WO1997024140A1 - Interaction de proteines hla avec des membres de la famille hsp70 de proteines - Google Patents

Interaction de proteines hla avec des membres de la famille hsp70 de proteines Download PDF

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Publication number
WO1997024140A1
WO1997024140A1 PCT/US1997/000117 US9700117W WO9724140A1 WO 1997024140 A1 WO1997024140 A1 WO 1997024140A1 US 9700117 W US9700117 W US 9700117W WO 9724140 A1 WO9724140 A1 WO 9724140A1
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peptide
hla
hsp70
ofthe
binding
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PCT/US1997/000117
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English (en)
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Carol Clayberger
Alan M. Krensky
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The Board Of Trustees Of Leland Stanford Junior University
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Priority to AU18236/97A priority Critical patent/AU1823697A/en
Publication of WO1997024140A1 publication Critical patent/WO1997024140A1/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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • 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

Definitions

  • T-cell Because ofthe crucial role that the T-cell plays at the center of a major component ofthe immune system, it remains of great importance to be able to understand how T-cells are selected, activated or tolerized. By understanding the role that various participants play in T-cell activation, there will be opportunities to regulate the immune system, either enhancing the immune response, where one is dealing with vaccines, pathogens, neoplasia, or the like, or diminishing the immune response, where one is dealing with autoimmunity or organ transplantation.
  • HLA A2 peptides can regulate cytolysis by human allogeneic T lymphocytes.
  • WO93/17699 describes the activity of peptides from the HLA- A and -B ⁇ l 25 and ⁇ 2 helices in modulating CTL activity. HLA-B2702.75-84 peptide is described.
  • the HLA-B2702.75-84 peptide has been shown to be effective in modulating immune system activity, particularly the activity of T-cell function, particularly activation and T-cell mediated responses.
  • the underlying molecular mechanism by which the HLA-B2702.75-84 peptide exerted immunomodulating activity was not understood.
  • the present invention has advanced the state ofthe art by identifying the molecular interaction involved in HLA- B2702.75-84 activity. By identifying the pertinent molecular interaction, the present invention provides a basis for identifying new immunomodulating agents which are more effective or more selective than HLA-B2702.75-84 and methods for modulating immune system activity using such identified compounds.
  • the present invention is based, in part, on the identification of one ofthe mechanism by which immune responses, particularly those involving T-cells, are potentiated
  • data are provided showing that members of the HSP70 class of proteins interact with peptides corresponding to members ofthe HLA proteins to inhibit immune responsiveness mediated by T-cells, for example Cytotoxic T-cell mediated killing (CTL) and T-cell differentiation
  • CTL Cytotoxic T-cell mediated killing
  • the present invention is further based on identifying how fragments of HLA peptides, particularly the HLA-B2702 75-84 peptide, interact with a member ofthe HLA70 family of proteins to modulate immune responses
  • the ability of HLA-B2702.75-84 to block immune functions was shown to be mediated by the interaction of HLA-B2702.75-84 with a member ofthe HSP70 family of proteins This observation is important because the target of HLA-B2702 75-84 was previously unknown in the art
  • the identified HLA-B2702 75-84/HSP70 interaction ofthe present invention can be used as a basis for making and identifying agents which can modulate immune responses.
