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EP0666867A1 - Reactifs et procedes de traitement de l'arthrite rhumatoide - Google Patents

Reactifs et procedes de traitement de l'arthrite rhumatoide

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Publication number
EP0666867A1
EP0666867A1 EP93921548A EP93921548A EP0666867A1 EP 0666867 A1 EP0666867 A1 EP 0666867A1 EP 93921548 A EP93921548 A EP 93921548A EP 93921548 A EP93921548 A EP 93921548A EP 0666867 A1 EP0666867 A1 EP 0666867A1
Authority
EP
European Patent Office
Prior art keywords
dqw2
patients
antigen
rheumatoid arthritis
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP93921548A
Other languages
German (de)
English (en)
Other versions
EP0666867A4 (fr
Inventor
Michael B. Brenner
Harout Dersimonian
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Brigham and Womens Hospital Inc
Original Assignee
Brigham and Womens Hospital Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Brigham and Womens Hospital Inc filed Critical Brigham and Womens Hospital Inc
Publication of EP0666867A1 publication Critical patent/EP0666867A1/fr
Publication of EP0666867A4 publication Critical patent/EP0666867A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention is directed to reagents which are capable of preventing, suppressing, or treating immune-related disease such as autoimmune disease.
  • the invention is directed to peptides derived from major histocompatibility complex HLA molecules, T cell receptors, and other autologous antigens responsible for autoimmunity in rheumatoid arthritis.
  • the invention is also related to antibodies directed against the MHC antigen- presenting molecule.
  • the HLA system of antigens is controlled by genes located on the short arm of chromosome 6 in the major histocompatibility complex (MHC) region. This region has well-defined counterparts in other mammals such as mouse, dog, horse, pig, rat, and guinea pig.
  • MHC major histocompatibility complex
  • the HLA antigens are composed of two major classes: class I and class II. Class I and class II antigens differ in their biochemistry, cellular distribution, expression, and function. For a detailed examination of these features, see Dorf, M.E., ed., The Role of the Major Histocompatibility Complex in Immunobiology, New York: Garland STPM
  • class I and class II molecules consist of a 44,000 kd heavy chain noncovalently bound to
  • Class I molecules are ubiquitously distributed and appear on the surface of all cells, whereas class II molecules are restricted to B-lymphocytes, monocytes, macrophages, dendritic cells, Langerhans cells of the skin, and activated T cells.
  • Class I molecules transport endogenous antigens, such as viral antigens, through the Golgi apparatus to the cell surface, whereas class II molecules transport endocytosed exogenous antigens, such as bacterial antigens, through the lysosomal/endosomal system.
  • Class I molecules interact primarily with cytotoxic (CD8 + ) T-cells, whereas class II molecules interact with helper (CD4 + ) T-lymphocytes (Bjorkman, P.J., et al , Ann. Rev. Biochem. 59:253- 288 (1990)).
  • HLA DQ antigens are relevant to the present invention, since the association with autoimmune disease presented herein is with this HLA antigen.
  • the HLA D region on the short arm of chromosome 6 has been subdivided into several subregions, including DR, DQ and DP. The subdivision is classically defined by serologic and cellular typing methods, and recently has been defined by biochemical and molecular analyses.
  • HLA-Al is the 1 allele at the HLA-A locus.
  • Alleles that have been tentatively assigned to a given locus but are not yet officially recognized are designated by a "w" (for "workshop") placed before the number, for example, HLA-DQw2.
  • w for "workshop" placed before the number, for example, HLA-DQw2.
  • the HLA system is extremely polymorphic, having multiple different alleles at each known locus. For example, there are at least 23 distinct alleles at the HLA-A locus, and at least 47 distinct alleles at the HLA-B locus. Each allele determines a product.
  • the products of the HLA-A, -B, -C, -D, -DR, -DQ and -DP alleles are cell surface molecules that bear the antigenic determinants.
  • haplotype Since one chromosome is inherited from each parent, there are two HLA haplotypes. Because all HLA genes are co-dominant, both alleles at a given HLA locus are expressed, and two complete sets of HLA antigens can be detected on cells.
  • the HLA type of an individual is usually given as the phenotype and designates all HLA antigens possessed by the individual, for example, HLA-Al, -A2, -B7, -B12, -Cwl, -Cw2, -Dw2,
  • the DQ a and ⁇ chains have 234 and 229 amino acids, respectively.
  • the DQ genomic subregion contains two chain genes and two ⁇ chain genes.
  • T cells The thymus derived lymphocytes (T cells) mediate two general types of immunologic functions: effector and regulatory. Effector functions include delayed hypersensitivity, allograft rejection, tumor immunity, and graft-versus- host reactivity. These effector functions reflect the ability of T lymphocytes to secrete proteins called lymphokines, and the ability to kill other cells (referred to as "cytotoxicity").
  • the regulatory functions of T cells are represented by their ability to amplify cell-mediated cytotoxicity by other T cells and immunoglobulin production by B cells. T cells are antigen-specific. The specificity is the result of receptor molecules that are capable of recognizing antigen.
  • the receptor consists of two chains: an acidic chain (molecular weight 45,000-55,000) and a more basic ⁇ chain (molecular weight 40,000-50,000), which are linked by a disulfide bond on the T cell surface. Both and ⁇ chains are integral membrane proteins. Both chains can be divided into variable and constant region domains. The variable region interacts with antigen, and thus the amino acid sequences of the variable region of different antigen-specific T cell clones are different. The amino acid sequences of the constant region from these same clones are similar. This general structure is analogous to the variable and constant region domains noted for immunoglobulin molecules.
  • the genetic loci encoding the T cell receptor genes can be divided into the following regions: variable region genes (V), diversity segment genes (D), joining region genes (J), and constant region genes (C).
  • V, D and J regions represent gene clusters. There are approximately 100 different V region genes for the a chain and around 80 different V region genes for the ⁇ chain. There are at least two D region genes for the ⁇ chain. There are around 100 J region genes for the a chain and 13 J region genes for the ⁇ chain. There is 1 C region gene for the a chain and 2 C region genes for the ⁇ chain. Rearrangements occur among these genes in a fashion analogous to that demonstrated for immunoglobulin genes. This provides the diversity to account for the existence in each individual of more than 1 million distinct T cell clones, each of which has a different antigen specificity.
  • the / ⁇ T cell antigen receptor heterodimer does not recognize soluble antigen alone.
  • the antigen receptor recognizes antigen in conjunction with products of MHC genes.
  • recognition occurs in conjunction with class II molecules.
  • recognition is in conjunction with class I molecules.
  • large soluble antigens are processed by an appropriate accessory cell, such as a macrophage or dendritic cell.
  • the sequence of events involved in T cell recognition of antigen is as follows.
  • the antigen is phagocytosed by an antigen-presenting cell, internalized, processed, and then a peptide is expressed on the cell surface in conjunction with class I or class II MHC molecules.
  • the T cell antigen receptor heterodimer then recognizes the antigen plus the MHC gene product.
