WO1994025610A1 - Compositions et procedes d'immunotherapie au moyen du domaine alpha-3 d'une molecule d'histocompatibilite majeure de classe i - Google Patents

Compositions et procedes d'immunotherapie au moyen du domaine alpha-3 d'une molecule d'histocompatibilite majeure de classe i Download PDF

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WO1994025610A1
WO1994025610A1 PCT/US1994/005065 US9405065W WO9425610A1 WO 1994025610 A1 WO1994025610 A1 WO 1994025610A1 US 9405065 W US9405065 W US 9405065W WO 9425610 A1 WO9425610 A1 WO 9425610A1
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polypeptide
composition
domain
cell
positive
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PCT/US1994/005065
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Mark L. Tykocinski
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Tykocinski Mark L
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C07K2319/91—Fusion polypeptide containing a motif for post-translational modification containing a motif for glycosylation
    • C07K2319/912—Fusion polypeptide containing a motif for post-translational modification containing a motif for glycosylation containing a GPI (phosphatidyl-inositol glycane) anchor

Definitions

  • This invention relates to the alpha-3 (" ⁇ - 3 ”) domain, dissociated from the alpha-1 ("( ') and alpha-2 (“ ⁇ . 2 ”) domains, of a class I major histocompatibility molecule, and its uses .
  • compositions and methods for nonspecific immunosuppression have broad clinical relevance for the treatment of alloimmune and autoimmune diseases.
  • Numerous methods for generalized nonspecific immunosuppression have been described in the literature, for example, X- irradiation, cytotoxic drugs, cyclosporin A, and corticosteroids. More selective methods for nonspecific immunosuppression, affecting narrower subsets of the immune cell repertoire, have also been reported, for example, anti-CD4 antibodies (targeting CD4-positive T- cells) for autoimmune and alloimmune disease therapy, and anti-IgE antibodies (targeting IgE-positive B-cells) for allergy therapy.
  • anti-CD4 antibodies targeting CD4-positive T- cells
  • anti-IgE antibodies targeting IgE-positive B-cells
  • CD8-positive T-cells are effectors of cytotoxicity in certain diseases, such as alloimmune, autoimmune, and viral diseases. Most CD8-positive T-cells require cell surface CD8 to function as a coreceptor alongside the T- cell receptor, for activation of proliferation and triggering of cytotoxicity when stimulated by antigen- presenting cells (referred to hereinafter as "APCs") .
  • APCs antigen-presenting cells
  • CD8-positive T-cells require cell surface CD8 to function as a coreceptor alongside the T- cell receptor, for activation of proliferation and triggering of cytotoxicity when stimulated by antigen- presenting cells (referred to hereinafter as "APCs") .
  • APCs antigen-presenting cells
  • antisense RNA-mediated inhibition of CD8 expression in a CD8-positive human T-cell clone prevents antigen-specific T-cell activation of proliferation and triggering of cytotoxicity in this clone (Hambor, 168 J___ Exp
  • CD8-positive T-cells for example, Dembic, 320 Nature 232, 1986; Dembic, 326 Nature 510, 1987; and Gabert, 50 Cell 545, 1987.
  • CD8-dependent T-cells Such CD8-positive T-cells requiring CD8 coreceptor function are generally referred to as "CD8- dependent T-cells.”
  • CD8 coreceptor function is dependent upon its binding to a class I major histocompatibility complex (referred to hereinafter as "MHC-I") molecule on the APC.
  • MHC-I major histocompatibility complex
  • Anti-CD8 antibodies can interfere with the activation of CD8-dependent T-cells.
  • this antibody-mediated interference is a consequence of both the competitive blocking of CD8:MHC-I interaction (for example, Swain, 78 Proc. Natl . Acad. Sci . USA 7101, 1981; Landegren, 155 J. Exp. Med. 1579, 1982; Spits, 134 J. Immunol. 2294, 1985; Schimonkevitz, 135 J. Immunol . 892, 1985; Goldstein, 138 J. Immunol.
  • MHC-I molecules are each composed of an alpha (hereinafter referred to as "a” ) heavy chain noncovalently associated with a ⁇ 2 -microglobulin (hereinafter referred to as " ⁇ 2 m") light chain.
  • the a heavy chain has three extracellular domains (designated a. , 2 , and o- 3 ) , and a transmembrane and cytoplasmic domain (for example, Tykocinski, 133 J. of Immunology 2261, 1984) .
  • X-ray crystallographic data has established that the polymorphic ot and ⁇ - 2 domains interlock to form a nominal antigen peptide binding structure.
  • the non-polymorphic a 3 domain bridges the interlocked a 1 : a 2 unit and the membrane, and is structurally homologous to immunoglobulin constant region domains (Bjorkman, 329 Nature 506, 1987) .
  • the noncovalently associated ⁇ 2 m light chain contacts both the ⁇ - L td- ; , and ⁇ - 3 structural units of the MHC-I heavy chain.
  • the ⁇ ! 3 domain noncovalently binds ⁇ 2 va and CD8.
