WO1996040235A1 - Procede d'utilisation de superantigenes pour cibler des sous-populations de lymphocytes t - Google Patents

Procede d'utilisation de superantigenes pour cibler des sous-populations de lymphocytes t Download PDF

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WO1996040235A1
WO1996040235A1 PCT/US1996/008193 US9608193W WO9640235A1 WO 1996040235 A1 WO1996040235 A1 WO 1996040235A1 US 9608193 W US9608193 W US 9608193W WO 9640235 A1 WO9640235 A1 WO 9640235A1
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superantigen
cells
derivative
cell
bacterial
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PCT/US1996/008193
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Jerome R. Bill
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Nexstar Pharmaceuticals, Inc.
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Priority to AU59600/96A priority Critical patent/AU5960096A/en
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    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus

Definitions

  • This invention relates to the field of immunology. Specifically, this invention relates to the use of superantigens to specifically target pathogenic subsets of T cells for subsequent inactivation or deletion by an additional agent or agents.
  • This application thus, describes a method for treating diseases mediated by T cells having a limited V ⁇ profile by inactivating or deleting certain V ⁇ defined subpopulations of T cells which comprise the pathogenic T cells.
  • the vertebrate immune system evolved to provide specific protection from infection by foreign micro- and macroorganisms.
  • the immune system responds to antigens in one of two ways: (1) humoral antibody responses, mediated through B lymphocytes, or B cells, and (2) cell-mediated immune responses, mediated through T lymphocytes, or T cells.
  • the B cell response results in the production of antibodies that circulate in the bloodstream and bind specifically to the foreign antigen that induced them.
  • the binding of the antibody to the antigen either directly inactivates the antigen, marks it for ingestion by phagocytic cells or marks it for destruction by cytotoxic cells. It is now known that for most antigens the production of antibodies by B cells requires T cell help.
  • the T cell response results in the activation and proliferation of antigen-specific T cells that, in addition to helping B cells, react directly with foreign antigens on the surface of host cells, either killing the host cell if the antigen is an infecting virus or inducing other host cells, such as macrophages, to destroy the antigen (Nossal (1993) Sci. Amer. 269:52; Janeway (1993) Sci. Amer. 269:72; Marrack and Kappler (1993) Sci. Amer. 269:80).
  • This invention is concerned primarily with T cell immunity.
  • this application describes a method for dealing with T cell responses which have gone awry resulting in, for example, autoimmune diseases.
  • the attack by the immune system on host cells can result in a large number of disorders, including neural diseases such as multiple sclerosis and myasthenia gravis, diseases of the joints such as rheumatoid arthritis, attacks on nucleic acids as observed with systemic lupus erythematosus, and diseases associated with various organs such as psoriasis, juvenile onset diabetes, Sjogren's disease and thyroid disease.
  • T cells have antigen-specific receptors on their surfaces, termed T cell antigen receptors (TCR).
  • TCR T cell antigen receptors
  • MHC major histocompatibility complex
  • MHC antigens are expressed on the cells of all higher vertebrates. In man they are called HLA antigens (human-leukocyte-associated antigens) because they were first demonstrated on leukocytes.
  • HLA antigens human-leukocyte-associated antigens
  • MHC antigens stimulate different subpopulations of T cells. MHC class II molecules are involved in most responses to extracellular pathogens, while MHC class I molecules are involved when the pathogen is cell-associated, i.e., when a virus or a malignant cell is involved.
  • MHC class I When MHC class I is involved, antibody stimulation does not result; rather, the interaction of MHC class I processed antigen and T-cell leads to lysis of cells infected with the pathogen. Processed antigen peptide fits in a cleft on an MHC molecule
  • T cells recognize antigens on the surface of cells only in association with self- MHC glycoproteins expressed on the cell surface.
  • the ability of the T cell to bind to the processed antigen and MHC complex is dependent on the T cell receptor (TCR).
  • TCR consists of two protein chains, usually cc and ⁇ chains. Each chain is composed of a constant and a variable domain. The variable domains are encoded in two ( ⁇ ) or three ( ⁇ ) different gene segments (variable (V), diversity (D), joining (J)) (Siu et al. (1984) Cell 37:393; Yanagi et al (1985) Proc. Natl. Acad. Sci. USA 82:3430).