  • T cell activation The events required for T cell activation are as follows. Interaction between the antigen receptor complex on the T cell and antigen plus MHC gene products on accessory cells generates biologically active metabolites from membrane inositol. Another signal, initiated by interleukin-1, is sometimes required. In order for T cell activation (as measured by T cell proliferation) to proceed to completion, secretion of interleukin-2 must occur, and enhanced expression of the receptors for interleukin-2 must also occur. Interleukin-2 acts as an autocoid as it expands the particular T cell clone. Once T cells are activated by the signals required for antigen-specific activation, they can release lymphokines. These lymphokines act in an antigen-nonspecific fashion on other populations of mononuclear cells.
  • autoimmune disease may result from a wide spectrum of genetic and immunologic abnormalities which differ from one individual to another and may express themselves early or later in life depending on the presence or absence, respectively, of many superimposed exogenous or endogenous accelerating factors.
  • the disease process may be caused, among other things, by sensitized T lymphocytes. These lymphocytes produce tissue lesions by poorly understood mechanisms which may involve the release of destructive lymphokines or which attract other inflammatory cells to the lesion.
  • Rheumatoid arthritis a major autoimmune disease, is characterized by the presence in serum of autoantibodies directed against the Fc portion of IgG.
  • Such autoantibodies usually of IgM or IgG isotype, combine with IgG to form immune complexes that are considered to participate in the associated synovitis and vasculitis via activation of the complement cascade and attraction of polymorphonuclear cells to the sites of their deposition.
  • Some rheumatoid factors cross-react with other autoantigens, such as DNA, histones, and cytoskeletal elements.
  • Joint pathology characterized by inflammation and joint destruction, is the result of a complex interaction of cellular events (inflammatory cells, immunocompetent cells, and synovial lining cells) and their secreted products
  • the synovial tissue (pannus) in rheumatoid arthritis has the appearance of a hypercellular lymphoid organ in which the predominant lymphocytes are T cells, which make up 80% of the synovial tissue lymphocytes (Bankhurst et al. , Arthritis and Rheumatism 79:555 (1976); Kurosaka, J. Exp. Med. 758: 1191 (1983)).
  • T cell function In addition to the preponderance of T cells in the synovial pannus and an increased number of these T cells being activated in vivo, evidence for defects in T cell function and proportions have been described. Importantly, therapeutic measures that alter T cell function, such as total nodal lymphoid irradiation, significantly improve the clinical disease state, but are associated with marked toxicity (Kotzin et al , N. Engl. J. Med. 305:969 (1981)).
  • HLA molecules and T cell receptors allow the immune system to distinguish self from foreign antigens through a selection process occurring in the thymus (Dorf, M.E., ed., The Role of the Major Histocompatibility Complex in Immunobiology , New York: Garland STPM Press, 1981; Sullivan, K.A., et al , The HLA System and Its Detection," in: Rose, N.R., et al , eds., Manual of Clinical Laboratory and Immunology, pp. 835-846, Washington, DC, American Soc. for Microbiology, 1986; Braun, W.E., Clin.
  • HLA alleles have been significantly associated with a number of diseases (Braun, W.E., HLA and Disease: A Comprehensive Review, Boca Raton, FL, CRC Press, 1979; Tiwari, J.L., et al , HLA and Disease, New York, Springer-Verlag, 1985). These include HLA-DR2 in narcolepsy,
  • HLA-B27 in ankylosing spondylitis, and sero-negative arthropathies and HLA-DR3 with -DR4 in diabetes mellitus.
  • class II structural polymorphism may mediate disease susceptibility (Nepom, G.T., et al , Ann. Rev. Immunol. 9:493-525 (1991)).
  • Polymorphic residues in the class II molecule might differentially bind a putative autoantigen peptide and present it to the responding T lymphocytes.
  • the spectrum of expressed T cell receptors might be different in individuals with different class II genotypes.
  • Autoreactive T cell receptors capable of recognizing an autoantigen class II complex would more likely be selected by thymic epithelium which expressed the nonsusceptible class II antigen.
  • homologous regions could serve as an autoantigenic target with an immune response to the (bacterium or) virus leading to attack of self-cells expressing relevant class (I or) II products" (Nepom, G.T., et al.. Ann. Rev. Immunol. 9:493-525 (1991)).
  • rheumatoid arthritis has strong associations with class II antigens, Dw4 as defined by homozygous typing cells (HTC), and DR4 defined serologically.
  • Amino acids 70-74 of the DR ⁇ chains of DR1 (Dwl), DR4 (Dw4), DR14 (Dwl4), and DR15 (Dwl5) are essentially the same as the segment of the gpl 10 protein of the Epstein-Barr virus (except for a minor substitution of arginine for lysine at position 71) (Braun, W.E., HLA and Disease: A Comprehensive Review, Boca Raton, FL, CRC Press, 1979). For these reasons, scientific investigators, searching for the causative agent for rheumatoid arthritis, have postulated that immune disregulation associated with the disease may be associated with specific genes of the major histocompatibility complex.
  • the disease may be associated with clonally expanded T cells detectable as a skewing of the peripheral ⁇ T cell antigen receptor repertoire.
  • U.S. Patent No. 4,886,783 describes a method of diagnosing autoimmune diseases, including rheumatoid arthritis, based upon an expansion of V/3 gene usage in the total population of T lymphocytes in rheumatoid arthritis patients.
  • PCT International Publication No. 90/06758 which broadly covers monoclonal antibodies reactive with epitopes on the T cell antigen receptor that can be used in the diagnosis and treatment of many immune related diseases, associates rheumatoid arthritis with an increased percentage of T cells which express V ⁇ l, V/33, V/39, or V01O T cell receptor variable regions in a patient sample. Methods of diagnosing and treating rheumatoid arthritis patients with monoclonal antibodies specific for these gene products are described.
  • Copending patent application 07/750,986 describes the detection of a subset of individuals afflicted with rheumatoid arthritis using a reagent having specificity for a unique sequence within the variable region of the a chain of the T cell receptor for antigen, and specifically for V ⁇ .12.1.
  • the application discloses the expression of the V ⁇ .12.1 gene product on CD8 + T lymphocytes that is expanded in a subset of rheumatoid arthritis patients when compared to the expression of V ⁇ .12.1 on CD8 + lymphocytes of normal, healthy subjects that do not have the immune abnormality. This expression thereby enables the detection of this subset of patients with reagents that bind specifically to V ⁇ .12.1 sequences.
  • Approaches based on this principle include vaccination with whole T cells, passive blockade using antibodies which bind to the TCR, passive blockade using antibodies that bind to the MHC portion of the complex, administration of antibodies reactive with the T cell markers (e.g., CD4 + , CD8 + ), and the use of peptides which mimic the antigen of interest and compete for binding to the MHC or the TCR molecule.
