (26,40-42), the human S antigen (for autoimmune uveitis) (29,43), type II collagen (for rheumatoid arthritis) (44), thyroglobulin (for thyroiditis) (45), Hepatitis B surface antigen (48-50), HIV gpl20 (51), residues 109- 145 of chorionic gonadotropin (52), malaria sporozoite antigen (53) and allergy antigens such as Amb aV, Amb tV, allergen M and antigen 5 (54,56,57,59), as well as any other source for a peptide ligand (64) which can be substituted or modified as described herein to have the effect of selectively inducing apoptosis of T cells without concom
  • the identification of a wild type peptide ligand for the T cell receptor which causes the T cell diseases and disorders described herein and which can be modified and/or substituted according to the methods of the present invention can be carried out as follows:
  • the T cells are taken from the peripheral blood of a subject diagnosed with a T cell associated disorder and maintained in culture and exposed to a variety of antigens (see, e.g., 23-43). After antigen exposure, T cells are assayed for activation or cycling according to assays standard in the art.
  • An antigen that is identified as having an increased activating or cycling-inducing effect on the subject's T cells, as compared to the activating or cycling-inducing effect of the antigen on the T cells of a subject which is not diagnosed with the T cell associated disorder of interest, can then be exposed to the cultured T cells in the form of a peptide to determine which peptide region of the antigen is the activating/cycling peptide ligand.
  • immunodominant pep tides of the antigen can be identified according to methods standard in the art and such immunodominant peptides can be used to partially screen 11 the antigen for identifying the peptide region containing the activating/cycling peptide ligand.
  • the amino acid sequence can be determined according to methods standard in the art. Once the amino acid sequence is known, substitutions and/or modifications in the wild type amino acid sequence can be made, resulting in variant peptide ligands which can be screened according to the methods provided herein for the ability to induce apoptosis without release of T cell-derived, non-death inducing cytokines.
  • the variant peptide ligands of the present invention can be screened for the ability to induce apoptosis without release of T cell-derived, non-death inducing cytokines by T-cells which recognize the variant peptide ligand as follows: Resting T cells can be isolated from a subject diagnosed with a particular T cell- associated disorder which is to be treated by the variant peptide ligand to be screened.
  • T cell clones can then be prepared by 1) activating the T cells by repetitively stimulating the resting T cells with the wild type agonist peptide ligand from which the variant peptide ligand was produced; 2) inducing cell cycling by growing the activated T cells in the presence of IL-2 or other T cell growth cytokines (e.g., interleukin-4, interleukin-7, interleukin-10, interleukin-12 and interleukin-15); and 3) carrying out limited dilution cloning according to standard methods.
  • IL-2 or other T cell growth cytokines e.g., interleukin-4, interleukin-7, interleukin-10, interleukin-12 and interleukin-15
  • the T cell clones can then be activated with wild type peptide ligand for two days, stimulated with IL-2 for 3-10 days to become cycling T cells and used to test the variant peptide ligands of this invention for the ability to induce death of the cycling, cloned T cells without stimulating the production of non-death inducing cytokines.
  • the present invention provides a method for screening a variant peptide ligand for the ability to induce apoptosis of T cells without release of T cell-derived, non-death inducing cytokines by T-cells which recognize the peptide ligand, comprising: a) contacting cycling T cells which recognize the variant peptide ligand with the variant peptide ligand; b) detecting the presence or absence of apoptosis of the T cells; and c) detecting the presence or absence of release of non-death inducing 12 cytokines by the T cells, whereby the presence of apoptosis of the T cells and the absence of release of non-death inducing cytokines by the T cells indicates a variant peptide ligand having the ability to induce apoptosis of T cells without release of non- death inducing cytokines by T cells which recognize the variant peptide ligand.
  • the presence or absence of apoptosis of the T cells can be determined by a variety of methods, such as flow cytometry, exposure of phosphatidylserine, incorporation of propidium iodide and/or labeling of fragmented DNA according to protocols described herein and as are well known in the art for detecting molecular or cellular changes associated with apoptosis (8, 70-74).