  • T cells When T cells are stimulated by an antigen, they divide and differentiate into activated effector cells that are responsible for various cell- mediated immune reactions. At least three different reactions are carried out by T cells: (1) cytolytic T cells specifically kill foreign or virus-infected vertebrate cells; (2) helper T cells help B lymphocytes; and (3) suppressor T cells suppress the responses of specific cells. It has been shown that a novel class of antigens, termed
  • “superantigens” are able to directly stimulate T cells by binding to a particular subset of V ⁇ elements. That is, the variable domain of the ⁇ chain of the TCR alone determines whether or not a particular superantigen will stimulate a particular T cell (Kappler et al. (1987) Cell 49:263; Kappler et al. (1987) Cell 49:273; MacDonald et al. (1988) Nature 332:40; Pullen et al. (1988) Nature 335:796; Kappler et al. (1988) Nature 332:35; Abe et al. (1988) J. Immunol. 140:4132; White et al.
  • Staphylococcus aureus (S. aureus), a common human pathogen, produces several enterotoxins, designated as SEA (Staphylococcal enterotoxin A) through SEE (Staphylococcal enterotoxin E), which can be responsible for food poisoning and occasionally shock in humans (Marrack & Kappler (1990) supra; Bohach et al. (1990) Crit. Rev. Microbio. 117:251).
  • Some S. aureus isolates also produce toxic shock syndrome toxin- 1 (TSST-1), which has been implicated in the majority of cases of human toxic shock syndrome, as well as the related exfoliative toxin (ExT), which is associated with the scalded skin syndrome.
  • TSST-1 toxic shock syndrome toxin- 1
  • Streptococcus pyrogenes or group A streptococcus, another common human pathogen of the skin and pharynx, also produces toxins with superantigenic properties (Abe etal. (1991) J. Immun. 146:3747). These toxins have been designated Streptococcal erythrogenic toxins A-C (SPEA-C).
  • amino acid sequences of the S. aureus toxins exhibit some homology, but also exhibit marked differences (See, Betley et al. (1988) J.
  • S. aureus toxins have the ability to stimulate powerful T cell proliferation responses in the presence of mouse cells bearing MHC class II type molecules (White et al. (1989) supra).
  • the S. aureus proteins selectively stimulate murine and human T cells bearing particular V ⁇ elements.
  • T cells rarely recognize peptide antigens bound to self-MHC molecules
  • individual T cell clones can respond to toxins bound not only to various allelic forms of MHC but also to different class II isotypes and even xenogeneic class II molecules. Observations such as these reinforce the concept that superantigens bind at a relatively conserved site outside the allelically variable groove where conventional peptide antigens bind.
  • Superantigens may contribute to autoimmune diseases in which components of the immune system attack normal tissue.
  • the process of deletion of T cells responsive to self, potentially harmful self-reactive T cells, is called tolerance or negative selection (Kappler et al. (1987) supra; Kappler et al. (1988) supra; Von Boehmer et al. (1988) Immunol. Rev. 101:21).
  • the immune system usually deletes self-reactive T cells, with good, but not complete efficiency, thus, some self-reactive cells appear to escape the surveillance mechanism. It is possible that the ability of superantigens to arouse 20 percent of a person's T cell repertoire leads to undesirable replication of the few circulating T cells that are capable of recognizing self (Kotzin et al.
  • T cells bearing certain V ⁇ types have been implicated in various autoimmune conditions, such as arthritis and multiple sclerosis. These destructive cells might have been initially activated by a superantigen that binds to the identified V ⁇ types (Kotzin et al. (1993) supra), leading to disease.
  • T cells specific for self- antigens play a critical role in the initiation of autoimmune diseases.
  • rheumatoid arthritis the linkage of the disease to the DR4 and DR1 alleles of the class II genes of MHC, and the finding of oligoclonal activated CD4 + T cells in synovial fluid and tissue of affected joints (Stastny et al. (1976) Engl. J. Med. 298:869; Gibofsky et al. (1978) J. Exp. Med. 148:1728; McMichael et al. (1977) Arth. Rheum. 20:1037; Schiff et al. (1982) Ann. Rheum.
  • CD4 + T cells recognize a complex consisting of an MHC class II molecule and a bound protein fragment (peptide) of about 15 amino acids. It is commonly accepted that the ability of a particular MHC molecule to bind particular peptides underlies its association with an increased or decreased propensity for disease.
  • Current therapy for most autoimmune diseases consists of non-specific immune suppression (by the administration of, e.g., cyclosporin, steroids) or of non-specific elimination of rapidly dividing cells (by the administration of, e.g., cytoxan, methotrexate).
  • T cells responsible for disease differ from beneficial T cells by their TCR.
  • the initial problem is to identify those T cells bearing aberrant or pathogenic T cell receptors. Assuming all receptors have an equal likelihood of causing disease, identifying those responsible is a daunting task.