  • CD8 and ⁇ 2 m binding sites are on opposite sides of the ⁇ - 3 domain, it was not previously known whether the binding of CD8 and ⁇ 2 m to the ⁇ - 3 domain is interdependent; more specifically, whether ⁇ 2 m association is required for CD8 association, and whether additional contact sites within the x and/or a 2 domains are required for effective CD8 binding to MHC-I.
  • the MHC-I ⁇ - 3 domain can be produced as an independent functional unit, dissociated from the ⁇ 17 2 , transmembrane and cytoplasmic domains of the class I MHC heavy chain, as well as from ⁇ 2 m.
  • the ⁇ 3 domain unit folds properly in the absence of its natural flanking sequences within the MHC-I heavy chain and in the absence of ⁇ 2 m, and in so doing, it retains the capacity to bind to CD8 (the subunit, and perhaps the ⁇ -subunit) .
  • CD8 the subunit, and perhaps the ⁇ -subunit
  • ⁇ 2 m association is not obligatorily required for CD8 association with the MHC-I 3 domain.
  • the ⁇ : 3 domain minimal unit structure provides a suitable reagent for binding CD8 molecules and CD8-positive cells in therapeutic and diagnostic contexts.
  • the invention features polypeptide compositions which share in common the presence of a complete MHC-I 3 domain, dissociated from the normally contiguous ⁇ x and 2 domains able to bind CD8.
  • the compositions included in the invention include both soluble and membrane-binding a 3 - containing polypeptide (referred to hereinafter as " ⁇ - 3 polypeptides") variants formed by standard procedures, as described below.
  • Soluble ⁇ - 3 polypeptides consist of either the MHC-I ⁇ - 3 domain alone (monomeric or multimeric) , or this domain covalently linked (for example, in a chimeric polypeptide) to a second molecular unit which confers to the chimeric molecule desired properties.
  • the second molecule can be any one of a number of antigenic polypeptides that are well known to those in the art, including the maltose binding protein and viral hemagglutinin sequences.
  • antigenic polypeptides simplify purification of the ⁇ 3 polypeptide by affinity chromatography. If they include a protease cleavage site, they can be readily proteolytically removed from the functional 3 domain unit following purification.
  • Another example of a second molecule is a toxin; such ⁇ - 3 polypep ⁇ tide:toxin chimeras can be used effectively for eliminating CD8-positive cells from cellular populations.
  • Other antigenic polypeptides include those which stabilize the ⁇ - 3 -domain, e.g. , an Fc region which prolongs the half-life of the ⁇ 3 domain in serum. If such a polypeptide contains at least three histidines then it can be purified on a nickel-SEPHAROSE column.
  • Membrane-binding 3 polypeptides are chimeras consisting of the MHC-I ⁇ . 3 domain linked to a membrane anchor, for example a glycosyl-phosphatidylinositol
  • GPS global positioning system
  • the invention also includes expression vectors for producing such c 3 polypeptides or chimeras.
  • c 3 polypeptides or chimeras are known that many eukaryotic polypeptides will not fold properly in prokaryotes, the MHC-I ⁇ - 3 domain does.
  • ⁇ - 3 polypeptides can be produced in both prokaryotes and eukaryotes. This constitutes a significant advantage for ⁇ - 3 polypeptides over alternative CD8-binding molecules, such as antibodies, in that the former can be produced in a cost-effective fashion via prokaryotic expression systems .
  • the invention features therapeutic methods that employ ⁇ - 3 polypeptides. Some of these methods take advantage of the capacity of the ⁇ - 3 polypeptide or chimeras to bind to CD8 at cellular surfaces and effectively compete for the functional interaction between this cell surface-associated CD8 on T-cells and MHC-I on APCs. When administered in vivo to a patient suffering from CD8- positive T-cell-mediated cytodestruction, the a 3 polypeptide will competitively inhibit the activation and triggering of cytotoxicity in CD8-positive T-cells. Thus, this method provides an effective immunosuppressive therapy directed specifically at the CD8-positive T-cell subset.
  • cytotoxic CD8-positive T-cells are pathogenic in a wide range of diseases, including alloimmune, autoimmune, and certain infectious diseases such as human immunodeficiency virus infection, competitive blockade of CD8-positive T-cell activation is of considerable therapeutic benefit.
  • the a 3 polypeptides are particularly beneficial in the context of acute pathogenic processes, for example, autoimmune disease flares, wherein recombinant ⁇ - 3 polypeptides can be adminis ⁇ tered to the patient in repeated doses.
  • the invention also features the use of ⁇ - 3 multimers to direct inhibitory signaling of CD8-positive T-cells directly through their surface CD8.
  • Anti-CD8 antibodies are known to mediate inhibitory signaling in CD8-positive T-cells.
  • Multimeric a 3 polypeptides mimic anti-CD8 antibodies in the direct nonspecific inhibition of CD8- positive T-cells.
  • the ⁇ ; 3 multimers offer advantages over antibodies in this context, including the absence of antigenic immunoglobulin sequences.
  • the ⁇ 3 multimers to be used do not contain any antigenic components.
  • the invention also features therapeutic methods for treating patients with lymphomas expressing CD8 at their surface.
  • ⁇ - 3 polypeptides can be used to target toxins, such as ricin, Pseudomonas and Staphylococcal endotoxins to CD8-positive lymphoma cells.