  • T cell markers e.g., CD4 + , CD8 +
  • T cells expressing the TCR ⁇ heterodimer can induce idiotypic and V gene family-specific antibodies that can regulate T cell function (Owhashi, M., et al, supra; Gascoigne, N.R.J., et al, Proc. Natl Acad. Sci. USA 84:2936 (1987); Kappler, J.W., et al, Nature 332:35 (1988); Kappler, J.W., et al. , Cell 49:263 (1987); MacDonald, H.R. , et al. , Nature 332:40 (1988)).
  • antibodies that recognize the TCR V/38 sequence have been effective in the prevention and treatment of autoimmunity in mice and rats (Owhashi, M., et al, supra; Acha-Orbea, H., et al, Cell 54:263-213 (1988); Urban, J., et al, Cell 54:511-592 (1988)).
  • Obtaining such antibodies selective for V region gene products has been dependent upon the availability of T cell clones that express TCR encoded by the relevant V gene family, and requires a laborious screening procedure using whole cells to establish specificity.
  • the protective state was termed "counter-autoimmunity. " This state involves T cell clones which can specifically proliferate in response to the vaccinating T cells, can suppress effector clones in vitro (non-specifically, presumably through release of a suppressive lymphokine), and can adoptively transfer counter-autoimmunity in vivo. Such counter-autoimmunity is accompanied by suppressed delayed hypersensitivity (DH) responses to the specific epitope and prevention or remission of clinical disease.
  • DH delayed hypersensitivity
  • T cell "vaccine” preparations to be useful in humans, must be both autologous and individually specific, that is, uniquely tailored for each patient. Furthermore, the presence of additional antigens on the surface of such T cells may result in a broader, possibly detrimental, immune response not limited to the desired T cell clones (Offner, H., et al, J. Neuroimmunol. 27: 13-22 (1989)).
  • agents and pharmaceutical compositions which have the properties of specificity for the targeted autoimmune response, predictability in their selection, convenience and reproducibility of preparation, and sufficient definition to permit precise control of dosage.
  • the present invention is based upon the applicants' discovery that a subset of rheumatoid arthritis patients have a marked expansion of Vc.12.1 bearing CD8+ T cells, and that a high frequency of these patients share the extended HLA DQw2 haplotype.
  • Molecular analysis of the T cell receptors in each of three unrelated patients with V ⁇ .12.1 elevation revealed antigen- driven clonal expansions.
  • the frequency of HLA DQw2 was 86%.
  • the invention is directed to reagents that block the normal interaction of HLA DQw2 antigen with T cell receptors, and particularly the T cell receptors bearing the V ⁇ l2.1 rearrangement, and to methods of using the reagents.
  • the methods of the present invention are directed to administering
  • DQw2 antibody to DQw2 rheumatoid arthritis patients and particularly rheumatoid arthritis patients bearing T cell receptors containing the V ⁇ .12.1 rearrangement.
  • the methods of the present invention use a monoclonal antibody against the DQw2 antigen.
  • reagents of the invention include peptides, preferably of around 8-24 amino acids, that represent alterations of the normally processed endogenous antigens that are causally related to autoimmunity in rheumatoid arthritis.
  • peptides may be derived from the DQw2 molecule itself which is being recognized as a "nonself" antigen and presented either by DQw2 itself as the MHC molecule on an effector cell or by another MHC antigen presenting molecule.
  • the peptide may be derived from the T cell receptor V ⁇ l2.1 gene product.
  • the autoantigen When the autoantigen is established to be a portion of DQw2 itself, this portion is altered such that it is recognized by the DQw2 on the antigen presenting cell and either displaces the toxic autoantigen, prevents further binding of the DQw2 present on the antigen presenting cell with the toxic autoantigen, or otherwise interferes with T cell activation. This competition or displacement thus prevents the further activation of T cells which were specific for the offending autoantigen.
  • the peptide may be derived from V ⁇ .12.1 and used for essentially the same purposes.
  • the offending autoantigen may be recovered and the peptide sequenced. Subsequently, altered sequence variants of the relevant autoantigen peptide may be prepared to either displace or compete with the true endogenous offending autoantigen.
  • the peptides so prepared may then be used as a treatment to ameliorate the symptoms of rheumatoid arthritis in DQw2 patients with rheumatoid diseases, particularly rheumatoid arthritis.
  • the invention is further directed to nucleic acid sequences corresponding to the peptides of the present invention which can be cloned into appropriate expression vectors such that the peptides are produced in sufficient amounts to be therapeutically useful.
  • the peptides can be synthesized by standard chemical synthetic procedures. Brief Description of the Figures
  • Figure 1. Major histocompatibility complex HLA genes.
  • Figure 2. Expansion of V ⁇ .12 bearing T cells in rheumatoid arthritis patients.
  • A Flow cytometric analysis was performed using directly conjugated antibodies. Heparinized blood was obtained from individuals and
  • PBMC peripheral blood mononuclear cells
  • Lymphocytes were gated on the basis of forward and side scatter profiles (not shown) and analyzed for fluorescence intensity in log scale. Ten thousand viable cells were analyzed by gating on lymphocytes excluding propidium iodide. The data was analyzed by dividing the dot plot into quadrants to represent unstained cells (lower left, quadrant 3), cells stained with FITC alone (OKT4 or OKT8) (lower right, quadrant 4), cells stained with FE alone (6D6) (upper left, quadrant 1), and cells that were co-stained with
  • FIG. 3 Predicted amino acid sequences of the V ⁇ l2 containing transcripts.
  • Nucleotide sequences of V ⁇ .12.1 containing transcripts was generated by direct (V ⁇ l2.1-specific) or by iPCR (all V ⁇ s) and cloned into M13 vector. Separation of CD4 + and CD8 + T cells was carried out on Ficoll- Paque (Pharmacia Fine Chemicals, " in Uppsala, Sweden) isolated PBMC and suspended in RPMI-1640 containing 5% FCS at 5xl0 5 /ml to mix with saturating amounts of anti-CD4 (OKT4) or anti-CD8 (OKT8 and 89.2) mAb.
  • Ficoll- Paque Pharmacia Fine Chemicals, " in Uppsala, Sweden
  • T cells are enriched to greater than 98% purity as verified routinely by staining the negatively selected populations by FACS (i.e., OKT4 or OKT8 depleted cell populations).
  • Second step of selection with anti-V ⁇ l2.1 (mAb 6D6) was carried out on CD4-depleted (CD8-enriched) cells in order to isolate V ⁇ l2.1/CD8 + T cells to make RNA and use in the iPCR method.
  • RNA from CD8 + or V ⁇ l2.1 + /CD8 + T cells was carried out according to Chomczynski et al. ⁇ Anal Bioch. 762: 156 (1987). Total RNA was quantitated and analyzed for intactness by resolving on a 1 % agarose mini-gel and visualized by staining with EtBr.
  • Complementary DNA (cDNA) for the direct PCR method was synthesized in a reverse transcription reaction using reverse transcriptase and 1-5 ug of total RNA by priming with 500 ng of oligo-(dT) g primer.