  • MHC associations with particular diseases suggest that the actual number of pathogenic TCRs may be small.
  • mice where this phenomenon has been more extensively studied (e.g., EAE, collagen arthritic), the responsible T cells express a very limited T cell receptor repertoire.
  • EAE collagen arthritic
  • the limited repertoire results from T cell recognition of only one or a few peptides derived from the incitatory antigen (e.g., MBP or collagen).
  • a stimulatory phase precedes the deletion/non- responsiveness induced by superantigens. During the stimulatory phase, in addition to T cell proliferation, a large quantity of cytokines is released.
  • European Patent Publication 340,109 entitled Anti-T-cell receptor determinants as autoimmune disease treatment
  • U.S. Patent No. 4,550,086, issued October 29, 1985 to Reinherz et al. entitled Monoclonal antibodies that recognize human T cells describe a method of detecting a particular sequence of the variable region gene of T cell receptors associated with a particular disease and treating the disease with antibodies to that sequence.
  • a method of diagnosing diseases based on the presence of T cells with a unique sequence in the V ⁇ region associated with a specific disease a method for detecting specific V ⁇ regions associated with rheumatoid arthritis (RA), specifically, V ⁇ 3, V ⁇ 9 and V ⁇ 10 and a method for the treatment of RA with monoclonal antibodies which recognize V ⁇ 3, V ⁇ 9 and V ⁇ 10 have been described as well (U.S. Patent 4,886,743; PCT Patent application publication WO 90/06758).
  • One application for the present invention involves a treatment for rheumatoid arthritis.
  • T cells expressing TCRs comprised of one or more of the V ⁇ s targeted by the superantigen SEB-namely V ⁇ s 3, 12. 14, 15 and 17
  • V ⁇ s 3 the V ⁇ s targeted by the superantigen SEB-namely V ⁇ s 3, 12. 14, 15 and 17
  • This invention is based on a novel concept for the treatment of subject animals having certain diseases.
  • the present invention is defined by the administration of a superantigen or superantigen derivative to mark certain T cell subpopulations based on their TCR V ⁇ expression for subsequent inactivation or deletion by an additional agent or agents.
  • the present invention is applicable to any disease caused by subpopulations of T cells, said subpopulations defined by the V ⁇ element which comprises their antigen receptor (TCR).
  • TCR antigen receptor
  • the present invention is useful to treat certain autoimmune diseases, but is not intended to be limited to these diseases.
  • a superantigen or superantigen derivative is administered in order to mark a V ⁇ defined subpopulation of T cells that includes pathogenic T cells.
  • the target subpopulation of T cells becomes phenotypically different from other T cells.
  • the targeted T cells proliferate, produce cytokines, and express or up-regulate certain surface proteins. It is possible, then, to exploit these phenotypic changes in order to inactivate or delete the marked T cell subpopulation.
  • cytotoxic agents such as methotrexate. For example, T cells which express Fas become susceptible to killing by Fas ligand.
  • V ⁇ subpopulations of T cells may result in release of toxic amount of cytokines.
  • administration of an additional agent or agents is contemplated in order to prevent or ameliorate cytokine release.
  • FIGURES Figure 1 shows tritiated thymidine incorporation by human peripheral blood lymphocytes (hPBLs) stimulated with toxins.
  • Figure 2 shows light scatter patterns of hPBLs with and without SEB stimulation.
  • Figure 3 shows an analysis of TCR expression in resting T cells versus proliferating T cells.
  • Figure 4 shows the cell viability and V ⁇ 3 TCR expression over time, in culture.
  • Figure 5 shows the effect of methotrexate on SEB induced T cell blasts.
  • Figure 6 shows the deletion of V ⁇ 3, 12 and 17 T cells by treatment with SEB and Methotrexate (MTX).
  • MTX Methotrexate
  • Figure 7 shows the dose of methotrexate required to prevent SEB induced T cell blasts.
  • Figure 8 shows the deletion of V ⁇ 2 T cells by TSST-1 + methotrexate/dexamethasone.
  • Figure 9 shows the prevention of IL-2 and TNF ⁇ production in response to SEB by pre-administration of dexamethasone (DEX).
  • DEX dexamethasone
  • Figure 10 shows the ability of dexamethasone to prevent superantigen induced toxicity in mice.
  • Figure 11 shows a primate system to study SEB mediated T cell deletion. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
  • the present invention describes a method for the treatment of certain diseases and medical conditions.