  • the presence or absence of apoptosis of T cells as a result of binding a variant peptide ligand of this invention is determined by comparison with the amount of apoptosis in a control population of T cells which have had no exposure to the variant peptide ligand.
  • an increase in the amount of apoptosis in a T cell population exposed to a variant peptide ligand relative to the amount of apoptosis in a T cell population having no exposure to the variant peptide ligand indicates the presence of apoptosis as a result of binding a variant peptide ligand.
  • the presence or absence of the production and release of non-death inducing cytokines by the T cells can be determined by methods standard in the art for measuring cytokines (e.g., ELISA and ELISPOT).
  • the presence or absence of the production and release of non-death inducing cytokines by the T cells as a result of binding a variant peptide ligand of this invention is determined by comparison with the amount of production and release of non-death inducing cytokines by a control population of T cells which have had no exposure to the variant peptide ligand.
  • a decrease in the amount of production and release of non-death inducing cytokines in a T cell population exposed to a variant peptide ligand relative to the amount of production and release of non-death inducing cytokines in a T cell population having no exposure to the variant peptide ligand indicates the absence of production and release of non-death inducing cytokines by the T cells as a result of binding a variant peptide ligand.
  • the variant peptide ligands identified according to the screening methods described herein to have the partial agonistic effect of this invention can be administered to a subject in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject along with the selected peptide ligand without causing any substantial undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.
  • the peptide ligand of this invention can be administered orally (46) or parenterally (48-50,52,53) to the subject (47).
  • Suitable carriers for oral administration of the peptide ligand include one or more substances which may also act as flavoring agents, lubricants, suspending agents, or as protectants.
  • Suitable solid carriers include calcium phosphate, calcium carbonate, magnesium stearate, sugars, starch, gelatin, cellulose, carboxypolymethylene, or cyclodextrans.
  • Suitable liquid carriers may be water, pyrogen free saline, pharmaceutically accepted oils, or a mixture of any of these.
  • the liquid can also contain other suitable pharmaceutical additions such as buffers, preservatives, flavoring agents, viscosity or osmo-regulators, stabilizers or suspending agents.
  • suitable liquid carriers include water with or without various additives, including carboxypolymethylene as a pH-regulated gel.
  • the peptide ligand may be contained in enteric coated capsules that release the peptide ligand into the intestine to avoid gastric breakdown.
  • a sterile solution or suspension is prepared in saline that may contain additives, such as ethyl 14 oleate or isopropyl myristate, and can be injected, for example, into subcutaneous or intramuscular tissues, as well as intravenously.
  • additives such as ethyl 14 oleate or isopropyl myristate
  • the peptide ligand may be microencapsulated with either a natural or a synthetic polymer into microparticles 4-8 ⁇ m in diameter, which target intestinal lymphoid tissues and produce a sustained release of ligand for up to four weeks (68,69).
  • the peptide ligand of this invention can be administered to the subject in amounts sufficient to selectively induce apoptosis of targeted T cell populations without concomitant production and release of non-death inducing cytokines by the targeted T cells.
  • Optimal dosages used will vary according to the individual being treated and ligand being used.
  • the amount of ligand will also vary among individuals on the basis of age, size, weight, condition, etc.
  • dosages are best optimized by the practicing physician and methods for determining dose amounts and regimens and preparing dosage forms are described, for example, in Remington's Pharmaceutical 'Sciences '(21).
  • suitable doses and dosage regimens can be determined by comparison to agents presently used in the treatment or prevention of autoimmune disorders, allergic disorders, graft rejection and other T cell related disorders.
  • the preferred or optimal dosage is the amount of peptide ligand which results in apoptosis of specific T cells without concomitant production of non-death inducing cytokines, in the absence of significant side effects.
  • the peptide ligand of this invention can be administered orally or parenterally in a dosage range of 0J to 100 mg/kg of body weight at about 3-10 day intervals, or more preferably, 3-5 day intervals, over a period of days, weeks or months, depending on the clinical response that is to be obtained.