  • HSP70 and the HLA- B2702 75-84 peptide, or a HLA-B2702 75-84 or HSP70 equivalent can be used to identify compounds which block the same interactions as that blocked by the HLA- B2702 75-84
  • peptide and protein modeling techniques can be used to study the specific interactions ofthe HLA-B2702 75-84 peptide with HSP70 to rationally design or rationally select agents for testing Such agents can be used as a therapeutic agent to inhibit immune responses in a fashion similar to that know for the HLA-B2702 75-84 peptide
  • one aspect ofthe present invention provides the specific interactions which mediate the immunorepressive activity ofthe HLA- B2702 75-84 peptide As described below, this interaction can be used 1) to identify and isolate new immunomodulating agents, 2) in methods to identify agents which block the association ofthe HLA-B2702 75-84 peptide with HSP70, and 3) as a target to rationally design immunomodulating agents
  • the present invention further provides methods for identifying agents which modulate immune activity by mimicking the HLA-B2702 75-84/HSP70 interaction
  • the immunomodulating activity ofthe HLA-B2702 75-84 peptide has been shown to be based on the ability ofthe HLA-B2702 75-84 peptide to bind to a member ofthe HSP70 family of proteins Knowledge of this interaction provides a basis of identifying new immunomodulating agents
  • Such new agents are said to mimic HLA-B2702 75-84/HSP70 interaction when the agent can reduce or block the association ofthe HLA-B2702 75-84 peptide with HSP70
  • Such agents can be selected as having an equivalent activity as the HLA-B2702 75-84 peptide, as having a more selective activity than the HLA-B2702 75-84 peptide, or as having a greater activity than the HLA-B2702 75-84 peptide
  • the HLA-B2702 75-84 peptide, a fragment thereof, or a protein containing the HLA-B2702 75-84 peptide (hereinafter collectively "the HLA-B2702 75-84 peptide"), is mixed with isolated HSP70, an isolated fragment of HSP70, or a cell capable of expressing HSP70 or a HSP70 fragment (hereinafter collectively "the HSP70"), in the presence and absence of an agent to be tested
  • the two mixtures are analyzed and compared to determine if the agent blocked or reduced the amount of binding ofthe HLA- B2702 75-84 peptide with the HSP70 Agents which block or decrease the binding of the HLA-B2702 75-84 peptide with the HSP70 will be identified as decreasing the amount of binding present in the sample containing the tested agent
  • an agent is said to block or decrease HLA- B2702 75-84/HSP70 binding when the presence ofthe agent prevents or reduces the amount of association ofthe HSP70 with the HLA-B2702 75-84 peptide
  • One class of agents will reduce or block the association by binding to the HSP70 while another class of agents will reduce or block the association by binding to the HLA- B2702 75-84 peptide
  • the HLA-B2702 75-84 peptide used in the present method can either be the entire HLA-B2702 75-84 peptide, with the amino acid sequence RENLRIALRY (Sequence ID NO 1 ), a fragment ofthe HLA-B2702 75-84 peptide which binds the HSP70, or a protein which contains the HLA-B2702 75-84 amino acid sequence, such as an isolated HLA subunit, a fusion protein containing the HLA-B2702 75-84 sequence, or a larger HLA fragment such as HLA-B2702 60-84
  • a synthetic peptide corresponding to the HLA-B2702 75-84 peptide is used
  • the HLA-B2702 75-84 containing peptide can contain more than one copy ofthe HLA-B2702 75-84 sequence, such as in a palidromic or tandem repeat
  • agents identified in the present method can be substituted for the HLA-B2702 75-84 peptide
  • an agent which is found to block HLA-B2702 75-84/HSP70 binding can be used in place ofthe HLA-B2702 75-84 peptide for further screening of compounds
  • the HSP70 used in the present method can be any isolated member ofthe HSP70 family of proteins so long as the member binds the HLA-B2702 75-84 peptide
  • the HSP70 family member can be used in its entirety or a fragment ofthe HSP70 protein which contains the HLA-B2702 75-84 binding site can be used Alternatively, a cell expressing the HSP70, or HSP70 fragment, can be used
  • an HSP70 family member refers to proteins currently known in the art which are members ofthe HSP70 family of proteins (for a review see The Biology of Heat Shock Proteins and Molecular Chaperones 1994 Cold Spring Harbor Laboratory Press.) These include, but are not limited to HSP70, a peptide which is constitutively expressed In some human cells but whose expression is highly enhanced under exposure to high temperature or stress and the constitutively expressed protein, HSC70
  • the HLA-B2702 75-84 and HSP70 peptides/proteins used in the present invention can be used in a variety of forms
  • the proteins can be used in a highly purified form, free of naturally occurring contaminants Alternatively, a crude preparation containing a mixture of cellular components as well as the HLA- B2702 75-84 and HSP70 proteins can be used So long as the association ofthe HSP70 with the agent to be tested and/or the HLA-B2702.75-84 peptide can be identified in the sample, the HLA-B2702 75-84 and HSP70 proteins are in a suitable form for use in the above described assay Additionally, the HLA-B2702 75-84 and/or HSP70 proteins can be modified to contain a detectable label/signal generation system to facilitate detection Methods for attaching agents such as florescence tags and secondary labeling agents such as biotin, are well known in the art
  • a variety of art known methods can be adapted and employed to detect whether an agent blocks or reduces the interaction ofthe HLA-B2702 75-84 peptide with the HSP70.