  • Medical conditions and diseases that may be treated according to the present invention are those that are induced or maintained, either in their inception or course of action, by subpopulations of T cells that are defined by the V ⁇ element which comprises their T cell antigen receptor (TCR).
  • TCR T cell antigen receptor
  • superantigens interact with amino acid residues that are on the outer walls of the binding cleft but bind to the exterior of the MHC TCR pocket.
  • Normal superantigens are capable, therefor, of activating a specific subset of TCRs. This feature may be taken advantage of in situations where the particular subsets of V ⁇ s that are marked by a superantigen may also be implicated in the causation or mediation of a disease or medical condition.
  • the present invention specifies the administration of an agent, preferably a superantigen or superantigen derivative, capable of marking certain T cell subpopulations, based upon their TCR V ⁇ expression, for subsequent inactivation or deletion by the administration of an additional agent or agents.
  • the superantigen or superantigen derivative administered acts to "mark" a given subset of V ⁇ containing T cells.
  • the V ⁇ s that are marked are those that are naturally associated with that superantigen. In such cases, the marking is generally the activation of the T cells that have been targeted.
  • the TCR V ⁇ s that are marked for subsequent inactivation or deletion include V ⁇ s other than those naturally targeted by the unmodified superantigen.
  • the marking of T cell subpopulations does not necessarily give rise to the activation of the T cells; marking in some other way may distinguish or target the selected subset of T cells so that they can be selectively inactivated or deleted by the agent or agents co-administered with the superantigen or superantigen derivative.
  • a cytotoxic agent is administered along with the superantigen or modified superantigen that will selectively kill that T cells that have been marked by the superantigen.
  • the cytotoxic agent will preferentially kill rapidly dividing activated T cells. In doing so, those selected T cell TCR V ⁇ subsets associated with the disease or medical condition are specifically killed, therefor acting as a method for the prevention or treatment of the disease or medical condition.
  • SEB activates T cells expressing TCRs comprised of V ⁇ 3, 12, 14, 15, 17 and 20.
  • T cells expressing these V ⁇ s will begin to proliferate and thus become susceptible to killing by cytotoxic agents. That is to say, SEB is used to kill certain T cells by causing them to proliferate in the presence of, for example, methotrexate.
  • the additional agent is an immunosuppressive agent, such as a corticosteroid.
  • a key event in the immune response of an animal is the interaction of MHC molecules with antigens to form a complex presented to T cells.
  • Animal is defined as an organism of the kingdom Animalia, as distinguished from plants by certain typical characteristics.
  • a subject animal is preferably a vertebrate, and more preferably a mammal, in whom it is desirable to modify the responsiveness of certain subpopulations of T cells.
  • T cells The "antigens" generally recognized by T cells consist not of intact proteins but of peptide fragments bound to a binding groove in an MHC molecules.
  • the T cell response to a particular antigen/MHC complex is highly specific and requires interaction of most or all of the components of both chains of the T cell receptor. Thus only a very small number of T cells respond to a given antigen.
  • Superantigen means a protein which upon binding to an MHC molecule is capable of interacting with subpopulations of T cells which express TCRs comprised of particular V ⁇ s—all other components of the TCR being essentially inconsequential to this interaction.
  • Superantigens differ from the antigens generally recognized by T cells in two important ways. First the superantigen generally binds to the MHC molecule as an intact protein (not as a peptide) and it binds to a different site than do peptide antigens. Second the T cell response requires the interaction of only the V ⁇ element of the T cell receptor—all other components of the TCR being essentially inconsequential. This means that the superantigen is able to react with a much larger number of T cells than are peptide antigens.
  • Superantigen species includes “superantigens” or “superantigen derivatives”.
  • Superantigen derivative means a molecule whose structure, at least, contains an amino acid sequence substantially identical to an amino acid sequence presented by a superantigen or portions of a superantigen required for binding to either the MHC or the T cell. It includes both mutated and modified superantigen derivatives.
  • “Mutated superantigen derivative,” or “fragment,” is defined as a molecule where the actual amino acid sequence of the mutation has been altered relative to the native form of the molecule.
  • the alteration may be a substitution, an addition or a deletion.
  • Modified superantigen derivative is defined as a molecule that contains an amino acid sequence identical to an amino acid sequence of superantigens, but contains modifications other than mutations not found in the superantigen molecule itself. Examples would include additions of carbohydrate or lipid moieties.