  • Administration of the peptide ligand can be stopped completely following a prolonged remission or 15 stabilization of disease signs and symptoms and readministered following a worsening of either the signs or symptoms of the disease, or following a significant change in immune status, as determined by routine follow-up immunological studies well known to a clinician in this field (e.g., a return to significant reactivity of T cells upon exposure to a particular antigen).
  • the efficacy of administration of a particular dose of peptide ligand in treating a T cell associated disorder as described herein can be determined by evaluating the particular aspects of the medical history, the signs, symptoms and objective laboratory tests that have a documented utility in evaluating pathophysiological activity of the particular T cell associated disorder being treated. These signs, symptoms and objective laboratory tests will vary depending on the particular disorder being treated, as will be well known to any clinician in this field.
  • a subject's frequency or severity of recurrences is shown to be improved; 2) the progression of the disease or disorder is shown to be stabilized; or 3) the need for use of other immunosuppressive medications is lessened, then a particular treatment can be considered efficacious.
  • clinical parameters and symptoms which can be monitored for efficacy can include the severity and number of attacks; or for continuously progressive disease, the worsening of symptoms and signs; the cumulative development of disability; the number or extent of brain lesions as determined by magnetic resonance imaging; and the use of immunosuppressive medications (65-67).
  • the efficacy of administration of a particular dose of a peptide ligand in preventing a T cell associated disorder in a subject not known to have a T cell associated disorder, but known to be at risk of developing a T cell associated disorder can be determined by evaluating standard signs, symptoms and objective laboratory tests, known to one of skill in the art, over time. This time interval may be long (i.e., years/decades). The determination of who would be at risk for the development of a T cell associated disorder would be made based on current knowledge of the known risk factors for a particular disorder familiar to clinicians and researchers in this field, such as a particularly strong family history of a disorder or exposure to or acquisition of factors or conditions which are likely to lead to development of a T cell associated disorder.
  • T cell clones and antigen presenting cells CD4 + A.E7 Thl cells (9J2) were maintained by bi-weekly stimulation with pigeon cytochrome c protein (5 ⁇ M) and irradiated splenocytes from BIO. A (H-2 a ) mice.
  • A.E7 cells stimulated in this manner two days previously were ficolled and transferred to fresh medium containing IL-2 (50-100 IU/ml recombinant human IL-2; Chiron, Emeryville, CA) or 10-15% T-StimTM (Collaborative Biomedical Products, Bedford, MA) and incubated for an additional 2 to 5 days to generate cycling A.E7 cells that are predisposed to apoptosis following TCR engagement (8).
  • P13.9 cells which are L cell transfectants expressing I-E k , ICAM-1, and B7J (CD80) molecules, were used as antigen-presenting cells (APC) (3).
  • Wild type (WT) peptide PCC (88-104) has the amino acid sequence: KAERADLIAYLKQATAK (SEQ ID NO:l).
  • KAERADLIAYLYQATAK SEQ ID NO:2
  • C99 KERADLIAYLCQATAK; SEQ ID NO:3
  • R99 KERADLIAYLRQATAK; SEQ ID NO:4
  • A99 KERADLIAYLAQATAK; SEQ ID NO:5
  • All peptides were synthesized by the Peptide Synthesis Facility, NIAID, NIH, Bethesda, MD.
  • Ungated cells were acquired for 30 seconds at a constant flow rate using a FACScan equipped with CellQuest software (Becton-Dickinson, Mountain View, CA) and the number of viable A.E7 cells in each sample was calculated as previously described (10, 11).
  • P13.9 cells were pre-loaded with 0J ⁇ M carboxyfluorescein diacetate-acetylester (CMFDA; Molecular Probes, Eugene, OR) to allow APC to be excluded from the analysis by gating on fluorescein-negative cells.
  • CMFDA carboxyfluorescein diacetate-acetylester
  • APC were stained with fluoresceinated-anti-CD4 before flow cytometry to differentiate them from APC.