  • Such methods include, but are not limited to, assays which employ a solid support, assays in solution phase, assays performed in a gel-type media, and assays which use a combination of these environments
  • An example of a solid phase assay would be one in which one or more ofthe HLA-B2702.75-84 or the HSP70 peptides are immobilized on a solid support and is incubated in a solution phase with the agent to be tested and the other peptide ofthe HLA-B2702 75-84/HSP70 pair
  • a secondary detection means, such as an antibody is then used to determine the amount ofthe second peptide which binds to the immobilized peptide
  • the second peptide ofthe HLA-B2702 75-84/HSP70 pair can be detectably labeled and its binding to the immobil
  • both peptides ofthe HLA-B2702 75-84/HSP70 binding pair can be in solution
  • the binding ofthe HLA-B2702 75-84 peptide to the HSP70 can be detected using a variety of methods, for example detecting mobility shifts using electrophoretic means
  • detecting mobility shifts using electrophoretic means One skilled in the art can readily appreciate how numerous assay-type formats which are known in the art for use in competitive assays can be modified to use the HLA-B2702 75-84 HSP70 pair
  • Direct binding to the HLA-B2702 75-84 peptide ofthe HSP70 can be used as first step in identifying agents which block HLA-B2702 75-84 HSP70 interaction
  • agents are first screened for the ability to bind HSP70
  • Agents which bind HSP70 are then screened for the ability to block HLA- B2702 75-84/HSP70 interaction, or for the ability to modulate a function ofthe immune system
  • Agents which are assayed in the above methods can be randomly selected or rationally selected or designed
  • an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association ofthe HLA-B2702 75-84 peptide with the HSP70
  • An example of randomly selected agents is the use a chemical library or a peptide combinatorial library
  • an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence ofthe target site and/or its conformation in connection with the agent's action
  • two sites of actions for agents ofthe present invention are the HLA protein and the HSP70 Agents can be rationally selected or rationally designed by utilizing the peptide sequences which make up the contact sites ofthe HLA-B2702 75-84/HSP70 pair
  • a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to the HLA-B2702 75-84 contact site found on the HSP70
  • Such an agent will reduce or block the association ofthe HLA-B2702 75-84 peptide with the HSP70 by binding to the HLA
  • the agents ofthe present invention can be peptides, including peptides containing modified amino acids, vitamin derivatives, as well as carbohydrates
  • a skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention or those used in the present assay methods
  • agents ofthe present invention are peptide agents whose amino acid sequences is chosen based on the amino acid sequence ofthe HLA-B2702 75-84 peptide or the HLA-B2702 75-84 contact site found on the HSP70
  • the HLA- B2702 75-84 contact site on HSP70 can readily be determined using art-known methodologies For example, tryptic digestion ofthe HSP70 protein can be performed and the various fragments ofthe HSP70 can be tested for their ability to bind the HLA- B2702 75-84 peptide
  • a modification of a bind and chew assay can be used in which the HLA-B2702.75-84 and HSP70 peptides are allowed to interact and the interactive pair is subject to protein digestion Regions ofthe HSP70 which are contacted by the HLA-B2702 75-84 peptide will be protected from digestion and can be later characterized to determine the amino acid sequence which is bound and protected
  • All ofthe peptide agents ofthe invention when an amino acid forms the C- terminus, may be in the form ofthe pharmaceutically acceptable salts or esters Salts may be, for example, Na ⁇ K ⁇ Ca +2 , Mg' 2 and the like, the esters are generally those of alcohols of 1-6C
  • Alternative peptide linking moieties can also be used to decrease the rate of degradation of peptide based agents
  • the following references describe preparation of peptide analogs which include these alternative-linking moieties Spatola, A F , Vega Data (March 1983), Vol.