  • V ⁇ s, V ⁇ s and J ⁇ s gene segments present in the V ⁇ l2.1 + /CD8 + T cells were identified by a second method that does not require prior sequence information on both ends of the DNA to be amplified.
  • This technique independently confirmed findings generated by the direct PCR method, and provided new sequence information on V ⁇ , J ⁇ and chain usage.
  • Inverse PCR (iPCR) was first established to amplify the flanking region of a known sequence within a given restriction fragment of genomic DNA. Recently, this method was shown to be applicable to the amplification of the full length cDNA of TCR ⁇ and ⁇ genes.
  • First strand cDNA was synthesized from RNA of T lymphocytes by priming with an olig-(dT) 8mer , using Moloney murine leukemia virus derived reverse transcriptase lacking RNase H activity.
  • RNase H was used to nick the mRNA strand of the RNA/DNA hybrid, generating a series of RNA primers that serve to synthesize the second strand DNA with E. coli DNA polymerase I and E. coli DNA ligase.
  • the double-stranded cDNA was blunt ended using the 3 '-5' exonuclease activity of T4 DNA polymerase and the DNA was then circularized using T4 DNA ligase.
  • This circular cDNA was used as the template for the iPCR amplification by using a pair of C ⁇ or C ⁇ primers, which are oriented in an outward direction from one another.
  • This method generated PCR products of approximately 700 bp for the TCR ⁇ chain, and for the TCR3 chain, a major (700 bp) and a minor (approximately 400 bp) corresponding to the expected VDJC/3 and DJC/3 transcript lengths.
  • This method generated up to 2xl0 6 primer specific cDNA clones starting with as little as 1 ug of total RNA
  • a library could be generated of TCR ⁇ or cDNA products from RNA isolated from as little as 2x10 of fresh peripheral T cells.
  • One of the two primers contained an artificial Sal I site and the other primer contains a Not I site, which facilitated DNA cloning and sequencing. Restriction sites in the PCR primers were used to generate sticky ends for cloning.
  • Appropriately sized DNA products were isolated from low melting point (LMP) agarose gels (BRL, Gaithersburg, MD) and ligated to vector with cloning sites.
  • LMP low melting point
  • FIG. 4 Expanded V ⁇ l2 + T cells in rheumatoid arthritis were previously activated. The cell surface expression of DR, IL-2R ⁇ and ⁇ chains, CD45RO, HLA-1 and transferrin receptor were analyzed by immunofluorescence staining as described in Figure 2. The graph represents relative expression amongst CD3 + , CD4 + , CD8 + and V ⁇ l2 + T cells.
  • LB3 .1 (anti-HLA DR), 84.19.9 (anti-IL-2R ⁇ ), Tu27 (anti-IL-2R ⁇ ), UCHL1 (anti-CD45RO), TS2/7 (anti-VLA-1), 5E9 (anti-transferrin receptor) were used at 1/100 ascites dilution with a second antibody (goat anti-Mouse immunoglobulin) conjugated with FITC or PE.
  • the present invention is directed to reagents and methods based on the human T cell receptor V ⁇ l2.1 rearrangement and the DQw2 haplotype for treating humans suffering from rheumatoid arthritis.
  • an object of the present invention is to provide compositions, pharmaceutical formulations, and methods useful for treating humans suffering from rheumatoid arthritis.
  • a still further object of the invention is to provide compositions, pharmaceutical formulations, and methods useful for preventing or attenuating the clinical symptoms of rheumatoid arthritis.
  • the invention is directed to a peptide comprising an amino acid sequence (I) that is analogous to an amino acid sequence (II) that is presented to a T lymphocyte, in rheumatoid arthritis patients, by an MHC- bearing antigen-presenting cell, wherein amino acid sequence II is derived from the DQw2 protein.
  • amino acid sequence (I) is intended a sequence which is recognized by and bound to a major histocompatibility antigen on an antigen presenting cell.
  • amino acid sequence (II) is a processed peptide derived from a naturally occurring protein in these patients which is treated by the patient's immune system as a nonself antigen, processed, and presented to a T lymphocyte by an MHC-bearing antigen presenting cell as a foreign antigen. This presentation thus activates specific T lymphocytes to complete an immune response against the patient's own protein.
  • amino acid sequence I is designed to bind the major histocompatibility antigen present on the antigen presenting cell, displacing the processed self peptide or preventing the binding of the processed self peptide to the major histocompatibility molecule.
  • the peptide of the present invention is derived from a persistent foreign antigen, including, but not limited to, viral antigens that are the result of a persistent viral infection.
  • amino acid sequence I is also designed to bind the major histocompatibility antigen present on the antigen presenting cell, and displacing the foreign antigen or preventing the binding of the foreign antigen to the major histocompatibility molecule or the functional interaction with the T-cell.
  • the peptide is also designed to bind to the MHC molecule but to prevent interaction of the MHC/antigen complex with a relevant T lymphocyte so as to prevent the activation and clonal expansion of this T lymphocyte.
  • the alteration in the amino acid sequence of the MHC binding antigen is designed either to prevent the binding of the complex to the T cell receptor or to allow the binding of the receptor but to prevent further activation.
  • amino acid sequence comprising the peptide of this invention can be used alone, or bound to, or contained within the sequence of, a longer peptide.
  • the longer peptide may carry additional sequences derived from the parent protein of interest or may include sequences of an unrelated peptide, such as a carrier protein, used to enhance the binding of the amino acid sequence of interest to the MHC antigen or to enhance the interaction of the complex with a T lymphocyte.
  • the peptide contains an amino acid sequence (I) derived from the MHC HLA DQw2 protein. Because this protein is expressed in the subset of rheumatoid arthritis patients with a clonal expansion of T cells bearing the V ⁇ l2.1 rearrangement, the invention is specifically directed to the use of the DQw2 derived amino acid sequence I in patients bearing the V ⁇ l2.1 clonal expansion.
  • the use of the peptide is not limited to such patients, but is presumed to be effective in treating any rheumatoid arthritis patient bearing the DQw2 haplotype.
  • the MHC protein is MHC HLA DQw2 itself.
  • the DQw2 peptide is presented to a T lymphocyte by the MHC HLA DQw2 present on an antigen presenting cell.
  • amino acid sequence II, derived from DQw2 is thus presented by the DQw2 MHC protein.
  • analogs of this peptide are designed to bind to DQw2 and to thus prevent interaction with or block activation of the T cell presented with the DQw2 peptide by DQw2 itself.
  • the MHC protein is other than DQw2 and may be any of the other relevant major histocompatibility proteins found on antigen presenting cells and which function to present processed DQw2 antigens to T lymphocytes.
  • the peptide is bound to HLA DQw2, which peptide stimulates V ⁇ l2.1-bearing T cells.
  • This peptide may be derived from any self or foreign protein if it has the properties of stimulating V ⁇ 12.1 -bearing T cells.
  • the peptide contains an amino acid sequence I that is analogous to an amino acid sequence in the ⁇ chain of a T cell receptor which bears the V ⁇ l2.1 rearrangement.