  • Cytokine measurement Supernatants from the apoptosis assays were harvested at 24 hours and assayed for cytokine production by ELISA. IL-2 and IL-3 were detected using antibodies purchased from Pharmingen, San Diego, CA, according to the manufacturer's instructions. IFN- ⁇ was measured by ELISA as previously described (6). Data are expressed as the mean of quadruplicate wells calculated as a percentage of the response to 100 ⁇ M WT peptide.
  • RNA analysis by semi-quantitative RT-PCR A.E7 cells were incubated with P13.9 cells pre-pulsed for 2 h with 100 ⁇ M of the indicated peptides and total RNA was isolated using RNAzol according to the manufacturer's instructions (Tel-test, Inc, Friendswood, TX).
  • the reverse transcriptase (RT) reaction was performed using 18 random hexamer primers and part of each sample was subjected to polymerase chain reaction (PCR) amplification using ⁇ -actin primers and 32 P-labeled nucleotides (12). Serial dilutions of RT products were used to ensure comparisons were in the linear range.
  • ⁇ -actin After normalizing the input cDNA to the ⁇ -actin signal, ⁇ -actin, Fas-L, TNF ⁇ , IL-2, IFN- ⁇ and Bcl-X primers were then used to amplify their respective cDNA under the same conditions.
  • the PCR protocol was 95°C for 30 seconds, 55°C for 30 seconds and 72°C for 60 seconds, for 30 cycles.
  • Sequences of the primers are as follows: ⁇ -Actin, 5' primer: 5 'GAT GAC GAT ATC GCT GCG CTG3' (SEQ ID NO:6); ⁇ - Actin, 3' primer: 5'GTA CGA CCA GAG GCA TAC AGG3' (SEQ ID NO:7); mIL-2, 5' primer: 5'ATG TAC AGC ATG CAG CTC GCA TC3' (SEQ ID NO:8); mIL-2, 3' primer: 5'GGC TTG TTG AGA TGA TGC TTT GAC A3' (SEQ ID NO:9); mFas-L, 5' primer: 5'CTG GTG GCT CTG GTT GGA AT3' (SEQ ID NO: 10); mFas-L, 3' primer: 5'GTT TAG GGG CTG GTT GTT GC3' (SEQ ID NO:l 1); mTNF ⁇ , 5' primer: 5'ATG AGC ACA G
  • Protein biochemistry 2-5 x 10 6 P13.9 APC were incubated for 2-4 hours in 200 ⁇ l complete medium alone (no peptide) or with the variant peptides added at a concentration of 100 ⁇ M. Pulsed APC were then centrifuged together with 1-1.25 x 10 7 cycling A.E7 cells in Eppendorf tubes and warmed to 37°C, as described (6).
  • T cells + APC were lysed (6) and lysates were immunoprecipitated with anti-CD3e (500A2 mAb; Pharmingen), anti-ZAP-70 (rabbit antiserum; NICHD, NTH, Bethesda, MD), or anti-TCR- ⁇ (rabbit antiserum; DNAX, Palo Alto, CA,) (13).
  • Immunoprecipitates were resolved by SDS-PAGE (12% under reducing conditions), transferred to nitrocellulose and blotted with anti-phosphotyrosine antibody (6). Blots were developed using SuperSignalTM chemiluminescent substrate (Pierce, Rockford, IL).
  • Variant ligands induce apoptosis without the production of activation cytokines such as IL-2, IL-3, or IFN- ⁇ .
  • cytokines such as IL-2, IL-3, or IFN- ⁇ .
  • many peptides with single amino acid substitutions at the major epitopic residue (lysine 99) of the agonist 88-104 peptide of pigeon cytochrome c (PCC 88-104) have been previously screened for their effect on A.E7 cells that were actively cycling under the influence of IL-2.
  • Certain variants of PCC 88-104 with substitutions at position 99 have been previously found to induce variant signaling in unactivated, resting A.E7 cells (3). These substitutions do not affect binding to the I-E k molecule and therefore are assumed only to alter TCR recognition of peptide-MHC ligand (3, 14).