  • Antipeptide peptides are another type of rationally designed peptide agent of the present invention
  • Antipeptide agents are peptides whose amino-acid sequence is specifically selected so as to interact with a target peptide sequence
  • Antipeptides can be designed using art-known methods, for example, see T K Gartner et al. , Biochem. Biophys. Res. Commun. 180 1446-1452, (1991)
  • agents ofthe present invention are antibodies immunoreactive with critical positions ofthe HLA-B2702 75-84 peptide or with the HSP70 Antibody agents are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions ofthe HLA-B2702 75-84 peptide or HSP70 which are intended to be targeted by the antibodies Critical regions include, but are not limited to, the contact sites involved in the association ofthe HLA- B2702 75-84 with the HSP70 and sights which provide steric interference upon binding
  • Antibody agents are prepared by immunizing suitable mammalian hosts in appropriate immunization protocols using the peptide haptens alone, if they are of sufficient length, or, if desired, or if required to enhance immunogenicity, conjugated to suitable carriers.
  • Methods for preparing immunogenic conjugates with earners such as BSA, KLH, or other carrier proteins are well known in the art In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective, in other instances linking reagents such as those supplied by Pierce Chemical Co , Rockford, LL, may be desirable to provide accessibility to the hapten
  • the hapten peptides can be extended at the amino or carboxy terminus with a Cys residue or interspersed with cysteine residues, for example, to facilitate linking to carrier Administration ofthe immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art.
  • Immortalized cell Unes which secrete the desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known. (See Harlow: Antibodies Cold Spring Harbor Press NY 1989) The immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the peptide hapten or is the HSP70 or HLA-B2702.74-85 peptide.
  • the cells can be cultured either in vitro or by production in ascites fluid.
  • the desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments ofthe monoclonals or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies.
  • Use of immunologically reactive fragments, such as the Fab, Fab', of F(ab') 2 fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.
  • the antibodies or fragments may also be produced, using current technology, by recombinant means. Regions that bind specifically to the desired regions of receptor can also be produced in the context of chimeras with multiple species origin. The antibodies thus produced are useful not only modulators of immune function, but are also useful in immunoassays.
  • C Uses for agents which block the association of HLA-B2702 75-84 with a member ofthe HSP70 family of proteins
  • the HLA-B2702 75-84 peptide has been shown to modulate a variety of biological responses, particularly those involving the immune system
  • the HLA-B2702.75-84 peptide has been shown to be a potent inhibitor of CTL mediated killing and has found use in increasing allograft tolerance and reducing the severity of autoimmune disorders such as rheumatoid arthritis
  • agents which bind to members ofthe HSP70 family have been shown to modulate overlapping biological responses, and in particular the immunosuppressive compound, deoxyspergualin, has been shown to bind HSP70 (Nadler Science 258 484-486 (1992)) Therefore, agents which block or reduce HLA-B2702 75-84 HSP70 binding can be used to modulate immune system responsiveness in the same fashion as shown for the HLA-B2702.75-84 peptide and immunomodulatory agents which bind HSP70
  • immune system activity such as T-cell mediated responses, can be modulated by administering to a subject
  • immune system activity refers to the wide variety of cellular events in which cells ofthe immune system participate
  • the HLA- B2702 75-84 peptide has been shown to selectively inhibit events mediated by T-cells and T-cell activation, and in particular CTL mediated killing
  • Examples of situations where it is desirable to modulated such activity includes, but are not limited to, transplant surgery and autoimmune disorders In each of these situations, it is desirable to selectively reduce T-cell responsiveness
  • an agent is said to modulate a immune system activity, or reduce the severity of a pathological condition mediated by the immune system, when the agent prevents the normal immune activity ofthe subject
  • an agent is said to modulate graft rejection when the agent reduces the rate of onset of graft rejection or reduces the severity of graft rejection
  • agents ofthe present invention can be provided alone, or in combination with another agent that modulates a function ofthe immune system