  • Cytotoxic agent means any natural or synthetic substance having the ability to kill cells or having a destructive action upon cells, for example, methotrexate, etc. It specifically includes substances that have a predilection for activated T cells. Activation of T cells leads to several phenotypic changes which may render them more susceptible to cytotoxic agents. The most obvious examples of such phenotypic changes include the state of rapid cell division and the expression or up-regulation of surface molecules such as Fas. Rapidly dividing cells are more susceptible to a number of cytotoxic agents and the expression of Fas makes the cells susceptible to killing by Fas ligand.
  • Immunosuppressive agent means any natural or synthetic substance having the ability to reduce, for example, the response to autoimmune disease, an immune response by way of interfering with lymphocyte growth.
  • immunosuppressive agents can include antimetabolites, irradiation or antibodies.
  • Subpopulation means a subset of T cells as defined by a specific
  • T cells expressing particular V ⁇ elements are known in the art as a T cell subpopulation.
  • the present invention is embodied in a method for the co- administration of a superantigen with one or more cytotoxic agents to modify the responsiveness of pathogenic subsets of T cells with minimal accompanying release of potentially toxic immune mediators in the prevention or treatment of, for example, autoimmune diseases.
  • Staphylococcal enterotoxin B SEB
  • which engages V ⁇ 3, 12, 14, 15 and 17 TCRs that have been implicated in rheumatoid arthritis is administered in conjunction with methotrexate and dexamethasone.
  • Methotrexate is commonly used in rheumatoid arthritis though its precise mechanism of action is unknown. It is demonstrated, in Example 3 and Fig. 6, in vitro methotrexate administration in combination with SEB prevents the generation of T cell blasts and leads to the disappearance of most V ⁇ 3, 12, 14 and 17 cells in human peripheral blood leukocytes (hPBLs). However, interleukin 2 (IL-2) as well as other cytokines are still produced. In order to reduce the production of IL-2 and tumor necrosis factor alpha (TNF ⁇ ), an immunosuppressive agent, for example, dexamethasone, is added to the regimen.
  • an immunosuppressive agent for example, dexamethasone
  • the goal of the instant invention is the elimination of subsets of T cells which include pathogenic T cells, without unduly incapacitating the overall immune system or causing unacceptable toxicity.
  • the dose of methotrexate is expected to be well tolerated by most subjects, particularly, as only a single dose may be required. Preliminary experiments show methotrexate does not prevent the release of IL-2. Even though this and other cytokines play a beneficial role in immune responses, their release in large quantities is potentially toxic. An example of this toxicity is that which occurs in Toxic Shock Syndrome.
  • the therapeutic composition of the present invention is preferably administered parenterally by injection or by continuous infusion by an implanted pump. Also, other effective administration forms, for example, parenteral slow- release formulations, are also envisioned..
  • the preferred carrier is physiological saline solution, but it is contemplated that other pharmaceutically acceptable carriers may also be used.
  • the specific dose is calculated according to the approximate body weight of the patient. Further, the refinement of calculations necessary to determine the appropriate dosage for treatment involving each of the formulations is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them without undue experimentation.
  • hPBLs human peripheral blood lymphocytes
  • MHC class II molecules e.g., monocytes, dendritic cells and macrophages
  • the amount of proliferation is quantitated by measuring the amount of tritiated ( 3 H) thymidine incorporated into newly synthesized DNA following a three day stimulation.
  • Venous blood is obtained from human subjects and the mononuclear cells (hPBLs) are isolated by centrifugation over ficol using standard techniques. 10 5 hPBLs are incubated in round bottom 96 well plates in a final volume of 100 ⁇ l of IMDM containing 10 % fetal calf serum and the indicated molar concentration of Staphylococcal enterotoxin B (Sigma, St. Louis, MO) or toxic shock syndrome toxin (Toxin Technologies, Sarasota, FL) for three days in a humidified atmosphere of 10 % CO at 37° C. Cultures are then pulsed with 0.5 ⁇ Ci 3 H-thymidine (Amersham,
  • hPBLs are stained with fluorochrome-conjugated monoclonal antibodies specific for various cell-surface molecules and are examined by flow cytometry.
  • Fig.2 shows the light scatter characteristics of hPBLs that have been cultured in vitro for three days.
  • the Y axis shows the ability of the cells to scatter light in the forward direction. This ability directly correlates with the size of the cells.
  • the X axis shows the ability of cells to scatter light at a 90° angle and correlates with the cells' granularity.
  • Bitmap A is where the small resting B and T cells are found; bitmap C represents the dead and dying cells; and bitmap B is where T cell blasts are found.
  • panel A there is no population of blast cells, however there is a poorly defined population of cells to the right of B. These cells are shown, using monoclonal antibodies, to be primarily monocytes.