  • T cell apoptosis as well as IL-2, IL-3 and IFN ⁇ production in cycling A.E7 cells, was measured after stimulation for 24 hours with PI 3.9 APC in the presence of 100 ⁇ M WT peptide or variant peptides in concentrations ranging from 0.0001 to 100 ⁇ M ( Figure 1 A). All values were expressed as a percentage of the response obtained with 100 ⁇ M WT peptide. The actual 100% maximal values were as follows: apoptosis: 55% dead cells at 24 hours; IL-2: 1.4 ng/ml; IL-3: >250 ng/ml; and IFN- ⁇ : 120 ng/ml.
  • the wild-type (WT) PCC 88- 104 peptide induced both cell death and the secretion of IL-2, IL-3 and IFN- ⁇ .
  • WT wild-type
  • two of the variant peptides tested Y99 and C99, induced as much cell death as the WT peptide, 20 but failed to induce IL-2, IL-3, or IFN- ⁇ production.
  • T cell death under these circumstances has the morphological and nuclear fragmentation characteristics of apoptosis (8).
  • Other variant peptides, such as A99 and R99 did not stimulate either apoptosis or cytokine secretion.
  • Y99 and C99 are true partial agonists for A.E7 cells, because they induce apoptosis but not other typical effector functions.
  • R99 and A99 failed to induce these responses at any dose and therefore are non-agonists with respect to the responses examined.
  • T cell death induced by partial agonists employs the Fas and TNF pathways. Because T cell apoptosis can result from the action of Fas or tumor necrosis factor- alpha (TNF- ⁇ ) (15-18), a role for these molecules in death induced by the Y99 and C99 partial agonists was assessed. Stimulation of cycling A.E7 cells for two hours with the apoptogenic WT, Y99, or C99 peptides (100 ⁇ l) induced the mRNAs for TNF- ⁇ and Fas-L but not for Bcl-X, a protein that prevents apoptosis (19).
  • Fas-L and TNF blocking experiments cycling A.E7 cells were stimulated with APC pre-pulsed with 2.5 nM WT peptide, 100 ⁇ M C99 peptide or 100 ⁇ M Y99 21 peptide, in medium alone or in the presence of either Fas-Fc (10 ⁇ g/ml), TNFR-Fc (10 ⁇ g/ml) or both Fas-Fc and TNFR-Fc.
  • Fas-Fc 10 ⁇ g/ml
  • TNFR-Fc 10 ⁇ g/ml
  • Fas-Fc and TNFR-Fc both Fas-Fc and TNFR-Fc
  • Tyrosine phosphorylation patterns induced by variant ligands In resting T cells, partial agonist or antagonist ligands generate unique patterns of protein tyrosine phosphorylation (2, 3, 6). Hence, whether apoptosis-inducing variant peptides produce characteristic TCR-associated phosphorylation patterns in cycling T cells was determined.
  • CD3e, ZAP-70, or TCR- ⁇ and their associated proteins were immunoprecipitated from lysates prepared from cycling A.E7 cells that had been stimulated for 10 minutes with PI 3.9 APC pre-pulsed with WT or the variant peptide ligands.
  • the WT peptide In cycling A.E7 cells, the WT peptide induced prominent tyrosine-phosphorylated species, including the three isoforms of phospho- ⁇ (pi 8, p21 and p23), phosphorylated CD3e and phosphorylated ZAP-70. This is the same pattern as that obtained when resting A.E7 cells are stimulated with WT peptide (3). However, no correlation was found between the pattern of TCR- ⁇ tyrosine phosphorylation and programmed cell death. The apoptosis-inducing partial agonist, C99 and the non-agonist ligands, A99 and R99, all produced a modest increase in the p21 form of phospho- ⁇ .
  • the partial agonist Y99 surprisingly failed to induce a detectable increase in p21 phospho- ⁇ , even upon examination by direct anti- ⁇ immunoprecipitation. None of the variant peptide ligands induced detectable levels of the other species of phospho- ⁇ (pi 8 and p23), phosphorylated CD3e, or phosphorylated ZAP-70.
  • Ligands that deliver death-inducing signals induce TCR capping.