  • an agent ofthe present invention used to that reduce graft rejection can be administered in combination with other immunosuppressive agents
  • two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time
  • agents ofthe present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes Alternatively, or concurrently, administration may be by the oral route
  • the dosage administered will be dependent upon the age, health, and weight ofthe recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired
  • the present invention further provides compositions containing one or more agents ofthe present invention While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill ofthe art Typical dosages comprise 0 1 to 100 mg/kg/body wt The preferred dosages compnse 1 to 100 mg/kg body wt The most preferred dosages comprise 10 to 100 mg kg/body wt
  • a composition comprising an agent ofthe present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing ofthe active compounds into preparations which can be used pharmaceutically for delivery to the site of action
  • suitable formulations for parenteral administration include aqueous solutions ofthe active compounds in water-soluble form, for example, water-soluble salts
  • suspensions ofthe active compounds as appropriate oily injection suspensions may be administered
  • suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides
  • Aqueous injection suspensions may contain substances which increase the viscosity ofthe suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran
  • the suspension may also contain stabilizers Liposomes can also be used to encapsulate the agent for delivery into the cell
  • the pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration Indeed, all three types of formulation may be used simultaneously to achieve systemic administration ofthe active ingredient
  • Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof
  • the present invention further provides methods for increasing the affinity ofthe agents ofthe present invention, as well as other known agents which block or reduce HLA-B2702 75-84/HSP70 interaction Specifically the affinity of an agent which blocks the HLA-B2702 75-84/HSP70 interaction can be increased by covalently linking the agent to a second agent which has a equal or higher affinity for either HSP70 or HLA Such a second agent will bind to another site on either the HLA or HSP70 molecule and bring the HLA-B2702 75-84/HSP70 blocking agent into close proximity to the target site Such second agents can be, but are not limited to, antibody and peptide agents The second agent can be covalently attached to the
  • HLA-B2702 75-84/HSP70 blocking agent using art know methods Methods which employ linkers are particularly well suited for this use
  • a CTL line was labeled with 35 S-methionine and cysteine. The cells were then divided into two groups. One group was incubated at 30°C for 60 min with the following peptides:
  • Biotin-(CH)12-YRLALRLNERRENLRIALRY (The B2702 palindrome dimer) (Seq. LD No:2)
  • Biotin (CH)12-YGRLNRLSERRESLRNLRGY) (The B7 palindrome dimer)(Seq. ID No:3) The cell/peptide complexes were washed extensively and solubilized with
  • the other group of cells was first lysed in CHAPS and then incubated with the biotin-peptide-streptavidin complex. All samples were washed extensively, boiled in SDS, and separated by SDS-PAGE and subjected to fluorography A prominent band of approximately 75-80kD was detected in the lanes with the B2702 peptide, but not the B7 peptide, nor the streptavidin alone lanes, for both methods of preparation The same band was observed under the same conditions with activated CTL, the cell tumor PEER, and the EBV transformed ceil line JY.
  • the peptides synthesized include (Table 1) peptide B0701 75- 84, peptide B2702 75-84 and peptide B2705 75-84 corresponding to amino acids 75-84 ofthe alpha 1 helix ofthe HLA- B0701, HLA-B2702 and HLA-B2705 molecules Amino acid residues which comprise the "public epitope” Bw4a (B2702) or Bw6a (B0701), respectively, are underlined in Table 1 HLA class I heavy chain sequences are from Zemmour and Parham (Zemmour, J , et al.
  • B2702 84- 75/75-84 and B2702 84-75/75-84 represent inverted repeat dimers of amino acids 75-84 ofthe respective HLA class I molecule and peptide B2702 75-84/75-84 is a direct repeat of amino acids 75-84 B2702 84-75T/75-84, B2702 84-75/75-84T and B2702 84-75T/75-84T contain single or double threonine substitutions (see Table 1)
  • Peripheral blood lymphocytes were isolated from venous blood of healthy donors via Hypaque-Ficoll density centrifugation Cytotoxic T cell lines (CTL) were established from PBL by stimulation with irradiated B- lymphoblastoid cell lines (B-LCL) (allogeneic stimulation) (Buxton, S E , et al. J. Exp. Med. (1992) 175 809-820) Long-term CTL cultures were carried in T cell conditioned medium (Krensky, A M., Proc. Natl. Acad. Sci.
  • CTL Metabolic Radiolabelmg.