  • ⁇ . 3 :toxin chimera conjugates can be produced using chimeric coding sequences in prokaryotic expression systems. Such conjugates can be used in several clinical contexts.
  • a preferred clinical application is the in vitro purging of CD8-positive lymphoma cells from bone marrow in the course of autologous bone marrow transplantation.
  • In vivo tumor therapeutic applications involve situati'ons wherein transient deficits in the peripheral CD8-positive T-cell population will not be problematic, for example, patients who are to undergo immunosuppressive therapy prior to autologous bone marrow transplantation.
  • the invention also includes use of a 3 polypep ⁇ tide:toxin conjugates to eliminate normal CD8-positive T- cells from cellular populations both in vitro and in vivo.
  • a preferred in vitro method is pre-treating allogeneic bone marrow to be transplanted, in order to eliminate alloreactive CD8-positive T-cells that can contribute to graft-versus-host disease.
  • This therapy can be combined with anti-CD4 antibody therapy that is currently used to eliminate CD4-positive T-cells from immune cell populations.
  • the invention also concerns diagnostic methods for detecting CD8 polypeptides.
  • the method utilizes ⁇ . 3 polypeptides as detecting reagents.
  • a major advantage of ⁇ - 3 polypeptides over the more conventional antibodies used in this context is the economy of producing large amounts of ⁇ 3 polypeptide via prokaryotic expression systems .
  • Soluble CD8 is known to be elevated in the course of a variety of infectious and other diseases.
  • ⁇ - 3 polypeptides can be incorporated into ELISA or other conventional assays for the detection of soluble CD8 in serum samples from patients suffering from diseases with this diagnostic marker.
  • ⁇ - 3 polypeptides can also be used in cellular diagnostic assays to detect CD8-positive cells.
  • Biotinylated or fluoresceinated 3 polypeptide derivatives are suitable for such detecting reagents.
  • ⁇ - 3 polypeptides can be tagged in conventional ways to permit their use for the in vivo detection of solid tumor masses containing CD8-positive tumor cells.
  • the invention also allows production of anti- ⁇ : 3 domain-specific antibodies using the isolated ⁇ 3 domain unit as an immunogen. This provides an effective means for producing such antibodies and circumvents the generation of ex. and a 2 domain-specific antibodies when the complete extracellular domain of MHC-I is used as an immunogen.
  • this invention features methods for purification of polypeptides comprising CD8 from mixtures of proteins.
  • ⁇ - 3 can be used as a ligand on solid-phase supports for affinity purification procedures.
  • the invention features a method for treating a patient suffering from an autoimmune, alloimmune, or viral disease with aberrant CD8-positive T-cell cytotoxicity, by administering to the patient a reagent comprising an MHC-I ⁇ - 3 domain (and not the , or ⁇ 2 domains) , to thereby block activation of the cell.
  • the reagent comprises, consists of, or consists essentially of, a compound selected from an isolated ⁇ - 3 domain; an ⁇ : 3 domain linked to an antigenic polypeptide; an 3 domain linked to biotin or fluorescein; an ⁇ - 3 domain linked to a toxin; an ⁇ 3 domain linked to a GPI or Fc moiety; and an ⁇ - 3 domain linked to a hydrophobic polypeptide transmembrane extension.
  • the reagent preferably competitively inhibits binding of natural MHC-I to CD8 ⁇ . or ⁇ .
  • consisting essentially of is meant to include those peptides or chimeras in which the CD8 binding portion of ⁇ -3 is provided free from the other MHC ⁇ - regions, although short regions of about 5 - 10 amino acids may be present so long as they do not affect the CD8 binding ability of the peptide.
  • the invention features a method for treating a patient with a CD8-positive T-cell lymphoma, by eradicating tumor cells with an a 3 :toxin conjugate, either in vitro or in vivo.
  • the method includes contacting the conjugate with the target cells to be killed under suitable killing conditions well known to those in the art.
  • the method is a method for purging CD8-positive lymphoma cells from a bone marrow specimen to be used for autologous bone marrow transplantation; other methods include the use of an ⁇ - 3 :toxin conjugate in vivo to eradicate tumor cells.
  • the invention features diagnostic methods for detecting CD8 in vitro or in vivo by detecting binding of the ⁇ - 3 peptide to CD8 + cells or soluble CD8. Such a method is useful for detection of the presence and amount of CD8 conjugates used in therapy. See Hambor et al . , supra.
  • the ⁇ - 3 polypeptide is incorporated into an ELISA assay to detect soluble CD8 in patients' sera.
  • the ⁇ - 3 polypeptide is conjugated to a reporter molecule such as biotin, fluorescein, or radionuclide and used to detect CD8-positive normal or tumor cells in vitro or in vivo.
  • the invention features a method for producing 3 polypeptides using an expression vector comprising sequence encoding a MHC-I 3 domain.
  • the vector is selected from a prokaryotic expression vector and a eukaryotic expression vector.
  • the invention features a method for producing MHC-I ⁇ - 3 domain-specific polyclonal and monoclonal antibodies by use of the ⁇ - 3 domain only as an immunogen.
  • the invention features a method for purifying polypeptides comprising CD8 sequences, by using the ⁇ - 3 domain bound to a solid support for affinity purification.