  • Successful activation of resting T cells with an agonist peptide causes the redistribution of the TCR into polarized focal aggregates on the membrane, i.e. "capping" (20).
  • TCR polarization was observed at points of contact with the APC after stimulation with the WT peptide and the apoptosis-inducing Y99 and C99 peptides but not by A99 peptide which, by itself, does not induce cell death.
  • the aggregation of the TCR correlated closely with actin polymerization detected after intracellular staining of A.E7+APC cells with Texas Red-conjugated X phalloidin.
  • no distinct capping or TCR polarization was seen after stimulation with the variants R99 and A99 that fail to cause apoptosis, even though they induced readily detectable ⁇ -chain phosphorylation.
  • Treatment of multiple sclerosis with a variant peptide ligand of the present invention as an example of treatment of a T cell associated disorder with a variant peptide ligand of the present invention.
  • a subject diagnosed with active relapsing/remitting or chronic progressive multiple sclerosis (MS), using established MS diagnostic criteria, can be treated with a variant peptide ligand of this invention as follows: T cells from the subject can be demonstrated to be reactive in increased numbers with myelin basic protein or other myelin antigens, preferably immunodominant peptides 84-102 or 143-168 according to standard in vitro assays, as described herein.
  • the subject's T cells can then be cloned as described herein and the same wild type peptides can then be used to identify T cell clones which are reactive against these peptides by exposing the cloned T cells to these wild type peptides and assaying the cloned T cells for reactivity to the peptide as described herein.
  • these clones can be used in screening assays as described herein to identify variant ligand peptides of this invention which have the ability to induce apoptosis in the cloned T cell populations without concomitant release of non-death inducing cytokines.
  • those variant peptides which induce apoptosis without concomitant release of non-death inducing cytokines in a majority of the T cell clones are selected for upscaled production for clinical use.
  • the variant peptides identified to function as described 23 above can be produced in large quantities by standard chemical protocols and sterilized for administration to the subject.
  • the variant peptide ligands can be administered to the subject by oral or parenteral route in appropriate pharmaceutically acceptable delivery vehicles in a dosage range of OJ to 100 mg/kg body weight.
  • the optimal dosage can be determined according to the protocols described herein and can depend on the particular pharmacological formula as well as the active concentration of the variant peptide ligand based on the average value that was needed to induce apoptosis in the majority of the T cell clones described above.
  • the variant peptide ligand can be administered every 3-5 days, depending on the clinical setting and the subject's response. Administration of the ligand can be continued for days, weeks or months, until a favorable clinical response is observed unless complications develop or an exacerbation of the disease occurs.
  • Clinical improvement can be determined according to standards recommended by the International Workshop on Outcomes Assessment sponsored by the National Multiple Sclerosis Society (65). Such standards can include an improved score on the Kurtzke expanded disability status scale (EDSS) and decreased numbers of lesions as detected by cranial magnetic resonance imaging (especially using quantitative imaging with gadolinium enhancement and T2 weighted images).
  • EDSS Kurtzke expanded disability status scale
  • Assessment of immunological responses in the subject's T cells can also be carried out, using the subject's post-treatment T cells and the original wild-type agonist peptides in ELISPOT assays. A decrease in the frequency of responding T cells in the ELISPOT assay would indicate the establishment in the subject of a type of immunological tolerance induced by the peptide ligand treatment.
  • HLA-DRJa is the dominant restriction molecule for the cytotoxic T cell response to myelin basic protein in DRJDw2 individuals. J. Immunol. 145:2880-2885.