  • the disease-causing peptide does not require alteration. Instead, the peptide portion which is presented by a major histocompatibility complex antigen on an antigen presenting cell, is derived from an appropriate protein but is not altered in amino acid sequence. Instead, the peptide is administered in the appropriate dose so as to induce tolerance, which entails diminished T cell activity.
  • the present invention is broadly directed to a method for preventing, ameliorating, or otherwise treating rheumatoid arthritis by the administration of any of the peptides described above in amounts sufficient for such amelioration, prevention, or treatment.
  • the reagents of the present invention when utilized in the methods of the present invention, may act on a number of levels. Accordingly, the efficacy of the reagents includes, but is not limited to, preventing antigen presentation, preventing T cell activation, or inducing a state of anergy in the T cell population.
  • the present invention is thus directed to a method for preventing antigen presentation in patients with rheumatoid arthritis, said patients expressing the MHC HLA DQw2 allele, comprising administering any of the peptides described above in amounts sufficient to prevent the presentation.
  • the invention is further directed to a method for preventing T cell activation in patients with rheumatoid arthritis, which patients express the MHC HLA DQw2 allele, comprising administering any of the peptides described above in amounts sufficient to prevent the activation.
  • the methods of the present invention are also broadly useful for preventing or ameliorating a chronic inflammatory response in patients with rheumatoid arthritis and expressing the MHC HLA DQw2 allele, the response resulting from the persistent activation of T lymphocytes by an MHC-bearing antigen presenting cell, particularly where the MHC is the DQw2 gene product.
  • the method comprises administering any of the proteins described above in amounts sufficient to prevent the chronic inflammatory response.
  • the reagents and methods of the present invention are also useful to induce tolerance which entails diminished T cell activity. This tolerance is induced by the administration of peptides derived from the appropriate antigens but not bearing alterations in amino acid sequence that is different from the offending protein.
  • anergy which is the inability of the recipient T cell to react to the antigen, is induced.
  • the anergy results from the tolerization of the recipient to a particular antigen, which is induced by administration of excessive amounts of the antigen.
  • the peptide may also contain an amino acid sequence I derived from the ⁇ chain of a T cell receptor bearing the V ⁇ 12.1 rearrangement.
  • a peptide is prepared which is analogous to the portion of the V ⁇ l2.1 which is presented by an MHC antigen to a T lymphocyte by an MHC-bearing antigen presenting cell.
  • the MHC molecule may be DQw2 or any other histocompatibility antigen that functions to present antigen to T lymphocytes.
  • the invention is specifically directed to the use of such a peptide in the subset of patients bearing clonal expansions of T cells containing receptors with the V ⁇ l2.1 rearrangement.
  • the invention is directed to all of the hereinbefore described methods wherein the peptide comprises an amino acid sequence I derived from the V ⁇ 12.1 rearrangement.
  • treatment with the peptides of the present invention that are derived from DQw2 is not restricted to patients with rheumatoid arthritis who bear the V ⁇ l2.1 clonal expansion. It is understood that the presentation of DQw2 as an antigen to T lymphocytes may occur wherein the T lymphocyte does not necessarily bear the V ⁇ 12.1 rearrangement but where the DQw2, recognized by a T lymphocyte as nonself, may be recognized by a T lymphocyte other than the lymphocyte with the V ⁇ l2.1 rearrangement such that said lymphocyte becomes clonally expanded.
  • the invention is also generally directed to methods using antibody reagents wherein said antibodies are developed against the DQw2 protein itself. Accordingly, the invention is directed to a method for preventing antigen presentation in patients with rheumatoid arthritis, which patients express the MHC HLA DQw2 allele, comprising administering an antibody against the MHC HLA DQw2 protein, which is effective in blocking the interaction of a DQw2-bearing antigen-presenting cell with a T lymphocyte.
  • the present invention is directed to a method for preventing T lymphocyte activation by use of the above method, a method for preventing or ameliorating a chronic inflammatory response in patients with rheumatoid arthritis and expressing the MHC HLA DQw2 allele, which response results from the persistent activation of T lymphocytes by an MHC-bearing antigen- presenting cell, by the application of the above method, and more generally, a method for treating rheumatoid arthritis in these patients.
  • the antibody may be used to bind the DQw2 molecule when it functions as an antigen-bearing MHC found on an antigen- presenting cell or may be developed against a DQw2 peptide which functions as the presented antigen.
  • the DQw2 functions to present a peptide derived from the processing of an endogenous protein and which is recognized by the T-lymphocytes as foreign.
  • the antibody may be directed against any of those portions of DQw2 which, when bound by antibody, prevent an interaction with a relevant T lymphocyte, which interaction is necessary for the subsequent activation of that T lymphocyte.
  • the antibody When the antibody is developed against the specific region of DQw2 which is presented as an antigen, the antibody likewise functions to prevent the interaction of the relevant T lymphocyte with the DQw2 peptide which has become recognized as a foreign antigen.
  • the DQw2 peptide is processed by an MHC molecule on an antigen-presenting cell.
  • This MHC molecule may be either DQw2 itself or may be one of the other histo ⁇ compatibility antigens which functions in presenting a relevant antigen to a T lymphocyte for a normal immune response.
  • V ⁇ l2.1 protein V ⁇ l2.1 protein.
  • the methods are not restricted to patients bearing
  • T cells with this rearrangement are practiced in any patient with rheumatoid arthritis, which patient expresses the DQw2 haplotype.
  • the invention also encompasses administration of the antibody of the present invention to patients wherein the processed peptide which is recognized as nonself may be any of a variety of autoantigens other than DQw2 and V ⁇ l2.1.
  • this autoantigen is presented by an antigen-presenting cell bearing the protein gene product of the DQw2 locus.
  • the methods of the present invention may be practiced either with polyclonal or monoclonal antibodies.
  • treatment is meant to include both prophylactic treatment to prevent an autoimmune disease having the symptoms of rheumatoid arthritis (or the manifestation of clinical symptoms thereof) as well as the therapeutic treatment, i.e. , the suppression or any measurable alleviation of one or more symptoms after the onset of a disease presenting the symptoms of rheumatoid arthritis.
  • autoantigen is intended for the purposes of the invention any substance normally found within a mammal that, in an abnormal situation, is no longer recognized as part of the mammal itself by the lymphocytes or antibodies of that mammal, and is therefore attacked by the immunoregulatory system as though it were a foreign substance.
  • MHC or “major histocompatibility complex” is meant a complex series of mammalian cell surface proteins present on the surface of activated T cells, macrophages and other immune cells.
  • the MHC plays a central role in many aspects of immunity, both in presenting histocompatibility (or transplantation) antigens and in regulating the immune response against conventional (foreign) antigens.
  • the human MHC genes are located on human chromosome 6 and the mouse MHC genes are located in the H-2 genetic locus on mouse chromosome 17.
  • the relevant MHC expression products are those that present DQw2 peptide in rheumatoid arthritis patients and in the DQw2 expression product as an antigen-presenting MHC molecule.