  • CTL were used on day 4 or day 5 after allogeneic stimulation 3 x 10 ⁇ cells/ml were pre-incubated in methionine/cysteine free RPMI 1640 (ICN Biomedical Inc , Costa Mesa, C A) supplemented with 5 % dialyzed FCS, 2 mM L-glutamine and 1.4 mM sodium pyruvate for 1 h Then [ 35 S] methionine/cysteine (50 ⁇ Ci/ml of ProMix, Amersham) was added to the culture and the cells further incubated for 4 h After radiolabeling, cells were washed twice in ice-cold PBS
  • EDTA diluted 1/10 in dH2 ⁇ and supplemented with 0 05 % deoxycholic acid, 0 01 % SDS, and a 1/10 dilution of TNEN containing 0.5 M NaCl
  • Radiolabeled cell lysates were incubated with 5 ⁇ l of normal rabbit serum and 250 ⁇ l of a 10 % solution of protein A positive S. aureus cells (Boehringer Mannheim) for 2-4 h at 4°C (pre-cl earing) S.
  • aureus cell were spun out and the precleared lysates immunoprecipitated with 5 ⁇ g of purified monoclonal antibody or 10 ⁇ l of rabbit antiserum or ascites fluid and 50 ⁇ l of packed ProteinG-Sepharose beads (Pharmacia Biotech Inc , Alameda, CA) for 1 5 h at 4°C Immune complexes were collected by centrifugation and washed sequentially in one ml of each TNEN supplemented with SDS (0 1 %) and deoxycholic acid (0 5 %), TNEN, diluted 1/10 in dH2 ⁇ and supplemented with 0 5 M NaCl
  • Electrophoresis Precipitated proteins were separated by reducing SDS-PAGE (Lammli, U K Nature (1970) 227 680-685) or isoelectric focusing electrophoresis (LEF) (Yang, S Y (1987) 332-335) Western Blot Analysis.
  • Peptide binding proteins were precipitated from unlabeled cell lysates as described above After separation on SDS-PAGE, proteins were transferred to PVDF membrane (Millipore Corporation, Bedford, MA) Immunodetection using enhanced chemiluminescence (ECL, Amersham) was performed following the manufacturer's instruction The membrane was probed first with anti-HSP70 antibody (C92, 1 2000) and horseradish peroxidase conjugated anti- mouse antibody (1 5000) After ECL-detection, the membrane was stripped off bound antibodies by incubating in a solution of 100 mM ⁇ -mercaptoethanol/2 % SDS/62 5 mM Tris-HCl pH 6 8 for 30 min at 50°C Then the membrane was re-probed using anti-HSC70 antibody (1B5, 1 5000) and horseradish peroxidase conjugated anti-rat antibody (1 5000)
  • the T cell inhibitory peptide B2702 binds to proteins with apparent molecular masses of 74kD and 70kD. Synthetic peptides corresponding to amino acid sequences ofthe alpha 1 helix of HLA class I molecules which overlap the "public epitope"
  • Bw4/Bw6 were found to inhibit T lymphocyte function in vitro (Clayberger, C , et al. J. Immunol. (1990) 144 4172-4176, Clayberger, C , et al. Transplantation Proc (1993) 25 477-478, Krensky, A M , et al. Curr. Opm. Immunol. (1994) 6 791-796) and to result in allograft tolerance in transplantation models (Nisco, S , et al. J. Immunol. ( 1994) 152 3786-3792, Buelow, R , et al. Transplantation ( 1995) 59 455- 460, Cuturi, M -C , et al.
  • peptide binding proteins were isolated from radiolabeled T cell lysates using Streptavidin-agarose beads conjugated with biotinylated peptides Precipitations were performed with the inverted repeat dimeric peptides, B2702 84-75/75-84 and B0701 84-75/75-84, in parallel Two proteins with molecular masses of 70kD and 74kD were identified which bound to the inhibitory peptide B2702, but not to the non-inhibitory peptide B0701 Additional bands of 78kD and 50kD were found with both the B2702 and B7 peptide and also with Streptavidin-agarose in the absence of any peptide, indicating that their binding is not peptide dependent When the lysis buffer was supplemented with ATP, the B2702 peptide no longer bound to the 74kD and 70kD proteins The non-hydrolys
  • Heat-shock treatment of cells before metabolic labeling resulted in a dramatic increase in intensity ofthe 70kD band and a modest increase for the 74kD band.
  • the weak signal observed for the 70kD protein under normal growth conditions therefore, reflects lower quantities ofthe radiolabeled 70kD protein present in cells grown under normal conditions, rather than lower affinity ofthe B2702 peptide for the 70kD protein compared to the 74kD protein.