  • Example 1 Production of recombinant - -.maltose-binding protein chimeric polypeptide
  • the ⁇ - 3 domain of human MHC-I was produced as a fusion protein, ' linked to the maltose-binding protein (referred to hereinafter as "MalE") , in order to simplify purification of the a 3 polypeptide.
  • MalE fusion proteins can be readily purified on amylose columns.
  • the ⁇ - 3 :MalE polypeptide was expressed with a prokaryotic vector system which targets the polypeptides of interest to the outer cytoplasmic wall and the less reducing microenvironment of the periplasmic space.
  • the specific example here provides an indication of the region referred to herein as ⁇ - 3 MHCI . See Bjorkman et al . , 329 Nature 506, 1987, hereby incorporated by reference herein. Those in the art will recognize that slight variations (+5 amino acids can be readily devised using similar methodology.
  • M13mpl8/A2.1 (from J. Nuchtern, N.I.H. see Roller and Borr, 134 J. Immunol. 2727,1985) , a cDNA encoding full- length HLA-A2.1, was used as a source of a MHC-I a 3 domain.
  • An EcoRI insert containing the entire HLA-A2.1 cDNA was subcloned into pBluescript II KS (Stratagene) to generate pHLA-A2.1/BT.
  • a partial cDNA encoding the ⁇ - 3 domain of HLA-A2.1 only was generated by PCR using 5' -TAGGATCCAT- GGACGCCCCCAAAAC-3' and 5' -TAGAATTCTCACCATCTCAGGTGAGG-3 ' as 5' and 3' primers, respectively, and pHLA-A2.l/BT plasmid as template.
  • a start codon, as well as a BamHI (GGATCC) restriction endonuclease site were incorporated into the 5' -end of the 5' primer, and a stop codon and another restriction endonuclease site (EcoRI) were incorporated into the 5'-end of the 3' primer.
  • PCR reactions and thermocycling were carried out using standard procedures (see example 2 below) .
  • the digested ⁇ - 3 cDNA insert was subcloned between the BamHI and EcoRI sites in the multiple cloning site of the baculovirus vector pVL1393 (Invitrogen, San Diego, CA) , re-mobilized with BamHI/Bglll digestion, and subcloned into the prokaryotic expression vector pMALp (New England Biolabs, Beverly, MA) which carries the MalE coding sequence adjacent to the multiple cloning site of the vector.
  • pMALp New England Biolabs, Beverly, MA
  • the resulting ⁇ - 3 :MalE expression vector was designated pA2.1 ⁇ - 3 :Malp.
  • EDTA/10 mM EGTA supplemented with 50 mg lysozyme (Sigma) .
  • Coomassie Blue staining documented the production of an intact fusion protein of approximately 46 kD, as expected, which contrasted with the molecular weight of nonfused MalE produced by the vector (approximately 30 kD) .
  • the identity of the ⁇ : 3 :MalE fusion protein was confirmed by additional methods. When bound to plastic, both the fusion protein and the native nonfused peptide MalE could be detected with a rabbit antiserum directed against MalE.
  • a biotinylated form of monoclonal antibody ml00038 (anti-HLA I ABC HC) (Olympus) , which has specificity for the ⁇ - 3 domain MHC- I, could bind the fusion protein product, but not negative control proteins.
  • the 3 domain can also be readily expressed in other systems, including the Bacullovirus and yeast expression systems.
  • Example 2 Production of recombinant sCD8o- For purposes of sCD8 ⁇ - production, we chose the eukaryotic glutamine synthetase amplification/expression
  • GS GS
  • pT8Fl includes a cDNA encoding full-length human CD8 ⁇ - (see, Litman, 40 Cell 237, 1985) .
  • ⁇ - 3 :MalE The interaction between plate-bound ⁇ 3 :MalE and sCD8 ⁇ - was characterized.
  • Immulon 4 microtiter wells were coated with varying amounts of ⁇ - 3 :MalE, blocked with BSA, and incubated with 15 I-sCD8 ⁇ - at 37°C.
  • sCD ⁇ 1 was radio-iodinated using 0.8 mCi Na- 125 I (Amersham) using a standard lactoperoxidase labeling method.
  • Variable amounts of either ⁇ - 3 /MBP or a control protein were bound to wells of Immulon 4 strips (Dynatech) for 16h at 4°C.
  • Wells were then washed with de-ionized, distilled water and blocked with 0.5% BSA in DPBS for 2h at 37°C. After an additional washing step, wells were incubated with 125 I-sCD8 ⁇ ! in 0.5% BSA/DPBS in a total volume of 0.1 ml for 5h at 37°C. In some experiments, either nonradioactive "cold" sCD ⁇ . or monoclonal antibody (100 ug/ml) were added. After incubation, wells were aspirated, washed twice with 0.1% Tween 20 in DPBS, separated from other wells on the strip, and counted in a gamma counter.
  • Example 4 Monoclonal antibodies directed against either sCD8 ⁇ - or MHC ⁇ -3 inhibit binding
  • the hybridomas OKT8, W6/32, BBM.l were obtained from the American Tissue Type Culture collection.