  • Class II MHC molecules are membrane glycoproteins that form part of the MHC. Class II MHC molecules are found mainly on cells of the immune system, including B cells, macrophages, brain astrocytes, epidermal Langerhans's cells, dendritic cells, thymic epithelium, and helper T cells. Class II MHC molecules are involved in regulating the immune response during tissue graft rejection, stimulation of antibody production, graft versus host reactions, and in the recognition of self (or autologous) antigens, among other phenomena.
  • T cells immune system cells derived from stem cells located within hematopoietic (i.e., blood forming) tissues.
  • T cells express either the CD4 antigen (and are then called CD4+ T cells) or the CD8 antigen (in which case they are called CD8+ T cells) on their cell surface.
  • CD4+ T cells express either the CD4 antigen (and are then called CD4+ T cells) or the CD8 antigen (in which case they are called CD8+ T cells) on their cell surface.
  • CD8+ T cells CD8 antigens by peripheral (circulating) T cells correlates with the function and specificity of the T cell.
  • T cell receptor or "TCR” is meant the antigen recognition receptor present on the surface of T cells.
  • TCR is, therefore, the receptor that binds a molecule which the immune system recognizes— and presents— as an antigen (whether the molecule is foreign or autologous, the latter being the case in an autoimmune disease).
  • a majority of T cells express a TCR composed of a disulfide bond and heterodimer protein containing one ⁇ and one ⁇ chain, whereas a minority of T cells express two different chains
  • the TCR is composed of an ⁇ and a ⁇ chain, each of which comprises a variable and a constant region.
  • the variable in turn comprises a variable, a diversity, and a joining segment.
  • the junction among the variable, diversity and joining segment is postulated to be the site of antigen recognition by T cells.
  • T cells initiate the immune response when antigen presenting cells (APC), such as mononuclear phagocytes (macrophages, monocytes), Langerhans's cells and follicular dendritic cells, initially take up, process (digest) and present antigenic fragments of their polypeptide on their cell surface (in connection with their MHC).
  • APC antigen presenting cells
  • CD4+ T cells recognize antigen molecules exclusively when the protein is processed and peptide fragments thereof are presented by APCs that express class II MHC molecules.
  • T cell recognition of an antigen reflects a trimolecular interaction between the TCR, MHC molecules and peptides processed by APCs via a cleft or pocket in the three-dimensional structure of the class II MHC molecule
  • the T cell receptor relevant to the present invention may be, but is not limited to, receptor expressing the V ⁇ l2.1 ⁇ variable region rearrangement.
  • the T cell receptor of interest to the present invention is clonally expanded in a subset of rheumatoid arthritis patients. Accordingly, the present invention is relevant to clonally expanded populations of T cells, identified by sequencing of T cell receptor cDNA, according to the exemplary material herein, which clonal expansion has resulted from interaction with an autologous antigen (autoantigen).
  • autologous antigen autologous antigen
  • MHC HLA DQw2 HLA DQw2 or “DQw2” is intended that allele of a protein encoded by the DQ region in the major histocompatibility complex of genes according to Figure 1 herein. Three alleles at the DQ locus are currently known to exist. Of these three polymorphic alleles, the second has been tentatively designated as DQw2.
  • amino acid sequence (I) is intended a sequence analog, which sequence is analogous to a sequence derived from a naturally occurring protein.
  • the amino acid sequence (I) may be from about 8-24 amino acids in length. The significance of this amino acid sequence is that it is an analog of an amino acid sequence normally found in the body and which is presented by the MHC to a T lymphocyte.
  • the amino acid sequence is analogous to that sequence in rheumatoid arthritis patients which is presented by MHC and is responsible for the T cell inflammatory response that is associated with rheumatoid arthritis.
  • amino acid sequence II is intended the naturally occurring peptide that is presented to a T lymphocyte in rheumatoid arthritis patients and which is presented to T lymphocytes in such patients and is responsible for the inflammatory response characteristic of rheumatoid arthritis.
  • amino acid sequence II is that portion of the DQw2 protein sequence which is presented to the T lymphocyte by the MHC.
  • amino acid sequence II is that portion of the receptor sequence which is presented to a T lymphocyte by an MHC antigen, particularly the DQw2 MHC antigen.
  • analog or “analogous” is intended for the purpose of the invention an amino acid sequence which is similar to a second amino acid sequence, but which alters the function of the first amino acid sequence.
  • a variant of DQw2 as the antigen being presented, is altered in sequence such that it will bind to the MHC but will prevent the activation of the T lymphocyte recognizing the true DQw2 sequence either by preventing binding to the T cell receptor or, while not preventing binding, preventing subsequent steps that lead to T cell activation. In this manner, the T lymphocyte which normally recognizes DQw2 as a foreign antigen is inhibited from activation and subsequent expansion and cytotoxic effects.
  • Antibodies may be prepared against soluble DQw2, DQw2 complexed with antigen, or DQw2 complexed with antigen as part of an antigen presenting cell/MHC/antigen complex.
  • antibodies may be prepared against DQw2 peptides complexed with reagents rendering said peptides immunogenic.
  • the amino acid sequence of the DQw2 allele is known in the art (see Figure 3, Wu et al, Human Immunol 27:305-322 (1990), which is incorporated herein by reference).
  • Antibodies may be prepared in accordance with conventional techniques, particularly employing the monoclonal antibody techniques as described, for example, in U.S. Patent Nos.4,690,893; 4,713,325; 4,714,681; 4,716,111; and 4,720,459. Any of a number of techniques may be employed for identifying the presence of a T cell receptor binding to the particular monoclonal antibody or antiserum.
  • a wide variety of labels have been used for detection, such as particles, enzymes, chromophors, fluorophors, chemiluminescents, and the like. Any particular label or technique which is employed is not critical to this invention, and any convenient technique may be employed.
  • the techniques may either be competitive or non-competitive methodologies, including sandwich methodologies.
  • the cells will usually be lysed to provide membrane-free proteins in accordance with conventional techniques. Cellular debris may be removed and the protein extracted and harvested. Alternatively, intact cells may be employed and detected by fluorescence activated cell sorting or the like.
  • mice or other lower mammals may be immunized, and the genes encoding the variable regions of the antibodies specific for the region of interest isolated and manipulated by joining to an appropriate human constant region, and optionally, the complementarity determining regions used to replace the complementarity determining regions of a human antibody by genetic engineering.
  • the resulting chimeric construct comprising a lower mammalian variable region or CDR and a human constant region may then be removed into a microorganism or mammalian host cell in culture, particularly a lymphocyte, and the hybrid antibodies expressed.
  • mouse antibodies where tolerance can be achieved or some degree of immune suppression may be involved.
  • the antibody may be formulated with conventional pharmaceutically or pharmacologically acceptable vehicles for administration, conveniently by injection.
  • Vehicles include deionized water, saline, phosphate buffered saline, Ringer's solution, dextrose solution, Hank's solution, etc.
  • Other additives may include additives to provide isotonicity, buffers, preservatives, and the like.