  • Panel B shows the emergence of a large population of T cell blasts in region B following stimulation with SEB.
  • T cells it is possible to demonstrate that T cells expressing particular V ⁇ s are selectively enriched in the blast population and depleted from the resting population. Because neither the resting cell population nor the blast population are completely pure and because CD4 + T cells respond moderately better to superantigen stimulation, two-color analysis using fluorochrome labeled antibodies to CD4 and to specific V ⁇ s is routinely performed. Fig. 3 shows the results of such an analysis.
  • Human peripheral blood lymphocytes are stimulated in vitro for 3 days with 1 ng/ml of SEB (3.3 x 10 "12 M) and stained with phycoeiythrin conjugated monoclonal antibody to CD4 (DAKO, Carpinteria, CA) and with FITC conjugated monoclonal antibody to V ⁇ 3 (T Cell Diagnostics, Cambridge, MA) and analyzed on a Coulter flow cytometer.
  • SEB 3.3 x 10 "12 M
  • DAKO phycoeiythrin conjugated monoclonal antibody to CD4
  • FITC conjugated monoclonal antibody to V ⁇ 3 T Cell Diagnostics, Cambridge, MA
  • the panel on the left shows the light scatter profile of the resulting cells.
  • Bitmaps A, B, and C encircle the populations of resting cells, proliferating cells (blasts), and dying cells respectively.
  • the lower right panel shows an analysis of the resting cells.
  • V ⁇ 3 cells fall in quadrants 1 and 2 while the V ⁇ 3 cells fall in quadrants 2 and 4.
  • V ⁇ 3 cells comprise 4.1 % of the CD4 + cells.
  • V ⁇ 3 cells are shown to comprise 30.5 % of the blast cells.
  • hPBLs are cultured at 10 6 /ml for the number of days indicated in Fig. 4 and each aliquot analyzed by flow cytometry.
  • Cell viability is determined by the number of cells per ml that fall in the A and B bitmaps described in Example 2 and for V ⁇ 3 (T Cell Diagnostics, Cambridge, MA) and phycoerythrin conjugated anti-CD4 (DAKO, Carpinteria, CA). Cell viability falls off immediately to about 65 % at day 1 through 3. Cell viability then drops to about 30 % by day 5, where it remains until day 9.
  • V ⁇ 3 T cells among the resting CD4 + T cells is examined. This percentage remains constant at about 8 % over the entire 9 day experiment. Accordingly, this assay can be used to examine the effects of superantigens on human T cells in culture.
  • An alternative approach is to subvert what is normally a stimulatory effect on T cells in order to induce the cells to suicide. This is analogous to current therapy for autoimmune and neoplastic diseases that exploit the rapid proliferation of pathogenic cells in order to render them more susceptible to toxic agents.
  • methotrexate MTX
  • High dose methotrexate works by inhibiting folate metabolism, and thus, DNA synthesis.
  • the mechanism of low dose methotrexate, as used in rheumatoid arthritis is unknown, but there is evidence to support that it may involve interference with adenosine metabolism (Kremer ( 1994) J. Rheumatol.21: 1 ).
  • Fig. 5 The effect of methotrexate on the emergence of a population of T cell blasts following a 3 day in vitro culture of hPBLs in the presence of SEB is illustrated in Fig. 5.
  • hPBLs are cultured at 10 6 /ml either alone, in the presence of 1 ng/ml SEB, or in the presence of 1 ng/ml SEB + 0.1 ⁇ g/ml methotrexate.
  • the left and center panels in Fig. 5 reiterate the findings shown above in Fig. 3. That is, culture with SEB leads to the appearance in bitmap B of a distinct population of blasts.
  • methotrexate to this regimen nearly abolishes this blast population (right panel) while leading to the presence of more dead and dying cells in bitmap C.
  • Two-color flow cytometric analysis of hPBL was performed following in vitro culture under the following conditions: PBL alone, hPBL cultured at 10 6 /ml in IMDM plus 10% fetal bovine serum for 4 days; SEB alone, SEB added on day 1 at 1 ng/ml; and SEB+MTX+DEX, MTX (0.1 ⁇ g/ml) (Sigma, St. Louis, MO) and DEX (100 ⁇ g/ml) (Gensia (Elkins-Sinn), Irvine, CA) are added on day 0 and SEB (1 ng/ml) is added on day 1. On day 4 all cells are stained with anti-V ⁇ 2, 3, 12 and 17 and anti-CD4 as described in Example 3.