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Abstract

La présente invention concerne une méthode de traitement ou de prévention des troubles associés aux lymphocytes T chez un sujet. La méthode consiste à administrer audit sujet un ligand peptidique du récepteur des lymphocytes T qui induit l'apoptose de ces derniers sans provoquer la libération, par les lymphocytes T qui reconnaissent ledit ligand, de cytokines n'entraînant pas la mort cellulaire. L'invention concerne également une méthode d'amélioration de la tolérance immunitaire d'un receveur de greffe, qui consiste à administrer audit receveur un ligand peptidique du récepteur des lymphocytes T qui induit l'apoptose de ces derniers sans provoquer la libération, par les lymphocytes T qui reconnaissent ledit ligand, de cytokines n'entraînant pas la mort cellulaire. L'invention concerne, en outre, une méthode qui induit l'apoptose des lymphocytes T sans provoquer la libération par ces derniers, de cytokines n'entraînant pas la mort cellulaire et qui consiste à mettre en contact les lymphocytes T avec un ligand peptidique du récepteur des lymphocytes T qui induit l'apoptose de ces derniers sans provoquer la libération par lesdits lymphocytes, de cytokines n'entraînant pas la mort cellulaire. L'invention concerne également une méthode améliorant la greffe de cellules de moelle osseuse, qui consiste à induire l'apoptose des lymphocytes T sans provoquer la libération par ces derniers, de cytokines n'entraînant pas la mort cellulaire et à mettre en contact des lymphocytes T avec un ligand peptidique du récepteur des lymphocytes T qui induit l'apoptose de ces derniers sans provoquer la libération par lesdits lymphocytes de cytokines n'entraînant pas la mort cellulaire.
PCT/US1999/001912 1998-01-29 1999-01-29 Variants de ligands peptidiques induisant selectivement l'apoptose WO1999038526A1 (fr)

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Cited By (6)

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US7442778B2 (en) 2004-09-24 2008-10-28 Amgen Inc. Modified Fc molecules
US8143380B2 (en) 2004-07-08 2012-03-27 Amgen Inc. Therapeutic peptides
EP2594288A1 (fr) 2006-04-21 2013-05-22 Amgen Inc. Formulations peptide-anticorps thérapeutiques lyophilisées
US9114175B2 (en) 2005-08-12 2015-08-25 Amgen Inc. Modified Fc molecules
US9145450B2 (en) 1998-10-23 2015-09-29 Amgen Inc. Thrombopoietic compounds
CN113056555A (zh) * 2018-08-22 2021-06-29 塞莱克特生物疗法有限公司 凋亡易感细胞的调节

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WO1996036881A2 (fr) * 1995-05-16 1996-11-21 Cancer Research Campaign Technology Limited Evaluation preliminaire d'inhibiteurs potentiels des interactions entre les recepteurs des lymphocytes t (rlt) et le complexe majeur d'histocompatibilite (cmh)

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9534032B2 (en) 1998-10-23 2017-01-03 Amgen Inc. Thrombopoietic compounds
US9145450B2 (en) 1998-10-23 2015-09-29 Amgen Inc. Thrombopoietic compounds
US8143380B2 (en) 2004-07-08 2012-03-27 Amgen Inc. Therapeutic peptides
US7442778B2 (en) 2004-09-24 2008-10-28 Amgen Inc. Modified Fc molecules
US9114175B2 (en) 2005-08-12 2015-08-25 Amgen Inc. Modified Fc molecules
US11266744B2 (en) 2005-08-12 2022-03-08 Amgen Inc. Modified Fc molecules
US10188740B2 (en) 2005-08-12 2019-01-29 Amgen Inc. Modified Fc molecules
EP2594287A1 (fr) 2006-04-21 2013-05-22 Amgen Inc. Formulations peptide-anticorps thérapeutiques lyophilisées
EP2594285A1 (fr) 2006-04-21 2013-05-22 Amgen Inc. Formulations peptide-anticorps thérapeutiques lyophilisées
EP2594284A1 (fr) 2006-04-21 2013-05-22 Amgen Inc. Formulations peptide-anticorps thérapeutiques lyophilisées
EP2594286A1 (fr) 2006-04-21 2013-05-22 Amgen Inc. Formulations peptide-anticorps thérapeutiques lyophilisées
EP2594288A1 (fr) 2006-04-21 2013-05-22 Amgen Inc. Formulations peptide-anticorps thérapeutiques lyophilisées
CN113056555A (zh) * 2018-08-22 2021-06-29 塞莱克特生物疗法有限公司 凋亡易感细胞的调节

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