  • the antibody may be administered parenterally, typically intravenous or intramuscularly, as a bolus, intermittently or in a continuous regimen.
  • Typical doses for adult humans will be in the range of about 1 ng-100 mg/kg body weight.
  • a preferred dosage is between about 10 ng and 10 mg/kg, and a more preferred range is between 100 ng and 1 mg/kg.
  • Doses for children or other animal species may be extrapolated from the adult human doses based on relative body weight.
  • a random set of overlapping peptides is synthesized from the variable domain of V ⁇ 12.1.
  • DQw2 peptides random sets of overlapping peptides are synthesized from each of the following domains: ⁇ l, ⁇ 2, ⁇ l, and ⁇ 2.
  • peptides are identified by releasing them from DQw2 cells of rheumatoid arthritis patients such as by acid elution or other denaturing measures as used for release of class I peptides.
  • the peptides are separated by high pressure liquid chromatography and subjected to microsequencing for determination of the amino acid sequence.
  • the peptide sequence to be synthesized is part of a sequence of an immunogen of interest associated with an autoimmune disease.
  • the peptide sequence is that sequence which is presented by a major histocompatibility antigen to a T lymphocyte in order to induce activation of that T lymphocyte.
  • the oligonucleotide comprising the subject peptide may be from any site of the immunogen sequence, that is, N terminal or C terminal proximal or central, where the oligopeptide sequence will normally be substantially homologous with from about 8-24 amino acids of the immunogen sequence, although longer sequences may also be employed.
  • the difference in homology between a natural sequence and the oligopeptide which is employed will be not more than three lesions, usually not more than one lesion, which may be insertions, deletions, or conservative or non-conservative substitutions.
  • compositions of this invention will include usually at least one sequence of an immunogen of interest, including the subject peptide, and may include two or more oligopeptide sequences containing sequences of from about 8-24 amino acids present in the immunogen, depending on the number of presented amino acid sequences present in the immunogen. Thus, if there are a plurality of these sequences present in the immunogen, all or fewer than all of the sequences may be employed in a single composition.
  • the immunogen in preparing the subject compositions, one would select an immunogen related to the particular manifestation of autoimmunity in a patient. For example, in a patient with the V ⁇ l2.1 rearrangement and clonal expansion, the immunogen would be selected from this protein product. Similarly, in DQw2 positive rheumatoid arthritis patients, the immunogen would be selected from the DQw2 molecule. Alternatively, in DQw2 positive rheumatoid arthritis patients with a clonal expansion of a T lymphocyte containing a specific T cell receptor rearrangement, the immunogen would be said T cell receptor chain containing said rearrangement. Alternatively, the immunogen of interest may be a peptide in DQw2 positive or V ⁇ l2.1 positive patients which is other than these two proteins, but which is presented by the DQw2
  • MHC or other MHC to V ⁇ l2.1 positive T lymphocytes or other lymphocytes.
  • the subject oligopeptides may be administered in a variety of ways, by themselves or in conjunction with various additives.
  • Various carriers may be employed which are physiologically acceptable such as water, alcohol, saline, phosphate buffered saline, sugar, mineral oil, etc.
  • Other additives may also be included as stabilizers, detergents, flavoring agents, thickeners, etc.
  • the amount of active ingredient administered will vary widely depending upon the particular composition, the particular host, the number and frequency of administrations, the manner of administration, age, health, etc.
  • Peptides based on the sequence of DQw2, V ⁇ l2.1, or other clonally expanded T cell rearrangements and autologous peptides causing such clonally expanded T cells in rheumatoid arthritis patients can be synthesized using well known solid phase methods (Merrifield, R.B., Fed. Proc. Am. Soc. Exp. Biol. 27:412 (1962) and J. Am. Chem. Soc. 55:2149 (1963); Mitchel, A.R., et al,
  • these peptides can be obtained as the expression products after incorporation of DNA sequences encoding the immunogen (or fragments or analogs thereof) into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired peptides individually or as part of fusion peptides or proteins.
  • Peptide analogs can be designed using the known amino acid sequences encoded by the genes of interest as disclosed below, using the synthetic or recombinant techniques described above and the methods of, e.g., Eyler, E.H., in Adv. Exp. Med. Biol. 95:259-281 (1978).
  • a peptide having a sequence based upon the amino acid sequence of DQw2 or V ⁇ l2.1 can be chemically synthesized using the above-described techniques.
  • the peptide can be tested for disease-suppressive activity when administered to a mammal using, for example, the experimental protocol of Howell, M.D., et al , Science 246:66% (1989) or Vanderbark, A. A., et al , Nahire 341:541 (1989).
  • amino acid sequence variants of the peptide can be prepared by mutations in the DNA which encodes the synthesized peptide. Such variants include, for example, deletions from, or insertions and substitutions of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity.
  • these variants ordinarily are prepared by site- directed mutagenesis of nucleotides in the DNA encoding the peptide molecule, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.
  • the variants typically exhibit the same qualitative biological activity as the nonvariant peptide.
  • Preparation of a peptide variant in accordance herewith is preferably achieved by site-specific mutagenesis of DNA that encodes an earlier prepared variant or a nonvariant version of the relevant protein or peptide.
  • Site-specific mutagenesis allows the production of peptide variants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • the technique of site-specific mutagenesis is well known in the art, as exemplified by Adelman et al. , DNA 2: 183 (1983).
  • Typical vectors useful in site-directed mutagenesis include vectors such as the M 13 phage, for example, as disclosed by Messing et al.
  • phages are readily commercially available and their use is generally well known to those skilled in the art.
  • plasmid vectors that contain a single-stranded phage origin of replication may be employed to obtain single-stranded DNA.
  • site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector that includes within its sequence a DNA sequence that encodes the relevant peptide.
  • An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example, by the method of Crea et al. , Proc. Natl. Acad. Sci. (USA) 75:5765 (1978).
  • This primer is then annealed with the single-stranded protein-sequence-containing vector, and subjected to DNA- polymerizing enzymes such as E. coli polymerase I Klenow fragment, to complete the synthesis of the mutation-bearing strand.
  • a mutated sequence and the second strand bears the desired mutation.
  • This heteroduplex vector is then used to transform appropriate cells and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.
  • the mutated protein region may be removed and placed in an appropriate vector for protein production, generally an expression vector of the type that may be employed for transformation of an appropriate host.
  • An example of a terminal insertion includes a fusion of a signal sequence, whether heterologous or homologous to the host cell, to the N- terminus of the peptide molecule to facilitate the secretion of mature peptide molecule from recombinant hosts.
  • variants are those in which at least one amino acid residue in the peptide molecule, and preferably only one, has been removed and a different residue inserted in its place. Such substitutions preferably are made in accordance with the following list when it is desired to modulate finely the characteristics of a peptide molecule.
  • Substantial changes in functional or immunological properties are made by selecting substitutions that are less conservative that those in the above list, that is, by selecting residues that differ more significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the bulk of the side chain.