  • results for PBLs from 2 subjects are shown in Fig. 6. In all cases the results are expressed as the percent of CD4 + T cells which express a given V ⁇ . V ⁇ 2 expressing T cells are not stimulated by SEB and are included in the instant protocol as a control.
  • V ⁇ 2 resting and total T cells actually increases slightly upon SEB stimulation with or without the addition of MTX and DEX. This is to be expected and reflects the loss from these populations of the SEB stimulated V ⁇ 3, 12, 14 and 17 T cells. Conversely, the percent of V ⁇ 3, 12 and 17 resting T cells decreases with SEB stimulation and SEB + MTX + DEX stimulation. As expected, the percent of V ⁇ 3, 12 and 17 T cells is increased in the blast population, together accounting for 40-50 % of the total blasts. Less than 100 % is indicative of the fact that V ⁇ 14, 15 and 20 expressing T cells are also stimulated by SEB.
  • FIG. 7 A dose-response titration for the ability of MTX to prevent the appearance of T cell blasts in the presence of 1 ng/ml SEB is shown in Fig. 7.
  • the experiment is performed as described above by culturing hPBLs for 3 days in the presence of SEB and MTX and determining the number of T cell blasts per 10,000 resting cells by flow cytometry using light scatter profiles.
  • the experiment is conducted either without (W/O) or with (W) dexamethasone (100 ⁇ g/1) present.
  • a dose of 0.1 ⁇ g/ml of MTX is sufficient to decrease the blasts to background levels, corresponding to a concentration of 2.2 x 10 '7 M - a concentration that compares with serum levels in rheumatoid arthritis patients following conventional low dose MTX therapy (Kremer et al. (1986) Arthritis and Rheumatology 22:832).
  • MTX + DEX in combination with a superantigen to achieve deletion of T cells is not unique to SEB, as shown in Fig. 8.
  • TSST-1 Toxin Technologies, Sarasota, FL
  • V ⁇ 2 T cells specifically deleting V ⁇ 2 T cells.
  • superantigens stimulate a much larger percentage of total T cells than do conventional antigens, they also stimulate the release of much larger amounts of cytokines. While these cytokines play an essential role in the response to conventional antigens, superantigens stimulate the release of toxic levels.
  • Preliminary experiments demonstrate that although the addition of MTX prevents the accumulation of T cell blasts in response to superantigen stimulation, it does not prevent cytokine release.
  • DEX prevents cytokine release but is not very effective in preventing the accumulation of T cell blasts. Consequently, these two therapeutic agents are combined in the practice of the instant invention.
  • Example 2 demonstrates that DEX does not interfere with the ability of MTX to delete superantigen activated T cells. The following protocol gives rise to data that demonstrate that dexamethasone inhibits superantigen mediated cytokine release in vitro and that prevents toxicity in an in vivo mouse model.
  • IL-2 interleukin 2
  • TNF ⁇ interleukin 2
  • SEB syntheticly-produced at Supragen, Lakewood, CO
  • MTX is added at 0.1 ⁇ g/ml
  • DEX is added at 100 ⁇ g/ml.
  • IL-2 is measured by assaying culture supernatants for the ability to promote the growth of the IL-2 dependent cell line HT-2 as described in Kappler et al. ((1981) J. Exp. Med. 151:1198). Briefly, 4000 HT-2 cells were added to 2- fold dilution's of culture supernatant in a 96 well plate with a final volume of 100 ⁇ l of dumdum plus 10 % fetal bovine serum. Following overnight culture HT-2 cell viability is assessed visually and then quantitated by the MTT assay (Mosmann (1983) J. Immunol. Meth. 65:55). The minimal amount of IL-2 required to maintain greater than 90 % survival of the HT-2 cells is 10 units. Levels of TNF ⁇ (R & D Systems, Minneapolis, MN) are determined by ELISA following the manufacturers instructions.
  • Example 4 A System to Test the Method in Rhesus Macaques
  • Fig. 11 shows a preliminary experiment using PBLs from a Rhesus Macaque (PBLs provided by Dr. Peter Didier, Tulane Regional Primate Center, Covington, LA). The experiment is carried out as described for human PBLs. Briefly, monkey PBLs are isolated over ficol and put into culture at 10 6 /ml in IMDM plus 10 % FCS for three days. Where indicated in the figure, SEB is added at 0.1 ng/ml and MTX at 0.1 ⁇ g/ml. This preliminary experiment demonstrates several critical features.