  • Medium for the hybridoma cells was DMEM (Whittaker) without glutamine and supplemented with 4.5 gm glucose, 10% FCS (Sigma) , pyruvate, nonessential amino acids, glutamate and asparagine, penicillin/streptomycin, and glutamine.
  • Monoclonal antibodies were purified from mouse ascites using a protein A/Affi-gel Immunoglobulin Purification Kit (Bio-Rad) .
  • a protein A/Affi-gel Immunoglobulin Purification Kit Bio-Rad
  • CD8 immunosorbant approximately 50 mg of protein A-purified anti-Leu2a was coupled to 15 gm cyanogen bromide-activated Sepharose 4B CL (Pharmacia) using standard methods.
  • Applicant has demonstrated specific, saturable binding between an ⁇ - 3 polypeptide and a soluble form of CD8 ⁇ - at an appreciable affinity of interaction.
  • the specificity of this interaction was confirmed using monoclonal antibodies directed against either CD8 ⁇ : or against the ⁇ - 3 domain.
  • monoclonal antibodies directed against other sites on MHC-I and ⁇ 2 m did not block binding, confirming the conclusion that the a 3 domain is functioning as an independent unit.
  • ⁇ 3 polypeptides are smaller molecules, can be readily produced in quantity using prokaryotic expression systems, are less immunogenic, and directly compete for the precise binding site on CD8 for native MHC-I.
  • ⁇ . 3 polypeptides as described in the present invention, offer a number of advantages over soluble MHC-I molecules that incorporate the ⁇ and ⁇ - 2 domains along with the ⁇ - 3 domain for purposes of modulating cell surface CD8 function, for example, in contrast to ⁇ - ⁇ - ⁇ multidomain polypeptides, ⁇ - 3 (single domain) polypeptides are smaller, are more effective competitive inhibitors, can be readily produced in quantity using prokaryotic expression systems, do not require ⁇ 2 m for proper transport and folding, and lack the polymorphic a_ and ⁇ - 2 domains, thereby avoiding allo- immunogenicity problems in patients.
  • Second mole ⁇ cules can be selected which convey to the a 3 polypeptides desired properties.
  • the second molecule can be a toxin or radionuclide to be delivered to transformed (tumor) or nontransformed, for example, alloreactive, CD8-positive cells.
  • the second molecules can include fluoro- chromes, biotin (for binding avidin conjugates) , radionuclides, or any of a large array of epitope tags or antigenic polypeptides that are well-known to those familiar with the art.
  • CD8-positive cytotoxic T- cells are candidates for immunosuppressive therapy using c ⁇ 3 polypeptides.
  • diseases caused by other (non-T) CD8-positive cells in the body for example, subsets of natural killer cells and dendritic cells
  • patients with such diseases would also be candidates for immunosuppressive therapy using a 3 polypeptides and could be treated with 3 polypeptides by methods which parallel those provided in the present invention.
  • Pathogenic CD8-positive cytotoxic T-cells occur in a variety of disease conditions, including alloimmune, autoimmune, and infectious diseases.
  • CD8-positive T-cells are also known to contribute significantly to allograft cytodestruction in the course of sub-acute graft rejection
  • CD8-positive T-cells are also known to contribute to autoimmune cytodestruction in diseases such as multiple sclerosis and inflammatory bowel disease
  • CD8-positive T-cells have also been implicated in the extensive cytodestruction of both human immunodeficiency virus-infected and non-infected CD4- positive T-cells that occurs in the course of acquired immunodeficiency diseases.
  • ⁇ 3 polypeptides can be used to abrogate the pathogenic effects of CD8-positive cytotoxic T-cells in these diseases.
  • the first step is to identify a patient in need of cytotoxic T-cell immunosuppressive therapy.
  • Preferred diagnostic methods for accomplishing this are well known to those familiar with the art, and entail the determination of whether a patient suffers from one of the diseases known to be aggravated by CD8-positive T-cell- mediated cytodestruction.
  • ⁇ - 3 polypeptides can be used to competitively inhibit CD8-positive T-cell activation and triggering of cytotoxicity. Such therapy is of particular benefit in those clinical settings where a transient halt to an acute flare-up of a disease process is needed.
  • Pharmaceutical compositions comprising ot 3 polypeptides are administered to patients by methods appropriate for polypeptide pharmaceuticals, and such methods are well-known to those familiar with the art.
  • strategies for optimizing in vivo dosing schedules for patients to be treated are well-known.
  • the amount to be administered can be determined by routine experimentation and optimization, and is generally between 0.1 and 1000 ⁇ g/kg animal/day.
  • ⁇ . 3 polypeptides can be used to induce a persistent non-responsive state in those cells. It is known that cross- linking of surface CD8 on T-cells using anti-CD8 antibodies leads to a non-responsive state in the cells. Multimeric ⁇ - 3 polypeptides can be used effectively in place of anti-CD8 antibodies to cross-link surface CD8, for purposes of inducing immune non-responsiveness in the CD8- bearing cell . Different forms of multimeric ⁇ 3 polypeptides can be used in this context, for example, chimeric polypeptides incorporating two or more ⁇ .