  • the 74kD and 7 OkD peptide binding proteins correspond to the constitutively expressed (HSC70) and heat-inducible (HSP70) members of the HSP70 family.
  • the B2702 peptide binding proteins are members ofthe heat-shock protein 70 (HSP70) family.
  • HSP70 heat-shock protein 70
  • Different members ofthe HSP70 family were precipitated with respective antibodies and compared to the B2702 peptide binding proteins on SDS-PAGE and LEF.
  • Two glucose-regulated family members, grp78 and grp75, were tested and found to differ in molecular mass and isoelectric point from the 74kD and 70kD peptide binding proteins.
  • HSC70 and HSP70 are two highly related family members which differ in their expression pattern. HSC70 is expressed constitutively (therefore called HSC70) in all cells and only moderately upregulated upon heat-shock.
  • HSP70 also called the inducible family member
  • HSP70 is expressed constitutively only in some human cells, but its expression is highly enhanced upon exposure to high temperature.
  • the 74kD and 70kD peptide binding proteins closely resemble the HSC70 and HSP70 proteins in electrophoretic mobility on SDS-PAGE and LEF and display identical response to heat.
  • the 74kD peptide binding protein was recognized by 1B5 (specific for HSC70) and the 70kD peptide binding protein was recognized by C92 (specific for HSP70).
  • the 74kD and 70kD peptide binding proteins appeared to correspond to HSC70 and HSP70 proteins, respectively.
  • HSP70 family members are known to bind peptides and the binding motifs have been described recently (Fourie, A M , et al. J. Biol. Chem.
  • HLA-B2702 and HLA-B2705 are closely related proteins which differ by only three amino acids in the alpha 1 helix (Table 1) Synthetic peptides corresponding to that region were investigated in m vitro T cell assays and the B2705 peptide was found ineffective while the B2702 peptide inhibited T cell function (Clayberger, C , et al. Transplantation Proc.
  • immunophilins Two classes of immuno suppressant binding proteins, collectively called immunophilins, have been identified and the binding ofthe immunosuppressant to its immunophilin is required to mediate the immunosuppressive effect
  • the immunophilins of both CsA and FK506 are abundantly expressed proteins which are conserved throughout evolution They are found in different isoforms and are localized to various subcellular compartments In addition, both exhibit rotamase activity and are postulated to be involved in protein folding, reduction of protein aggregates and protein translocation (Fruman, D A , et al. FASEB J. ( 1994) 8 391 -400)
  • HSP70 proteins Glass, M-J , el al.
  • the HLA-derived peptide inhibits allospecific cytotoxic T cells and inhibits T cell proliferation, while DSG does not inhibit cytolysis
  • the HLA-derived peptide effects are exerted at the level ofthe T cell, not at the level ofthe antigen-presenting cell as demonstrated for DSG (Hoeger, P H , et al. J. Immunol. (1994) 153.3908-3916)
  • HSC/HSP70 in the peptide's immunomodulation does not involve antigen presentation
  • HSC/HSP70 proteins Consistent with the broad expression of HSC/HSP70 proteins, the 74kD/70kD peptide binding proteins were found in whole cell lysates of all cell types studied, including B-LCL, T cell tumor lines, HeLa, a pre-erythroid cell line (K562), and an endothelial cell line (SK-HEPl )
  • the T cell specific peptide effect must, therefore, be due to an additional mechanism not found in other cell types
  • Indicative of such a T cell specific mechanism is the observation that HSC/HSP70 proteins were isolated from T lymphocytes and CTL, only, when intact cells were incubated with B2702 peptides

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Abstract

La présente invention concerne l'identification de l'interaction moléculaire, laquelle est à la base de l'activité immunosuppressive du peptide HLA-B2702.75-84. L'invention dévoile notamment que le peptide HLA-B2702.75-84 se fixe à des membres de la famille HSP70 de protéines. D'après cette observation, on décrit des procédés d'identification d'agents que l'on peut utiliser pour moduler l'activité du système immunitaire.