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Abstract

L'invention concerne un procédé permettant de traiter des pathologies induites par des lymphocytes T pathogènes, dont la majorité expriment des récepteurs superficiels constitués d'une ou de quelques régions Vβ, à l'aide d'un superantigène utilisé pour marquer les lymphocytes T exprimant lesdites régions Vβ, en vue de leur inactivation ou de leur suppression par un agent cytotoxique. Ce procédé n'altère pas les lymphocytes T qui expriment des récepteurs superficiels comprenant d'autres Vβs et laisse par conséquent le système immunitaire intact dans sa globalité. Ce procédé peut par exemple s'utiliser dans le traitement de maladies auto-immunes.
PCT/US1996/008193 1995-06-07 1996-05-31 Procede d'utilisation de superantigenes pour cibler des sous-populations de lymphocytes t WO1996040235A1 (fr)

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AU59600/96A AU5960096A (en) 1995-06-07 1996-05-31 Method to use superantigens to target subpopulations of t ce lls

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US47254795A 1995-06-07 1995-06-07
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998029444A1 (fr) 1996-12-30 1998-07-09 Yissum Research Development Company Of The Hebrew University Of Jerusalem Exotoxines pyrogenes a large spectre, antagonistes et vaccins
EP0994717A1 (fr) * 1997-01-24 2000-04-26 Autoimmune, Inc. Traitement des maladies auto-immunes a l'aide d'une tolerance induite en combinaison avec du methotrexate
WO2003031471A1 (fr) * 2001-10-09 2003-04-17 The Government Of The United States Of America, As Represented By The Department Of Health And Human Services Procedes et compositions pour l'elaboration et la purification d'enterotoxine b staphylococcique de recombinaison (rseb)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AUPN870396A0 (en) * 1996-03-14 1996-04-04 Australian National University, The Treatment of auto-immune insulin-dependent diabetes mellitus

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Title
CANCER RESEARCH, Volume 44, issued November 1984, CESARIO et al., "Effect of Antineoplastic Agents on Interferon Production in Human Peripheral Blood Mononuclear Cells", pages 4962-4966. *
EUROPEAN JOURNAL IMMUNOLOGY, Volume 22, issued 1992, GONZALO et al., "Expansion and Clonal Deletion of Peripheral T Cells Induced by Bacterial Superantigen is Independent of the Interleukin-2 Pathway", pages 1007-1011. *
EUROPEAN JOURNAL IMMUNOLOGY, Volume 23, issued 1993, LUSSOW et al., "Peripheral Clonal Deletion of Superantigen-Reactive T Cells is Enhanced by Cortisone", pages 578-581. *
J. EXP. MED., Volume 176, issued July 1992, VANIER et al., "Cyclosporin A Markedly Enhances Superantigen-Induced Peripheral T Cell Deletion and Inhibits Anergy Induction", pages 37-46. *
J. EXP. MED., Volume 177, issued May 1993, GONZALO et al., "Glucocorticoid-Mediated Control of the Activation and Clonal Deletion of Peripheral T Cells In Vivo", pages 1239-1246. *
JOURNAL OF NEUROIMMUNOLOGY, Volume 43, issued 1993, SOOS et al., "Treatment of PL/J Mice with the Superantigen, Staphylococcal Enterotoxin B, Prevents Development of Experimental Allergic Encephalomyelitis", pages 39-43. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 269, No. 51, issued 23 December 1994, MEHINDATE et al., "Induction of Chemokine Gene Expression by Major Histocompatibility Complex Class II Ligands in Human Fibroblast-Like Synoviocytes Differential Regulation by Interleukin-4 and Dexamethasone", pages 32063-32069. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998029444A1 (fr) 1996-12-30 1998-07-09 Yissum Research Development Company Of The Hebrew University Of Jerusalem Exotoxines pyrogenes a large spectre, antagonistes et vaccins
US7189398B2 (en) 1996-12-30 2007-03-13 Yissum Research Development Company Of The Hebrew University Of Jerusalem Broad spectrum pyrogenic exotoxins antagonists and vaccines
EP0994717A1 (fr) * 1997-01-24 2000-04-26 Autoimmune, Inc. Traitement des maladies auto-immunes a l'aide d'une tolerance induite en combinaison avec du methotrexate
EP0994717A4 (fr) * 1997-01-24 2000-07-26 Autoimmune Inc Traitement des maladies auto-immunes a l'aide d'une tolerance induite en combinaison avec du methotrexate
WO2003031471A1 (fr) * 2001-10-09 2003-04-17 The Government Of The United States Of America, As Represented By The Department Of Health And Human Services Procedes et compositions pour l'elaboration et la purification d'enterotoxine b staphylococcique de recombinaison (rseb)

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