  • Multimeric ⁇ 3 polypeptides can be administered to a patient alone, or in combination with other immuno- supressive agents, for example, anti-CD4 antibodies in order to immunosuppress both CD4- and CD8-positive T-cell subsets simultaneously.
  • Cytodestruction of CD8-positive T-cells can also be achieved using a 3 polypeptides.
  • the ⁇ - 3 polypeptides are used as targeting ligands to direct a cytotoxic molecule selectively to CD8-positive T-cells.
  • Any of a number of cytotoxic molecules can be covalently linked with an 3 polypeptide for this purpose, for example, ricin, or Staphylococcal endotoxin, or a radionuclide.
  • ⁇ . 3 :toxin conjugates can be used therapeutically in vivo in several ways.
  • CD8-positive tumor cells such as CD8-positive lymphoma cells
  • CD8-positive lymphoma cells can be eliminated in a patient suffering from such a tumor, by infusing ⁇ - 3 :toxin conjugates into the patient.
  • Clinical protocols have been developed for the optimal use of polypeptide :toxin conjugates for therapy of cancers, such as for the use of anti-tumor antibody:toxin conjugates; methods for using ⁇ 3 :polypeptide conjugates for cancer therapy parallel these methods.
  • 3 polypeptide: oxin conjugates have application for non- cancer diseases.
  • 3 polypeptide:toxin conjugates can be used to nonspecifically eliminate CD8- positive T-cells to interfere with an acute disease flare in an autoimmune disease such as multiple sclerosis.
  • ⁇ . 3 polypeptide:toxin conjugates in the treatment of alloimmune and certain infectious diseases, such as human immunodeficiency virus infection, will be apparent to those familiar with the art .
  • One particularly useful ⁇ - 3 polypeptide is that incorporating the ⁇ - 3 domain sequence exemplified above of HLA-A2.1 and maltose binding protein sequence. It will be obvious to those skilled in the art how to design alternative ⁇ 3 polypeptides that retain CD8 binding capacity.
  • ⁇ - 3 domain There is flexibility in the boundaries for the ⁇ - 3 domain that can be chosen within HLA-A2.1 sequence, since all that is required for efficient CD8-binding function is sufficient sequence within the ⁇ - 3 sequence to generate the ⁇ -pleated sheets and intervening sequences, together including the immunoglobulin-fold of the 3 domain. Similarly, there is flexibility in the choice of second molecules to be appended to the ⁇ - 3 domain within ⁇ - 3 polypeptides, since the ⁇ - 3 domain does not require extraneous sequences in order to assume its proper folded structure.
  • the physical binding assay described in the previous example can be used as a general method, according to this invention, to evaluate the function of any chosen ⁇ ; 3 polypeptide in vitro.
  • ⁇ . 3 polypeptides can also be used as effective in vitro therapeutics. In the latter case, the ⁇ 3 polypeptides are also being used as reagents to suppress cytotoxic CD ⁇ - positive T-cell function.
  • Purified GPI-modified proteins have the property of being reincorporable back into cell membranes. Any protein can be produced in a GPI-modified form by transfecting cells with an expression vector comprising a coding sequence for the protein of interest linked in-frame to the coding sequence for the 3'-end of a protein that is naturally GPI-modified and comprises the GPI modification signal sequence, for example, human decay-accelerating factor.
  • Alternative 3'-end sequences from GPI-modified proteins can be employed in this way in order to situate the ⁇ - 3 domain at variable distances from the cell membrane.
  • GPI chimeras can be readily purified by immunoaffinity, using anti - 3 antibodies or soluble CD ⁇ , as disclosed in the present invention, conjugated to a solid phase matrix.
  • Alternative methods for exogenous reincorporation into membranes can be employed. Standard methods include micellar transfer in the presence of low concentrations of detergent, for example, 0.004% NP-40. Reincorporation can also be efficiently accomplished, as disclosed in the present invention, by ⁇ - 3 :GPI protein transfer in the absence of detergent.
  • Detergent-free ⁇ - 3 :GPI can be readily prepared by eluting the ⁇ - 3 :GPI from the affinity column in the presence of CHAPS, a dialyzable detergent, and then dialyzing the CHAPS away from the ⁇ 3 :GPI. Detergent-free protein transfer can be used more generally for proteins other than 3 polypeptides.
  • Alternative 3 polypeptides and methods can be used for coating cells.
  • cells can be pre-coated with biotin:lipid conjugates or directly biotinylated.
  • ⁇ - 3 :streptavidin chimeras as disclosed in the present invention, can be combined with the biotin-bearing cells in order to coat them. High levels of surface a 3 can be obtained by this method.
  • ⁇ - 3 polypeptides can also be expressed at cell surfaces by gene transfer. Any one of a number of expression vector systems can be used for this purpose, for example, episomal vectors and retroviral vectors.
  • Alternative membrane-anchoring domains can be incorporated into a 3 polypeptides.
  • those comprising a GPI- anchoring domain, a non-MHC transmembrane domain comprising a hydrophobic polypeptide sequence, with or without an associated cytoplasmic extension a MHC transmembrane domain, which can include the transmembrane domain naturally associated with the ⁇ . 3 domain being used, or else, the transmembrane domain from a different class I MHC molecule, with or without an associated cytoplasmic extension.