PCT/US1997/000117 1996-01-02 1997-01-02 Interaction de proteines hla avec des membres de la famille hsp70 de proteines WO1997024140A1 (fr)

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AU18236/97A AU1823697A (en) 1996-01-02 1997-01-02 Interaction of hla proteins with members of the hsp70 family of proteins

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US58191796A 1996-01-02 1996-01-02
US08/581,917 1996-01-02
US63586096A 1996-04-22 1996-04-22
US08/635,860 1996-04-22

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998022141A2 (fr) * 1996-11-19 1998-05-28 Sangstat Medical Corporation Effets renforces pour therapeutique associee a l'haptene
EP1024701A1 (fr) * 1997-10-16 2000-08-09 Fordham University Procedes et compositions pour le traitement de maladies auto-immunes a l'aide de proteines de choc thermique
EP1135405A1 (fr) * 1998-11-20 2001-09-26 Vincent Guerriero PROTEINES CODANT POUR L'ADN QUI INHIBENT LA FONCTION Hsp70
US6696545B1 (en) 1997-04-11 2004-02-24 Sangstat Medical Corporation Cytomodulating lipophilic peptides for modulating immune system activity and inhibiting inflammation
US7094413B2 (en) 2002-01-24 2006-08-22 Sangstat Medical Corporation Combined therapy for treatment of HIV infection
US7267822B2 (en) 1997-04-11 2007-09-11 Genzyme Corporation Cytomodulating lipophilic peptides for modulating immune system activity and inhibiting inflammation
US8105608B2 (en) 2000-03-31 2012-01-31 Purdue Research Foundation Method of treatment using ligand-immunogen conjugates

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993017699A1 (fr) * 1992-03-02 1993-09-16 The Board Of Trustees Of The Leland Stanford Junior University Regulation de l'activite de lymphocytes a l'aide de peptides hla

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993017699A1 (fr) * 1992-03-02 1993-09-16 The Board Of Trustees Of The Leland Stanford Junior University Regulation de l'activite de lymphocytes a l'aide de peptides hla

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CURR. OPIN. IMMUNOL., 1994, Vol. 6, KRENSKY et al., "The Induction of Tolerance to Alloantigens Using HLA-based Synthetic Peptides", pages 791-796. *
NATURE, December 1987, Vol. 330, CLAYBERGER et al., "HLA-A2 Peptides Can Regulate Cytolysis by Human Allogeneic T Lymphocytes", pages 763-765. *
TRANS. PROC., 1993, Vol. 25, No. 1, CLAYBERGER et al., "Peptides Corresponding to T-cell Receptor - HLA Contact Regions Inhibit Class I-restricted Immune Responses", pages 477-478. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998022141A2 (fr) * 1996-11-19 1998-05-28 Sangstat Medical Corporation Effets renforces pour therapeutique associee a l'haptene
WO1998022141A3 (fr) * 1996-11-19 1999-01-07 Sangstat Medical Corp Effets renforces pour therapeutique associee a l'haptene
US6696545B1 (en) 1997-04-11 2004-02-24 Sangstat Medical Corporation Cytomodulating lipophilic peptides for modulating immune system activity and inhibiting inflammation
US7267822B2 (en) 1997-04-11 2007-09-11 Genzyme Corporation Cytomodulating lipophilic peptides for modulating immune system activity and inhibiting inflammation
EP1024701A1 (fr) * 1997-10-16 2000-08-09 Fordham University Procedes et compositions pour le traitement de maladies auto-immunes a l'aide de proteines de choc thermique
EP1024701A4 (fr) * 1997-10-16 2000-12-06 Univ Fordham Procedes et compositions pour le traitement de maladies auto-immunes a l'aide de proteines de choc thermique
EP1135405A1 (fr) * 1998-11-20 2001-09-26 Vincent Guerriero PROTEINES CODANT POUR L'ADN QUI INHIBENT LA FONCTION Hsp70
EP1135405A4 (fr) * 1998-11-20 2002-10-29 Vincent Guerriero PROTEINES CODANT POUR L'ADN QUI INHIBENT LA FONCTION Hsp70
US8105608B2 (en) 2000-03-31 2012-01-31 Purdue Research Foundation Method of treatment using ligand-immunogen conjugates
US7094413B2 (en) 2002-01-24 2006-08-22 Sangstat Medical Corporation Combined therapy for treatment of HIV infection

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AU1823697A (en) 1997-07-28

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