  • substitutions that in general are expected to those in which (a) glycine and/or proline is substituted by another amino acid or is deleted or inserted; (b) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (c) a cysteine residue is substituted for (or by) any other residue; (d) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) a residue having an electronegative charge, e.g., glutamyl or aspartyl; or (e) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having such a side chain, e
  • deletions and insertions, and substitutions in particular are not expected to produce radical changes in the characteristics of the peptide molecule.
  • substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays.
  • a variant typically is made by site-specific mutagenesis of the peptide molecule-encoding nucleic acid, expression of the variant nucleic acid in recombinant cell culture, and, optionally, purification from the cell culture, for example, by immunoaffinity adsorption on an anti-peptide antibody column (to absorb the variant by binding it to at least one epitope).
  • TCR V ⁇ l2.1 + T cells The relative percentage of TCR V ⁇ l2.1 + T cells in rheumatoid arthritis patients was assessed using V ⁇ l2.1-specific mAb 6D6 in both CD4 + and CD8 + subsets by two-color staining and flow cytometry.
  • V ⁇ 12.1 -normal contained percentages of TCR V ⁇ l2.1 + , CD8 + T cells (mean of 3.6%, range 1.0% to 7.0%) which was similar to those found in normal subjects (Figure 2) (Der Simonian et al, J. Exp. Med. 174:639 (1991)).
  • V ⁇ l2.1-elevated had much higher percentages of V ⁇ l2.1 + T cells (mean value of 22%, range 8.0% to 43 %) in the CD8 + subset.
  • This group of patients contained greater than 7.5 % of V ⁇ l2.1 bearing CD8 + T cells (above two standard deviations (7.3%) from the mean (3.6%) of V ⁇ 12.1 -normal and healthy controls).
  • Each of the nine patients in this V ⁇ l2.1-elevated group contained 43%, 28%, 26%, 22%, 21 %, 20%, 18%, 12%, 10% and 8.5 % V ⁇ l2.1 bearing CD8 + T cells, respectively.
  • V ⁇ l2.1 + T cells The high percentages of V ⁇ l2.1 + T cells in patients was a relatively stable phenomenon as tested over one or two year period showed consistent levels. For instance, the V ⁇ l2.1 percentage in patient #3 (MG) was 26.5% (10/89), 22.5% (5/91), and 28.5 % (6/91) and for patient #4 (AL) was 26.5% (6/91) and 19% (11/91).
  • T o gain insight into the basis for the V ⁇ l2.1 + T cell expansion in rheumatoid arthritis, V ⁇ l2.1 transcripts from positively selected CD8 + T cells were cloned and sequenced. In each of the three patients analyzed, distinct, repeated V ⁇ l2.1 containing sequences corresponding to functional TCR a-chain transcripts were identified. For example, in patient 1 where 43% of the
  • CD8 + T cells were V ⁇ l2.1 + , all 15 DNA clones analyzed had identical sequences. Similarly, 9 of 16 (56%) V ⁇ l2.1 containing DNA clones were identical in patient #2, where 26 % of COB' T cells were V ⁇ l2.1 + . In patient 3, where 28% of CD8 + T cells were V ⁇ l2.1 + , 2 distinct sequences which were repeated were identified. One sequence was represented in 16 of 23
  • V ⁇ l2.1 + T cells expressed mostly IL-2R ⁇ - chain. No significant number of freshly isolated V ⁇ l2.1 + T lymphocytes expressed the "high affinity" IL-2R ad-chains.
  • transferrin receptor on V ⁇ l2.1 + T cells also was analyzed. However, no detectable levels were found.
  • CD45RO was expressed on majority of the V ⁇ l2.1 + T cells indicating a memory phenotype.
  • V ⁇ l2.1 + T cells (10% to 30%) expressed HLA-1 and HLA-DR, suggesting ongoing activation for a fraction of these cells.
  • MHC alleles present in the V ⁇ l2.1-elevated group were examined and compared to the group of rheumatoid arthritis patients lacking expansion of V ⁇ l2.1 bearing T cells ( Figure 5). Both the V ⁇ 12.1 -elevated and the V ⁇ l2.1-normal group expressed HLA-DR1 and -DR4 alleles as expected for adult rheumatoid arthritis patients.
  • the frequency of HLA DQw2 was increased as it was found in 6 of 7 (86%) of the patients ( Figure 5).
  • the frequency for the DQw2 (DQB/0201) encoding allele, linked with DR3-(DQA1/0501) and DR7-(DQA1/0201) extended haplotype is approximately one in three (depending on the ethnic background), and a little less than 1 in 3 of the V ⁇ l2.1-normal group were DQw2 positive.
  • T cells in rheumatoid arthritis that were encoded by V ⁇ l2.1 gene segment rearrangement to Jl 1 genes containing the pro-tyr dipeptide. These populations were readily detected using the simple screening technique of mAb staining and flow cytometry, which allowed examination of a relatively large number of subjects. The expansions found were present in approximately 1 of 6 (19%) patients and only in the CD811 population of (peripheral blood) T cells. It is widely assumed that the clinical diagnosis of rheumatoid arthritis may include at least several specific diseases.
  • V ⁇ 12.1 -elevated group of rheumatoid arthritis patients represents a subset defined by an immunologic aberration, the expansion of V ⁇ l2.1 bearing CDB T cells and the increased association of the HLA DQw2 haplotype.
  • This group of patients did not differ from the V ⁇ l2.1-normal group of R3 patients in their clinical parameters (such as ESR, PCV).
  • HLA-00 molecules in the expansion of COB' T cells may be important in autoimmune regulation or immunoglobulin production as demonstrated in a different system by Salgame et al , Proc. Natl. Acad. Sci. USA 88:2598 (1991). Whether the V ⁇ l2.1 + T cell population or the antigen to which it responds are critical to the process of rheumatoid arthritis remains to be determined. However, this finding of striking clonal expansions that account for one fifth to nearly one half of the CD8 T cells in the periphery of a group of patients with rheumatoid arthritis is highly relevant to the diagnosis of rheumatoid arthritis and yields insight into the pathophysiology of the disease process.
  • ADDRESSEE Sterne, Kessler, Goldstein & Fox

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Abstract

L'invention porte sur des agents et des procédés de traitement de l'arthrite rhumatoïde. Le procédé consiste à administer des anticorps dirigés contre le complexe majeur d'histocompatibilité HLA DQw2 à des patients souffrant d'arthrite rhumatoïde de type DQw2. Dans un autre mode de réalisation, on développe des peptides imitant les self-peptides présentés aux lymphocytes T de ces patients. Ces peptides empêchent l'activation des lymphocytes T stimulée par des self-peptides dérivés de DQw2, Vα12,1, un allèle dont on retrouve l'expression sur des clones de lymphocytes T de ces patients, et d'autres antigènes autologues.
EP93921548A 1992-09-14 1993-09-14 Reactifs et procedes de traitement de l'arthrite rhumatoide. Withdrawn EP0666867A4 (fr)

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