  • a cell bearing an ⁇ - 3 polypeptide can be used in a number of therapeutic contexts to modulate CD8- positive cells. Both in vitro and in vivo applications will be readily apparent to those familiar with the art.
  • the present invention discloses methods for producing ⁇ - 3 polypeptides.
  • ⁇ - 3 polypeptides can be produced using prokaryotic expression vectors.
  • prokaryotic expression vectors unlike many other eukaryotic proteins, one is not restricted to eukaryotic expression vectors for production of this particular eukaryotic protein.
  • the possibility of producing ⁇ - 3 polypeptides by prokaryotic means makes these reagents economical .
  • Numerous prokaryotic and eukaryotic expression systems are generally available and can be readily applied to the production of ⁇ - 3 polypeptides.
  • ⁇ ; 3 polypeptides can be purified from these cells by conventional protein purification methods.
  • soluble CD8 can be readily bound to a solid phase matrix, and in turn, the CD ⁇ :solid phase matrix conjugate can be used for affinity purification of ⁇ - 3 polypeptides.
  • polypeptide tags can be linked in-frame to ⁇ 3 domain sequences.
  • epitope tags can be appended to ⁇ - 3 domain sequences to permit immunoaffinity purification with epitope-specific antibodies.
  • An example of a commonly used epitope tag is a short sequence derived from a viral hemagglutinin.
  • Other epitope tags are generally available or can be readily designed.
  • three or more histidines can be appended to either the amino or carboxy terminus of the a 3 polypeptide, in order to permit its rapid one-step purification by standard nickel-sepharose chromatography.
  • Protease cleavage sites can be inserted in between either epitope tags or poly-histidine stretches and ⁇ - 3 polypeptides to permit dissociation of the two following purification.
  • the present invention discloses an efficient method for producing ⁇ - 3 -specific monoclonal and polyclonal antibodies.
  • the findings described in the present invention disclose that at least one "pan-HLA" antibody is reactive with an a 3 domain epitope.
  • the ⁇ - 3 polypeptides are combined with the target T-cells in vitro in order to inactivate and/or eliminate the pathogenic T-cells from a cell population prior to infusion of the cell population into a patient.
  • ⁇ - 3 polypeptides relate to hematopoietic stem cell transplantation, including, but not restricted to, bone marrow transplantation.
  • one significant therapeutic application is in autologous bone marrow transplantation to be performed in cancer patients who require bone marrow reconstitution after undergoing chemotherapeutic and/or radiotherapeutic insults to their hematopoieitic stem cell pool.
  • lymphoma or leukemia patients to be so treated require purging of CD8- positive lymphoma or leukemia cells from their bone marrow prior to its autologous reinfusion.
  • a 3 polypeptide :toxin conjugates offer a preferred method for purging marrow of CD8-positive lymphoma cells. Methods for pre-treating marrow, for example, with antibody:toxin conjugates, have been well-described in the literature, and these methods can be readily adapted for use with ⁇ 3 polypeptide:toxin cojugates.
  • Another significant therapeutic application is in allogeneic bone marrow transplantation to be performed in treating patients with a wide variety of disease conditions, including cancer.
  • the therapeutic goal is to eliminate alloreactive CD ⁇ -positive T-cells from the donor marrow, in order to abrogate graft- versus-host cytotoxicity mediated by these cells.
  • the cells can be inactivated using a 3 polypeptide multimers or destroyed using ⁇ . 3 polypeptide:toxin conjugates.
  • ⁇ - 3 polypeptides as effective CD8- binding reagents has diagnostic implications.
  • ⁇ - 3 polypeptides can be substituted for anti-CD8 antibodies or anti-CD8 antibody derivatives in a wide range of diagnostic assays performed in vitro or in vivo.
  • Advantages for ⁇ . 3 polypeptides over antibodies have been described herein. From the standpoint of diagnostic assays which require large quantities of detecting reagent, a particular advantage for a 3 polypeptides is that they can be produced in quantity using inexpensive prokaryotic expression systems.
  • a preferred diagnostic method utilizing a 3 polypeptides is to detect and quantify CD8-positive cells in mononuclear cell samples from patients. For example, ratios of CD4:CD8 positive cells are determined in diagnosing and monitoring the clinical course of acquired immunodeficiency disease.
  • CD ⁇ -positive cells in the mononuclear cell populations can be detected by flow cytometry using 3 polypeptide:fluorescein conjugates. Methods for obtaining mononuclear cell populations from the peripheral blood of patients, as well as methods for processing cells for flow cytometry, are well-established in the literature.
  • a 3 polypeptides can be tagged with a variety of other well- characterized reporters, such as biotin, streptavidin, peroxidase, and used in diagnostic assays.
  • One preferred method involves chimeric polypeptides in which a tagged a 3 polypeptide reagent consists of a chimeric polypeptide, comprising an a 3 polypeptide linked in-frame, with or without a bridging polypeptide spacer, to streptavidin.
  • Other polypeptide:streptavidin chimeras have been reported, and these have been shown to retain biotin- binding capacity.
  • Another preferred diagnostic method utilizing ⁇ - 3 polypeptides is to detect and quantify CD ⁇ -positive lymphoma cells in vivo.
  • ⁇ 3 polypeptides for this purpose can be tagged with any of a number of radionuclides that are suited for in vivo detection by standard radiological techniques.
  • solid lymphoma masses can be detected at various sites throughout the body of a patient, for example, bone marrow, spleen, liver, brain.
  • antibodies are generally used for such diagnostic tumor mass detection purposes.
  • a 3 polypeptides have special advantages over antibodies for the detection of CD ⁇ -positive lymphoma masses, for example, the former are smaller and hence will penetrate tissues in a more optimal fashion.
  • the present invention discloses that not only soluble, but also cell surface-associated ⁇ - 3 polypeptides retain CD ⁇ -binding capacity.
  • 3 polypeptides can be incorporated into the surfaces of cells in order to modify their functional interactions with CD ⁇ -bearing cells.
  • ⁇ - 3 polypeptides can be incorporated into antigen- presenting cell surfaces in order to modify their interactions with CD ⁇ -positive T-cells. Such incorporation will enhance adhesive interactions between the two cell types.
  • 3 polypeptides can inhibit activation of the CD8-positive T-cells, functioning like multimeric ⁇ - 3 polypeptides.
  • ⁇ - 3 polypeptides are suitable for cell surface coating.
  • a preferred ⁇ - 3 polypeptide for this purpose is a glycosyl-phosphatidylinositol (GPI)- modified form of a 3 .
  • GPI glycosyl-phosphatidylinositol

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Abstract

L'invention se rapporte à une composition et à un procédé d'utilisation de cette composition, laquelle contient un polypeptide possédant le domaine α3 mais dépourvu des domaines α1 et α2 d'un polypeptide à chaîne lourde du complexe d'histocompatibilité majeure de classe I.
PCT/US1994/005065 1993-05-05 1994-05-03 Compositions et procedes d'immunotherapie au moyen du domaine alpha-3 d'une molecule d'histocompatibilite majeure de classe i WO1994025610A1 (fr)

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AU69080/94A AU6908094A (en) 1993-05-05 1994-05-03 Compositions and methods for immunotherapy with the alpha-3 domain of a class i major histocompatibility molecule

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997008328A1 (fr) * 1995-08-30 1997-03-06 The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services Elimination selective des lymphocites t reconnaissant des cibles specifiques preselectionnees
EP1394181A1 (fr) * 2002-09-02 2004-03-03 Boehringer Ingelheim International GmbH Adn vaccines
US9139809B2 (en) 2009-01-08 2015-09-22 Albert Einstein College Of Medicine Of Yeshiva University Bacterial vaccines with cell wall-associated ceramide-like glycolipids and uses thereof
US9371352B2 (en) 2013-02-08 2016-06-21 Vaccinex, Inc. Modified glycolipids and methods of making and using the same
US9603922B2 (en) 2007-02-21 2017-03-28 Vaccinex, Inc. Modulation of NKT cell activity with antigen-loaded CD1d molecules
US9809654B2 (en) 2002-09-27 2017-11-07 Vaccinex, Inc. Targeted CD1d molecules

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1993008817A1 (fr) * 1991-11-08 1993-05-13 The Board Of Trustees Of The Leland Stanford Junior University Peptides du domaine de liaison de cd8

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1993008817A1 (fr) * 1991-11-08 1993-05-13 The Board Of Trustees Of The Leland Stanford Junior University Peptides du domaine de liaison de cd8

Non-Patent Citations (2)

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Title
A. KRENSKY ET AL.: "Peptides corresponding to the CD8 binding region of HLA class I block the differentiation of cytotoxic T lymphocyte precursors.", TRANSPLANTATION PROCEEDINGS, vol. 25, no. 1, February 1993 (1993-02-01), NEW YORK NY, USA, pages 483 - 484 *
J. FAYEN ET AL.: "Soluble human CD8alpha binds to a recombinant fusion protein which incorporates the class I MHC alpha3 domain.", THE JOURNAL OF IMMUNOLOGY, vol. 150, no. 8(2), 15 April 1993 (1993-04-15), BALTIMORE MD, USA, pages 288A *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997008328A1 (fr) * 1995-08-30 1997-03-06 The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services Elimination selective des lymphocites t reconnaissant des cibles specifiques preselectionnees
EP1394181A1 (fr) * 2002-09-02 2004-03-03 Boehringer Ingelheim International GmbH Adn vaccines
US9809654B2 (en) 2002-09-27 2017-11-07 Vaccinex, Inc. Targeted CD1d molecules
US9603922B2 (en) 2007-02-21 2017-03-28 Vaccinex, Inc. Modulation of NKT cell activity with antigen-loaded CD1d molecules
US9139809B2 (en) 2009-01-08 2015-09-22 Albert Einstein College Of Medicine Of Yeshiva University Bacterial vaccines with cell wall-associated ceramide-like glycolipids and uses thereof
US9371352B2 (en) 2013-02-08 2016-06-21 Vaccinex, Inc. Modified glycolipids and methods of making and using the same
US10111950B2 (en) 2013-02-08 2018-10-30 Vaccinex, Inc. Modified glycolipids and methods of making and using the same

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